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Kim J, Kim S, Uddin S, Lee SS, Park S. Microfabricated Stretching Devices for Studying the Effects of Tensile Stress on Cells and Tissues. BIOCHIP JOURNAL 2022; 16:366-375. [DOI: 10.1007/s13206-022-00073-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/16/2022] [Revised: 07/05/2022] [Accepted: 07/12/2022] [Indexed: 11/02/2022]
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Yang H, Hou C, Xiao W, Qiu Y. The role of mechanosensitive ion channels in the gastrointestinal tract. Front Physiol 2022; 13:904203. [PMID: 36060694 PMCID: PMC9437298 DOI: 10.3389/fphys.2022.904203] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2022] [Accepted: 07/19/2022] [Indexed: 11/13/2022] Open
Abstract
Mechanosensation is essential for normal gastrointestinal (GI) function, and abnormalities in mechanosensation are associated with GI disorders. There are several mechanosensitive ion channels in the GI tract, namely transient receptor potential (TRP) channels, Piezo channels, two-pore domain potassium (K2p) channels, voltage-gated ion channels, large-conductance Ca2+-activated K+ (BKCa) channels, and the cystic fibrosis transmembrane conductance regulator (CFTR). These channels are located in many mechanosensitive intestinal cell types, namely enterochromaffin (EC) cells, interstitial cells of Cajal (ICCs), smooth muscle cells (SMCs), and intrinsic and extrinsic enteric neurons. In these cells, mechanosensitive ion channels can alter transmembrane ion currents in response to mechanical forces, through a process known as mechanoelectrical coupling. Furthermore, mechanosensitive ion channels are often associated with a variety of GI tract disorders, including irritable bowel syndrome (IBS) and GI tumors. Mechanosensitive ion channels could therefore provide a new perspective for the treatment of GI diseases. This review aims to highlight recent research advances regarding the function of mechanosensitive ion channels in the GI tract. Moreover, it outlines the potential role of mechanosensitive ion channels in related diseases, while describing the current understanding of interactions between the GI tract and mechanosensitive ion channels.
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Affiliation(s)
- Haoyu Yang
- Experimental Center of Basic Medicine, College of Basic Medical Sciences, Army Medical University, Chongqing, China
| | - Chaofeng Hou
- Experimental Center of Basic Medicine, College of Basic Medical Sciences, Army Medical University, Chongqing, China
| | - Weidong Xiao
- Department of General Surgery, Xinqiao Hospital, Army Medical University, Chongqing, China
| | - Yuan Qiu
- Department of General Surgery, Xinqiao Hospital, Army Medical University, Chongqing, China
- *Correspondence: Yuan Qiu,
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Takeuchi K, Ogawa H, Kuramitsu N, Akaike K, Goto A, Aoki H, Lassar A, Suehara Y, Hara A, Matsumoto K, Akiyama H. Colchicine protects against cartilage degeneration by inhibiting MMP13 expression via PLC-γ1 phosphorylation. Osteoarthritis Cartilage 2021; 29:1564-1574. [PMID: 34425229 PMCID: PMC8542595 DOI: 10.1016/j.joca.2021.08.001] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/30/2021] [Revised: 07/17/2021] [Accepted: 08/10/2021] [Indexed: 02/02/2023]
Abstract
OBJECTIVE Low molecular weight compounds that reduce the expression of MMP13 at the mRNA level might serve as disease-modifying osteoarthritis (OA) drugs (DMOADs). The objective of this study was to identify a candidate DMOAD that targets MMP13 expression. DESIGN High-throughput screening was performed to identify compounds that suppress inflammatory cytokine-induced MMP13 expression. Ingenuity pathway analysis (IPA) using isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic analysis was conducted to identify signaling pathways related to cytokines. MMP13 expression in chondrocytes was evaluated through RT-qPCR and western blotting analyses. Additionally, 10-week-old mice were subjected to destabilization of the medial meniscus (DMM) surgery to induce OA and were sacrificed 12 weeks post-surgery for pathological examination. OA was evaluated using the OARSI scoring system. RESULTS Colchicine was identified as a DMOAD candidate as it inhibited inflammatory cytokine-induced MMP13 expression in vitro, and the colchicine-administered mice with DMM presented significantly lower OARSI scores (adjusted P: 0.0242, mean difference: 1.6, 95% confidence interval (CI) of difference: 0.1651-3.035) and significantly lower synovial membrane inflammation scores (adjusted P: 0.0243, mean difference: 0.6, 95% CI of difference: 0.06158-1.138) than mice with DMM. IPA further revealed that components of the Rho signaling pathways are regulated by cytokines and colchicine. IL-1β and TNF-α activate RAC1 and SRC signals, respectively, leading to the phosphorylation of PLC-γ1 and synergistic induction of MMP13 expression. Most notably, colchicine abrogates inflammatory cytokine-induced phosphorylation of PLC-γ1, leading to the induction of MMP13 expression. CONCLUSIONS Colchicine is a potential DMOAD candidate that inhibits MMP13 expression and consequent cartilage degradation by disrupting the SRC/RAC1-phospho-PLCγ1-Ca2+ signaling pathway.
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Affiliation(s)
- K Takeuchi
- Department of Orthopaedic Surgery, Gifu University Graduate School of Medicine, Yanagido 1-1, Gifu, Gifu, 501-1194, Japan
| | - H Ogawa
- Department of Orthopaedic Surgery, Gifu University Graduate School of Medicine, Yanagido 1-1, Gifu, Gifu, 501-1194, Japan; Department of Orthopaedic Surgery, Ogaki Tokushukai Hospital, Hayashi-machi 6-85-1, Ogaki, Gifu, 503-0015, Japan.
| | - N Kuramitsu
- Department of Orthopaedic Surgery, Gifu University Graduate School of Medicine, Yanagido 1-1, Gifu, Gifu, 501-1194, Japan
| | - K Akaike
- Department of Orthopaedic Surgery, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo, 113-8431, Japan
| | - A Goto
- Department of Orthopaedic Surgery, Gifu University Graduate School of Medicine, Yanagido 1-1, Gifu, Gifu, 501-1194, Japan
| | - H Aoki
- Department of Tissue and Organ Development, Regeneration and Advanced Medical Science, Gifu Graduate School of Medicine, Yanagido 1-1, Gifu, Gifu, 501-1194, Japan
| | - A Lassar
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA, 02115, USA
| | - Y Suehara
- Department of Orthopaedic Surgery, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo, 113-8431, Japan
| | - A Hara
- Department of Tumor Pathology, Gifu University Graduate School of Medicine, Yanagido 1-1, Gifu, Gifu, 501-1194, Japan
| | - K Matsumoto
- Department of Orthopaedic Surgery, Gifu University Graduate School of Medicine, Yanagido 1-1, Gifu, Gifu, 501-1194, Japan
| | - H Akiyama
- Department of Orthopaedic Surgery, Gifu University Graduate School of Medicine, Yanagido 1-1, Gifu, Gifu, 501-1194, Japan
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Reed DA, Zhao Y, Han M, Mercuri LG, Miloro M. Mechanical Loading Disrupts Focal Adhesion Kinase Activation in Mandibular Fibrochondrocytes During Murine Temporomandibular Joint Osteoarthritis. J Oral Maxillofac Surg 2021; 79:2058.e1-2058.e15. [PMID: 34153254 PMCID: PMC8500914 DOI: 10.1016/j.joms.2021.05.001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2021] [Revised: 05/03/2021] [Accepted: 05/03/2021] [Indexed: 02/08/2023]
Abstract
PURPOSE Mechanical overloading is a key initiating condition for temporomandibular joint (TMJ) osteoarthritis (OA). The integrin-focal adhesion kinase (FAK) signaling axis is implicated in the mechanobiological response of cells through phosphorylation at Tyr397 (pFAK) but poorly defined in TMJ health and disease. We hypothesize that mechanical overloading disrupts TMJ homeostasis through dysregulation of FAK signaling. MATERIALS AND METHODS To assess if FAK and pFAK are viable clinical targets for TMJ OA, peri-articular tissues were collected from patients with TMJ OA receiving a total TMJ replacement. To compare clinical samples with preclinical in vivo studies of TMJ OA, the joints of c57/bl6 mice were surgically destabilized and treated with and without inhibitor of pFAK (iFAK). FAK signaling and TMJ OA progression was evaluated and compared using RT-PCR, western blot, immunohistochemistry, and histomorphometry. To evaluate mechanical overloading in vitro, primary murine mandibular fibrochondrocytes were seeded in a 4% agarose-collagen scaffold and loaded in a compression bioreactor with and without iFAK. RESULTS FAK/pFAK was mostly absent from the articular cartilage layer in the clinical sample and suppressed on the central condyle and elevated on the lateral and medial condyle in murine TMJ OA. In vitro, compressive loading lowered FAK/pFAK levels and elevated the expression of TGFβ, NG2, and MMP-13. iFAK treatment suppressed MMP13 and Col6 and elevated TGFβ, NG2, and ACAN in a load independent manner. In vivo, iFAK treatment moderately attenuated OA progression and increased collagen maturation. CONCLUSION These data illustrate that FAK/pFAK is implicated in the signaled dysfunction of excessive mechanical loading during TMJ OA and that iFAK treatment can moderately attenuate the progression of cartilage degeneration in the mandibular condyle.
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Affiliation(s)
- David A. Reed
- Department of Oral Biology, University of Illinois at Chicago, Chicago IL,Corresponding author: David A. Reed,
| | - Yan Zhao
- Department of Oral Biology, University of Illinois at Chicago, Chicago IL
| | - Michael Han
- Department of Oral and Maxillofacial Surgery, University of Illinois at Chicago, Chicago IL
| | - Louis G. Mercuri
- Department of Orthopaedic Surgery, Rush University, Chicago IL, Adjunct Professor, Department of Bioengineering, University of Illinois at Chicago, Chicago, IL
| | - Michael Miloro
- Department of Oral and Maxillofacial Surgery, University of Illinois at Chicago, Chicago IL
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Pang Y, Zhao Y, Wang Y, Wang X, Wang R, Liu N, Li P, Ji M, Ye J, Sun T, Li J, Ma D, Lu F, Ji C. TNFAIP8 promotes AML chemoresistance by activating ERK signaling pathway through interaction with Rac1. JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH : CR 2020; 39:158. [PMID: 32795319 PMCID: PMC7427779 DOI: 10.1186/s13046-020-01658-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/25/2020] [Accepted: 07/28/2020] [Indexed: 12/18/2022]
Abstract
Background Chemoresistance is emerging as a major barrier to successful treatment in acute myeloid leukemia (AML), and evasion of apoptosis is among the fundamental underlying mechanisms. Therefore, unraveling molecular networks that drive this process constitutes an urgent unmet need. Herein, we aim to characterize the role and molecular mechanism of the tumor necrosis factor ɑ-induced protein 8 (TNFAIP8), a novel anti-apoptotic molecule, in AML chemoresistance. Methods The expression levels of TNFAIP8 were assessed in AML patients and cell lines by RT-qPCR and western blots. The transcriptional regulation of TNFAIP8 was analyzed with luciferase reporter assay and ChIP followed by RT-qPCR. Functional experiments were conducted to evaluate the effects of TNFAIP8 on apoptosis, drug sensitivity and proliferation of AML cells. Potential effects of TNFAIP8 on the activation of extracellular signal-regulated kinase (ERK) pathway were detected by western blots. CoIP and P21-activated kinase (PAK) pull-down assay were performed to ascertain the upstream target. The overall effects of TNFAIP8 on AML were examined in murine models. Results Upregulated TNFAIP8 expression was first confirmed in human AML patients and cell lines. E74 like ETS transcription factor 1 (ELF1) was then identified to contribute to its aberrant expression. Through manipulating TNFAIP8 expression, we described its role in protecting AML cells from apoptosis induced by chemotherapeutic agents and in promoting drug resistance. Notably, the leukemia-promoting action of TNFAIP8 was mediated by sustaining activity of the ERK signaling pathway, through an interaction with Rac family small GTPase 1 (Rac1). In addition, in vivo experiments confirmed that TNFAIP8 suppression lowered leukemia infiltration and improved survival. Conclusion Our data provide a molecular basis for the role of TNFAIP8 in chemoresistance and progression of AML and highlight the unique function of TNFAIP8 as an attractive therapeutic target.
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Affiliation(s)
- Yihua Pang
- Department of Hematology, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, China
| | - Yanan Zhao
- Department of Hematology, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, China
| | - Yan Wang
- Department of Hematology, Taian central hospital, Taian, 271000, Shandong, China
| | - Xinlu Wang
- Department of Hematology, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, China
| | - Ruiqing Wang
- Department of Hematology, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, China
| | - Na Liu
- Department of Hematology, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, China
| | - Peng Li
- Department of Hematology, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, China
| | - Min Ji
- Department of Hematology, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, China
| | - Jingjing Ye
- Department of Hematology, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, China
| | - Tao Sun
- Department of Hematology, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, China
| | - Jingxin Li
- Department of Physiology, School of Basic Medical Sciences, Shandong University, Jinan, 250012, Shandong, China
| | - Daoxin Ma
- Department of Hematology, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, China
| | - Fei Lu
- Department of Hematology, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, China.
| | - Chunyan Ji
- Department of Hematology, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, China.
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Fang Y, Wu D, Birukov KG. Mechanosensing and Mechanoregulation of Endothelial Cell Functions. Compr Physiol 2019; 9:873-904. [PMID: 30873580 PMCID: PMC6697421 DOI: 10.1002/cphy.c180020] [Citation(s) in RCA: 125] [Impact Index Per Article: 20.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Vascular endothelial cells (ECs) form a semiselective barrier for macromolecules and cell elements regulated by dynamic interactions between cytoskeletal elements and cell adhesion complexes. ECs also participate in many other vital processes including innate immune reactions, vascular repair, secretion, and metabolism of bioactive molecules. Moreover, vascular ECs represent a unique cell type exposed to continuous, time-dependent mechanical forces: different patterns of shear stress imposed by blood flow in macrovasculature and by rolling blood cells in the microvasculature; circumferential cyclic stretch experienced by the arterial vascular bed caused by heart propulsions; mechanical stretch of lung microvascular endothelium at different magnitudes due to spontaneous respiration or mechanical ventilation in critically ill patients. Accumulating evidence suggests that vascular ECs contain mechanosensory complexes, which rapidly react to changes in mechanical loading, process the signal, and develop context-specific adaptive responses to rebalance the cell homeostatic state. The significance of the interactions between specific mechanical forces in the EC microenvironment together with circulating bioactive molecules in the progression and resolution of vascular pathologies including vascular injury, atherosclerosis, pulmonary edema, and acute respiratory distress syndrome has been only recently recognized. This review will summarize the current understanding of EC mechanosensory mechanisms, modulation of EC responses to humoral factors by surrounding mechanical forces (particularly the cyclic stretch), and discuss recent findings of magnitude-specific regulation of EC functions by transcriptional, posttranscriptional and epigenetic mechanisms using -omics approaches. We also discuss ongoing challenges and future opportunities in developing new therapies targeting dysregulated mechanosensing mechanisms to treat vascular diseases. © 2019 American Physiological Society. Compr Physiol 9:873-904, 2019.
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Affiliation(s)
- Yun Fang
- Department of Medicine, University of Chicago, Chicago, Illinois, USA,Correspondence to
| | - David Wu
- Department of Medicine, University of Chicago, Chicago, Illinois, USA
| | - Konstantin G. Birukov
- Department of Anesthesiology, University of Maryland Baltimore School of Medicine, Baltimore, Maryland, USA
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Jung D, Alshaikh A, Ratakonda S, Bashir M, Amin R, Jeon S, Stevens J, Sharma S, Ahmed W, Musch M, Hassan H. Adenosinergic signaling inhibits oxalate transport by human intestinal Caco2-BBE cells through the A 2B adenosine receptor. Am J Physiol Cell Physiol 2018; 315:C687-C698. [PMID: 30020825 DOI: 10.1152/ajpcell.00024.2017] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Most kidney stones (KS) are composed of calcium oxalate, and small increases in urine oxalate affect the stone risk. Intestinal oxalate secretion mediated by anion exchanger SLC26A6 (PAT1) plays a crucial role in limiting net absorption of ingested oxalate, thereby preventing hyperoxaluria and related KS, reflecting the importance of understanding regulation of intestinal oxalate transport. We previously showed that ATP and UTP inhibit oxalate transport by human intestinal Caco2-BBE cells (C2). Since ATP is rapidly degraded to adenosine (ADO), we examined whether intestinal oxalate transport is regulated by ADO. We measured [14C]oxalate uptake in the presence of an outward Cl gradient as an assay of Cl-oxalate exchange activity, ≥49% of which is PAT1-mediated in C2 cells. We found that ADO significantly inhibited oxalate transport by C2 cells, an effect completely blocked by the nonselective ADO receptor antagonist 8- p-sulfophenyltheophylline. ADO also significantly inhibited oxalate efflux by C2 cells, which is important since PAT1 mediates oxalate efflux in vivo. Using pharmacological antagonists and A2B adenosine receptor (A2B AR) siRNA knockdown studies, we observed that ADO inhibits oxalate transport through the A2B AR, phospholipase C, and PKC. ADO inhibits oxalate transport by reducing PAT1 surface expression as shown by biotinylation studies. We conclude that ADO inhibits oxalate transport by lowering PAT1 surface expression in C2 cells through signaling pathways including the A2B AR, PKC, and phospholipase C. Given higher ADO levels and overexpression of the A2B AR in inflammatory bowel disease (IBD), our findings have potential relevance to pathophysiology of IBD-associated hyperoxaluria and related KS.
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Affiliation(s)
- Daniel Jung
- Department of Medicine, The University of Chicago , Chicago, Illinois
| | - Altayeb Alshaikh
- Department of Medicine, The University of Chicago , Chicago, Illinois
| | | | - Mohamed Bashir
- Department of Medicine, The University of Chicago , Chicago, Illinois
| | - Ruhul Amin
- Department of Medicine, The University of Chicago , Chicago, Illinois
| | - Sohee Jeon
- Department of Medicine, The University of Chicago , Chicago, Illinois
| | - Jan Stevens
- Department of Medicine, The University of Chicago , Chicago, Illinois
| | - Sapna Sharma
- Department of Medicine, The University of Chicago , Chicago, Illinois
| | - Wahaj Ahmed
- Department of Medicine, The University of Chicago , Chicago, Illinois
| | - Mark Musch
- Department of Medicine, The University of Chicago , Chicago, Illinois
| | - Hatim Hassan
- Department of Medicine, The University of Chicago , Chicago, Illinois
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Chen ZL, Yang J, Shen YW, Li ST, Wang X, Lv M, Wang BY, Li P, Zhao W, Qiu RY, Liu Y, Liu PJ, Yang J. AmotP130 regulates Rho GTPase and decreases breast cancer cell mobility. J Cell Mol Med 2018; 22:2390-2403. [PMID: 29377471 PMCID: PMC5867092 DOI: 10.1111/jcmm.13533] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2017] [Accepted: 12/14/2017] [Indexed: 12/17/2022] Open
Abstract
Angiomotin (Amot) is a newly discovered, multifunctional protein that is involved in cell migration and angiogenesis. However, the role of its isoform, AmotP130, in the regulation of cytoskeleton and metastasis of breast cancer, is unclear. The aim of this study was to investigate the role of AmotP130 in the reorganization of the actin cytoskeleton and the changes of morphology in breast cancer cells through the Rho pathway that influences the invasion and migration of cells. The results suggested that AmotP130 suppressed the invasion ability through remodelling the cytoskeleton of breast cancer cells, including the actin fibre organization and focal adhesion protein turnover. Global transcriptome changes in breast cancer cells following knockdown of AmotP130 identified pathways related with the cytoskeleton and cell motility that involved the Rho GTPase family. From database analyses, changes in the Rho GTPase family of proteins were identified as possible prognostic factors in patients with breast cancer. We have been suggested that AmotP130 suppressed the invasion ability through remodelling of the cytoskeleton of breast cancer cells, involving regulation of the Rho pathway. The cytoskeleton-related pathway components may provide novel, clinically therapeutic targets for breast cancer treatment.
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Affiliation(s)
- Zhe-Ling Chen
- Department of Medical Oncology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China
| | - Jiao Yang
- Department of Medical Oncology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China
| | - Yan-Wei Shen
- Department of Medical Oncology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China
| | - Shu-Ting Li
- Department of Medical Oncology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China
| | - Xin Wang
- Department of Oncology, Shangluo Central Hospital, Shangluo, Shaanxi, China
| | - Meng Lv
- Department of Medical Oncology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China
| | - Bi-Yuan Wang
- Department of Medical Oncology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China
| | - Pan Li
- Department of Medical Oncology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China
| | - Wen Zhao
- Department of Medical Oncology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China
| | - Rui-Yue Qiu
- Department of Medical Oncology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China
| | - Yu Liu
- Department of Biology & Biochemistry, University of Houston, Houston, TX, USA
| | - Pei-Jun Liu
- Center for Translational Medicine, The First Affiliated Hospital of Xian Jiaotong University, Xi'an, Shaanxi, China
| | - Jin Yang
- Department of Medical Oncology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China
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Toumpanakis D, Vassilakopoulou V, Sigala I, Zacharatos P, Vraila I, Karavana V, Theocharis S, Vassilakopoulos T. The role of Src & ERK1/2 kinases in inspiratory resistive breathing induced acute lung injury and inflammation. Respir Res 2017; 18:209. [PMID: 29237457 PMCID: PMC5729404 DOI: 10.1186/s12931-017-0694-7] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2017] [Accepted: 12/05/2017] [Indexed: 01/08/2023] Open
Abstract
Background Inspiratory resistive breathing (IRB), a hallmark of obstructive airway diseases, is associated with large negative intrathoracic pressures, due to strenuous contractions of the inspiratory muscles. IRB is shown to induce lung injury in previously healthy animals. Src is a multifunctional kinase that is activated in the lung by mechanical stress. ERK1/2 kinase is a downstream target of Src. We hypothesized that Src is activated in the lung during IRB, mediates ERK1/2 activation and IRB-induced lung injury. Methods Anaesthetized, tracheostomized adult rats breathed spontaneously through a 2-way non-rebreathing valve. Resistance was added to the inspiratory port to provide a peak tidal inspiratory pressure of 50% of maximum (inspiratory resistive breathing). Activation of Src and ERK1/2 in the lung was estimated during IRB. Following 6 h of IRB, respiratory system mechanics were measured by the forced oscillation technique and bronchoalveolar lavage (BAL) was performed to measure total and differential cell count and total protein levels. IL-1b and MIP-2a protein levels were measured in lung tissue samples. Wet lung weight to total body weight was measured and Evans blue dye extravasation was estimated to measure lung permeability. Lung injury was evaluated by histology. The Src inhibitor, PP-2 or the inhibitor of ERK1/2 activation, PD98059 was administrated 30 min prior to IRB. Results Src kinase was activated 30 min after the initiation of IRB. Src inhibition ameliorated the increase in BAL cellularity after 6 h IRB, but not the increase of IL-1β and MIP-2a in the lung. The increase in BAL total protein and lung injury score were not affected. The increase in tissue elasticity was partly inhibited. Src inhibition blocked ERK1/2 activation at 3 but not at 6 h of IRB. ERK1/2 inhibition ameliorated the increase in BAL cellularity after 6 h of IRB, blocked the increase of IL-1β and returned Evans blue extravasation and wet lung weight to control values. BAL total protein and the increase in elasticity were partially affected. ERK1/2 inhibition did not significantly change total lung injury score compared to 6 h IRB. Conclusions Src and ERK1/2 are activated in the lung following IRB and participate in IRB-induced lung injury.
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Affiliation(s)
- Dimitrios Toumpanakis
- 1st Department of Critical Care and Pulmonary Medicine and "Marianthi Simou" Applied Biomedical Research and Training Center, Medical School, University of Athens, 45-47 Ispilandou str, 10676, Athens, Greece
| | - Vyronia Vassilakopoulou
- 1st Department of Critical Care and Pulmonary Medicine and "Marianthi Simou" Applied Biomedical Research and Training Center, Medical School, University of Athens, 45-47 Ispilandou str, 10676, Athens, Greece
| | - Ioanna Sigala
- 1st Department of Critical Care and Pulmonary Medicine and "Marianthi Simou" Applied Biomedical Research and Training Center, Medical School, University of Athens, 45-47 Ispilandou str, 10676, Athens, Greece
| | - Panagiotis Zacharatos
- 1st Department of Critical Care and Pulmonary Medicine and "Marianthi Simou" Applied Biomedical Research and Training Center, Medical School, University of Athens, 45-47 Ispilandou str, 10676, Athens, Greece
| | - Ioanna Vraila
- 1st Department of Critical Care and Pulmonary Medicine and "Marianthi Simou" Applied Biomedical Research and Training Center, Medical School, University of Athens, 45-47 Ispilandou str, 10676, Athens, Greece
| | - Vassiliki Karavana
- 1st Department of Critical Care and Pulmonary Medicine and "Marianthi Simou" Applied Biomedical Research and Training Center, Medical School, University of Athens, 45-47 Ispilandou str, 10676, Athens, Greece
| | | | - Theodoros Vassilakopoulos
- 1st Department of Critical Care and Pulmonary Medicine and "Marianthi Simou" Applied Biomedical Research and Training Center, Medical School, University of Athens, 45-47 Ispilandou str, 10676, Athens, Greece.
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10
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Portillo JAC, Muniz-Feliciano L, Lopez Corcino Y, Lee SJ, Van Grol J, Parsons SJ, Schiemman WP, Subauste CS. Toxoplasma gondii induces FAK-Src-STAT3 signaling during infection of host cells that prevents parasite targeting by autophagy. PLoS Pathog 2017; 13:e1006671. [PMID: 29036202 PMCID: PMC5658194 DOI: 10.1371/journal.ppat.1006671] [Citation(s) in RCA: 42] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2017] [Revised: 10/26/2017] [Accepted: 09/26/2017] [Indexed: 02/01/2023] Open
Abstract
Targeting of Toxoplasma gondii by autophagy is an effective mechanism by which host cells kill the protozoan. Thus, the parasite must avoid autophagic targeting to survive. Here we show that the mammalian cytoplasmic molecule Focal Adhesion Kinase (FAK) becomes activated during invasion of host cells. Activated FAK appears to accompany the formation of the moving junction (as assessed by expression the parasite protein RON4). FAK activation was inhibited by approaches that impaired β1 and β3 integrin signaling. FAK caused activation of Src that in turn mediated Epidermal Growth Factor Receptor (EGFR) phosphorylation at the unique Y845 residue. Expression of Src-resistant Y845F EGFR mutant markedly inhibited ROP16-independent activation of STAT3 in host cells. Activation of FAK, Y845 EGFR or STAT3 prevented activation of PKR and eIF2α, key stimulators of autophagy. Genetic or pharmacologic inhibition of FAK, Src, EGFR phosphorylation at Y845, or STAT3 caused accumulation of the autophagy protein LC3 and LAMP-1 around the parasite and parasite killing dependent on autophagy proteins (ULK1 and Beclin 1) and lysosomal enzymes. Parasite killing was inhibited by expression of dominant negative PKR. Thus, T. gondii activates a FAK→Src→Y845-EGFR→STAT3 signaling axis within mammalian cells, thereby enabling the parasite to survive by avoiding autophagic targeting through a mechanism likely dependent on preventing activation of PKR and eIF2α.
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Affiliation(s)
- Jose-Andres C. Portillo
- Department of Medicine, Division of Infectious Disease and HIV Medicine, Case Western Reserve University School of Medicine, Cleveland, OH, United States of America
| | - Luis Muniz-Feliciano
- Department of Medicine, Division of Infectious Disease and HIV Medicine, Case Western Reserve University School of Medicine, Cleveland, OH, United States of America
- Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, United States of America
| | - Yalitza Lopez Corcino
- Department of Medicine, Division of Infectious Disease and HIV Medicine, Case Western Reserve University School of Medicine, Cleveland, OH, United States of America
- Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, United States of America
| | - So Jung Lee
- Department of Medicine, Division of Infectious Disease and HIV Medicine, Case Western Reserve University School of Medicine, Cleveland, OH, United States of America
| | - Jennifer Van Grol
- Department of Medicine, Division of Infectious Disease and HIV Medicine, Case Western Reserve University School of Medicine, Cleveland, OH, United States of America
- Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, United States of America
| | - Sarah J. Parsons
- Department of Microbiology and Cancer Center, University of Virginia, Charlottesville, VA, United States of America
| | - William P. Schiemman
- Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, United States of America
| | - Carlos S. Subauste
- Department of Medicine, Division of Infectious Disease and HIV Medicine, Case Western Reserve University School of Medicine, Cleveland, OH, United States of America
- Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, United States of America
- * E-mail:
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11
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Ito J, Uchida H, Machida N, Ohtake K, Saito Y, Kobayashi J. Inducible and neuronal nitric oxide synthases exert contrasting effects during rat intestinal recovery following fasting. Exp Biol Med (Maywood) 2017; 242:762-772. [PMID: 28195513 DOI: 10.1177/1535370217694434] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
We investigated the effects of endogenous inducible (iNOS) and neuronal nitric oxide synthase on recovery from intestinal mucosal atrophy caused by fasting-induced apoptosis and decreased cell proliferation during refeeding in rats. Rats were divided into five groups, one of which was fed ad libitum, and four of which underwent 72 h of fasting, followed by refeeding for 0, 6, 24, and 48 h, respectively. iNOS and neuronal nitric oxide synthase mRNA and protein levels in jejunal tissues were measured, and mucosal height was histologically evaluated. Apoptotic indices, interferon-γ (IFN-γ) transcription levels, nitrite levels (as a measure of nitric oxide [NO] production),8-hydroxydeoxyguanosine formation (indicating reactive oxygen species [ROS] levels), crypt cell proliferation, and the motility indices (MI) were also estimated. Associations between mucosal height and NOS protein levels were determined using Spearman's rank correlation test. Notably, we observed significant increases in mucosal height and in neuronal nitric oxide synthase mRNA and protein expression as refeeding time increased. Indeed, there was a significant positive correlation between neuronal nitric oxide synthase protein level and mucosal height during the 48-h refeeding period ( r = 0.725, P < 0.01). Conversely, iNOS mRNA and protein expression decreased according to refeeding time, with a significant negative correlation between iNOS protein level and mucosal height being recorded during the 48-h refeeding period ( r = -0.898, P < 0.01). We also noted a significant negative correlation between jejunal neuronal nitric oxide synthase and iNOS protein concentrations over this same period ( r = -0.734, P < 0.01). Refeeding also restored the decreased jejunal MI caused by fasting. Our finding suggests that refeeding likely repairs fasting-induced jejunal atrophy by suppressing iNOS expression and subsequently inhibiting NO, ROS, and IFN-γ as apoptosis mediators, and by promoting neuronal nitric oxide synthase production and inducing crypt cell proliferation via mechanical stimulation. Impact statement Besides providing new data confirming the involvement of iNOS and nNOS in intestinal mucosal atrophy caused by fasting, this study details their expression and function during recovery from this condition following refeeding. We demonstrate a significant negative correlation between iNOS and nNOS levels during refeeding, and associate this with cell proliferation and apoptosis in crypts and villi. These novel findings elucidate the relationship between these NOS isoforms and its impact on recovery from intestinal injury. A mechanism is proposed comprising the up-regulation of nNOS activity by mechanical stimulation due to the presence of food in the intestine, restricting iNOS-associated apoptosis and promoting cell proliferation and gut motility. Our investigation sheds light on the molecular basis behind the repercussions of total parenteral nutrition on intestinal mucosal integrity, and more importantly, the beneficial effects of early enteral feeding.
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Affiliation(s)
- Junta Ito
- Division of Pathophysiology, Faculty of Pharmaceutical Science, Department of Clinical Dietetics and Human Nutrition, Josai University, Saitama 350-0295, Japan
| | - Hiroyuki Uchida
- Division of Pathophysiology, Faculty of Pharmaceutical Science, Department of Clinical Dietetics and Human Nutrition, Josai University, Saitama 350-0295, Japan
| | - Naomi Machida
- Division of Pathophysiology, Faculty of Pharmaceutical Science, Department of Clinical Dietetics and Human Nutrition, Josai University, Saitama 350-0295, Japan
| | - Kazuo Ohtake
- Division of Pathophysiology, Faculty of Pharmaceutical Science, Department of Clinical Dietetics and Human Nutrition, Josai University, Saitama 350-0295, Japan
| | - Yuki Saito
- Division of Pathophysiology, Faculty of Pharmaceutical Science, Department of Clinical Dietetics and Human Nutrition, Josai University, Saitama 350-0295, Japan
| | - Jun Kobayashi
- Division of Pathophysiology, Faculty of Pharmaceutical Science, Department of Clinical Dietetics and Human Nutrition, Josai University, Saitama 350-0295, Japan
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12
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Cei D, Costa J, Gori G, Frediani G, Domenici C, Carpi F, Ahluwalia A. A bioreactor with an electro-responsive elastomeric membrane for mimicking intestinal peristalsis. BIOINSPIRATION & BIOMIMETICS 2016; 12:016001. [PMID: 27918289 DOI: 10.1088/1748-3190/12/1/016001] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/17/2023]
Abstract
This study describes an actuated bioreactor which mimics the pulsatile contractile motion of the intestinal barrier using electro-responsive elastomers as smart materials that undergo deformation upon electrical stimulation. The device consists of an annular dielectric elastomer actuator working as a radial artificial muscle able to rhythmically contract and relax a central cell culture well. The bioreactor maintained up to 4 h of actuation at a frequency of 0.15 Hz and a strain of 8%-10%, to those of the cyclic contraction and relaxation of the small intestine. In vitro tests demonstrated that the device was biocompatible and cell-adhesive for Caco-2 cells, which formed a confluent monolayer following 21 days of culture in the central well. In addition, cellular adhesion and cohesion were maintained after 4 h of continuous cyclic strain. These preliminary results encourage further investigations on the use of dielectric elastomer actuation as a versatile technology that might overcome the limitations of commercially available pneumatic driving systems to obtain bioreactors that can cyclically deform cell cultures in a biomimetic fashion.
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Affiliation(s)
- Daniele Cei
- Research Center 'E.Piaggio' and Department of Information Engineering, University of Pisa, Largo L Lazzarino, I-56126 Pisa, Italy
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13
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Shiratsuchi H, Wang Z, Chen G, Ray P, Lin J, Zhang Z, Zhao L, Beer D, Ray D, Ramnath N. Oncogenic Potential of CYP24A1 in Lung Adenocarcinoma. J Thorac Oncol 2016; 12:269-280. [PMID: 27793774 DOI: 10.1016/j.jtho.2016.10.010] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2016] [Revised: 10/06/2016] [Accepted: 10/07/2016] [Indexed: 12/20/2022]
Abstract
INTRODUCTION We have previously demonstrated that a subset of lung cancer cells express higher CYP24A1 mRNA, a metabolizing enzyme for 1,25-D3, compared to benign tumors or surrounding normal lung and that high CYP24A1 mRNA expression is associated with poor prognosis in resected lung adenocarcinoma (AC). We hypothesized that CYP24A1 has oncogenic potential and increased CYP24A1 expression may contribute to tumor growth, whereas, CYP24A1 targeting may reduce tumor burden. METHODS Two low CYP24A1 expressing human lung cancer cell lines (SK-LU-1 and Calu-6) were stably transfected either with an empty lentiviral vector or with the CYP24A1 expressing vector. Over-expression of mRNA and protein levels of CYP24A1 in SK-LU-1 and Calu-6 were confirmed using qRT-PCR and immunoblotting respectively. Next, effects of targeting CYP24A1 were examined in lung cancer cells (A549 and H441), which express higher basal levels of CYP24A1. Finally, we studied the effects of stable knockdown of CYP24A1 in xenograft models. RESULTS Over-expression of CYP24A1 correlated with accelerated cell growth and invasion compared to control vector-transfected cells. CYP24A1 over-expression also increased RAS protein expression. Knockdown of CYP24A1 using either si- or shRNA reduced CYP24A1 mRNA and protein expression and significantly decreased cell proliferation (30-60%) and reduced mitochondrial DNA content compared to non-targeting (NT) si-/shRNA transfected/transduced cells. Transfection with CYP24A1 siRNA also decreased total RAS protein, thus reducing phosphorylated AKT. Importantly, stable knockdown of CYP24A1 in A549 and H441 lung tumor xenograft models resulted in tumor growth delay and smaller tumor size as evident from tumor bioluminescence and tumor volume measurement studies. Such observations were correlated with decreased tumor cell proliferation as evidenced by reduced Ki67 and Cyclin D staining. CONCLUSIONS Our data suggest that CYP24A1 has oncogenic properties mediated by increasing RAS signaling, targeting of which may provide an alternate strategy to treat a subset of lung AC.
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Affiliation(s)
- Hiroe Shiratsuchi
- Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan
| | - Zhuwen Wang
- Section of Thoracic Surgery, Department of Surgery, University of Michigan, Ann Arbor, Michigan
| | - Guoan Chen
- Section of Thoracic Surgery, Department of Surgery, University of Michigan, Ann Arbor, Michigan
| | - Paramita Ray
- Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan
| | - Jules Lin
- Section of Thoracic Surgery, Department of Surgery, University of Michigan, Ann Arbor, Michigan
| | - Zhuo Zhang
- Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan
| | - Lili Zhao
- Department of Biostatistics, University of Michigan, Ann Arbor, Michigan
| | - David Beer
- Section of Thoracic Surgery, Department of Surgery, University of Michigan, Ann Arbor, Michigan
| | - Dipankar Ray
- Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan
| | - Nithya Ramnath
- Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan; Ann Arbor Veterans Administration Medical Center, Ann Arbor, Michigan.
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14
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Arvans D, Jung YC, Antonopoulos D, Koval J, Granja I, Bashir M, Karrar E, Roy-Chowdhury J, Musch M, Asplin J, Chang E, Hassan H. Oxalobacter formigenes-Derived Bioactive Factors Stimulate Oxalate Transport by Intestinal Epithelial Cells. J Am Soc Nephrol 2016; 28:876-887. [PMID: 27738124 DOI: 10.1681/asn.2016020132] [Citation(s) in RCA: 62] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2016] [Accepted: 08/13/2016] [Indexed: 12/12/2022] Open
Abstract
Hyperoxaluria is a major risk factor for kidney stones and has no specific therapy, although Oxalobacter formigenes colonization is associated with reduced stone risk. O. formigenes interacts with colonic epithelium and induces colonic oxalate secretion, thereby reducing urinary oxalate excretion, via an unknown secretagogue. The difficulties in sustaining O. formigenes colonization underscore the need to identify the derived factors inducing colonic oxalate secretion. We therefore evaluated the effects of O. formigenes culture conditioned medium (CM) on apical 14C-oxalate uptake by human intestinal Caco-2-BBE cells. Compared with control medium, O. formigenes CM significantly stimulated oxalate uptake (>2.4-fold), whereas CM from Lactobacillus acidophilus did not. Treating the O. formigenes CM with heat or pepsin completely abolished this bioactivity, and selective ultrafiltration of the CM revealed that the O. formigenes-derived factors have molecular masses of 10-30 kDa. Treatment with the protein kinase A inhibitor H89 or the anion exchange inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid completely blocked the CM-induced oxalate transport. Knockdown of the oxalate transporter SLC26A6 also significantly restricted the induction of oxalate transport by CM. In a mouse model of primary hyperoxaluria type 1, rectal administration of O. formigenes CM significantly reduced (>32.5%) urinary oxalate excretion and stimulated (>42%) distal colonic oxalate secretion. We conclude that O. formigenes-derived bioactive factors stimulate oxalate transport in intestinal cells through mechanisms including PKA activation. The reduction in urinary oxalate excretion in hyperoxaluric mice treated with O. formigenes CM reflects the in vivo retention of biologic activity and the therapeutic potential of these factors.
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Affiliation(s)
- Donna Arvans
- Department of Medicine, The University of Chicago, Chicago, Illinois
| | - Yong-Chul Jung
- Department of Medicine, The University of Chicago, Chicago, Illinois
| | - Dionysios Antonopoulos
- Department of Medicine, The University of Chicago, Chicago, Illinois.,Biosciences Division, Argonne National Laboratory, Argonne, Illinois
| | - Jason Koval
- Biosciences Division, Argonne National Laboratory, Argonne, Illinois
| | - Ignacio Granja
- Litholink Corporation, Laboratory Corporation of America Holdings, Chicago, Illinois; and
| | - Mohamed Bashir
- Department of Medicine, The University of Chicago, Chicago, Illinois
| | - Eltayeb Karrar
- Department of Medicine, The University of Chicago, Chicago, Illinois
| | | | - Mark Musch
- Department of Medicine, The University of Chicago, Chicago, Illinois
| | - John Asplin
- Litholink Corporation, Laboratory Corporation of America Holdings, Chicago, Illinois; and
| | - Eugene Chang
- Department of Medicine, The University of Chicago, Chicago, Illinois
| | - Hatim Hassan
- Department of Medicine, The University of Chicago, Chicago, Illinois;
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15
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Song H, Liang W, Xu S, Li Z, Chen Z, Cui W, Zhou J, Wang Q, Liu F, Fan W. A novel role for integrin-linked kinase in periodic mechanical stress-mediated ERK1/2 mitogenic signaling in rat chondrocytes. Cell Biol Int 2016; 40:832-9. [PMID: 27154044 DOI: 10.1002/cbin.10622] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2016] [Accepted: 05/03/2016] [Indexed: 12/14/2022]
Abstract
In recent years, a variety of studies have been performed to investigate the cellular responses of periodic mechanical stress on chondrocytes. Integrin β1-mediated ERK1/2 activation was proven to be indispensable in periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. However, other signal proteins responsible for the mitogenesis of chondrocytes under periodic mechanical stress remain incompletely understood. In the current investigation, we probed the roles of integrin-linked kinase (ILK) signaling in periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. We found that upon periodic mechanical stress induction, ILK activity increased significantly. Depletion of ILK with targeted shRNA strongly inhibited periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. In addition, pretreatment with a blocking antibody against integrin β1 resulted in a remarkable decrease in ILK activity in cells exposed to periodic mechanical stress. Furthermore, inhibition of ILK with its target shRNA significantly suppressed ERK1/2 activation in relation to periodic mechanical stress. Based on the above results, we identified ILK as a crucial regulator involved in the integrin β1-ERK1/2 signal cascade responsible for periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis.
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Affiliation(s)
- Huanghe Song
- Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, 300 Guang Zhou Road, Nanjing, 210029, China
| | - Wenwei Liang
- Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, 300 Guang Zhou Road, Nanjing, 210029, China
| | - Shun Xu
- Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, 300 Guang Zhou Road, Nanjing, 210029, China
| | - Zeng Li
- Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, 300 Guang Zhou Road, Nanjing, 210029, China
| | - Zhefeng Chen
- Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, 300 Guang Zhou Road, Nanjing, 210029, China
| | - Weiding Cui
- Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, 300 Guang Zhou Road, Nanjing, 210029, China
| | - Jinchun Zhou
- Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, 300 Guang Zhou Road, Nanjing, 210029, China
| | - Qing Wang
- Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, 300 Guang Zhou Road, Nanjing, 210029, China
| | - Feng Liu
- Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, 300 Guang Zhou Road, Nanjing, 210029, China
| | - Weimin Fan
- Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, 300 Guang Zhou Road, Nanjing, 210029, China
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16
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Kumar A, Baycin-Hizal D, Wolozny D, Pedersen LE, Lewis NE, Heffner K, Chaerkady R, Cole RN, Shiloach J, Zhang H, Bowen MA, Betenbaugh MJ. Elucidation of the CHO Super-Ome (CHO-SO) by Proteoinformatics. J Proteome Res 2015; 14:4687-703. [PMID: 26418914 DOI: 10.1021/acs.jproteome.5b00588] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Chinese hamster ovary (CHO) cells are the preferred host cell line for manufacturing a variety of complex biotherapeutic drugs including monoclonal antibodies. We performed a proteomics and bioinformatics analysis on the spent medium from adherent CHO cells. Supernatant from CHO-K1 culture was collected and subjected to in-solution digestion followed by LC/LC-MS/MS analysis, which allowed the identification of 3281 different host cell proteins (HCPs). To functionally categorize them, we applied multiple bioinformatics tools to the proteins identified in our study including SignalP, TargetP, SecretomeP, TMHMM, WoLF PSORT, and Phobius. This analysis provided information on the presence of signal peptides, transmembrane domains, and cellular localization and showed that both secreted and intracellular proteins were constituents of the supernatant. Identified proteins were shown to be localized to the secretory pathway including ones playing roles in cell growth, proliferation, and folding as well as those involved in protein degradation and removal. After combining proteins predicted to be secreted or having a signal peptide, we identified 1015 proteins, which we termed as CHO supernatant-ome (CHO-SO), or superome. As a part of this effort, we created a publically accessible web-based tool called GO-CHO to functionally categorize proteins found in CHO-SO and identify enriched molecular functions, biological processes, and cellular components. We also used a tool to evaluate the immunogenicity potential of high-abundance HCPs. Among enriched functions were catalytic activity and structural constituents of the cytoskeleton. Various transport related biological processes, such as vesicle mediated transport, were found to be highly enriched. Extracellular space and vesicular exosome associated proteins were found to be the most enriched cellular components. The superome also contained proteins secreted from both classical and nonclassical secretory pathways. The work and database described in our study will enable the CHO community to rapidly identify high-abundance HCPs in their cultures and therefore help assess process and purification methods used in the production of biologic drugs.
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Affiliation(s)
- Amit Kumar
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University , Baltimore, Maryland 21218, United States.,Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases , National Institute of Health, Building 14A, Bethesda, Maryland 20892, United States
| | - Deniz Baycin-Hizal
- Antibody Discovery and Protein Engineering, MedImmune LLC , 1 MedImmune Way, Gaithersburg, Maryland 20878, United States
| | - Daniel Wolozny
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University , Baltimore, Maryland 21218, United States
| | - Lasse Ebdrup Pedersen
- The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , DK-2970 Hørsholm, Denmark
| | - Nathan E Lewis
- Department of Biology, Brigham Young University , Provo, Utah 84602, United States.,Department of Pediatrics, University of California , San Diego, California 92093, United States
| | - Kelley Heffner
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University , Baltimore, Maryland 21218, United States
| | - Raghothama Chaerkady
- Institute of Basic Biomedical Sciences, Mass Spectrometry and Proteomics Facility, Johns Hopkins University School of Medicine , 733 North Broadway Street, Baltimore, Maryland 21205, United States
| | - Robert N Cole
- Institute of Basic Biomedical Sciences, Mass Spectrometry and Proteomics Facility, Johns Hopkins University School of Medicine , 733 North Broadway Street, Baltimore, Maryland 21205, United States
| | - Joseph Shiloach
- Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases , National Institute of Health, Building 14A, Bethesda, Maryland 20892, United States
| | - Hui Zhang
- Department of Pathology, Johns Hopkins School of Medicine , 400 North Broadway Street, Baltimore, Maryland 21287, United States
| | - Michael A Bowen
- Antibody Discovery and Protein Engineering, MedImmune LLC , 1 MedImmune Way, Gaithersburg, Maryland 20878, United States
| | - Michael J Betenbaugh
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University , Baltimore, Maryland 21218, United States
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17
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Basson MD, Zeng B, Downey C, Sirivelu MP, Tepe JJ. Increased extracellular pressure stimulates tumor proliferation by a mechanosensitive calcium channel and PKC-β. Mol Oncol 2015; 9:513-526. [PMID: 25454347 PMCID: PMC4487881 DOI: 10.1016/j.molonc.2014.10.008] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2014] [Revised: 10/17/2014] [Accepted: 10/20/2014] [Indexed: 01/31/2023] Open
Abstract
Large tumors exhibit high interstitial pressure heightened by growth against the constraining stroma. Such pressures could stimulate tumor proliferation via a mechanosensitive ion channel. We studied the effects of 0-80 mmHg increased extracellular pressure for 24 h on proliferation of SW620, Caco-2, and CT-26 colon; MCF-7 breast; and MLL and PC3 prostate cancer cells, and delineated its mechanism in SW620 cells with specific inhibitors and siRNA. Finally, we compared NF-kB, phospho-IkB and cyclin D1 immunoreactivity in the high pressure centers and low pressure peripheries of human tumors. Pressure-stimulated proliferation in all cells. Pressure-driven SW620 proliferation required calcium influx via the T-type Ca(2+) channel Cav3.3, which stimulated PKC-β to invoke the IKK-IkB-NF-kB pathway to increase proliferation and S-phase fraction. The mitotic index and immunoreactivity of NF-kB, phospho-IkB, and cyclin D1 in the center of 28 large human colon, lung, and head and neck tumors exceeded that in tumor peripheries. Extracellular pressure increases [Ca(2+)]i via Cav3.3, driving a PKC-β- IKK- IkB-NF-kB pathway that stimulates cancer cell proliferation. Rapid proliferation in large stiff tumors may increase intratumoral pressure, activating this pathway to stimulate further proliferation in a feedback cycle that potentiates tumor growth. Targeting this pathway may inhibit proliferation in large unresectable tumors.
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Affiliation(s)
- Marc D Basson
- Department of Surgery, Michigan State University College of Human Medicine, 1200 E Michigan Ave, Lansing Charter Township, MI 48912, USA.
| | - Bixi Zeng
- Department of Surgery, Michigan State University College of Human Medicine, 1200 E Michigan Ave, Lansing Charter Township, MI 48912, USA
| | - Christina Downey
- Department of Surgery, Michigan State University College of Human Medicine, 1200 E Michigan Ave, Lansing Charter Township, MI 48912, USA
| | - Madhu P Sirivelu
- Department of Surgery, Michigan State University College of Human Medicine, 1200 E Michigan Ave, Lansing Charter Township, MI 48912, USA
| | - Jetze J Tepe
- Department of Pharmacology, Michigan State University, 1355 Bogue Street, B440 Life Sciences Building, East Lansing, MI 48824, USA
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18
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Samak G, Gangwar R, Crosby LM, Desai LP, Wilhelm K, Waters CM, Rao R. Cyclic stretch disrupts apical junctional complexes in Caco-2 cell monolayers by a JNK-2-, c-Src-, and MLCK-dependent mechanism. Am J Physiol Gastrointest Liver Physiol 2014; 306:G947-58. [PMID: 24722904 PMCID: PMC4042113 DOI: 10.1152/ajpgi.00396.2013] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The intestinal epithelium is subjected to various types of mechanical stress. In this study, we investigated the impact of cyclic stretch on tight junction and adherens junction integrity in Caco-2 cell monolayers. Stretch for 2 h resulted in a dramatic modulation of tight junction protein distribution from a linear organization into wavy structure. Continuation of cyclic stretch for 6 h led to redistribution of tight junction proteins from the intercellular junctions into the intracellular compartment. Disruption of tight junctions was associated with redistribution of adherens junction proteins, E-cadherin and β-catenin, and dissociation of the actin cytoskeleton at the actomyosin belt. Stretch activates JNK2, c-Src, and myosin light-chain kinase (MLCK). Inhibition of JNK, Src kinase or MLCK activity and knockdown of JNK2 or c-Src attenuated stretch-induced disruption of tight junctions, adherens junctions, and actin cytoskeleton. Paracellular permeability measured by a novel method demonstrated that cyclic stretch increases paracellular permeability by a JNK, Src kinase, and MLCK-dependent mechanism. Stretch increased tyrosine phosphorylation of occludin, ZO-1, E-cadherin, and β-catenin. Inhibition of JNK or Src kinase attenuated stretch-induced occludin phosphorylation. Immunofluorescence localization indicated that phospho-MLC colocalizes with the vesicle-like actin structure at the actomyosin belt in stretched cells. On the other hand, phospho-c-Src colocalizes with the actin at the apical region of cells. This study demonstrates that cyclic stretch disrupts tight junctions and adherens junctions by a JNK2, c-Src, and MLCK-dependent mechanism.
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Affiliation(s)
| | | | | | | | | | | | - RadhaKrishna Rao
- Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee
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19
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Kaito Y, Kataoka R, Takechi K, Mihara T, Tamura M. Nox1 activation by βPix and the role of Ser-340 phosphorylation. FEBS Lett 2014; 588:1997-2002. [PMID: 24792722 DOI: 10.1016/j.febslet.2014.04.025] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2014] [Revised: 04/04/2014] [Accepted: 04/14/2014] [Indexed: 11/19/2022]
Abstract
Rac is an activating factor for Nox1, an O2(-)-generating NADPH oxidase, expressed in the colon and other tissues. Rac requires a GDP-GTP exchange factor for activation. Nox1 activation by βPix has been demonstrated in cell lines. We examined the effects of βPix and its phosphomimetic mutant on endogenous Nox1 in Caco-2 cells transfected with Noxo1 and Noxa1. βPix expression enhanced O2(-) production in resting cells and cells stimulated with EGF or phorbol ester. βPix(S340E) further enhanced O2(-) production, while βPix(S340A) eliminated the βPix effect. βPix(S340E), but not βPix(S340A), had higher affinity and GEF activity for Rac than wild-type βPix. These results suggest that βPix phosphorylation at Ser-340 upregulates Nox1 through Rac activation, confirming Rac as a trigger for acute Nox1-dependent ROS production.
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Affiliation(s)
- Yuuki Kaito
- Department of Applied Chemistry, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan
| | - Ryosuke Kataoka
- Department of Applied Chemistry, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan
| | - Kento Takechi
- Department of Applied Chemistry, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan
| | - Tatsuya Mihara
- Department of Applied Chemistry, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan
| | - Minoru Tamura
- Department of Applied Chemistry, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
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20
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Kovalenko PL, Yuan L, Sun K, Kunovska L, Seregin S, Amalfitano A, Basson MD. Regulation of epithelial differentiation in rat intestine by intraluminal delivery of an adenoviral vector or silencing RNA coding for Schlafen 3. PLoS One 2013; 8:e79745. [PMID: 24244554 PMCID: PMC3823574 DOI: 10.1371/journal.pone.0079745] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2013] [Accepted: 09/23/2013] [Indexed: 12/15/2022] Open
Abstract
Although we stimulate enterocytic proliferation to ameliorate short gut syndrome or mucosal atrophy, less effort has been directed at enterocytic differentiation. Schlafen 3 (Slfn3) is a poorly understood protein induced during IEC-6 enterocytic differentiation. We hypothesized that exogenous manipulation of Slfn3 would regulate enterocytic differentiation in vivo. Adenoviral vector coding for Slfn3 cDNA (Ad-GFP-Slfn3) or silencing RNA for Slfn3 (siSlfn3) was introduced intraluminally into rat intestine. We assessed Slfn3, villin, sucrase-isomaltase (SI), Dpp4, and Glut2 by qRT-PCR, Western blot, and immunohistochemistry. We also studied Slfn3 and these differentiation markers in atrophic defunctionalized jejunal mucosa and the crypt-villus axis of normal jejunum. Ad-GFP-Slfn3 but not Ad-GFP increased Slfn3, villin and Dpp4 expression in human Caco-2 intestinal epithelial cells. Injecting Ad-GFP-Slfn3 into rat jejunum in vivo increased mucosal Slfn3 mRNA three days later vs. intraluminal Ad-GFP. This Slfn3 overexpression was associated with increases in all four differentiation markers. Injecting siSlfn3 into rat jejunum in vivo substantially reduced Slfn3 and all four intestinal mucosal differentiation markers three days later, as well as Dpp4 specific activity. Endogenous Slfn3 was reduced in atrophic mucosa from a blind-end Roux-en-Y anastomosis in parallel with differentiation marker expression together with AKT and p38 signaling. Slfn3 was more highly expressed in the villi than the crypts, paralleling Glut2, SI and Dpp4. Slfn3 is a key intracellular regulator of rat enterocytic differentiation. Understanding how Slfn3 works may identify targets to promote enterocytic differentiation and maintain mucosal function in vivo, facilitating enteral nutrition and improving survival in patients with mucosal atrophy or short gut syndrome.
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Affiliation(s)
- Pavlo L. Kovalenko
- Department of Surgery, Michigan State University, East Lansing, Michigan, United States of America
| | - Lisi Yuan
- Department of Surgery, Michigan State University, East Lansing, Michigan, United States of America
- Research Service, John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan, United States of America
| | - Kelian Sun
- Department of Surgery, Michigan State University, East Lansing, Michigan, United States of America
| | - Lyudmyla Kunovska
- Department of Surgery, Michigan State University, East Lansing, Michigan, United States of America
| | - Sergey Seregin
- Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America
| | - Andrea Amalfitano
- Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America
| | - Marc D. Basson
- Department of Surgery, Michigan State University, East Lansing, Michigan, United States of America
- Research Service, John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan, United States of America
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Tange S, Zhou Y, Nagakui-Noguchi Y, Imai T, Nakanishi A. Initiation of human astrovirus type 1 infection was blocked by inhibitors of phosphoinositide 3-kinase. Virol J 2013; 10:153. [PMID: 23680019 PMCID: PMC3750554 DOI: 10.1186/1743-422x-10-153] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2012] [Accepted: 04/23/2013] [Indexed: 01/31/2023] Open
Abstract
BACKGROUND Upon initial contact with a virus, host cells activate a series of cellular signaling cascades that facilitate viral entry and viral propagation within the cell. Little is known about how the human astrovirus (HAstV) exploits signaling cascades to establish an infection in host cells. Recent studies showed that activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is important for HAstV infection, though the involvement of other signaling cascades remains unclear. METHODS A panel of kinase blockers was used to search for cellular signaling pathways important for HAstV1 infection. To determine their impact on the infectious process, we examined viral gene expression, RNA replication, and viral RNA and capsid protein release from host cells. RESULTS Inhibitors of phosphoinositide 3-kinase (PI3K) activation interfered with the infection, independent of their effect on ERK 1/2 activation. Activation of the PI3K signaling cascade occurred at an early phase of the infection, judging from the timeframe of Akt phosphorylation. PI3K inhibition at early times, but not at later times, blocked viral gene expression. However, inhibiting the downstream targets of PI3K activation, Akt and Rac1, did not block infection. Inhibition of protein kinase A (PKA) activation was found to block a later phase of HAstV1 production. CONCLUSIONS Our results reveal a previously unknown, essential role of PI3K in the life cycle of HAstV1. PI3K participates in the early stage of infection, possibly during the viral entry process. Our results also reveal the role of PKA in viral production.
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Affiliation(s)
- Shoichiro Tange
- Section of Gene Therapy, Department of Aging Intervention, National Center for Geriatrics and Gerontology, 35, Gengo, Morioka, Obu, Aichi 474-8522, Japan
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22
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Amin R, Sharma S, Ratakonda S, Hassan HA. Extracellular nucleotides inhibit oxalate transport by human intestinal Caco-2-BBe cells through PKC-δ activation. Am J Physiol Cell Physiol 2013; 305:C78-89. [PMID: 23596171 DOI: 10.1152/ajpcell.00339.2012] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Nephrolithiasis remains a major health problem in Western countries. Seventy to 80% of kidney stones are composed of calcium oxalate, and small changes in urinary oxalate affect risk of kidney stone formation. Intestinal oxalate secretion mediated by the anion exchanger SLC26A6 plays an essential role in preventing hyperoxaluria and calcium oxalate nephrolithiasis, indicating that understanding the mechanisms regulating intestinal oxalate transport is critical for management of hyperoxaluria. Purinergic signaling modulates several intestinal processes through pathways including PKC activation, which we previously found to inhibit Slc26a6 activity in mouse duodenal tissue. We therefore examined whether purinergic stimulation with ATP and UTP affects oxalate transport by human intestinal Caco-2-BBe (C2) cells. We measured [¹⁴C]oxalate uptake in the presence of an outward Cl⁻ gradient as an assay of Cl⁻/oxalate exchange activity, ≥50% of which is mediated by SLC26A6. We found that ATP and UTP significantly inhibited oxalate transport by C2 cells, an effect blocked by the PKC inhibitor Gö-6983. Utilizing pharmacological agonists and antagonists, as well as PKC-δ knockdown studies, we observed that ATP inhibits oxalate transport through the P2Y₂ receptor, PLC, and PKC-δ. Biotinylation studies showed that ATP inhibits oxalate transport by lowering SLC26A6 surface expression. These findings are of potential relevance to pathophysiology of inflammatory bowel disease-associated hyperoxaluria, where supraphysiological levels of ATP/UTP are expected and overexpression of the P2Y₂ receptor has been reported. We conclude that ATP and UTP inhibit oxalate transport by lowering SLC26A6 surface expression in C2 cells through signaling pathways including the P2Y₂ purinergic receptor, PLC, and PKC-δ.
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Affiliation(s)
- Ruhul Amin
- Department of Medicine, University of Chicago, Chicago, IL 60637, USA
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23
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Higashida C, Kiuchi T, Akiba Y, Mizuno H, Maruoka M, Narumiya S, Mizuno K, Watanabe N. F- and G-actin homeostasis regulates mechanosensitive actin nucleation by formins. Nat Cell Biol 2013; 15:395-405. [DOI: 10.1038/ncb2693] [Citation(s) in RCA: 85] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2012] [Accepted: 01/14/2013] [Indexed: 12/12/2022]
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Terada N, Saitoh Y, Ohno N, Komada M, Yamauchi J, Ohno S. Involvement of Src in the membrane skeletal complex, MPP6-4.1G, in Schmidt-Lanterman incisures of mouse myelinated nerve fibers in PNS. Histochem Cell Biol 2013; 140:213-22. [PMID: 23306908 DOI: 10.1007/s00418-012-1073-6] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/15/2012] [Indexed: 11/26/2022]
Abstract
Schmidt-Lanterman incisures (SLIs) are a specific feature of myelinated nerve fibers in the peripheral nervous system (PNS). In this study, we report localization of a signal transduction protein, Src, in the SLIs of mouse sciatic nerves, and its phosphorylation states in Y527 and Y418 (P527 and P418, respectively) under normal conditions or deletion of a membrane skeletal protein, 4.1G. In adult mouse sciatic nerves, Src was immunolocalized in SLIs as a cone-shape, as well as in paranodes and some areas of structures reminiscent of Cajal bands. By immunostaining in normal nerves, P527-Src was strongly detected in SLIs, whereas P418-Src was much weaker. Developmentally, P418-Src was detected in SLIs of early postnatal mouse sciatic nerves. The staining patterns for P527 and P418 in normal adult nerve fibers were opposite to those in primary culture Schwann cells and a Schwannoma cell line, RT4-D6P2T. In 4.1G-deficient nerve fibers, which had neither 4.1G nor the membrane protein palmitoylated 6 (MPP6) in SLIs, the P418-Src immunoreactivity in SLIs was clearly detected at a stronger level than that in the wild type. An immunoprecipitation study revealed Src interaction with MPP6. These findings indicate that the Src-MPP6-4.1G protein complex in SLIs has a role in signal transduction in the PNS.
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Affiliation(s)
- Nobuo Terada
- Department of Occupational Therapy, School of Health Sciences, Shinshu University School of Medicine, Matsumoto City, Nagano, Japan.
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Shaw D, Gohil K, Basson MD. Intestinal mucosal atrophy and adaptation. World J Gastroenterol 2012; 18:6357-6375. [PMID: 23197881 PMCID: PMC3508630 DOI: 10.3748/wjg.v18.i44.6357] [Citation(s) in RCA: 89] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/06/2012] [Revised: 11/06/2012] [Accepted: 11/14/2012] [Indexed: 02/06/2023] Open
Abstract
Mucosal adaptation is an essential process in gut homeostasis. The intestinal mucosa adapts to a range of pathological conditions including starvation, short-gut syndrome, obesity, and bariatric surgery. Broadly, these adaptive functions can be grouped into proliferation and differentiation. These are influenced by diverse interactions with hormonal, immune, dietary, nervous, and mechanical stimuli. It seems likely that clinical outcomes can be improved by manipulating the physiology of adaptation. This review will summarize current understanding of the basic science surrounding adaptation, delineate the wide range of potential targets for therapeutic intervention, and discuss how these might be incorporated into an overall treatment plan. Deeper insight into the physiologic basis of adaptation will identify further targets for intervention to improve clinical outcomes.
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Kovalenko PL, Flanigan TL, Chaturvedi L, Basson MD. Influence of defunctionalization and mechanical forces on intestinal epithelial wound healing. Am J Physiol Gastrointest Liver Physiol 2012; 303:G1134-G1143. [PMID: 22997197 PMCID: PMC3517654 DOI: 10.1152/ajpgi.00321.2012] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/08/2012] [Accepted: 09/15/2012] [Indexed: 01/31/2023]
Abstract
The influence on mucosal healing of luminal nutrient flow and the forces it creates are poorly understood. We hypothesized that altered deformation and extracellular pressure mediate, in part, the effects of defunctionalization on mucosal healing. We created patent or partially obstructing defunctionalizing jejunal Roux-en-Y anastomoses in rats to investigate mucosal healing in the absence or presence of luminal nutrient flow and measured luminal pressures to document partial obstruction. We used serosal acetic acid to induce ulcers in the proximal, distal, and defunctionalized intestinal segments. After 3 days, we assessed ulcer area, proliferation, and phosphorylated ERK. In vitro, we measured proliferation and migration in Caco-2 and IEC-6 intestinal epithelial cells subjected to cyclic strain, increased extracellular pressure, or strain and pressure together. Defunctionalization of intestine without obstruction reduced phosphorylated ERK, slowed ulcer healing, and inhibited mucosal proliferation. This outcome was blocked by PD-98059. Partial obstruction delayed ulcer healing but stimulated proliferation independently of ERK. In vitro, strain increased Caco-2 and IEC-6 proliferation and reduced migration across collagen but reduced proliferation and increased migration across fibronectin. In contrast, increased pressure and the combination of pressure and strain increased proliferation and reduced migration independently of substrate. PD-98059 reduced basal migration but increased migration under pressure. These results suggest that loss of the repetitive distension may decrease mucosal healing in defunctionalized bowel, while increased luminal pressure above anastomoses or in spastic bowel disease could further inhibit mucosal healing, despite peristaltic repetitive strain. ERK may mediate the effects of repetitive deformation but not the effects of pressure.
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Affiliation(s)
- Pavlo L Kovalenko
- Dept. of Surgery, College of Human Medicine, Michigan State Univ., Lansing, MI 48912, USA
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Kovalenko PL, Kunovska L, Chen J, Gallo KA, Basson MD. Loss of MLK3 signaling impedes ulcer healing by modulating MAPK signaling in mouse intestinal mucosa. Am J Physiol Gastrointest Liver Physiol 2012; 303:G951-G960. [PMID: 22917630 PMCID: PMC3469692 DOI: 10.1152/ajpgi.00158.2012] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/20/2012] [Accepted: 08/21/2012] [Indexed: 01/31/2023]
Abstract
Mixed-lineage kinase 3 (MLK3) activates multiple MAPK pathways and can initiate apoptosis, proliferation, migration, or differentiation in different cell types. However, whether MLK3 signaling regulates intestinal epithelial cell sheet migration in vivo is not known. We sought to investigate whether MLK3 signaling is important in intestinal mucosal healing and epithelial cell motility in vivo and in vitro. In vivo, we compared the healing of jejunal mucosal ulcers induced in MLK3 knockout (KO) mice with healing in wild-type (WT) mice. Ulcer healing was 20.8% less at day 3 (P < 0.05) and 18.9% less at day 5 (P < 0.05) in MLK3 KO than WT mice. Within the intestinal mucosa of MLK3 KO mice, ERK and JNK signaling were reduced, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) level was increased, and p38 signaling was unchanged. Parallel in vitro studies using an MLK inhibitor assessed the role of MLK signaling in human Caco-2 intestinal epithelial migration across collagen substrates. The MLK inhibitor reduced closure of circular wounds in Caco-2 monolayers. MLK inhibition reduced ERK and JNK, but not p38, signaling in Caco-2 cells. Although PTEN is increased after MLK inhibition, it does not influence MLK-mediated cell migration. These findings indicate that disruption of MLK3 signaling impairs ulcer healing by suppressing ERK and JNK signaling in vitro and in mouse intestinal mucosa in vivo. These results reveal a novel role for MLK3 signaling in the regulation of intestinal epithelial migration in vivo and suggest that MLK3 may be an important target for the regulation of intestinal mucosal healing.
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Affiliation(s)
- Pavlo L Kovalenko
- Department of Surgery, Michigan State University, East Lansing, Michigan 48912, USA
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Hassan HA, Cheng M, Aronson PS. Cholinergic signaling inhibits oxalate transport by human intestinal T84 cells. Am J Physiol Cell Physiol 2012; 302:C46-58. [PMID: 21956166 PMCID: PMC3328906 DOI: 10.1152/ajpcell.00075.2011] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2011] [Accepted: 09/28/2011] [Indexed: 02/06/2023]
Abstract
Urolithiasis remains a very common disease in Western countries. Seventy to eighty percent of kidney stones are composed of calcium oxalate, and minor changes in urinary oxalate affect stone risk. Intestinal oxalate secretion mediated by anion exchanger SLC26A6 plays a major constitutive role in limiting net absorption of ingested oxalate, thereby preventing hyperoxaluria and calcium oxalate urolithiasis. Using the relatively selective PKC-δ inhibitor rottlerin, we had previously found that PKC-δ activation inhibits Slc26a6 activity in mouse duodenal tissue. To identify a model system to study physiologic agonists upstream of PKC-δ, we characterized the human intestinal cell line T84. Knockdown studies demonstrated that endogenous SLC26A6 mediates most of the oxalate transport by T84 cells. Cholinergic stimulation with carbachol modulates intestinal ion transport through signaling pathways including PKC activation. We therefore examined whether carbachol affects oxalate transport in T84 cells. We found that carbachol significantly inhibited oxalate transport by T84 cells, an effect blocked by rottlerin. Carbachol also led to significant translocation of PKC-δ from the cytosol to the membrane of T84 cells. Using pharmacological inhibitors, we observed that carbachol inhibits oxalate transport through the M(3) muscarinic receptor and phospholipase C. Utilizing the Src inhibitor PP2 and phosphorylation studies, we found that the observed regulation downstream of PKC-δ is partially mediated by c-Src. Biotinylation studies revealed that carbachol inhibits oxalate transport by reducing SLC26A6 surface expression. We conclude that carbachol negatively regulates oxalate transport by reducing SLC26A6 surface expression in T84 cells through signaling pathways including the M(3) muscarinic receptor, phospholipase C, PKC-δ, and c-Src.
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Affiliation(s)
- Hatim A Hassan
- Section of Nephrology, Dept. of Medicine, The Univ. of Chicago, 5841 S. Maryland Ave., MC5100, Chicago, IL 60637, USA.
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Ren K, Ma Y, Huang Y, Liang W, Liu F, Wang Q, Cui W, Liu Z, Yin G, Fan W. Periodic mechanical stress activates MEK1/2-ERK1/2 mitogenic signals in rat chondrocytes through Src and PLCγ1. Braz J Med Biol Res 2011; 44:1231-42. [DOI: 10.1590/s0100-879x2011007500150] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2011] [Accepted: 10/25/2011] [Indexed: 12/18/2022] Open
Affiliation(s)
| | - Yimin Ma
- Nanjing Medical University, China
| | | | | | - Feng Liu
- Nanjing Medical University, China
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Chaturvedi LS, Marsh HM, Basson MD. Role of RhoA and its effectors ROCK and mDia1 in the modulation of deformation-induced FAK, ERK, p38, and MLC motogenic signals in human Caco-2 intestinal epithelial cells. Am J Physiol Cell Physiol 2011; 301:C1224-C1238. [PMID: 21849669 PMCID: PMC3213924 DOI: 10.1152/ajpcell.00518.2010] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2010] [Accepted: 08/11/2011] [Indexed: 12/12/2022]
Abstract
Repetitive deformation enhances intestinal epithelial migration across tissue fibronectin. We evaluated the contribution of RhoA and its effectors Rho-associated kinase (ROK/ROCK) and mammalian diaphanous formins (mDia1) to deformation-induced intestinal epithelial motility across fibronectin and the responsible focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), p38, and myosin light chain (MLC) signaling. We reduced RhoA, ROCK1, ROCK2, and mDia1 by smart-pool double-stranded short-interfering RNAs (siRNA) and pharmacologically inhibited RhoA, ROCK, and FAK in human Caco-2 intestinal epithelial monolayers on fibronectin-coated membranes subjected to 10% repetitive deformation at 10 cycles/min. Migration was measured by wound closure. Stimulation of migration by deformation was prevented by exoenzyme C3, Y27632, or selective RhoA, ROCK1, and ROCK2 or mDia1 siRNAs. RhoA, ROCK inhibition, or RhoA, ROCK1, ROCK2, mDia1, and FAK reduction by siRNA blocked deformation-induced nuclear ERK phosphorylation without preventing ERK phosphorylation in the cytoplasmic protein fraction. Furthermore, RhoA, ROCK inhibition or RhoA, ROCK1, ROCK2, and mDia1 reduction by siRNA also blocked strain-induced FAK-Tyr(925), p38, and MLC phosphorylation. These results suggest that RhoA, ROCK, mDia1, FAK, ERK, p38, and MLC all mediate the stimulation of intestinal epithelial migration by repetitive deformation. This pathway may be an important target for interventions to promote mechanotransduced mucosal healing during inflammation.
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Yu G, Dymond M, Yuan L, Chaturvedi LS, Shiratsuchi H, Durairaj S, Marsh HM, Basson MD. Propofol's effects on phagocytosis, proliferation, nitrate production, and cytokine secretion in pressure-stimulated microglial cells. Surgery 2011; 150:887-896. [PMID: 21676422 PMCID: PMC3837575 DOI: 10.1016/j.surg.2011.04.002] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2010] [Accepted: 04/22/2011] [Indexed: 01/05/2023]
Abstract
BACKGROUND Intracranial hypertension complicates severe traumatic brain injury frequently and might be associated with poor outcomes. Traumatic brain injury induces a neuroinflammatory response by microglial activation and upregulation of proinflammatory cytokines, such as interleukin-1β, tumor necrosis factor alpha, and interleukin-6. To elucidate the effect of increased intracranial pressure on microglial function, we studied the effects of increased extracellular pressure on primary human microglial cell phagocytosis, proliferation, cytokine secretion, and total nitrate production. In addition, because many patients receive propofol during anesthesia or intensive care unit sedation, we evaluated whether propofol alters the effects of pressure. METHODS Human microglial cells were pretreated with (2.5-20 μg/mL) propofol or Intralipid as a vehicle control were incubated at ambient atmospheric pressure or at 15 or 30 mm Hg increased pressure for 2 h for phagocytosis assays or 24 h for proliferation, cytokine secretion, and total nitrate production studies. Phagocytosis was determined by incorporation of intracellular fluorescent latex beads. Tumor necrosis factor alpha, interleukin-1β, and interleukin-6 were assayed by sandwich enzyme-linked immunosorbent assay and total nitrate by Greiss reagent. RESULTS Increased extracellular pressure stimulated phagocytosis versus untreated microglial cells or cells treated with an Intralipid vehicle control. Propofol also stimulated microglial phagocytosis at ambient pressure. Increased pressure, however, decreased phagocytosis in the presence of propofol. Pressure also increased microglial tumor necrosis factor-α and interleukin-1β secretion and propofol pretreatment blocked the pressure-stimulated effect. Interleukin-6 production was not altered either by pressure or by propofol. Pressure also induced total nitrate secretion, and propofol pretreatment decreased basal as well as pressure-induced microglial nitrate production. CONCLUSION Extracellular pressures consistent with increased intracranial pressure after a head injury activate inflammatory signals in human primary microglial cells in vitro, stimulating phagocytosis, proliferation, tumor necrosis factor-α, interleukin-1β, and total nitrate secretion but not affecting interleukin-6. Such inflammatory events may contribute to the worsened prognosis of traumatic brain injury after increased intracranial pressure. Because propofol alleviated these potentially proinflammatory effects, these results suggest that the inflammatory cascade activated by intracranial pressure might be targeted by propofol in patients with increased intracranial pressure after traumatic brain injury.
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Affiliation(s)
- Guangxiang Yu
- Department of Anesthesiology, Wayne State University, Detroit, MI
| | - Michael Dymond
- Department of Anesthesiology, Wayne State University, Detroit, MI
- Department of Surgery, Michigan State University, Lansing, MI
- John D. Dingell VA Medical Center
| | - Lisi Yuan
- Department of Surgery, Michigan State University, Lansing, MI
| | - Lakshmi S Chaturvedi
- Department of Anesthesiology, Wayne State University, Detroit, MI
- John D. Dingell VA Medical Center
| | | | | | - H. Michael Marsh
- Department of Anesthesiology, Wayne State University, Detroit, MI
- John D. Dingell VA Medical Center
| | - Marc D. Basson
- Department of Anesthesiology, Wayne State University, Detroit, MI
- Department of Surgery, Michigan State University, Lansing, MI
- John D. Dingell VA Medical Center
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Owen KA, Abshire MY, Tilghman RW, Casanova JE, Bouton AH. FAK regulates intestinal epithelial cell survival and proliferation during mucosal wound healing. PLoS One 2011; 6:e23123. [PMID: 21887232 PMCID: PMC3160839 DOI: 10.1371/journal.pone.0023123] [Citation(s) in RCA: 62] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2011] [Accepted: 07/11/2011] [Indexed: 12/29/2022] Open
Abstract
Background Following damage to the intestinal epithelium, restoration of epithelial barrier integrity is triggered by a robust proliferative response. In other tissues, focal adhesion kinase (FAK) regulates many of the cellular processes that are critical for epithelial homeostasis and restitution, including cell migration, proliferation and survival. However, few studies to date have determined how FAK contributes to mucosal wound healing in vivo. Methodology and Principal Findings To examine the role of FAK in intestinal epithelial homeostasis and during injury, we generated intestinal epithelium (IE)-specific conditional FAK knockout mice. Colitis was induced with dextran-sulfate-sodium (DSS) and intestinal tissues were analyzed by immunohistochemistry and immunoblotting. While intestinal development occurred normally in mice lacking FAK, FAK-deficient animals were profoundly susceptible to colitis. The loss of epithelial FAK resulted in elevated p53 expression and an increased sensitivity to apoptosis, coincident with a failure to upregulate epithelial cell proliferation. FAK has been reported to function as a mechanosensor, inducing cyclin D1 expression and promoting cell cycle progression under conditions in which tissue/matrix stiffness is increased. Collagen deposition, a hallmark of inflammatory injury resulting in increased tissue rigidity, was observed in control and FAK knockout mice during colitis. Despite this fibrotic response, the colonic epithelium in FAK-deficient mice exhibited significantly reduced cyclin D1 expression, suggesting that proliferation is uncoupled from fibrosis in the absence of FAK. In support of this hypothesis, proliferation of Caco-2 cells increased proportionally with matrix stiffness in vitro only under conditions of normal FAK expression; FAK depleted cells exhibited reduced proliferation concomitant with attenuated cyclin D1 expression. Conclusions In the colon, FAK functions as a regulator of epithelial cell survival and proliferation under conditions of mucosal injury and a mechanosensor of tissue compliance, inducing repair-driven proliferation in the colonic epithelium through upregulation of cyclin D1.
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Affiliation(s)
- Katherine A. Owen
- Department of Cell Biology, University of Virginia Health System, Charlottesville, Virginia, United States of America
| | - Michelle Y. Abshire
- Department of Microbiology, University of Virginia Health System, Charlottesville, Virginia, United States of America
| | - Robert W. Tilghman
- Department of Microbiology, University of Virginia Health System, Charlottesville, Virginia, United States of America
| | - James E. Casanova
- Department of Cell Biology, University of Virginia Health System, Charlottesville, Virginia, United States of America
- Department of Microbiology, University of Virginia Health System, Charlottesville, Virginia, United States of America
- * E-mail: (AHB); (JEC)
| | - Amy H. Bouton
- Department of Microbiology, University of Virginia Health System, Charlottesville, Virginia, United States of America
- * E-mail: (AHB); (JEC)
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Zebda N, Dubrovskyi O, Birukov KG. Focal adhesion kinase regulation of mechanotransduction and its impact on endothelial cell functions. Microvasc Res 2011; 83:71-81. [PMID: 21741394 DOI: 10.1016/j.mvr.2011.06.007] [Citation(s) in RCA: 83] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2011] [Revised: 06/19/2011] [Accepted: 06/20/2011] [Indexed: 01/06/2023]
Abstract
Vascular endothelial cells lining the blood vessels form the interface between the bloodstream and the vessel wall and as such they are continuously subjected to shear and cyclic stress from the flowing blood in the lumen. Additional mechanical stimuli are also imposed on these cells in the form of substrate stiffness transmitted from the extracellular matrix components in the basement membrane, and additional mechanical loads imposed on the lung endothelium as the result of respiration or mechanical ventilation in clinical settings. Focal adhesions (FAs) are complex structures assembled at the abluminal endothelial plasma membrane which connect the extracellular filamentous meshwork to the intracellular cytoskeleton and hence constitute the ideal checkpoint capable of controlling or mediating transduction of bidirectional mechanical signals. In this review we focus on focal adhesion kinase (FAK), a component of FAs, which has been studied for a number of years with regards to its involvement in mechanotransduction. We analyzed the recent advances in the understanding of the role of FAK in the signaling cascade(s) initiated by various mechanical stimuli with particular emphasis on potential implications on endothelial cell functions.
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Affiliation(s)
- Noureddine Zebda
- Section of Pulmonary and Critical Care, Lung Injury Center, Department of Medicine, The University of Chicago, IL 60637, USA
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Schlegel N, Meir M, Spindler V, Germer CT, Waschke J. Differential role of Rho GTPases in intestinal epithelial barrier regulation in vitro. J Cell Physiol 2011; 226:1196-203. [PMID: 20945370 DOI: 10.1002/jcp.22446] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Maintenance of intestinal epithelial barrier functions is crucial to prevent systemic contamination by microbes that penetrate from the gut lumen. GTPases of the Rho-family such as RhoA, Rac1, and Cdc42 are known to be critically involved in the regulation of intestinal epithelial barrier functions. However, it is still unclear whether inactivation or activation of these GTPases exerts barrier protection or not. We tested the effects of Rho GTPase activities on intestinal epithelial barrier functions by using the bacterial toxins cytotoxic necrotizing factor 1 (CNF-1), toxin B, C3 transferase (C3 TF), and lethal toxin (LT) in an in vitro model of the intestinal epithelial barrier. Incubation of cell monolayers with CNF-1 for 3 h induced exclusive activation of RhoA whereas Rac1 and Cdc42 activities were unchanged. As revealed by FITC-dextran flux and measurements of transepithelial electrical resistance (TER) intestinal epithelial permeability was significantly increased under these conditions. Inhibition of Rho kinase via Y27632 blocked barrier destabilization of CNF-1 after 3 h. In contrast, after 24 h of incubation with CNF-1 only Rac1 and Cdc42 but not RhoA were activated which resulted in intestinal epithelial barrier stabilization. Toxin B to inactivate RhoA, Rac1, and Cdc42 as well as Rac1 inhibitor LT increased intestinal epithelial permeability. Similar effects were observed after inhibition of RhoA/Rho kinase signaling by C3 TF or Y27632. Taken together, these data demonstrate that both activation and inactivation of RhoA signaling increased paracellular permeability whereas activation of Rac1 and Cdc42 correlated with stabilized barrier functions.
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Affiliation(s)
- Nicolas Schlegel
- Department of Surgery I, University of Würzburg, Würzburg, Germany
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Yuan L, Sanders MA, Basson MD. ILK mediates the effects of strain on intestinal epithelial wound closure. Am J Physiol Cell Physiol 2011; 300:C356-C367. [PMID: 21084641 PMCID: PMC3043633 DOI: 10.1152/ajpcell.00273.2010] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2010] [Accepted: 11/15/2010] [Indexed: 01/02/2023]
Abstract
The intestinal epithelium is subjected to repetitive deformation during normal gut function by peristalsis and villous motility. Such repetitive strain promotes intestinal epithelial migration across fibronectin in vitro, but signaling mediators for this are poorly understood. We hypothesized that integrin-linked kinase (ILK) mediates strain-stimulated migration in intestinal epithelial cells cultured on fibronectin. ILK kinase activity increased rapidly 5 min after strain induction in both Caco-2 and intestinal epithelial cell-6 (IEC-6) cells. Wound closure in response to strain was reduced in ILK small interfering RNA (siRNA)-transfected Caco-2 cell monolayers when compared with control siRNA-transfected Caco-2 cells. Pharmacological blockade of phosphatidylinositol-3 kinase (PI3K) or Src or reducing Src by siRNA prevented strain activation of ILK. ILK coimmunoprecipitated with focal adhesion kinase (FAK), and this association was decreased by mutation of FAK Tyr925 but not FAK Tyr397. Strain induction of FAK Tyr925 phosphorylation but not FAK Tyr397 or FAK Tyr576 phosphorylation was blocked in ILK siRNA-transfected cells. ILK-Src association was stimulated by strain and was blocked by the Src inhibitor PP2. Finally, ILK reduction by siRNA inhibited strain-induced phosphorylation of myosin light chain and Akt. These results suggest a strain-dependent signaling pathway in which ILK association with FAK and Src mediates the subsequent downstream strain-induced motogenic response and suggest that ILK induction by repetitive deformation may contribute to recovery from mucosal injury and restoration of the mucosal barrier in patients with prolonged ileus. ILK may therefore be an important target for intervention to maintain the mucosa in such patients.
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Affiliation(s)
- Lisi Yuan
- Dept. of Surgery, Michigan State University, East Lansing, MI 48912, USA
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Lactobacillus plantarum surface layer adhesive protein protects intestinal epithelial cells against tight junction injury induced by enteropathogenic Escherichia coli. Mol Biol Rep 2010; 38:3471-80. [PMID: 21086172 DOI: 10.1007/s11033-010-0457-8] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2010] [Accepted: 11/09/2010] [Indexed: 01/01/2023]
Abstract
Lactobacillus plantarum (LP) has previously been used for the treatment and prevention of intestinal disorders and disease. However, the role of the LP surface layer adhesive protein (SLAP) in inhibition of epithelial cell disruption is not fully understood. The aim of the present study was to investigate the protective effects of purified SLAP on Caco-2 cells infected with enteropathogenic Escherichia coli (EPEC). The role of ERK in LP-mediated inhibition of tight junction (TJ) injury was also evaluated in order to determine the molecular mechanisms underlying the protective effects of LP in epithelial cells. SLAP was extracted and purified from LP cells using a porcine stomach mucin-Sepharose 4B column. SLAP-mediated inhibition of bacterial adhesion was measured using a competition-based adhesion assay. Expression of TJ-associated proteins, maintenance of TJ structure, and levels of extracellular signal regulated kinase (ERK) and ERK phosphorylation were assessed in SLAP-treated cells by a combination of real-time PCR, western blotting, and immunofluorescence microscopy. Cell permeability was analyzed by measurement of trans-epithelial electrical resistance (TER) and dextran permeability. The effect of SLAP on levels of apoptosis in epithelial cells was assessed by flow cytometry. Results from these experiments revealed that treatment with SLAP decreased the level of adhesion of EPEC to Caco-2 cells. SLAP treatment also enhanced expression of TJ proteins at both the mRNA and protein levels and affected F-actin distribution. Although ERK levels remained unchanged, ERK phosphorylation was increased by SLAP treatment. Caco-2 cells treated with SLAP exhibited increased TER and decreased macromolecular permeability, which was accompanied by a decrease in the level of apoptosis. Together, these results suggest that LP-produced SLAP protects intestinal epithelial cells from EPEC-induced injury, likely through a mechanism involving ERK activation.
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Nong L, Yin G, Ren K, Tang J, Fan W. Periodic mechanical stress enhances rat chondrocyte area expansion and migration through Src-PLCγ1-ERK1/2 signaling. Eur J Cell Biol 2010; 89:705-11. [DOI: 10.1016/j.ejcb.2010.04.005] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2009] [Revised: 04/20/2010] [Accepted: 04/20/2010] [Indexed: 10/19/2022] Open
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Rapier R, Huq J, Vishnubhotla R, Bulic M, Perrault CM, Metlushko V, Cho M, Tay RTS, Glover SC. The extracellular matrix microtopography drives critical changes in cellular motility and Rho A activity in colon cancer cells. Cancer Cell Int 2010; 10:24. [PMID: 20667086 PMCID: PMC2919527 DOI: 10.1186/1475-2867-10-24] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2010] [Accepted: 07/28/2010] [Indexed: 11/29/2022] Open
Abstract
We have shown that the microtopography (mT) underlying colon cancer changes as a tumor de-differentiates. We distinguish the well-differentiated mT based on the increasing number of "pits" and poorly differentiated mT on the basis of increasing number of "posts." We investigated Rho A as a mechanosensing protein using mT features derived from those observed in the ECM of colon cancer. We evaluated Rho A activity in less-tumorogenic (Caco-2 E) and more tumorigenic (SW620) colon cancer cell-lines on microfabricated pits and posts at 2.5 μm diameter and 200 nm depth/height. In Caco-2 E cells, we observed a decrease in Rho A activity as well as in the ratio of G/F actin on surfaces with either pits or posts but despite this low activity, knockdown of Rho A led to a significant decrease in confined motility suggesting that while Rho A activity is reduced on these surfaces it still plays an important role in controlling cellular response to barriers. In SW620 cells, we observed that Rho A activity was greatest in cells plated on a post microtopography which led to increased cell motility, and an increase in actin cytoskeletal turnover.
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Van Grol J, Subauste C, Andrade RM, Fujinaga K, Nelson J, Subauste CS. HIV-1 inhibits autophagy in bystander macrophage/monocytic cells through Src-Akt and STAT3. PLoS One 2010; 5:e11733. [PMID: 20661303 PMCID: PMC2908694 DOI: 10.1371/journal.pone.0011733] [Citation(s) in RCA: 104] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2010] [Accepted: 06/16/2010] [Indexed: 11/18/2022] Open
Abstract
Autophagy is a homeostatic mechanism of lysosomal degradation. Defective autophagy has been linked to various disorders such as impaired control of pathogens and neurodegeneration. Autophagy is regulated by a complex array of signaling pathways that act upstream of autophagy proteins. Little is known about the role of altered regulatory signaling in disorders associated with defective autophagy. In particular, it is not known if pathogens inhibit autophagy by modulation of upstream regulatory pathways. Cells infected with HIV-1 blocked rapamycin-induced autophagy and CD40-induced autophagic killing of Toxoplasma gondii in bystander (non-HIV-1 infected) macrophage/monocytic cells. Blockade of autophagy was dependent on Src-Akt and STAT3 triggered by HIV-1 Tat and IL-10. Neutralization of the upstream receptors VEGFR, beta-integrin or CXCR4, as well as of HIV-1 Tat or IL-10 restored autophagy in macrophage/monocytic cells exposed to HIV-1-infected cells. Defective autophagic killing of T. gondii was detected in monocyte-derived macrophages from a subset of HIV-1(+) patients. This defect was also reverted by neutralization of Tat or IL-10. These studies revealed that a pathogen can impair autophagy in non-infected cells by activating counter-regulatory pathways. The fact that pharmacologic manipulation of cell signaling restored autophagy in cells exposed to HIV-1-infected cells raises the possibility of therapeutic manipulation of cell signaling to restore autophagy in HIV-1 infection.
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Affiliation(s)
- Jennifer Van Grol
- Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America
- Division of Infectious Diseases and HIV Medicine, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America
- Department of Ophthalmology and Visual Sciences, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America
| | - Cecilia Subauste
- McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
| | - Rosa M. Andrade
- Department of Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America
| | - Koh Fujinaga
- Division of Infectious Diseases and HIV Medicine, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America
| | - Julie Nelson
- Center for AIDS Research, University of North Carolina, Chapel Hill, North Carolina, United States of America
| | - Carlos S. Subauste
- Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America
- Division of Infectious Diseases and HIV Medicine, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America
- Department of Ophthalmology and Visual Sciences, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America
- * E-mail:
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Elias BC, Bhattacharya S, Ray RM, Johnson LR. Polyamine-dependent activation of Rac1 is stimulated by focal adhesion-mediated Tiam1 activation. Cell Adh Migr 2010; 4:419-30. [PMID: 20448461 DOI: 10.4161/cam.4.3.12043] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Integrin receptors cluster on the cell surface and bind to extra cellular matrix (ECM) proteins triggering the formation of focal contacts and the activation of various signal transduction pathways that affect the morphology, motility, gene expression and survival of adherent cells. Polyamine depletion prevents the increase in autophosphorylation of focal adhesion kinase (FAK) and Src during attachment. Rac activity also shows a steady decline, and its upstream guanine nucleotide exchange factor (GEF), Tiam1 also shows a reduction in total protein level when cells are depleted of polyamines. When Tiam1 and Rac1 interaction was inhibited by NSC-23766, there was not only a decrease in Rac1 activity as expected but also a decrease in FAK auto-phosphorylation. Inhibition of Src activity by PP2 also reduced FAK autophosphorylation, which implies that Src modulates FAK autophosphorylation. From the data obtained in this study we conclude that FAK and Src are rapidly activated upon fibronectin mediated signaling leading to Tiam1-mediated Rac1 activation and that intracellular polyamines influence the signaling strength by modulating interaction of Src with Tiam1 using focal adhesion kinase as a scaffolding site.
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Affiliation(s)
- Bertha C Elias
- Department of Physiology, University of Tennessee Health Science Center, Memphis, TN, USA
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Yuan L, Yu Y, Sanders MA, Majumdar APN, Basson MD. Schlafen 3 induction by cyclic strain regulates intestinal epithelial differentiation. Am J Physiol Gastrointest Liver Physiol 2010; 298:G994-G1003. [PMID: 20299602 PMCID: PMC4865113 DOI: 10.1152/ajpgi.00517.2009] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/23/2009] [Accepted: 03/12/2010] [Indexed: 01/31/2023]
Abstract
The intestinal epithelium is subjected to repetitive deformation during normal gut function by peristalsis and villous motility. In vitro, cyclic strain promotes intestinal epithelial proliferation and induces an absorptive phenotype characterized by increased dipeptidyl dipeptidase (DPPIV) expression. Schlafen 3 is a novel gene recently associated with cellular differentiation. We sought to evaluate whether Schlafen 3 mediates the effects of strain on the differentiation of intestinal epithelial cell (IEC)-6 in the absence or presence of cyclic strain. Strain increased Schlafen 3 mRNA and protein. In cells transfected with a control-nontargeting siRNA, strain increased DPPIV-specific activity. However, Schlafen 3 reduction by siRNA decreased basal DPPIV and prevented any stimulation of DPPIV activity by strain. Schlafen 3 reduction also prevented DPPIV induction by sodium butyrate (1 mM) or transforming growth factor (TGF)-beta (0.1 ng/ml), two unrelated differentiating stimuli. However, Schlafen-3 reduction by siRNA did not prevent the mitogenic effect of strain or that of EGF. Blocking Src and phosphatidyl inositol (PI3)-kinase prevented strain induction of Schlafen 3, but Schlafen 3 induction required activation of p38 but not ERK. These results suggest that cyclic strain induces an absorptive phenotype characterized by increased DPPIV activity via Src-, p38-, and PI3-kinase-dependent induction of Schlafen 3 in rat IEC-6 cells on collagen, whereas Schlafen 3 may also be a key factor in the induction of intestinal epithelial differentiation by other stimuli such as sodium butyrate or TGF-beta. The induction of Schlafen 3 or its human homologs may modulate intestinal epithelial differentiation and preserve the gut mucosa during normal gut function.
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Affiliation(s)
- Lisi Yuan
- 1200 E. Michigan Ave., Suite 655, Dept. of Surgery, Michigan State Univ., Lansing, MI 48912, USA
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Gayer CP, Craig DH, Flanigan TL, Reed TD, Cress DE, Basson MD. ERK regulates strain-induced migration and proliferation from different subcellular locations. J Cell Biochem 2010; 109:711-725. [PMID: 20069571 DOI: 10.1002/jcb.22450] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
Repetitive deformation like that engendered by peristalsis or villous motility stimulates intestinal epithelial proliferation on collagenous substrates and motility across fibronectin, each requiring ERK. We hypothesized that ERK acts differently at different intracellular sites. We stably transfected Caco-2 cells with ERK decoy expression vectors that permit ERK activation but interfere with its downstream signaling. Targeting sequences constrained the decoy inside or outside the nucleus. We assayed proliferation by cell counting and migration by circular wound closure with or without 10% repetitive deformation at 10 cycles/min. Confocal microscopy confirmed localization of the fusion proteins. Inhibition of phosphorylation of cytoplasmic RSK or nuclear Elk confirmed functionality. Both the nuclear-localized and cytosolic-localized ERK decoys prevented deformation-induced proliferation on collagen. Deformation-induced migration on fibronectin was prevented by constraining the decoy in the nucleus but not in the cytosol. Like the nuclear-localized ERK decoy, a Sef-overexpressing adenovirus that sequesters ERK in the cytoplasm also blocked the motogenic and mitogenic effects of strain. Inhibiting RSK or reducing Elk ablated both the mitogenic and motogenic effects of strain. RSK isoform reduction revealed isoform specificity. These results suggest that ERK must translocate to the nucleus to stimulate cell motility while ERK must act in both the cytosol and the nucleus to stimulate proliferation in response to strain. Selectively targeting ERK within different subcellular compartments may modulate or replace physical force effects on the intestinal mucosa to maintain the intestinal mucosal barrier in settings when peristalsis or villous motility are altered and fibronectin is deposited into injured tissue.
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Hao HF, Naomoto Y, Bao XH, Watanabe N, Sakurama K, Noma K, Tomono Y, Fukazawa T, Shirakawa Y, Yamatsuji T, Matsuoka J, Takaoka M. Progress in researches about focal adhesion kinase in gastrointestinal tract. World J Gastroenterol 2009; 15:5916-23. [PMID: 20014455 PMCID: PMC2795178 DOI: 10.3748/wjg.15.5916] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Focal adhesion kinase (FAK) is a 125-kDa non-receptor protein tyrosine. Growth factors or the clustering of integrins facilitate the rapid phosphorylation of FAK at Tyr-397 and this in turn recruits Src-family protein tyrosine kinases, resulting in the phosphorylation of Tyr-576 and Tyr-577 in the FAK activation loop and full catalytic FAK activation. FAK plays a critical role in the biological processes of normal and cancer cells including the gastrointestinal tract. FAK also plays an important role in the restitution, cell survival and apoptosis and carcinogenesis of the gastrointestinal tract. FAK is over-expressed in cancer cells and its over-expression and elevated activities are associated with motility and invasion of cancer cells. FAK has been proposed as a potential target in cancer therapy. Small molecule inhibitors effectively inhibit the kinase activity of FAK and show a potent inhibitory effect for the proliferation and migration of tumor cells, indicating a high potential for application in cancer therapy.
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Chaturvedi LS, Saad SA, Bakshi N, Marsh HM, Basson MD. Strain matrix-dependently dissociates gut epithelial spreading and motility. J Surg Res 2009; 156:217-223. [PMID: 19691992 PMCID: PMC2749895 DOI: 10.1016/j.jss.2009.03.050] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2009] [Revised: 03/18/2009] [Accepted: 03/22/2009] [Indexed: 01/26/2023]
Abstract
BACKGROUND Repetitive deformation enhances intestinal epithelial migration across tissue fibronectin (tFN) via Src but inhibits migration across collagen. Since cell spreading generally precedes motility, we compared the effects of cyclic strain on Caco-2 spreading and migration on tFN, collagen-I, and plasma fibronectin (pFN), and investigated the role of Src in deformation-influenced spreading and migration. MATERIALS AND METHODS Human Caco-2 intestinal epithelial cells on tFN, collagen-I or pFN were subjected to an average 10% strain at 10 cycles/min for 2 h. Src was inhibited with 10muM PP2 or Src was reduced with siRNA. Parallel studies assessed deformation effects on monolayer wound closure. RESULTS Deformation, Src-inhibition or reduction each inhibited spreading on tFN but Src-inhibition or reduction prevented further inhibition of spreading by deformation without preventing further inhibition of motility. Deformation did not alter spreading on collagen-I or pFN, but inhibited wound closure. CONCLUSIONS Although cell spreading generally precedes and parallels motility, repetitive deformation regulates motility independently of spreading. Since deformation activates Src, the ability of Src blockade to mimic strain-associated inhibition of spreading on tFN suggests that this effect occurs by a separate mechanism that may also require basal Src activity. Further delineation of the mechanisms by which strain disparately modulates spreading and motility may permit acceleration of mucosal healing by targeted interventions to separately promote spreading and epithelial motility.
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Affiliation(s)
- Lakshmi S. Chaturvedi
- Departments of Surgery, John D. Dingell VA Medical Center
- Departments of Surgery, Wayne State University
| | - Samira A. Saad
- Departments of Surgery, John D. Dingell VA Medical Center
- Departments of Surgery, Wayne State University
| | - Neil Bakshi
- Departments of Surgery, John D. Dingell VA Medical Center
- Departments of Surgery, Wayne State University
| | - Harold M. Marsh
- Departments of Surgery, John D. Dingell VA Medical Center
- Departments of Surgery, Wayne State University
| | - Marc D. Basson
- Departments of Surgery, Michigan State University
- Departments of Surgery, John D. Dingell VA Medical Center
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Gayer CP, Basson MD. The effects of mechanical forces on intestinal physiology and pathology. Cell Signal 2009; 21:1237-1244. [PMID: 19249356 PMCID: PMC2715958 DOI: 10.1016/j.cellsig.2009.02.011] [Citation(s) in RCA: 138] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2009] [Accepted: 02/17/2009] [Indexed: 12/18/2022]
Abstract
The epithelial and non-epithelial cells of the intestinal wall experience a myriad of physical forces including strain, shear, and villous motility during normal gut function. Pathologic conditions alter these forces, leading to changes in the biology of these cells. The responses of intestinal epithelial cells to forces vary with both the applied force and the extracellular matrix proteins with which the cells interact, with differing effects on proliferation, differentiation, and motility, and the regulation of these effects involves similar but distinctly different signal transduction mechanisms. Although normal epithelial cells respond to mechanical forces, malignant gastrointestinal epithelial cells also respond to forces, most notably by increased cell adhesion, a critical step in tumor metastasis. This review will focus on the phenomenon of mechanical forces influencing cell biology and the mechanisms by which the gut responds these forces in both the normal as well as pathophysiologic states when forces are altered. Although more is known about epithelial responses to force, information regarding mechanosensitivity of vascular, neural, and endocrine cells within the gut wall will also be discussed, as will, the mechanism by which forces can regulate epithelial tumor cell adhesion.
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Silbert O, Wang Y, Maciejewski BS, Lee H, Shaw SK, Sanchez–Esteban J. ROLES OF RhoA AND Rac1 ON ACTIN REMODELING AND CELL ALIGNMENT AND DIFFERENTIATION IN FETAL TYPE II EPITHELIAL CELLS EXPOSED TO CYCLIC MECHANICAL STRETCH. Exp Lung Res 2009; 34:663-80. [DOI: 10.1080/01902140802339615] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
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Desai LP, White SR, Waters CM. Mechanical stretch decreases FAK phosphorylation and reduces cell migration through loss of JIP3-induced JNK phosphorylation in airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 2009; 297:L520-9. [PMID: 19574423 DOI: 10.1152/ajplung.00076.2009] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
JNK is a nonreceptor kinase involved in the early events that signal cell migration after injury. However, the linkage to early signals required to initiate the migration response to JNK has not been defined in airway epithelial cells, which exist in an environment subjected to cyclic mechanical strain (MS). The present studies demonstrate that the JNK/stress-activated protein kinase-associated protein 1 (JSAP1; also termed JNK-interacting protein 3, JIP3), a scaffold factor for MAPK cascades that links JNK activation to focal adhesion kinase (FAK), are both associated and activated following mechanical injury in 16HBE14o- human airway epithelial cells and that both FAK and JIP3 phosphorylation seen after injury are decreased in cells subjected to cyclic MS. Overexpression of either wild-type (WT)-FAK or WT-JIP3 enhanced phosphorylation and kinase activation of JNK and reduced the inhibitory effect of cyclic MS. These results suggest that cyclic MS impairs signaling of cell migration after injury via a pathway that involves FAK-JIP3-JNK.
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Affiliation(s)
- Leena P Desai
- Dept. of Physiology, The Univ. of Tennessee Health Science Center, 894 Union Ave, Rm. 426, Memphis, TN 38163-0001, USA
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Walsh MF, Ampasala DR, Rishi AK, Basson MD. TGF-beta1 modulates focal adhesion kinase expression in rat intestinal epithelial IEC-6 cells via stimulatory and inhibitory Smad binding elements. BIOCHIMICA ET BIOPHYSICA ACTA 2009; 1789:88-98. [PMID: 19059368 PMCID: PMC2730956 DOI: 10.1016/j.bbagrm.2008.11.002] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/28/2008] [Revised: 10/30/2008] [Accepted: 11/06/2008] [Indexed: 12/21/2022]
Abstract
TGF-beta and FAK modulate cell migration, differentiation, proliferation and apoptosis, and TGF-beta promotes FAK transcription in intestinal epithelial cells via Smad-dependent and independent pathways. We utilized a 1320 bp FAK promoter-luciferase construct to characterize basal and TGF-beta-mediated FAK gene transcription in IEC-6 cells. Inhibiting JNK or Akt negated TGF-beta-stimulated promoter activity; ERK inhibition did not block the TGF-beta effect but increased basal activity. Co-transfection with Co-Smad4 enhanced the TGF-beta response while the inhibitory Smad7 abolished it. Serial deletions sequentially removing the four Smad binding elements (SBE) in the 5' untranslated region of the promoter revealed that the two most distal SBE's are positive regulators while SBE3 exerts a negative influence. Mutational deletion of two upstream p53 sites enhanced basal but did not affect TGF-beta-stimulated increases in promoter activity. TGF-beta increased DNA binding of Smad4, phospho-Smad2/3 and Runx1/AML1a to the most distal 435 bp containing 3 SBE and 2 AML1a sites by ChIP assay. However, although point mutation of SBE1 ablated the TGF-beta-mediated rise in SV40-promoter activity, mutation of AML1a sites did not. TGF-beta regulation of FAK transcription reflects a complex interplay between positive and negative non-Smad signals and SBE's, the last independent of p53 or AML1a.
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Affiliation(s)
- Mary F. Walsh
- Department of Surgery, Wayne State University, Detroit, Michigan
| | | | - Arun K. Rishi
- Barbara Ann Karmanos Cancer Institute, Detroit, Michigan
| | - Marc D. Basson
- Department of Surgery, John D. Dingell VA Medical Center, Detroit, Michigan
- Department of Surgery, Wayne State University, Detroit, Michigan
- Department of Anesthesiology, Wayne State University, Detroit, Michigan
- Department of Anatomy and Cell Biology, Wayne State University, Detroit, Michigan
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Gayer CP, Chaturvedi LS, Wang S, Craig DH, Flanigan T, Basson MD. Strain-induced proliferation requires the phosphatidylinositol 3-kinase/AKT/glycogen synthase kinase pathway. J Biol Chem 2009; 284:2001-2011. [PMID: 19047055 PMCID: PMC2629112 DOI: 10.1074/jbc.m804576200] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2008] [Revised: 11/24/2008] [Indexed: 12/26/2022] Open
Abstract
The intestinal epithelium is repetitively deformed by shear, peristalsis, and villous motility. Such repetitive deformation stimulates the proliferation of intestinal epithelial cells on collagen or laminin substrates via ERK, but the upstream mediators of this effect are poorly understood. We hypothesized that the phosphatidylinositol 3-kinase (PI3K)/AKT cascade mediates this mitogenic effect. PI3K, AKT, and glycogen synthase kinase-3beta (GSK-3beta) were phosphorylated by 10 cycles/min strain at an average 10% deformation, and pharmacologic blockade of these molecules or reduction by small interfering RNA (siRNA) prevented the mitogenic effect of strain in Caco-2 or IEC-6 intestinal epithelial cells. Strain MAPK activation required PI3K but not AKT. AKT isoform-specific siRNA transfection demonstrated that AKT2 but not AKT1 is required for GSK-3beta phosphorylation and the strain mitogenic effect. Furthermore, overexpression of AKT1 or an AKT chimera including the PH domain and hinge region of AKT2 and the catalytic domain and C-tail of AKT1 prevented strain activation of GSK-3beta, but overexpression of AKT2 or a chimera including the PH domain and hinge region of AKT1 and the catalytic domain and C-tail of AKT2 did not. These data delineate a role for PI3K, AKT2, and GSK-3beta in the mitogenic effect of strain. PI3K is required for both ERK and AKT2 activation, whereas AKT2 is sequentially required for GSK-3beta. Furthermore, AKT2 specificity requires its catalytic domain and tail region. Manipulating this pathway may prevent mucosal atrophy and maintain the mucosal barrier in conditions such as ileus, sepsis, and prolonged fasting when peristalsis and villous motility are decreased and the mucosal barrier fails.
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Affiliation(s)
- Christopher P Gayer
- Department of Surgery, John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan 48301, USA
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