|
1
|
Rezaul Islam M, Rauf A, Akash S, Kumer A, Hussain MS, Akter S, Gupta JK, Thameemul Ansari L, Mahfoj Islam Raj MM, Bin Emran T, Aljohani AS, Abdulmonem WA, Thiruvengadam R, Thiruvengadam M. Recent perspective on the potential role of phytocompounds in the prevention of gastric cancer. Process Biochem 2023; 135:83-101. [DOI: 10.1016/j.procbio.2023.11.006] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 09/05/2024]
|
|
2
|
Ashrafizadeh M, Rafiei H, Mohammadinejad R, Farkhondeh T, Samarghandian S. Anti-tumor activity of resveratrol against gastric cancer: a review of recent advances with an emphasis on molecular pathways. Cancer Cell Int 2021; 21:66. [PMID: 33478512 PMCID: PMC7818776 DOI: 10.1186/s12935-021-01773-7] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2020] [Accepted: 01/11/2021] [Indexed: 12/24/2022] Open
Abstract
Gastric cancer (GC) is one of the most common cancers with high malignancy. In spite of the great development in diagnostic tools and application of anti-tumor drugs, we have not witnessed a significant increase in the survival time of patients with GC. Multiple studies have revealed that Wnt, Nrf2, MAPK, and PI3K/Akt signaling pathways are involved in GC invasion. Besides, long non-coding RNAs and microRNAs function as upstream mediators in GC malignancy. GC cells have acquired resistance to currently applied anti-tumor drugs. Besides, combination therapy is associated with higher anti-tumor activity. Resveratrol (Res) is a non-flavonoid polyphenol with high anti-tumor activity used in treatment of various cancers. A number of studies have demonstrated the potential of Res in regulation of molecular pathways involved in cancer malignancy. At the present review, we show that Res targets a variety of signaling pathways to induce apoptotic cell death and simultaneously, to inhibit the migration and metastasis of GC cells.
Collapse
Affiliation(s)
- Milad Ashrafizadeh
- Faculty of Engineering and Natural Sciences, Sabanci University, Orta Mahalle, Üniversite Caddesi No. 27, Orhanlı, Tuzla, Istanbul, 34956, Turkey
- Sabanci University Nanotechnology Research and Application Center (SUNUM), Tuzla, Istanbul, 34956, Turkey
| | - Hossein Rafiei
- Department of Biology, Faculty of Sciences, Shiraz Branch, Islamic Azad University, Shiraz, Iran
| | - Reza Mohammadinejad
- Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran
| | - Tahereh Farkhondeh
- Medical Toxicology and Drug Abuse Research Center (MTDRC), Birjand University of Medical Sciences, Birjand, Iran
- Faculty of Pharmacy, Birjand University of Medical Sciences, Birjand, Iran
| | - Saeed Samarghandian
- Noncommunicable Diseases Research Center, Neyshabur University of Medical Sciences, Neyshabur, 9318614139, Iran.
| |
Collapse
|
|
3
|
de Araujo Junior RF, Eich C, Jorquera C, Schomann T, Baldazzi F, Chan AB, Cruz LJ. Ceramide and palmitic acid inhibit macrophage-mediated epithelial-mesenchymal transition in colorectal cancer. Mol Cell Biochem 2020; 468:153-168. [PMID: 32222879 PMCID: PMC7145792 DOI: 10.1007/s11010-020-03719-5] [Citation(s) in RCA: 37] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2019] [Accepted: 03/13/2020] [Indexed: 02/06/2023]
Abstract
Accumulating evidence indicates that ceramide (Cer) and palmitic acid (PA) possess the ability to modulate switching of macrophage phenotypes and possess anti-tumorigenic effects; however, the underlying molecular mechanisms are largely unknown. The aim of the present study was to investigate whether Cer and PA could induce switching of macrophage polarization from the tumorigenic M2- towards the pro-inflammatory M1-phenotype, and whether this consequently altered the potential of colorectal cancer cells to undergo epithelial–mesenchymal transition (EMT), a hallmark of tumor progression. Our study showed that Cer- and PA-treated macrophages increased expression of the macrophage 1 (M1)-marker CD68 and secretion of IL-12 and attenuated expression of the macrophage 2 (M2)-marker CD163 and IL-10 secretion. Moreover, Cer and PA abolished M2 macrophage-induced EMT and migration of colorectal cancer cells. At the molecular level, this coincided with inhibition of SNAI1 and vimentin expression and upregulation of E-cadherin. Furthermore, Cer and PA attenuated expression levels of IL-10 in colorectal cancer cells co-cultured with M2 macrophages and downregulated STAT3 and NF-κB expression. For the first time, our findings suggest the presence of an IL-10-STAT3-NF-κB signaling axis in colorectal cancer cells co-cultured with M2 macrophages, mimicking the tumor microenvironment. Importantly, PA and Cer were powerful inhibitors of this signaling axis and, consequently, EMT of colorectal cancer cells. These results contribute to our understanding of the immunological mechanisms that underlie the anti-tumorigenic effects of lipids for future combination with drugs in the therapy of colorectal carcinoma.
Collapse
Affiliation(s)
- Raimundo Fernandes de Araujo Junior
- Department of Morphology, Federal University of Rio Grande do Norte, Natal, RN, 59072-970, Brazil. .,Post-Graduation Programme in Structural and Functional Biology, Federal University of Rio Grande do Norte, Natal, RN, 59072-970, Brazil. .,Post-Graduation Programme in Health Science, Federal University of Rio Grande do Norte, Natal, RN, 59072-970, Brazil. .,Translational Nanobiomaterials and Imaging, Department of Radiology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands.
| | - Christina Eich
- Translational Nanobiomaterials and Imaging, Department of Radiology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Carla Jorquera
- Translational Nanobiomaterials and Imaging, Department of Radiology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Timo Schomann
- Percuros B.V., 2333 CL, Leiden, The Netherlands.,Translational Nanobiomaterials and Imaging, Department of Radiology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Fabio Baldazzi
- Translational Nanobiomaterials and Imaging, Department of Radiology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Alan B Chan
- Percuros B.V., 2333 CL, Leiden, The Netherlands.,Translational Nanobiomaterials and Imaging, Department of Radiology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Luis J Cruz
- Translational Nanobiomaterials and Imaging, Department of Radiology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| |
Collapse
|
|
4
|
The Role of Tyrosine Phosphorylation of Protein Kinase C Delta in Infection and Inflammation. Int J Mol Sci 2019; 20:ijms20061498. [PMID: 30917487 PMCID: PMC6471617 DOI: 10.3390/ijms20061498] [Citation(s) in RCA: 38] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2019] [Revised: 03/15/2019] [Accepted: 03/22/2019] [Indexed: 12/30/2022] Open
Abstract
Protein Kinase C (PKC) is a family composed of phospholipid-dependent serine/threonine kinases that are master regulators of inflammatory signaling. The activity of different PKCs is context-sensitive and these kinases can be positive or negative regulators of signaling pathways. The delta isoform (PKCδ) is a critical regulator of the inflammatory response in cancer, diabetes, ischemic heart disease, and neurodegenerative diseases. Recent studies implicate PKCδ as an important regulator of the inflammatory response in sepsis. PKCδ, unlike other members of the PKC family, is unique in its regulation by tyrosine phosphorylation, activation mechanisms, and multiple subcellular targets. Inhibition of PKCδ may offer a unique therapeutic approach in sepsis by targeting neutrophil-endothelial cell interactions. In this review, we will describe the overall structure and function of PKCs, with a focus on the specific phosphorylation sites of PKCδ that determine its critical role in cell signaling in inflammatory diseases such as sepsis. Current genetic and pharmacological tools, as well as in vivo models, that are used to examine the role of PKCδ in inflammation and sepsis are presented and the current state of emerging tools such as microfluidic assays in these studies is described.
Collapse
|
|
5
|
Visualizing bioactive ceramides. Chem Phys Lipids 2018; 216:142-151. [PMID: 30266560 DOI: 10.1016/j.chemphyslip.2018.09.013] [Citation(s) in RCA: 50] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2018] [Revised: 09/19/2018] [Accepted: 09/20/2018] [Indexed: 11/23/2022]
Abstract
In the last 30 years, ceramides have been found to mediate a myriad of biological processes. Ceramides have been recognized as bioactive molecules and their metabolizing enzymes are attractive targets in cancer therapy and other diseases. The molecular mechanism of action of cellular ceramides are still not fully established, with insights into roles through modification of lipid rafts, creation of ceramide platforms, ceramide channels, or through regulation of direct protein effectors such as protein phosphatases and kinases. Recently, the 'Many Ceramides' hypothesis focuses on distinct pools of subcellular ceramides and ceramide species as potential defined bioactive entities. Traditional methods that measure changes in ceramide levels in the whole cell, such as mass spectrometry, fluorescent ceramide analogues, and ceramide antibodies, fail to differentiate specific bioactive species at the subcellular level. However, a few ceramide binding proteins have been reported, and a smaller subgroup within these, have been shown to translocate to ceramide-enriched membranes, revealing these localized pools of bioactive ceramides. In this review we want to discuss and consolidate these works and explore the possibility of defining these binding proteins as new tools are emerging to visualize bioactive ceramides in cells. Our goal is to encourage the scientific community to explore these ceramide partners, to improve techniques to refine the list of these binding partners, making possible the identification of specific domains that recognize and bind ceramides to be used to visualize the 'Many Ceramides' in the cell.
Collapse
|
|
6
|
Hage-Sleiman R, Hamze AB, El-Hed AF, Attieh R, Kozhaya L, Kabbani S, Dbaibo G. Ceramide inhibits PKCθ by regulating its phosphorylation and translocation to lipid rafts in Jurkat cells. Immunol Res 2017; 64:869-86. [PMID: 26798039 DOI: 10.1007/s12026-016-8787-9] [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: 01/15/2023]
Abstract
Protein kinase C theta (PKCθ) is a novel, calcium-independent member of the PKC family of kinases that was identified as a central player in T cell signaling and proliferation. Upon T cell activation by antigen-presenting cells, PKCθ gets phosphorylated and activated prior to its translocation to the immunological synapse where it couples with downstream effectors. PKCθ may be regulated by ceramide, a crucial sphingolipid that is known to promote differentiation, growth arrest, and apoptosis. To further investigate the mechanism, we stimulated human Jurkat T cells with either PMA or anti-CD3/anti-CD28 antibodies following induction of ceramide accumulation by adding exogenous ceramide, bacterial sphingomyelinase, or Fas ligation. Our results suggest that ceramide regulates the PKCθ pathway through preventing its critical threonine 538 (Thr538) phosphorylation and subsequent activation, thereby inhibiting the kinase's translocation to lipid rafts. Moreover, this inhibition is not likely to be a generic effect of ceramide on membrane reorganization. Other lipids, namely dihydroceramide, palmitate, and sphingosine, did not produce similar effects on PKCθ. Addition of the phosphatase inhibitors okadaic acid and calyculin A reversed the inhibition exerted by ceramide, and this suggests involvement of a ceramide-activated protein phosphatase. Such previously undescribed mechanism of regulation of PKCθ raises the possibility that ceramide, or one of its derivatives, and may prove valuable in novel therapeutic approaches for disorders involving autoimmunity or excessive inflammation-where PKCθ plays a critical role.
Collapse
Affiliation(s)
- Rouba Hage-Sleiman
- Department of Biology, Faculty of Sciences, Lebanese University, Hadath, Lebanon
| | - Asmaa B Hamze
- Department of Biomedical Science, Faculty of Health Sciences, Global University, Batrakiyye, Beirut, Lebanon
| | - Aimée F El-Hed
- Department of Pediatrics and Adolescent Medicine, Center for Infectious Diseases Research, Department of Biochemistry and Molecular Genetics, Faculty of Medicine, American University of Beirut, PO Box 11-0236 Riad El Solh, Beirut, Lebanon
| | - Randa Attieh
- Department of Pediatrics and Adolescent Medicine, Center for Infectious Diseases Research, Department of Biochemistry and Molecular Genetics, Faculty of Medicine, American University of Beirut, PO Box 11-0236 Riad El Solh, Beirut, Lebanon
| | - Lina Kozhaya
- Department of Pediatrics and Adolescent Medicine, Center for Infectious Diseases Research, Department of Biochemistry and Molecular Genetics, Faculty of Medicine, American University of Beirut, PO Box 11-0236 Riad El Solh, Beirut, Lebanon
| | - Sarah Kabbani
- Department of Pediatrics and Adolescent Medicine, Center for Infectious Diseases Research, Department of Biochemistry and Molecular Genetics, Faculty of Medicine, American University of Beirut, PO Box 11-0236 Riad El Solh, Beirut, Lebanon
| | - Ghassan Dbaibo
- Department of Pediatrics and Adolescent Medicine, Center for Infectious Diseases Research, Department of Biochemistry and Molecular Genetics, Faculty of Medicine, American University of Beirut, PO Box 11-0236 Riad El Solh, Beirut, Lebanon.
| |
Collapse
|
|
7
|
Aburasayn H, Al Batran R, Ussher JR. Targeting ceramide metabolism in obesity. Am J Physiol Endocrinol Metab 2016; 311:E423-35. [PMID: 27382035 DOI: 10.1152/ajpendo.00133.2016] [Citation(s) in RCA: 74] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/06/2016] [Accepted: 07/04/2016] [Indexed: 12/12/2022]
Abstract
Obesity is a major health concern that increases the risk for insulin resistance, type 2 diabetes (T2D), and cardiovascular disease. Thus, an enormous research effort has been invested into understanding how obesity-associated dyslipidemia and obesity-induced alterations in lipid metabolism increase the risk for these diseases. Accordingly, it has been proposed that the accumulation of lipid metabolites in organs such as the liver, skeletal muscle, and heart is critical to these obesity-induced pathologies. Ceramide is one such lipid metabolite that accumulates in tissues in response to obesity, and both pharmacological and genetic strategies that reduce tissue ceramide levels yield salutary actions on overall metabolic health. We will review herein why ceramide accumulates in tissues during obesity and how an increase in intracellular ceramide impacts cellular signaling and function as well as potential mechanisms by which reducing intracellular ceramide levels improves insulin resistance, T2D, atherosclerosis, and heart failure. Because a reduction in skeletal muscle ceramide levels is frequently associated with improvements in insulin sensitivity in humans, the beneficial findings reported for reducing ceramides in preclinical studies may have clinical application in humans. Therefore, modulating ceramide metabolism may be a novel, exciting target for preventing and/or treating obesity-related diseases.
Collapse
Affiliation(s)
- Hanin Aburasayn
- Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada; and Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada
| | - Rami Al Batran
- Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada; and Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada
| | - John R Ussher
- Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada; and Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada
| |
Collapse
|
|
8
|
Pantham P, Viall CA, Chen Q, Kleffmann T, Print CG, Chamley LW. Antiphospholipid antibodies bind syncytiotrophoblast mitochondria and alter the proteome of extruded syncytial nuclear aggregates. Placenta 2015; 36:1463-73. [PMID: 26506561 DOI: 10.1016/j.placenta.2015.10.006] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/28/2015] [Revised: 08/28/2015] [Accepted: 10/11/2015] [Indexed: 12/21/2022]
Abstract
INTRODUCTION Antiphospholipid antibodies (aPL) are autoantibodies that increase the risk of women developing the hypertensive disorder pre-eclampsia. aPL are internalised by the syncytiotrophoblast and increase extrusion of necrotic multinucleated syncytial nuclear aggregates (SNAs), which may trigger endothelial dysfunction in pre-eclampsia. The mechanisms by which aPL alter death processes in the syncytiotrophoblast leading to extrusion of SNAs are unknown. METHODS First trimester human placentae (n = 10) were dissected into explants and cultured either with aPL (50 μg/mL), isotype-matched control antibody (50 μg/mL), or media for 24 h. Harvested SNAs underwent iTRAQ proteomic analysis. Mitochondria in syncytiotrophoblast treated with aPL labelled with FluoroNanogold were visualised using transmission electron microscopy (TEM). RESULTS aPL altered the expression of 72 proteins in SNAs. Thirteen proteins were involved in mitochondrial function. TEM demonstrated that aPL bind to mitochondria in the syncytiotrophoblast and may cause mitochondrial swelling. DISCUSSION aPL disrupt mitochondria increasing the extrusion of SNAs with an altered proteome from the syncytiotrophoblast. These altered SNAs may trigger endothelial dysfunction and pre-eclampsia in these pregnancies.
Collapse
Affiliation(s)
- Priyadarshini Pantham
- Department of Obstetrics & Gynaecology, The University of Auckland, Auckland, New Zealand; Department of Pediatrics, Section of Neonatology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
| | - Chez A Viall
- Department of Obstetrics & Gynaecology, The University of Auckland, Auckland, New Zealand
| | - Qi Chen
- Department of Obstetrics & Gynaecology, The University of Auckland, Auckland, New Zealand
| | - Torsten Kleffmann
- Centre for Protein Research, University of Otago, Dunedin, New Zealand
| | - Cristin G Print
- Department of Molecular Medicine and Pathology, The University of Auckland, Auckland, New Zealand; Bioinformatics Institute, The University of Auckland, Auckland, New Zealand
| | - Lawrence W Chamley
- Department of Obstetrics & Gynaecology, The University of Auckland, Auckland, New Zealand
| |
Collapse
|
|
9
|
Pantham P, Heazell AE, Mullard G, Begley P, Chen Q, Brown M, Dunn WB, Chamley LW. Antiphospholipid Antibodies Alter Cell-Death-Regulating Lipid Metabolites in First and Third Trimester Human Placentae. Am J Reprod Immunol 2015; 74:181-99. [DOI: 10.1111/aji.12387] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2014] [Accepted: 03/13/2015] [Indexed: 02/06/2023] Open
Affiliation(s)
- Priyadarshini Pantham
- Department of Obstetrics & Gynaecology; The University of Auckland; Auckland New Zealand
- Section of Neonatology; Department of Pediatrics; University of Colorado Anschutz Medical Campus; Aurora CO USA
| | - Alexander E.P. Heazell
- The Maternal and Fetal Health Research Centre; St. Mary's Hospital; The University of Manchester; Manchester UK
| | - Graham Mullard
- Centre for Advanced Discovery and Experimental Therapeutics; Manchester Biomedical Research Centre; The University of Manchester; Manchester UK
| | - Paul Begley
- Centre for Advanced Discovery and Experimental Therapeutics; Manchester Biomedical Research Centre; The University of Manchester; Manchester UK
| | - Qi Chen
- Department of Obstetrics & Gynaecology; The University of Auckland; Auckland New Zealand
| | - Maria Brown
- Centre for Advanced Discovery and Experimental Therapeutics; Manchester Biomedical Research Centre; The University of Manchester; Manchester UK
| | - Warwick B. Dunn
- Centre for Advanced Discovery and Experimental Therapeutics; Manchester Biomedical Research Centre; The University of Manchester; Manchester UK
- Centre for Endocrinology and Diabetes; Institute of Human Development; The University of Manchester; Manchester UK
- School of Biosciences; The University of Birmingham; Edgbaston Birmingham UK
| | - Lawrence W. Chamley
- Department of Obstetrics & Gynaecology; The University of Auckland; Auckland New Zealand
| |
Collapse
|
|
10
|
Saeki T, Inui H, Fujioka S, Fukuda S, Nomura A, Nakamura Y, Park EY, Sato K, Kanamoto R. Staurosporine synergistically potentiates the deoxycholate-mediated induction of COX-2 expression. Physiol Rep 2014; 2:2/8/e12143. [PMID: 25168879 PMCID: PMC4246598 DOI: 10.14814/phy2.12143] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Colorectal cancer is a major cause of cancer‐related death in western countries, and thus there is an urgent need to elucidate the mechanism of colorectal tumorigenesis. A diet that is rich in fat increases the risk of colorectal tumorigenesis. Bile acids, which are secreted in response to the ingestion of fat, have been shown to increase the risk of colorectal tumors. The expression of cyclooxygenase (COX)‐2, an inducible isozyme of cyclooxygenase, is induced by bile acids and correlates with the incidence and progression of cancers. In this study, we investigated the signal transduction pathways involved in the bile‐acid‐mediated induction of COX‐2 expression. We found that staurosporine (sts), a potent protein kinase C (PKC) inhibitor, synergistically potentiated the deoxycholate‐mediated induction of COX‐2 expression. Sts did not increase the stabilization of COX‐2 mRNA. The sts‐ and deoxycholate‐mediated synergistic induction of COX‐2 expression was suppressed by a membrane‐permeable Ca2+ chelator, a phosphoinositide 3‐kinase inhibitor, a nuclear factor‐κB pathway inhibitor, and inhibitors of canonical and stress‐inducible mitogen‐activated protein kinase pathways. Inhibition was also observed using PKC inhibitors, suggesting the involvement of certain PKC isozymes (η, θ, ι, ζ, or μ). Our results indicate that sts exerts its potentiating effects via the phosphorylation of p38. However, the effects of anisomycin did not mimic those of sts, indicating that although p38 activation is required, it does not enhance deoxycholate‐induced COX‐2 expression. We conclude that staurosporine synergistically enhances deoxycholate‐induced COX‐2 expression in RCM‐1 colon cancer cells. e12143 The expression of COX‐2, an inducible isozyme of cyclooxygenase, correlates with the incidence and progression of cancers, and bile acids have been shown to induce COX‐2 expression. We investigated the signal transduction pathways involved in the bile‐acid‐mediated induction of COX‐2 expression, and we found that staurosporine, a potent PKC inhibitor, synergistically potentiated the deoxycholate‐mediated induction of COX‐2 expression. Staurosporine exerted its potentiating effects via the phosphorylation of p38, and the involvement of certain PKC isozymes was suggested.
Collapse
Affiliation(s)
- Tohru Saeki
- Laboratory of Molecular Nutrition, Kyoto Prefectural University, Kyoto, Japan
| | - Haruka Inui
- Laboratory of Molecular Nutrition, Kyoto Prefectural University, Kyoto, Japan
| | - Saya Fujioka
- Laboratory of Molecular Nutrition, Kyoto Prefectural University, Kyoto, Japan
| | - Suguru Fukuda
- Laboratory of Molecular Nutrition, Kyoto Prefectural University, Kyoto, Japan
| | - Ayumi Nomura
- Laboratory of Molecular Nutrition, Kyoto Prefectural University, Kyoto, Japan
| | - Yasushi Nakamura
- Laboratory of Food Science, Kyoto Prefectural University, Kyoto, Japan
| | - Eun Young Park
- Laboratory of Food Science, Kyoto Prefectural University, Kyoto, Japan
| | - Kenji Sato
- Laboratory of Food Science, Kyoto Prefectural University, Kyoto, Japan
| | - Ryuhei Kanamoto
- Laboratory of Molecular Nutrition, Kyoto Prefectural University, Kyoto, Japan
| |
Collapse
|
|
11
|
Frazziano G, Moreno L, Moral-Sanz J, Menendez C, Escolano L, Gonzalez C, Villamor E, Alvarez-Sala JL, Cogolludo AL, Perez-Vizcaino F. Neutral sphingomyelinase, NADPH oxidase and reactive oxygen species. Role in acute hypoxic pulmonary vasoconstriction. J Cell Physiol 2011; 226:2633-40. [PMID: 21792922 DOI: 10.1002/jcp.22611] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The molecular mechanisms underlying hypoxic pulmonary vasoconstriction (HPV) are not yet properly understood. Mitochondrial electron transport chain (ETC) and NADPH oxidase have been proposed as possible oxygen sensors, with derived reactive oxygen species (ROS) playing key roles in coupling the sensor(s) to the contractile machinery. We have recently reported that activation of neutral sphingomyelinase (nSMase) and protein kinase C ζ (PKCζ) participate in the signalling cascade of HPV. Herein, we studied the significance of nSMase in controlling ROS production rate in rat pulmonary artery (PA) smooth muscle cells and thereby HPV in rat PA. ROS production (analyzed by dichlorofluorescein and dihydroethidium fluorescence) was increased by hypoxia in endothelium-denuded PA segments and their inhibition prevented hypoxia-induced voltage-gated potassium channel (K(V) ) inhibition and pulmonary vasoconstriction. Consistently, H(2) O(2) , or its analogue t-BHP, decreased K(V) currents and induced a contractile response, mimicking the effects of hypoxia. Inhibitors of mitochondrial ETC (rotenone) and NADPH oxidase (apocynin) prevented hypoxia-induced ROS production, K(V) channel inhibition and vasoconstriction. Hypoxia induced p47(phox) phosphorylation and its interaction with caveolin-1. Inhibition of nSMase (GW4869) or PKCζ prevented p47(phox) phosphorylation and ROS production. The increase in ceramide induced by hypoxia (analyzed by immunocytochemistry) was inhibited by rotenone. Exogenous ceramide increased ROS production in a PKCζ sensitive manner. We propose an integrated signalling pathway for HPV which includes nSMase-PKCζ-NADPH oxidase as a necessary step required for ROS production and vasoconstriction.
Collapse
Affiliation(s)
- Giovanna Frazziano
- Department of Pharmacology, School of Medicine, University Complutense of Madrid, Madrid, Spain
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
12
|
Baran Y, Bielawski J, Gunduz U, Ogretmen B. Targeting glucosylceramide synthase sensitizes imatinib-resistant chronic myeloid leukemia cells via endogenous ceramide accumulation. J Cancer Res Clin Oncol 2011; 137:1535-44. [PMID: 21833718 DOI: 10.1007/s00432-011-1016-y] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2011] [Accepted: 07/20/2011] [Indexed: 12/20/2022]
Abstract
PURPOSE Drug resistance presents a major obstacle for the treatment of some patients with chronic myeloid leukemia (CML). Pro-apoptotic ceramide mediates imatinib-induced apoptosis, and metabolism of ceramide by glucosylceramide synthase (GCS) activity, converting ceramide to glucosyl ceramide, might contribute to imatinib resistance. In this study, we investigated the role of ceramide metabolism by GCS in the regulation of imatinib-induced apoptosis in drug-sensitive and drug-resistant K562 and K562/IMA-0.2 and K562/IMA-1 human CML cells, which exhibit about 2.3- and 19-fold imatinib resistance, respectively. METHODS Cytotoxic effects of PDMP and imatinib were determined by XTT cell proliferation assay. Expression levels of GCS were determined by RT-PCR and western blot. Intracellular ceramide levels were determined by LC-MS. Cell viability analyses was conducted by Trypan blue dye exclusion assay. Cell cycle and apoptosis analyses were examined by flow cytometry. RESULTS We first showed that mRNA and protein levels of GCS are increased in drug-resistant K562/IMA as compared to sensitive K562 cells. Next, forced expression of GCS in sensitive K562 cells conferred resistance to imatinib-induced apoptosis. In reciprocal experiments, targeting GCS using its known inhibitor, PDMP, enhanced ceramide accumulation and increased cell death in response to imatinib in K562/IMA cells. CONCLUSION Our data suggest the involvement of GCS in resistance to imatinib-induced apoptosis, and that targeting GCS by PDMP increased imatinib-induced cell death in drug-sensitive and drug-resistant K562 cells via enhancing ceramide accumulation.
Collapse
MESH Headings
- Antineoplastic Agents/pharmacology
- Apoptosis/drug effects
- Benzamides
- Cell Cycle/drug effects
- Ceramides/metabolism
- Drug Resistance, Neoplasm
- Glucosyltransferases/antagonists & inhibitors
- Glucosyltransferases/physiology
- Humans
- Imatinib Mesylate
- K562 Cells
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology
- Morpholines/pharmacology
- Piperazines/pharmacology
- Pyrimidines/pharmacology
Collapse
Affiliation(s)
- Yusuf Baran
- Department of Molecular Biology and Genetics, Izmir Institute of Technology, Faculty of Science, Gulbahce Campus, 35430, Urla, Izmir, Turkey.
| | | | | | | |
Collapse
|
|
13
|
Deuse T, Koyanagi T, Erben RG, Hua X, Velden J, Ikeno F, Reichenspurner H, Robbins RC, Mochly-Rosen D, Schrepfer S. Sustained inhibition of epsilon protein kinase C inhibits vascular restenosis after balloon injury and stenting. Circulation 2010; 122:S170-8. [PMID: 20837910 DOI: 10.1161/circulationaha.109.927640] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
BACKGROUND ε protein kinase C (εPKC) is involved in vascular smooth muscle cell (VSMC) activation, but little is known about its function in vascular pathology. We aimed at assessing the role of εPKC in the development of restenosis. METHODS AND RESULTS Rat models of aortic balloon injury with or without subsequent stenting were used. Rats were treated with the selective ψεPKC activator ε receptor for activated protein kinase C (ψεRACK), the selective εPKC inhibitor εV1-2, or saline. Both down-stream cascades of the platelet-derived growth factor receptor via extracellular signal-regulated kinase and Akt, respectively, were evaluated in vivo and in VSMC cultures. Intimal hyperplasia with luminal obliteration developed in saline-treated balloon-injured rat aortas (20.3±8.0%), and ψεRACK significantly promoted neointima development (32.4±4.9%, P=0.033), whereas εV1-2 significantly inhibited luminal narrowing (9.2±4.3%, P=0.039). εPKC inhibition led to significantly reduced VSMC extracellular signal-regulated kinase phosphorylation in vivo, whereas Akt phosphorylation was not markedly affected. Neointimal proliferation in vivo and platelet-derived growth factor-induced VSMC proliferation/migration in vitro were significantly inhibited by εV1-2. The inhibition of the platelet-derived growth factor pathway was mediated by inhibiting down-stream extracellular signal-regulated kinase and Akt phosphorylation. In vitro, εV1-2 showed inhibitory properties on endothelial cell proliferation, but that did not prevent reendothelialization in vivo. εV1-2 showed proapoptotic effects on VSMC in vitro. After stent implantation, luminal restenosis (quantified by optical coherence tomography imaging) was significantly reduced with εV1-2 (8.0±2.0%) compared with saline (20.2±9.8%, P=0.028). CONCLUSIONS εPKC seems to be centrally involved in the development of neointimal hyperplasia. We suggest that εPKC inhibition may be mediated via inhibition of extracellular signal-regulated kinase and Akt activation. εPKC modulation may become a new therapeutic target against vascular restenosis.
Collapse
Affiliation(s)
- Tobias Deuse
- Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, CA 94305, USA
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
14
|
Lucki NC, Sewer MB. The interplay between bioactive sphingolipids and steroid hormones. Steroids 2010; 75:390-9. [PMID: 20138078 PMCID: PMC2854287 DOI: 10.1016/j.steroids.2010.01.020] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/14/2009] [Revised: 01/25/2010] [Accepted: 01/26/2010] [Indexed: 01/02/2023]
Abstract
Steroid hormones regulate various physiological processes including development, reproduction, and metabolism. These regulatory molecules are synthesized from cholesterol in endocrine organs - such as the adrenal glands and gonads - via a multi-step enzymatic process that is catalyzed by the cytochrome P450 superfamily of monooxygenases and hydroxysteroid dehydrogenases. Steroidogenesis is induced by trophic peptide hormones primarily via the activation of a cAMP/protein kinase A (PKA)-dependent pathway. However, other signaling molecules, including cytokines and growth factors, control the steroid hormone biosynthetic pathway. More recently, sphingolipids, including ceramide, sphingosine-1-phosphate, and sphingosine, have been found to modulate steroid hormone secretion at multiple levels. In this review, we provide a brief overview of the mechanisms by which sphingolipids regulate steroidogenesis. In addition, we discuss how steroid hormones control sphingolipid metabolism. Finally, we outline evidence supporting the emerging role of bioactive sphingolipids in various nuclear processes and discuss a role for nuclear sphingolipid metabolism in the control of gene transcription.
Collapse
Affiliation(s)
- Natasha C. Lucki
- School of Biology and Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, 310 Ferst Dr., Atlanta, GA 30332
| | - Marion B. Sewer
- Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California San Diego, 9500 Gilman Dr. MC0704, La Jolla, CA 92093
| |
Collapse
|
|
15
|
Joseph EK, Levine JD. Multiple PKCε-dependent mechanisms mediating mechanical hyperalgesia. Pain 2010; 150:17-21. [PMID: 20456866 DOI: 10.1016/j.pain.2010.02.011] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2009] [Revised: 01/11/2010] [Accepted: 02/05/2010] [Indexed: 12/14/2022]
Abstract
We have recently implicated mitochondrial mechanisms in models of neuropathic and inflammatory pain, in some of which a role of protein kinase Cepsilon (PKCepsilon) has also been implicated. Since mitochondria contain several proteins that are targets of PKCepsilon, we evaluated the role of mitochondrial mechanisms in mechanical hyperalgesia induced by proinflammatory cytokines that induce PKCepsilon-dependent nociceptor sensitization, and by a direct activator of PKCepsilon (psiepsilonRACK), in the rat. Prostaglandin E(2) (PGE(2))-induced hyperalgesia is short lived in naïve rats, while it is prolonged in psiepsilonRACK pre-treated rats, a phenomenon referred to as priming. Inhibitors of two closely related mitochondrial functions, electron transport (complexes I-V) and oxidative stress (reactive oxygen species), attenuated mechanical hyperalgesia induced by intradermal injection of psiepsilonRACK. In marked contrast, in a PKCepsilon-dependent form of mechanical hyperalgesia induced by prostaglandin E(2) (PGE(2)), inhibitors of mitochondrial function failed to attenuate hyperalgesia. These studies support the suggestion that at least two downstream signaling pathways can mediate the hyperalgesia induced by activating PKCepsilon.
Collapse
Affiliation(s)
- Elizabeth K Joseph
- Departments of Medicine and Oral Surgery, and Division of Neuroscience, University of California, San Francisco, USA
| | | |
Collapse
|
|
16
|
Gangoiti P, Camacho L, Arana L, Ouro A, Granado MH, Brizuela L, Casas J, Fabriás G, Abad JL, Delgado A, Gómez-Muñoz A. Control of metabolism and signaling of simple bioactive sphingolipids: Implications in disease. Prog Lipid Res 2010; 49:316-34. [PMID: 20193711 DOI: 10.1016/j.plipres.2010.02.004] [Citation(s) in RCA: 108] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2010] [Revised: 02/18/2010] [Accepted: 02/22/2010] [Indexed: 01/05/2023]
Abstract
Simple bioactive sphingolipids include ceramide, sphingosine and their phosphorylated forms sphingosine 1-phosphate and ceramide 1-phosphate. These molecules are crucial regulators of cell functions. In particular, they play important roles in the regulation of angiogenesis, apoptosis, cell proliferation, differentiation, migration, and inflammation. Decoding the mechanisms by which these cellular functions are regulated requires detailed understanding of the signaling pathways that are implicated in these processes. Most importantly, the development of inhibitors of the enzymes involved in their metabolism may be crucial for establishing new therapeutic strategies for treatment of disease.
Collapse
Affiliation(s)
- Patricia Gangoiti
- Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Bilbao, Spain
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
17
|
Arana L, Gangoiti P, Ouro A, Trueba M, Gómez-Muñoz A. Ceramide and ceramide 1-phosphate in health and disease. Lipids Health Dis 2010; 9:15. [PMID: 20137073 PMCID: PMC2828451 DOI: 10.1186/1476-511x-9-15] [Citation(s) in RCA: 155] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2009] [Accepted: 02/05/2010] [Indexed: 01/06/2023] Open
Abstract
Sphingolipids are essential components of cell membranes, and many of them regulate vital cell functions. In particular, ceramide plays crucial roles in cell signaling processes. Two major actions of ceramides are the promotion of cell cycle arrest and the induction of apoptosis. Phosphorylation of ceramide produces ceramide 1-phosphate (C1P), which has opposite effects to ceramide. C1P is mitogenic and has prosurvival properties. In addition, C1P is an important mediator of inflammatory responses, an action that takes place through stimulation of cytosolic phospholipase A2, and the subsequent release of arachidonic acid and prostaglandin formation. All of the former actions are thought to be mediated by intracellularly generated C1P. However, the recent observation that C1P stimulates macrophage chemotaxis implicates specific plasma membrane receptors that are coupled to Gi proteins. Hence, it can be concluded that C1P has dual actions in cells, as it can act as an intracellular second messenger to promote cell survival, or as an extracellular receptor agonist to stimulate cell migration.
Collapse
Affiliation(s)
- Lide Arana
- Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), PO Box 644, 48080 Bilbao, Spain
| | | | | | | | | |
Collapse
|
|
18
|
Subcellular Localization of Diacylglycerol-responsive Protein Kinase C Isoforms in HeLa Cells. B KOREAN CHEM SOC 2009. [DOI: 10.5012/bkcs.2009.30.9.1981] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
|
|
19
|
Abstract
Protein kinase C (PKC) isoforms comprise a family of lipid-activated enzymes that have been implicated in a wide range of cellular functions. PKCs are modular enzymes comprised of a regulatory domain (that contains the membrane-targeting motifs that respond to lipid cofactors, and in the case of some PKCs calcium) and a relatively conserved catalytic domain that binds ATP and substrates. These enzymes are coexpressed and respond to similar stimulatory agonists in many cell types. However, there is growing evidence that individual PKC isoforms subserve unique (and in some cases opposing) functions in cells, at least in part as a result of isoform-specific subcellular compartmentalization patterns, protein-protein interactions, and posttranslational modifications that influence catalytic function. This review focuses on the structural basis for differences in lipid cofactor responsiveness for individual PKC isoforms, the regulatory phosphorylations that control the normal maturation, activation, signaling function, and downregulation of these enzymes, and the intra-/intermolecular interactions that control PKC isoform activation and subcellular targeting in cells. A detailed understanding of the unique molecular features that underlie isoform-specific posttranslational modification patterns, protein-protein interactions, and subcellular targeting (i.e., that impart functional specificity) should provide the basis for the design of novel PKC isoform-specific activator or inhibitor compounds that can achieve therapeutically useful changes in PKC signaling in cells.
Collapse
Affiliation(s)
- Susan F Steinberg
- Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
| |
Collapse
|
|
20
|
Schwarz E, Prabakaran S, Whitfield P, Major H, Leweke FM, Koethe D, McKenna P, Bahn S. High throughput lipidomic profiling of schizophrenia and bipolar disorder brain tissue reveals alterations of free fatty acids, phosphatidylcholines, and ceramides. J Proteome Res 2008; 7:4266-77. [PMID: 18778095 DOI: 10.1021/pr800188y] [Citation(s) in RCA: 153] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
A mass spectrometry based high throughput approach was employed to profile white and gray matter lipid levels in the prefrontal cortex (Brodmann area 9) of 45 subjects including 15 schizophrenia and 15 bipolar disorder patients as well as 15 controls samples. We found statistically significant alterations in levels of free fatty acids and phosphatidylcholine in gray and white matter of both schizophrenia and bipolar disorder samples compared to controls. Also, ceramides were identified to be significantly increased in white matter of both neuropsychiatric disorders as compared to control levels. The patient cohort investigated in this study includes a number of drug naive as well as untreated patients, allowing the assessment of drug effects on lipid levels. Our findings indicate that while gray matter phosphatidylcholine levels were influenced by antipsychotic medication, this was not the case for phosphatidylcholine levels in white matter. Changes in free fatty acids or ceramides in either white or gray matter also did not appear to be influenced by antipsychotic treatment. To assess lipid profiles in the living patient, we also profiled lipids of 40 red blood cell samples, including 7 samples from drug naive first onset patients. We found significant alterations in the concentrations of free fatty acids as well as ceramide. Overall, our findings suggest that lipid abnormalities may be a disease intrinsic feature of both schizophrenia and bipolar disorder reflected by significant changes in the central nervous system as well as peripheral tissues.
Collapse
Affiliation(s)
- Emanuel Schwarz
- Institute of Biotechnology, University of Cambridge, Cambridge CB2 1QT, United Kingdom
| | | | | | | | | | | | | | | |
Collapse
|
|
21
|
Abstract
Most of the previous work on the sphingolipid ceramide has been devoted to its function as an apoptosis inducer. Recent studies, however, have shown that in stem cells, ceramide has additional nonapoptotic functions. In this article, ceramide signaling will be reviewed in light of 'systems interface biology': as an interconnection of sphingolipid metabolism, membrane biophysics and cell signaling. The focus will be on the metabolic interconversion of ceramide and sphingomyelin or sphingosine-1-phosphate. Lipid rafts and sphingolipid-induced protein scaffolds will be discussed as a membrane interface for lipid-controlled cell signaling. Ceramide/sphingomyelin and ceramide/sphingosine-1-phosphate-interdependent cell-signaling pathways are significant for the regulation of cell polarity, apoptosis and/or proliferation, and as novel pharmacologic targets in cancer and stem cells.
Collapse
Affiliation(s)
- Erhard Bieberich
- Institute of Molecular Medicine & Genetics, School of Medicine, Medical College of Georgia, 1120 15th Street, Room CB-2803, Augusta, GA 30912, USA
| |
Collapse
|
|
22
|
Abstract
Steroid hormones are essential regulators of a vast number of physiological processes. The biosynthesis of these chemical messengers occurs in specialized steroidogenic tissues via a multi-step process that is catalyzed by members of the cytochrome P450 superfamily of monooxygenases and hydroxysteroid dehydrogenases. Though numerous signaling mediators, including cytokines and growth factors control steroidogenesis, trophic peptide hormones are the primary regulators of steroid hormone production. These peptide hormones activate a cAMP/cAMP-dependent kinase (PKA) signaling pathway, however, studies have shown that crosstalk between multiple signal transduction pathways and signaling molecules modulates optimal steroidogenic capacity. Sphingolipids such as ceramide, sphingosine, sphingosine-1-phosphate, sphingomyelin, and gangliosides have been shown to control the steroid hormone biosynthetic pathway at multiple levels, including regulating steroidogenic gene expression and activity as well as acting as second messengers in signaling cascades. In this review, we provide an overview of recent studies that have investigated the role of sphingolipids in adrenal, gonadal, and neural steroidogenesis.
Collapse
Affiliation(s)
- Natasha C Lucki
- School of Biology and Parker H, Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, 310 Ferst Drive, Atlanta, GA 30332-0230, USA
| | | |
Collapse
|
|
23
|
Basu A, Sivaprasad U. Protein kinase Cepsilon makes the life and death decision. Cell Signal 2007; 19:1633-42. [PMID: 17537614 PMCID: PMC1986651 DOI: 10.1016/j.cellsig.2007.04.008] [Citation(s) in RCA: 103] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2007] [Accepted: 04/23/2007] [Indexed: 12/20/2022]
Abstract
Cancer is caused by dysregulation in cellular signaling systems that control cell proliferation, differentiation and cell death. Protein kinase C (PKC), a family of serine/threonine kinases, plays an important role in the growth factor signal transduction pathway. PKCepsilon, however, is the only PKCepsilon isozyme that has been considered as an oncogene. It can contribute to malignancy by enhancing cell proliferation or by inhibiting cell death. This review focuses on how PKCepsilon collaborates with other signaling pathways, such as Ras/Raf/ERK and Akt, to regulate cell survival and cell death. We have also discussed how PKCepsilon mediates its antiapoptotic signal by altering the level or function of pro- and antiapoptotic Bcl-2 family members.
Collapse
Affiliation(s)
- Alakananda Basu
- Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
| | | |
Collapse
|
|
24
|
Zeidan YH, Hannun YA. Activation of Acid Sphingomyelinase by Protein Kinase Cδ-mediated Phosphorylation. J Biol Chem 2007; 282:11549-61. [PMID: 17303575 DOI: 10.1074/jbc.m609424200] [Citation(s) in RCA: 115] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Although important for cellular stress signaling pathways, the molecular mechanisms of acid sphingomyelinase (ASMase) activation remain poorly understood. Previous studies showed that treatment of MCF-7 mammary carcinoma cells with the potent protein kinase C (PKC) agonist, phorbol 12-myristate 13-acetate (PMA), induces a transient drop in sphingomyelin concomitant with an increase in cellular ceramide levels (Becker, K. P., Kitatani, K., Idkowiak-Baldys, J., Bielawski, J., and Hannun, Y. A. (2005) J. Biol. Chem. 280, 2606-2612). Here we show that PMA selectively activates ASMase and that ASMase accounts for the majority of PMA-induced ceramide. Pharmacologic inhibition and RNA interference experiments indicated that the novel PKC, PKCdelta, is required for ASMase activation. Immunoprecipitation experiments revealed the formation of a novel PKCdelta-ASMase complex after PMA stimulation, and PKCdelta was able to phosphorylate ASMase in vitro and in cells. Using site-directed mutagenesis, we identify serine 508 as the key residue phosphorylated in response to PMA. Phosphorylation of Ser(508) proved to be an indispensable step for ASMase activation and membrane translocation in response to PMA. The relevance of the proposed mechanism of ASMase regulation is further validated in a model of UV radiation. UV radiation also induced phosphorylation of ASMase at serine 508. Moreover, when transiently overexpressed, ASMase(S508A) blocked the ceramide formation after PMA treatment, suggesting a dominant negative function for this mutant. Taken together, these results establish a novel direct biochemical mechanism for ASMase activation in which PKCdelta serves as a key upstream kinase.
Collapse
Affiliation(s)
- Youssef H Zeidan
- Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA
| | | |
Collapse
|
|
25
|
Wang J, Lv XW, Shi JP, Hu XS. Mechanisms involved in ceramide-induced cell cycle arrest in human hepatocarcinoma cells. World J Gastroenterol 2007; 13:1129-34. [PMID: 17373752 PMCID: PMC4146880 DOI: 10.3748/wjg.v13.i7.1129] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the effect of ceramide on the cell cycle in human hepatocarcinoma Bel7402 cells. Possible molecular mechanisms were explored.
METHODS: [3- (4, 5)-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, plasmid transfection, reporter assay, FACS and Western blotting analyses were employed to investigate the effect and the related molecular mechanisms of C2-ceramide on the cell cycle of Bel7402 cells.
RESULTS: C2-ceramide was found to inhibit the growth of Bel7402 cells by inducing cell cycle arrest. During the process, the expression of p21 protein increased, while that of cyclinD1, phospho-ERK1/2 and c-myc decreased. Furthermore, the level of CDK7 was downregulated, while the transcriptional activity of PPARγ was upregulated. Addition of GW9662, which is a PPARγ specific antagonist, could reserve the modulation action on CDK7.
CONCLUSION: Our results support the hypothesis that cell cycle arrest induced by C2-ceramide may be mediated via accumulation of p21 and reduction of cyclinD1 and CDK7, at least partly, through PPARγ activation. The ERK signaling pathway was involved in this process.
Collapse
Affiliation(s)
- Jing Wang
- Research Center for Eco-Environmental Sciences, The Chinese Academy of Sciences, Haidian District, Beijing 100085, China.
| | | | | | | |
Collapse
|
|
26
|
Peng CH, Huang CN, Hsu SP, Wang CJ. Penta-acetyl geniposide induce apoptosis in C6 glioma cells by modulating the activation of neutral sphingomyelinase-induced p75 nerve growth factor receptor and protein kinase Cdelta pathway. Mol Pharmacol 2006; 70:997-1004. [PMID: 16763091 DOI: 10.1124/mol.106.022178] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
In our previous studies, we demonstrated the apoptotic cascades protein kinase C (PKC) delta/c-Jun NH2-terminal kinase (JNK)/Fas/caspases induced by penta-acetyl geniposide [(Ac)5GP]. However, the upstream signals mediating PKCdelta activation have not yet been clarified. Ceramide, mainly generated from the degradation of sphingomyelin, was hypothesized upstream above PKCdelta in (Ac)5GP-transduced apoptosis. Furthermore, nerve growth factor (NGF)/p75 is supposed to be involved because(Ac)5GP-induced apoptosis was demonstrated previously in glioma cells. In the present study, (Ac)5GP was shown to activate neutral sphingomyelinase (N-SMase) immediately, with its maximum at 15 min. The NGF and p75 enhanced by (Ac)5GP was inhibited when added with GW4869, the N-SMase inhibitor, indicating NGF/p75 as the downstream signals of N-SMase/ceramide. To investigate whether N-SMase is involved in (Ac)5GP-transduced apoptotic pathway, cells were treated with (Ac)5GP added with or without GW4869. It showed that N-SMase inhibition blocked FasL expression and caspase 3 activation. Likewise, p75 antagonist peptide attenuated the FasL/caspase 3 expression. The PKCdelta translocation induced by (Ac)5GP was also eliminated by GW4869 and p75 antagonist peptide. To further confirm whether N-SMase activation plays an important role in (Ac)5GP-induced apoptosis, cells were analyzed the apoptotic rate by 4', 6-diamidino-2-phenylindole (DAPI) staining. (Ac)5GP-induced apoptosis was reduced 40 and 80% by 10 and 20 microM GW4869, respectively. It indicated that N-SMase activation is pivotal in (Ac)5GP-mediated apoptosis. In conclusion, SMase and NGF/p75 are suggested to mediate upstream above PKCdelta, thus transducing FasL/caspase cascades in (Ac)5GP-induced apoptosis.
Collapse
Affiliation(s)
- Chiung-Huei Peng
- Department of Nursing, Hungkuang University, Sha Lu, Taichung, Taiwan
| | | | | | | |
Collapse
|
|
27
|
Ricci C, Onida F, Ghidoni R. Sphingolipid players in the leukemia arena. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2006; 1758:2121-32. [PMID: 16904628 DOI: 10.1016/j.bbamem.2006.06.016] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/30/2005] [Revised: 06/12/2006] [Accepted: 06/19/2006] [Indexed: 01/21/2023]
Abstract
Sphingolipids function as bioactive mediators of different cellular processes, mostly proliferation, survival, differentiation and apoptosis, besides being structural components of cellular membranes. Involvement of sphingolipid metabolism in cancerogenesis was demonstrated in solid tumors as well as in hematological malignancies. Herein, we describe the main biological and clinical aspects of leukemias and summarize data regarding sphingolipids as mediators of apoptosis triggered in response to anti-leukemic agents and synthetic analogs as inducers of cell death as well. We also report the contribution of molecules that modulate sphingolipid metabolism to development of encouraging strategies for leukemia treatment. Finally we address how deregulation of sphingolipid metabolism is associated to occurrence of therapy resistance both in vitro and in vivo. Sphingolipids can be considered promising therapeutic tools alone or in combination with other compounds, as well as valid targets in the attempt to eradicate leukemia and overcome drug resistance.
Collapse
Affiliation(s)
- Clara Ricci
- Laboratory of Biochemistry and Molecular Biology, San Paolo University Hospital, Medical School, University of Milan, 20142 via A. di Rudinì, 8-Milan, Italy
| | | | | |
Collapse
|
|
28
|
Specific protein kinase C isoforms as transducers and modulators of insulin signaling. Mol Genet Metab 2006; 89:32-47. [PMID: 16798038 DOI: 10.1016/j.ymgme.2006.04.017] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/05/2006] [Revised: 04/23/2006] [Accepted: 04/23/2006] [Indexed: 12/14/2022]
Abstract
Recent studies implicate specific PKC isoforms in the insulin-signaling cascade. Insulin activates PKCs alpha, betaII, delta and zeta in several cell types. In addition, as will be documented in this review, certain members of the PKC family may also be activated and act upstream of PI3 and MAP kinases. Each of these isoforms has been shown one way or another either to mimic or to modify insulin-stimulated effects in one or all of the insulin-responsive tissues. Moreover, each of the isoforms has been shown to be activated by insulin stimulation or conditions important for effective insulin stimulation. Studies attempting to demonstrate a definitive role for any of the isoforms have been performed on different cells, ranging from appropriate model systems for skeletal muscle, liver and fat, such as primary cultures, and cell lines and even in vivo studies, including transgenic mice with selective deletion of specific PKC isoforms. In addition, studies have been done on certain expression systems such as CHO or HEK293 cells, which are far removed from the tissues themselves and serve mainly as vessels for potential protein-protein interactions. Thus, a clear picture for many of the isoforms remains elusive in spite of over two decades of intensive research. The recent intrusion of transgenic and precise molecular biology technologies into the research armamentarium has opened a wide range of additional possibilities for direct involvement of individual isoforms in the insulin signaling cascade. As we hope to discuss within the context of this review, whereas many of the long sought-after answers to specific questions are not yet clear, major advances have been made in our understanding of precise roles for individual PKC isoforms in mediation of insulin effects. In this review, in which we shall focus our attention on isoforms in the conventional and novel categories, a clear case will be made to show that these isoforms are not only expressed but are importantly involved in regulation of insulin metabolic effects.
Collapse
|
|
29
|
Miyaji M, Jin ZX, Yamaoka S, Amakawa R, Fukuhara S, Sato SB, Kobayashi T, Domae N, Mimori T, Bloom ET, Okazaki T, Umehara H. Role of membrane sphingomyelin and ceramide in platform formation for Fas-mediated apoptosis. ACTA ACUST UNITED AC 2005; 202:249-59. [PMID: 16009715 PMCID: PMC2213006 DOI: 10.1084/jem.20041685] [Citation(s) in RCA: 113] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Engagement of the Fas receptor (CD95) initiates multiple signaling pathways that lead to apoptosis, such as the formation of death-inducing signaling complex (DISC), activation of caspase cascades, and the generation of the lipid messenger, ceramide. Sphingomyelin (SM) is a major component of lipid rafts, which are specialized structures that enhance the efficiency of membrane receptor signaling and are a main source of ceramide. However, the functions of SM in Fas-mediated apoptosis have yet to be clearly defined, as the responsible genes have not been identified. After cloning a gene responsible for SM synthesis, SMS1, we established SM synthase–defective WR19L cells transfected with the human Fas gene (WR/Fas-SM(−)), and cells that have been functionally restored by transfection with SMS1 (WR/Fas-SMS1). We show that expression of membrane SM enhances Fas-mediated apoptosis through increasing DISC formation, activation of caspases, efficient translocation of Fas into lipid rafts, and subsequent Fas clustering. Furthermore, WR/Fas-SMS1 cells, but not WR/Fas-SM(−) cells, showed a considerable increase in ceramide generation within lipid rafts upon Fas stimulation. These data suggest that a membrane SM is important for Fas clustering through aggregation of lipid rafts, leading to Fas-mediated apoptosis.
Collapse
Affiliation(s)
- Michihiko Miyaji
- Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
| | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
30
|
Atten MJ, Godoy-Romero E, Attar BM, Milson T, Zopel M, Holian O. Resveratrol regulates cellular PKC alpha and delta to inhibit growth and induce apoptosis in gastric cancer cells. Invest New Drugs 2005; 23:111-9. [PMID: 15744586 DOI: 10.1007/s10637-005-5855-8] [Citation(s) in RCA: 71] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Resveratrol, a dietary phytoalexin, has emerged as a promising chemopreventive agent due to its antiproliferative and pro-apoptotic action toward cancer cells and its ability to inhibit tumor growth in animals. Gastric adenocarcinoma cells respond to resveratrol treatment with suppression of DNA synthesis, activation of nitric oxide synthase, induction of apoptosis and inhibition of total PKC and PKC alpha activity. Here we demonstrate that treatment of gastric adenocarcinoma SNU-1 cells with resveratrol results in time and concentration dependent accumulation of tumor suppressors p21(cip1/WAF-1) and p53 and is preceded by loss of membrane-associated PKC delta protein and a concomitant increase in cytosolic PKC alpha. Arrest of the cell cycle at transition of S to G(2)/M phases correlates with the profile of (3)H-thymidine incorporation and accumulation of p21(cip1/WAF-1) and was temporally dependent on increase of p53. SNU-1 cells respond to resveratrol treatment with up-regulation of both Fas and Fas-L proteins, whereas in KATO-III cells, with deleted p53, only Fas-L is increased after resveratrol treatment. Although Fas and Fas-L proteins in SNU-1 cells and Fas-L in KATO-III cells were elevated within 24 h of cell treatment with low concentrations of resveratrol, significant apoptotic response at these concentrations was observed only after 48 h. Altogether, our findings indicate that resveratrol engages PKC alpha and delta signals in gastric adenocarcinoma SNU-1 cells prior to up-regulation of antiproliferative and pro-apoptotic signals. The specific cell death signals engaged by resveratrol appear to be cell type dependent and suggest that resveratrol has chemopreventive potential even after mutational changes have occurred.
Collapse
Affiliation(s)
- Mary Jo Atten
- Department of Medicine Division of Gastroenterology, Cook County Hospital, Chicago, IL 60612, USA
| | | | | | | | | | | |
Collapse
|
|
31
|
Raval AP, Dave KR, Prado R, Katz LM, Busto R, Sick TJ, Ginsberg MD, Mochly-Rosen D, Pérez-Pinzón MA. Protein kinase C delta cleavage initiates an aberrant signal transduction pathway after cardiac arrest and oxygen glucose deprivation. J Cereb Blood Flow Metab 2005; 25:730-41. [PMID: 15716854 DOI: 10.1038/sj.jcbfm.9600071] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Protein kinase C (PKC) isozymes have been known to mediate a variety of complex and diverse cellular functions. deltaPKC has been implicated in mediating apoptosis. Using two models of cerebral ischemia, cardiac arrest in rats and oxygen glucose deprivation (OGD) in organotypic hippocampal slices, we tested whether an ischemic insult promoted deltaPKC cleavage during the reperfusion and whether the upstream pathway involved release of cytochrome c and caspase 3 cleavage. We showed that cardiac arrest/OGD significantly enhanced deltaPKC translocation and increased its cleavage at 3 h of reperfusion. Since deltaPKC is one of the substrates for caspase 3, we next determined caspase 3 activation after cardiac arrest and OGD. The maximum decrease in levels of procaspase 3 was observed at 3 h of reperfusion after cardiac arrest and OGD. We also determined cytochrome c release, since it is upstream of caspase 3 activation. Cytochrome c in cytosol increased at 1 h of reperfusion after cardiac arrest/OGD. Inhibition of either deltaPKC/caspase 3 during OGD and early reperfusion resulted in neuroprotection in CA1 region of hippocampus. Our results support the deleterious role of deltaPKC in reperfusion injury. We propose that early cytochrome c release and caspase 3 activation promote deltaPKC translocation/cleavage.
Collapse
Affiliation(s)
- Ami P Raval
- Department of Neurology and Neuroscience Program, Cerebral Vascular Disease Research Center, University of Miami School of Medicine, Florida 33101, USA.
| | | | | | | | | | | | | | | | | |
Collapse
|
|
32
|
Abstract
The brain is enriched with sphingolipids, which are important membrane constituents and major lipid signaling molecules that have a role in motor and cognitive behavior. Vitamin K has been implicated in brain sphingolipid metabolism for more than 30 years. The in vitro and in vivo studies to date suggest a role of vitamin K in the regulation of multiple enzymes involved in sphingolipid metabolism within the myelin-rich regions in the brain. However, the precise mechanisms of action are not well understood. Further, the physiological consequences of the observed effects of vitamin K on sphingolipid metabolism have not been systematically studied.
Collapse
Affiliation(s)
- Natalia A Denisova
- Nutrition and Neurocognition Laboratory, Jean Mayer US Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts 02111, USA.
| | | |
Collapse
|
|
33
|
Ramström C, Chapman H, Ekokoski E, Tuominen RK, Pasternack M, Törnquist K. Tumor necrosis factor alpha and ceramide depolarise the resting membrane potential of thyroid FRTL-5 cells via a protein kinase Czeta-dependent regulation of K+ channels. Cell Signal 2005; 16:1417-24. [PMID: 15381257 DOI: 10.1016/j.cellsig.2004.05.007] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2004] [Revised: 04/28/2004] [Accepted: 05/14/2004] [Indexed: 11/24/2022]
Abstract
Tumor necrosis factor alpha (TNFalpha) alters the electrophysiological properties of many cell types. In thyroid cells however, the effects have not yet been elucidated. Here, we report the effect of TNFalpha and its second messenger ceramide on the resting membrane potential (RMP) of thyroid FRTL-5 cells. In patch-clamp experiments, we showed that TNFalpha and ceramide depolarise the RMP by inhibiting an acid-sensitive inwardly rectifying potassium current. This depolarisation depended on the activation of protein kinase Czeta (PKCzeta), because it can be blocked by calphostin C, a PKC-inhibitory peptide and a specific inhibitor peptide for PKCzeta. The activation of PKCzeta was confirmed by Western blotting, in which a stimulation with TNFalpha led to the translocation of PKCzeta to the particulate fraction. We conclude that TNFalpha and ceramide depolarise the RMP of thyroid FRTL-5 cells by attenuating a Ba(2+)- and acid-sensitive potassium conductance via activation of PKCzeta.
Collapse
Affiliation(s)
- Cia Ramström
- Department of Biology, Abo Akademi University, BioCity, Artillerigatan 6, 20520, Turku, Finland
| | | | | | | | | | | |
Collapse
|
|
34
|
Conesa-Zamora P, Mollinedo F, Corbalán-García S, Gómez-Fernández JC. A comparative study of the effect of the antineoplastic ether lipid 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine and some homologous compounds on PKCα and PKCɛ. Biochim Biophys Acta Mol Cell Biol Lipids 2005; 1687:110-9. [PMID: 15708359 DOI: 10.1016/j.bbalip.2004.11.008] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2004] [Revised: 11/11/2004] [Accepted: 11/11/2004] [Indexed: 11/25/2022]
Abstract
The effects of the anti-neoplastic ether lipid ET-18-OCH3 and some structural homologues on the activity of protein kinase C alpha (PKC alpha) were studied and compared with the effects the same had on the activity of PKC epsilon. ET-18-OCH3 progressively inhibited the activity of PKC alpha as the concentration was increased up to 30 mol% of the total lipid, above which the effect was one of activation. The experiments carried out with the homologues showed that the methoxy group bound at the sn-2 position of the glycerol of ET-18-OCH3 is essential for both the initial inhibitory effect and the subsequent activation effect. On the other hand, variations in the type of bond linking substitutions in the sn-1 position, ether or ester, do not seem to play an important role in determining the activity of the enzyme. The effects were different on PKC epsilon since ET-18-OCH3 had a triphasic effect, activating the enzyme at low concentrations, inhibiting it at slightly higher concentrations and then activating it again at higher concentrations. In this case, when the homologues were used, it was observed that the presence of the methoxy group linked to the sn-2 position of glycerol and the type of bond linking substitutions to the sn-1 position were important for activating the enzyme, so that only homologues with ester bonds as LPC and PAPC were able to induce the initial activation step in a way similar to ET-18-OCH3. Substitution of the phosphocholine group of ET-18-OCH3 by phosphoserine led to a greater activation of PKC alpha, an effect that comes from the Ca(2+)-phospholipid binding site probably because of the specific interaction of this site with the phosphoserine group. The action of ET-18-OCH3 and its homologues, as demonstrated in this paper, may permit the selective inhibition or activation of PKC alpha and PKC epsilon by using the most suitable range of concentrations.
Collapse
Affiliation(s)
- Pablo Conesa-Zamora
- Departamento de Bioquímica y Biología Molecular A, Facultad de Veterinaria, Universidad de Murcia, Apartado de Correos 4021, E-30080-Murcia, Spain
| | | | | | | |
Collapse
|
|
35
|
Uchida Y, Itoh M, Taguchi Y, Yamaoka S, Umehara H, Ichikawa SI, Hirabayashi Y, Holleran WM, Okazaki T. Ceramide reduction and transcriptional up-regulation of glucosylceramide synthase through doxorubicin-activated Sp1 in drug-resistant HL-60/ADR cells. Cancer Res 2004; 64:6271-9. [PMID: 15342415 DOI: 10.1158/0008-5472.can-03-1476] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Treatment with doxorubicin (DOX) induced apoptosis with an increase of ceramide content in drug-sensitive HL-60 cells, but not in drug-resistant HL-60/ADR cells. In HL-60/ADR cells (but not in HL-60 cells), the levels of mRNA, protein, and activity in glucosylceramide synthase (GCS), which converts ceramide to glucosylceramide, were up-regulated in response to DOX. Thus, abrogation of apoptosis in HL-60/ADR cells might be involved in ceramide reduction through DOX-induced up-regulation of GCS function. Because we reported that a GC-rich/Sp1 promoter binding region was of importance in the regulation of GCS expression, the role of Sp1 in DOX-induced up-regulation of GCS and apoptosis was investigated. DOX induced Sp1 activation in HL-60/ADR cells, as assessed by Sp1 gel shift and promoter-luciferase reporter assays, whereas transfection of double-stranded oligodeoxynucleotides (ODNs) containing a GC-rich/Sp1 region (Sp1 decoy ODNs) inhibited DOX-induced Sp1 activation. In addition, DOX-increased mRNA and enzyme activity in GCS were inhibited by Sp1 decoy, in conjunction with corresponding elevations of ceramide content. Moreover, DOX-induced apoptotic cell death was significantly increased in Sp1 decoy ODN-transfected HL-60/ADR cells over mock-transfected HL-60/ADR cells. Together, the results suggest that transcriptional up-regulation of GCS through DOX-induced activation of Sp1 is one potential mechanism to regulate ceramide increase and apoptosis in HL-60/ADR cells.
Collapse
Affiliation(s)
- Yoshikazu Uchida
- Department of Hematology and Oncology, Clinical Sciences for Pathological Organs, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | | | | | | | | | | | | | | | | |
Collapse
|
|
36
|
Abboushi N, El-Hed A, El-Assaad W, Kozhaya L, El-Sabban ME, Bazarbachi A, Badreddine R, Bielawska A, Usta J, Dbaibo GS. Ceramide inhibits IL-2 production by preventing protein kinase C-dependent NF-kappaB activation: possible role in protein kinase Ctheta regulation. THE JOURNAL OF IMMUNOLOGY 2004; 173:3193-200. [PMID: 15322180 DOI: 10.4049/jimmunol.173.5.3193] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
The role of the sphingolipid ceramide in modulating the immune response has been controversial, in part because of conflicting data regarding its ability to regulate the transcription factor NF-kappaB. To help clarify this role, we investigated the effects of ceramide on IL-2, a central NF-kappaB target. We found that ceramide inhibited protein kinase C (PKC)-mediated activation of NF-kappaB. Ceramide was found to significantly reduce the kinase activity of PKCtheta as well as PKCalpha, the critical PKC isozymes involved in TCR-induced NF-kappaB activation. This was followed by strong inhibition of IL-2 production in both Jurkat T leukemia and primary T cells. Exogenous sphingomyelinase, which generates ceramide at the cell membrane, also inhibited IL-2 production. As expected, the repression of NF-kappaB activation by ceramide led to the reduction of transcription of the IL-2 gene in a dose-dependent manner. Inhibition of IL-2 production by ceramide was partially overcome when NF-kappaB nuclear translocation was reconstituted with activation of a PKC-independent pathway by TNF-alpha or when PKCtheta was overexpressed. Importantly, neither the conversion of ceramide to complex glycosphingolipids, which are known to have immunosuppressive effects, nor its hydrolysis to sphingosine, a known inhibitor of PKC, was necessary for its inhibitory activity. These results indicate that ceramide plays a negative regulatory role in the activation of NF-kappaB and its targets as a result of inhibition of PKC.
Collapse
Affiliation(s)
- Nour Abboushi
- Department of Biochemistry, American University of Beirut, Faculty of Medicine, Lebanon
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
37
|
Schultz A, Larsson C. Ceramide influences neurite outgrowth and neuroblastoma cell apoptosis regulated by novel protein kinase C isoforms. J Neurochem 2004; 89:1427-35. [PMID: 15189345 DOI: 10.1111/j.1471-4159.2004.02431.x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
We have previously seen that protein kinase C (PKC) epsilon induces neurite outgrowth and that PKCdelta and PKCtheta elicit apoptosis in neuroblastoma cells. In this study we investigate the effects of cell-permeable C(2)-ceramide on these events in SK-N-BE(2) neuroblastoma cells. C(2)-ceramide abolishes neurite formation induced by overexpression of PKCepsilon and, in cells overexpressing PKCdelta or PKCtheta, ceramide treatment leads to apoptosis. Exposure to C(2)-ceramide also suppressed neurite outgrowth induced by retinoic acid, but ceramide did not abrogate neurite induction by treatment with the ROCK inhibitor Y-27632, demonstrating that C(2)-ceramide is not a general inhibitor of neurite outgrowth. The neurite-suppressing effect occurs independently of cell-death. Furthermore, C(2)-ceramide relocated PKCepsilon and the isolated regulatory domain of PKCepsilon from the cytosol to the perinuclear region. In contrast, neither the localization of PKCdelta nor of PKCtheta was affected by C(2)-ceramide. Taken together, the data indicate that the neurite-inhibiting effect of C(2)-ceramide treatment may be caused by a re-localization of PKCepsilon and thus identify a functional consequence of ceramide effects on PKCepsilon localization.
Collapse
Affiliation(s)
- Anna Schultz
- Molecular Medicine, Lund University, 205-02 Malmö, Sweden
| | | |
Collapse
|
|
38
|
Peng CH, Tseng TH, Liu JY, Hsieh YH, Huang CN, Hsu SP, Wang CJ. Penta-acetyl geniposide-induced C6 glioma cell apoptosis was associated with the activation of protein kinase C-delta. Chem Biol Interact 2004; 147:287-96. [PMID: 15135084 DOI: 10.1016/j.cbi.2004.01.003] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/21/2004] [Indexed: 12/11/2022]
Abstract
Herbal medicine has been utilized to treat a variety of diseases, including cancer. On the other hand, disturbance of apoptosis is often observed in cancer cells. It has been reported that protein kinase C (PKC) isoforms are involved in the signaling of apoptosis. In the present study, we investigate the antitumor effect and possible mechanism of a herbal-originated product, (Ac)(5)GP. We demonstrate that (Ac)(5)GP treatment results in DNA fragmentation of C6 glioma cells dose-dependently. Stimulated by (Ac)(5)GP, PKCdelta and PKCzeta were activated and translocated to the cell membrane fraction. Flow cytometry analysis showed that PKCdelta, but not PKCzeta inhibition blocks the (Ac)(5)GP-induced apoptosis by decreasing the cell population of sub G1 peak. However, the mRNA levels of PKCdelta and PKCzeta were not altered by (Ac)(5)GP-induced glioma cell apoptosis. These results suggested that the treatment of (Ac)(5)GP induces apoptosis of tumor cells through the activation but not the synthesis of PKCdelta.
Collapse
Affiliation(s)
- Chiung-Huei Peng
- Institute of Biochemistry, Chung Shan Medical University, No. 110, Section 2, Chien Kuo N. Road, Taichung 402, Taichung, Taiwan, ROC
| | | | | | | | | | | | | |
Collapse
|
|
39
|
Matsunaga T, Kotamraju S, Kalivendi SV, Dhanasekaran A, Joseph J, Kalyanaraman B. Ceramide-induced Intracellular Oxidant Formation, Iron Signaling, and Apoptosis in Endothelial Cells. J Biol Chem 2004; 279:28614-24. [PMID: 15102832 DOI: 10.1074/jbc.m400977200] [Citation(s) in RCA: 84] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Sphingolipid ceramide (N-acetylsphingosine), a bioactive second messenger lipid, was shown to activate reactive oxygen species (ROS), mitochondrial oxidative damage, and apoptosis in neuronal and vascular cells. The proapoptotic effects of tumor necrosis factor-alpha, hypoxia, and chemotherapeutic drugs were attributed to increased ceramide formation. Here we investigated the protective role of nitric oxide (.NO) during hydrogen peroxide (H(2)O(2))-mediated transferrin receptor (TfR)-dependent iron signaling and apoptosis in C(2)-ceramide (C(2)-cer)-treated bovine aortic endothelial cells (BAECs). Addition of C(2)-cer (5-20 microm) to BAECs enhanced .NO generation. However, at higher concentrations of C(2)-cer (> or =20 microm), .NO generation did not increase proportionately. C(2)-cer (20-50 microm) also resulted in H(2)O(2)-mediated dichlorodihydrofluorescein oxidation, reduced glutathione depletion, aconitase inactivation, TfR overexpression, TfR-dependent uptake of (55)Fe, release of cytochrome c from mitochondria into cytosol, caspase-3 activation, and DNA fragmentation. N(w)-Nitro-l-arginine methyl ester (l-NAME), a nonspecific inhibitor of nitricoxide synthases, augmented these effects in BAECs at much lower (i.e. nonapoptotic) concentrations of C(2)-cer. The 26 S proteasomal activity in BAECs was slightly elevated at lower concentrations of C(2)-cer (< or =10 microm) but was greatly suppressed at higher concentrations (>10 microm). Intracellular scavengers of H(2)O(2), cell-permeable iron chelators, anti-TfR receptor antibody, or mitochondria-targeted antioxidant greatly abrogated C(2)-cer- and/or l-NAME-induced oxidative damage, iron signaling, and apoptosis. We conclude that C(2)-cer-induced H(2)O(2) and TfR-dependent iron signaling are responsible for its prooxidant and proapoptotic effects and that .NO exerts an antioxidative and cytoprotective role.
Collapse
Affiliation(s)
- Toshiyuki Matsunaga
- Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA
| | | | | | | | | | | |
Collapse
|
|
40
|
Chik CL, Li B, Karpinski E, Ho AK. Ceramide inhibits L-type calcium channel currents in GH3 cells. Mol Cell Endocrinol 2004; 218:175-83. [PMID: 15130522 DOI: 10.1016/j.mce.2003.10.048] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/18/2003] [Accepted: 10/16/2003] [Indexed: 11/28/2022]
Abstract
In this study, we investigated the effect of ceramide on the L-type Ca2+ channel (L-channel) in GH3 cells. We found that C6-ceramide, but not C6-dihydroceramide, the inactive analogue, had an inhibitory effect on BayK 8644-stimulated GH release. Using patch clamp analysis, C6- and C2-ceramide, but not C6-dihydroceramide, were found to inhibit the L-channel current. Increasing intracellular ceramide level with sphingomyelinase also inhibited the L-channel current. The inhibitory effect of ceramide on the L-channel current was attenuated by calphostin C, a myristolated pseudosubstrate protein kinase C (PKC) inhibitor, and lavendustin A, a tyrosine kinase inhibitor. Combined treatment with lavendustin A and the myristolated PKC inhibitor blocked the effect of ceramide on the L-channel current. These results indicate that ceramide, a lipid messenger of the sphingomyelin pathway, is an important regulator of the L-channel in GH3 cells and both tyrosine kinase and PKC are involved in this effect of ceramide.
Collapse
Affiliation(s)
- C L Chik
- Department of Medicine, 7-33 Medical Sciences Building, Edmonton, Alta., Canada T6G 2H7.
| | | | | | | |
Collapse
|
|
41
|
Rybin VO, Guo J, Sabri A, Elouardighi H, Schaefer E, Steinberg SF. Stimulus-specific Differences in Protein Kinase Cδ Localization and Activation Mechanisms in Cardiomyocytes. J Biol Chem 2004; 279:19350-61. [PMID: 14970215 DOI: 10.1074/jbc.m311096200] [Citation(s) in RCA: 94] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Protein kinase C (PKC) isoforms play key roles in the regulation of cardiac contraction, ischemic preconditioning, and hypertrophy/failure. Models of PKC activation generally focus on lipid cofactor-induced PKC translocation to membranes. This study identifies tyrosine phosphorylation as an additional mechanism that regulates PKC delta actions in cardiomyocytes. Using immunoblot analysis with antibodies to total PKC delta and PKC delta-pY(311), we demonstrate that PKC delta partitions between soluble and particulate fractions (with little Tyr(311) phosphorylation) in resting cardiomyocytes. Phorbol 12-myristate 13-acetate (PMA) promotes PKC delta translocation to membranes and phosphorylation at Tyr(311). H(2)O(2) also increases PKC delta-pY(311) in association with its release from membranes. Both PMA- and H(2)O(2)-dependent increases in PKC delta-pY(311) are mediated by Src family kinases, but they occur via different mechanisms. The H(2)O(2)-dependent increase in PKC delta-pY(311) results from Src activation and increased Src-PKC delta complex formation. The PMA-dependent increase in PKC delta-pY(311) results from a lipid cofactor-induced conformational change that renders PKC delta a better substrate for phosphorylation by precomplexed Src kinases (without Src activation). PKC delta-Y(311) phosphorylation does not grossly alter the kinetics of PMA-dependent PKC delta down-regulation. Rather, tyrosine phosphorylation regulates PKC delta kinase activity. PKC delta is recovered from the soluble fraction of H(2)O(2)-treated cardiomyocytes as a tyrosine-phosphorylated, lipid-independent enzyme with altered substrate specificity. In vitro PKC delta phosphorylation by Src also increases lipid-independent kinase activity. The magnitude of this effect varies, depending upon the substrate, suggesting that tyrosine phosphorylation fine-tunes PKC delta substrate specificity. The stimulus-specific modes for PKC delta signaling identified in this study allow for distinct PKC delta-mediated phosphorylation events and responses during growth factor stimulation and oxidant stress in cardiomyocytes.
Collapse
Affiliation(s)
- Vitalyi O Rybin
- Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA
| | | | | | | | | | | |
Collapse
|
|
42
|
Marchesini N, Osta W, Bielawski J, Luberto C, Obeid LM, Hannun YA. Role for mammalian neutral sphingomyelinase 2 in confluence-induced growth arrest of MCF7 cells. J Biol Chem 2004; 279:25101-11. [PMID: 15051724 DOI: 10.1074/jbc.m313662200] [Citation(s) in RCA: 130] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Recently, we reported that neutral sphingomyelinase 2 (nSMase2) functions as a bona fide neutral sphingomyelinase and that overexpression of nSMase2 in MCF7 breast cancer cells caused a decrease in cell growth (Marchesini, N., Luberto, C., and Hannun, Y. A. (2003) J. Biol. Chem. 278, 13775-13783). In this study, the role of endogenous nSMase2 in regulating growth arrest was investigated. The results show that endogenous nSMase2 mRNA was up-regulated approximately 5-fold when MCF7 cells became growth-arrested at confluence, and total neutral SMase activity was increased by 119 +/- 41% with respect to control. Cell cycle analysis showed that up-regulation of endogenous nSMase2 correlated with G(0)/G(1) cell cycle arrest and an increase in total ceramide levels (2.4-fold). Analysis of ceramide species showed that confluence caused selective increases in very long chain ceramide C(24:1) (370 +/- 54%) and C(24:0) (266 +/- 81%) during arrest. The role of endogenous nSMase2 in growth regulation and ceramide metabolism was investigated using short interfering RNA (siRNA)-mediated loss-of-function analysis. Down-regulation of nSMase2 with specific siRNA increased the cell population of cells in S phase of the cell cycle by 59 +/- 14% and selectively reverted the effects of growth arrest on the increase in levels of very long chain ceramides. Mechanistically, confluence arrest also induced hypophosphorylation of the retinoblastoma protein (6-fold) and induction of p21(WAF1) (3-fold). Down-regulation of nSMase2 with siRNA largely prevented the dephosphorylation of the retinoblastoma protein and the induction of p21(WAF1), providing a link between the action of nSMase2 and key regulators of cell cycle progression. Moreover, studies on nSMase2 localization in MCF7 cells showed that nSMase2 distributed throughout the cells in subconfluent, proliferating cultures. In contrast, nSMase2 became nearly exclusively located at the plasma membrane in confluent, contact-inhibited cells. Hence, we demonstrate for the first time that nSMase2 functions as a growth suppressor in MCF7 cells, linking confluence to the G(0)/G(1) cell cycle check point.
Collapse
Affiliation(s)
- Norma Marchesini
- Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA
| | | | | | | | | | | |
Collapse
|
|
43
|
Kajimoto T, Shirai Y, Sakai N, Yamamoto T, Matsuzaki H, Kikkawa U, Saito N. Ceramide-induced Apoptosis by Translocation, Phosphorylation, and Activation of Protein Kinase Cδ in the Golgi Complex. J Biol Chem 2004; 279:12668-76. [PMID: 14715667 DOI: 10.1074/jbc.m312350200] [Citation(s) in RCA: 90] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Protein kinase C (PKC), a Ca(2+)/phospholipid-dependent protein kinase, is known as a key enzyme in various cellular responses, including apoptosis. However, the functional role of PKC in apoptosis has not been clarified. In this study, we focused on the involvement of PKCdelta in ceramide-induced apoptosis in HeLa cells and examined the importance of spatiotemporal activation of the specific PKC subtype in apoptotic events. Ceramide-induced apoptosis was inhibited by the PKCdelta-specific inhibitor rottlerin and also was blocked by knockdown of endogenous PKCdelta expression using small interfering RNA. Ceramide induced the translocation of PKCdelta to the Golgi complex and the concomitant activation of PKCdelta via phosphorylation of Tyr(311) and Tyr(332) in the hinge region of the enzyme. Unphosphorylatable PKCdelta (mutants Y311F and Y332F) could translocate to the Golgi complex in response to ceramide, suggesting that tyrosine phosphorylation is not necessary for translocation. However, ceramide failed to activate PKCdelta lacking the C1B domain, which did not translocate to the Golgi complex, but could be activated by tyrosine phosphorylation. These findings suggest that ceramide translocates PKCdelta to the Golgi complex and that PKCdelta is activated by tyrosine phosphorylation in the compartment. Furthermore, we utilized species-specific knockdown of PKCdelta by small interfering RNA to study the significance of phosphorylation of Tyr(311) and Tyr(332) in PKCdelta for ceramide-induced apoptosis and found that phosphorylation of Tyr(311) and Tyr(332) is indispensable for ceramide-induced apoptosis. We demonstrate here that the targeting mechanism of PKCdelta, dual regulation of both its activation and translocation to the Golgi complex, is critical for the ceramide-induced apoptotic event.
Collapse
Affiliation(s)
- Taketoshi Kajimoto
- Laboratories of Molecular Pharmacology and Biochemistry, Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan
| | | | | | | | | | | | | |
Collapse
|
|
44
|
Ling M, Trollér U, Zeidman R, Lundberg C, Larsson C. Induction of neurites by the regulatory domains of PKCdelta and epsilon is counteracted by PKC catalytic activity and by the RhoA pathway. Exp Cell Res 2004; 292:135-50. [PMID: 14720513 DOI: 10.1016/j.yexcr.2003.08.013] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
We have shown that protein kinase C (PKC) epsilon, independently of its kinase activity, via its regulatory domain (RD), induces neurites in neuroblastoma cells. This study was designed to evaluate whether the same effect is obtained in nonmalignant neural cells and to dissect mechanisms mediating the effect. Overexpression of PKCepsilon resulted in neurite induction in two immortalised neural cell lines (HiB5 and RN33B). Phorbol ester potentiated neurite outgrowth from PKCepsilon-overexpressing cells and led to neurite induction in cells overexpressing PKCdelta. The effects were potentiated by blocking the PKC catalytic activity with GF109203X. Furthermore, kinase-inactive PKCdelta induced more neurites than the wild-type isoform. The isolated regulatory domains of novel PKC isoforms also induced neurites. Experiments with PKCdelta-overexpressing HiB5 cells demonstrated that phorbol ester, even in the presence of a PKC inhibitor, led to a decrease in stress fibres, indicating an inactivation of RhoA. Active RhoA blocked PKC-induced neurite outgrowth, and inhibition of the RhoA effector ROCK led to neurite outgrowth. This demonstrates that neurite induction by the regulatory domain of PKCdelta can be counteracted by PKCdelta kinase activity, that PKC-induced neurite outgrowth is accompanied by stress fibre dismantling indicating an inactivation of RhoA, and that the RhoA pathway suppresses PKC-mediated neurite outgrowth.
Collapse
Affiliation(s)
- Mia Ling
- Department of Laboratory Medicine, Molecular Medicine, Lund University, Malmö University Hospital, 205 02 Malmö, Sweden
| | | | | | | | | |
Collapse
|
|
45
|
Watanabe M, Kitano T, Kondo T, Yabu T, Taguchi Y, Tashima M, Umehara H, Domae N, Uchiyama T, Okazaki T. Increase of Nuclear Ceramide through Caspase-3-Dependent Regulation of the “Sphingomyelin Cycle” in Fas-Induced Apoptosis. Cancer Res 2004; 64:1000-7. [PMID: 14871831 DOI: 10.1158/0008-5472.can-03-1383] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Regardless of the existence of ceramide-related molecules, such as sphingomyelin (SM), neutral sphingomyelinase (nSMase), and SM synthase, in the nucleus, the regulation of ceramide in the nucleus is poorly understood in stress-induced apoptosis. In Fas-induced Jurkat T-cell apoptosis, we found a time- and dose-dependent increase of ceramide content in the nuclear and microsomal fractions. Fas-induced increase of ceramide content in the nucleus also was detected by confocal microscopy using anticeramide antibody. Activation of nSMase and inhibition of SM synthase were evident in the nuclear fraction after Fas cross-linking, whereas nSMase was activated, but SM synthase was not affected, in the microsomal fraction. Pretreatment with D-609, a putative SM synthase inhibitor, enhanced Fas-induced increase of ceramide in the nucleus and induction of apoptosis along with increase of Fas-induced inhibition of nuclear SM synthase. Fas-induced activation of caspase-3 was detected in the nuclear fraction and in whole cell lysate. A caspase-3 inhibitor, acetyl-Asp-Glu-Val-Asp-chloromethyl ketone, blocked not only Fas-induced increases of apoptosis and ceramide content but also Fas-induced activation of nSMase and inhibition of SM synthase in the nuclear fraction. Taken together, it is suggested that the nucleus is a site for ceramide increase and caspase-3 activation in Fas-induced Jurkat T-cell apoptosis and that caspase-3-dependent regulation of the "SM cycle" consisting of nSMase and SM synthase plays a role in Fas-induced ceramide increase in the nucleus.
Collapse
Affiliation(s)
- Mitsumasa Watanabe
- Department of Hematology, Graduate School of Medicine, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
46
|
Kaul S, Kanthasamy A, Kitazawa M, Anantharam V, Kanthasamy AG. Caspase-3 dependent proteolytic activation of protein kinase C delta mediates and regulates 1-methyl-4-phenylpyridinium (MPP+)-induced apoptotic cell death in dopaminergic cells: relevance to oxidative stress in dopaminergic degeneration. Eur J Neurosci 2003; 18:1387-401. [PMID: 14511319 DOI: 10.1046/j.1460-9568.2003.02864.x] [Citation(s) in RCA: 142] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
1-Methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), induces apoptosis in dopaminergic neurons; however, the cellular mechanisms underlying the degenerative process are not well understood. In the present study, we demonstrate that caspase-3 mediated proteolytic activation of protein kinase C delta (PKC delta) is critical in MPP+-induced oxidative stress and apoptosis. MPP+ exposure in rat dopaminergic neuronal cells resulted in time-dependent increases in reactive oxygen species generation, cytochrome c release, and caspase-9 and caspase-3 activation. Interestingly, MPP+ induced proteolytic cleavage of PKC delta (72-74 kDa) into a 41-kDa catalytic and a 38-kDa regulatory subunit, resulting in persistently increased kinase activity. The caspase-3 inhibitor Z-DEVD-fmk effectively blocked MPP+-induced PKC delta cleavage and kinase activity, suggesting that the proteolytic activation is caspase-3 mediated. Similar results were seen in MPP+-treated rat midbrain slices. Z-DEVD-fmk and the PKC delta specific inhibitor rottlerin almost completely blocked MPP+-induced DNA fragmentation. The superoxide dismutase mimetic, MnTBAP also effectively attenuated MPP+-induced caspase-3 activation, PKC delta cleavage, and DNA fragmentation. Furthermore, rottlerin attenuated MPP+-induced caspase-3 activity without affecting basal activity, suggesting positive feedback activation of caspase-3 by PKC delta. Intracellular delivery of catalytically active recombinant PKC delta significantly increased caspase-3 activity, further indicating that PKC delta regulates caspase-3 activity. Finally, over-expression of a kinase inactive PKC delta K376R mutant prevented MPP+-induced caspase activation and DNA fragmentation, confirming the pro-apoptotic function of PKC delta in dopaminergic cell death. Together, we demonstrate for the first time that MPP+-induced oxidative stress proteolytically activates PKC delta in a caspase-3-dependent manner to induce apoptosis and up-regulate the caspase cascade in dopaminergic neuronal cells.
Collapse
Affiliation(s)
- Siddharth Kaul
- Parkinson's Disorder Research Laboratory, Department of Biomedical Sciences, 2062 Veterinary Medicine Building, Iowa Sate University, Ames, IA 50011, USA
| | | | | | | | | |
Collapse
|
|
47
|
Knauf JA, Ouyang B, Croyle M, Kimura E, Fagin JA. Acute expression of RET/PTC induces isozyme-specific activation and subsequent downregulation of PKCɛ in PCCL3 thyroid cells. Oncogene 2003; 22:6830-8. [PMID: 14534528 DOI: 10.1038/sj.onc.1206829] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Most papillary thyroid carcinomas (PTC) have an isozyme-specific reduction of protein kinase C (PKC)epsilon, which occurs through a post-transcriptional mechanism. Here, we test whether the oncoprotein RET/PTC could be responsible for this effect, since RET/PTC rearrangements are quite prevalent in PTC and RET/PTC activates PLCgamma, an upstream modulator of PKCs. At 3 h after induction of RET/PTC1 or RET/PTC3 expression, there was evidence of PKCepsilon activation. Activation was restricted to PKCepsilon, as acute expression of RET/PTC did not change the subcellular distribution of other PKC isozymes expressed in PCCL3 cells. Prolonged RET/PTC expression (2-6 days) produced an isozyme-specific change in PKCepsilon subcellular localization and a decrease in total PKCepsilon levels. The expression of RET/PTC3(Y541F), which does not interact with PLCgamma, but signals normally through other RET effectors, had no effect on PKCepsilon distribution at any of the time points examined. However, downregulation of total PKCepsilon levels was only partially prevented by expression of RET/PTC(Y541F). Cells with decreased PKCepsilon following prolonged expression of RET/PTC were relatively resistant to doxorubicin-induced apoptosis. Based on our previous observation that PCCL3 cells expressing a dominant-negative PKCepsilon are also markedly resistant to apoptosis, we propose that selective downregulation of PKCepsilon following prolonged RET/PTC activation promotes cell survival and clonal expansion.
Collapse
Affiliation(s)
- Jeffrey A Knauf
- Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, Mail Location 0547, Cincinnati, OH 45267, USA.
| | | | | | | | | |
Collapse
|
|
48
|
Yakushiji K, Sawai H, Kawai S, Kambara M, Domae N. Characterization of C2-ceramide-resistant HL-60 subline (HL-CR): involvement of PKC delta in C2-ceramide resistance. Exp Cell Res 2003; 286:396-402. [PMID: 12749866 DOI: 10.1016/s0014-4827(03)00113-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
We have established a C2-ceramide-resistant HL-60 subline (HL-CR). HL-CR cells were resistant not only to C2-ceramide but also to various anticancer drugs. HL-CR cells did not respond to differentiation-inducing reagents including 1alpha,25-dihydroxyvitamin D(3), retinoic acid, and 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA induced apoptosis in HL-CR cells much slower than in parental HL-60 cells. As it was reported that PKC isozymes were involved in C2-ceramide-induced apoptosis, we investigated the role of PKC isozymes in C2-ceramide resistance in HL-CR cells. The protein level of PKC delta was lower in HL-CR cells than in parental HL-60 cells, whereas the levels of PKC alpha, betaI, epsilon, and zeta were rather higher in HL-CR cells than in parental cells. Translocation of PKC delta from membrane to cytosol was induced by C2-ceramide in HL-CR cells as well as in wild-type HL-60 cells. Furthermore, overexpression of PKC delta in HL-CR cells potentiated C2-ceramide- and TPA-induced apoptosis and growth inhibition. These results suggest a role for ceramide in apoptosis and differentiation in HL-60 cells, and also suggest that PKC delta might be involved in ceramide- and TPA-induced apoptosis.
Collapse
Affiliation(s)
- Kentaro Yakushiji
- Department of Preventive and Community Dentistry, Osaka Dental University, 8-1, Kuzuhahanazono-cho, Hirakata, Osaka 573-1121, Japan
| | | | | | | | | |
Collapse
|
|
49
|
Zhu XF, Liu ZC, Xie BF, Feng GK, Zeng YX. Ceramide induces cell cycle arrest and upregulates p27kip in nasopharyngeal carcinoma cells. Cancer Lett 2003; 193:149-54. [PMID: 12706871 DOI: 10.1016/s0304-3835(03)00050-8] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Ceramide mediates differentiation, growth arrest, apoptosis, proliferation, cytokine biosynthesis and secretion, and a variety of other cellular functions. However, little is known regarding ceramide signaling linked to the cell cycle. In the present study, the effect of ceramide on cell cycle in nasopharyngeal carcinoma cell line CNE2 was investigated. The results showed that ceramide inhibited cell proliferation and induced cell cycle arrest in G1 phase in CNE2 cells. Exposure of CNE2 cells to ceramide resulted in a dose-dependent up-regulation of the cyclin-dependent kinase inhibitor p27 and a decrease of phospho-Akt without reduced expression of total AKT protein. The activation of phosphatidylinositol-3-kinase (PI3K) and the protein expression of PTEN were unaffected following ceramide treatment. We concluded that ceramide induced cell cycle arrest in G1 phase in CNE2 cells and p27 up-regulation was involved in this process. In addition, up-regulation of p27 resulting from ceramide treatment may be due to the interruption of Akt, but decrease of phospho-Akt is independent of PI3K function or PTEN protein expression.
Collapse
Affiliation(s)
- Xiao-Feng Zhu
- Cancer Institute, Cancer Center, Sun Yat-sen University, 651 DongFeng Road East, Guangzhou 510060, China
| | | | | | | | | |
Collapse
|
|
50
|
Marchesini N, Luberto C, Hannun YA. Biochemical properties of mammalian neutral sphingomyelinase 2 and its role in sphingolipid metabolism. J Biol Chem 2003; 278:13775-83. [PMID: 12566438 DOI: 10.1074/jbc.m212262200] [Citation(s) in RCA: 156] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Neutral sphingomyelinase (N-SMase) is one of the key enzymes involved in the generation of ceramide; however, the gene(s) encoding for the mammalian N-SMase is still not well defined. Previous studies on the cloned nSMase1 had shown that the protein acts primarily as lyso-platelet-activating factor-phospholipase C. Recently the cloning of another putative N-SMase, nSMase2, was reported. In this study, biochemical characterization of the mouse nSMase2 was carried out using the overexpressed protein in yeast cells in which the inositol phosphosphingolipid phospholipase C (Isc1p) was deleted. N-SMase activity was dependent on Mg(2+) and was activated by phosphatidylserine and inhibited by GW4869. The ability of nSMase2 to recognize endogenous sphingomyelin (SM) as substrate was investigated by overexpressing nSMase2 in MCF7 cells. Mass measurements showed a 40% decrease in the SM levels in the overexpressor cells, and labeling studies demonstrated that nSMase2 accelerated SM catabolism. Accordingly, ceramide measurement showed a 60 +/- 15% increase in nSMase2-overexpressing cells compared with the vector-transfected MCF7. The role of nSMase2 in cell growth was next investigated. Stable overexpression of nSMase2 resulted in a 30-40% decrease in the rate of growth at the late exponential phase. Moreover, tumor necrosis factor induced approximately 50% activation of nSMase2 in MCF7 cells overexpressing the enzyme, demonstrating that nSMase2 is a tumor necrosis factor-responsive enzyme. In conclusion, these results 1) show that nSMase2 is a structural gene for nSMase, 2) suggest that nSMase2 acts as a bona fide N-SMase in cells, and 3) implicate nSMase2 in the regulation of cell growth and cell signaling.
Collapse
Affiliation(s)
- Norma Marchesini
- Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425, USA
| | | | | |
Collapse
|