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1
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Stupar M, Tan L, Kerr ED, De Voss CJ, Forde BM, Schulz BL, West NP. TcrXY is an acid-sensing two-component transcriptional regulator of Mycobacterium tuberculosis required for persistent infection. Nat Commun 2024; 15:1615. [PMID: 38388565 PMCID: PMC10883919 DOI: 10.1038/s41467-024-45343-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2023] [Accepted: 01/22/2024] [Indexed: 02/24/2024] Open
Abstract
The ability of Mycobacterium tuberculosis (Mtb) to persist in the host complicates and prolongs tuberculosis (TB) patient chemotherapy. Here we demonstrate that a neglected two-component system (TCS) of Mtb, TcrXY, is an autoregulated acid-sensing TCS that controls a functionally diverse 70-gene regulon required for bacterial persistence. Characterisation of two representatives of this regulon, Rv3706c and Rv3705A, implicate these genes as key determinants for the survival of Mtb in vivo by serving as important effectors to mitigate redox stress at acidic pH. We show that genetic silencing of the response regulator tcrX using CRISPR interference attenuates the persistence of Mtb during chronic mouse infection and improves treatment with the two front-line anti-TB drugs, rifampicin and isoniazid. We propose that targeting TcrXY signal transduction blocks the ability of Mtb to sense and respond to acid stress, resulting in a disordered program of persistence to render the organism vulnerable to existing TB chemotherapy.
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Affiliation(s)
- Miljan Stupar
- School of Chemistry and Molecular Biosciences, Australian Infectious Disease Research Centre, The University of Queensland, Brisbane, Australia
| | - Lendl Tan
- School of Chemistry and Molecular Biosciences, Australian Infectious Disease Research Centre, The University of Queensland, Brisbane, Australia
| | - Edward D Kerr
- School of Chemistry and Molecular Biosciences, Australian Infectious Disease Research Centre, The University of Queensland, Brisbane, Australia
| | - Christopher J De Voss
- School of Chemistry and Molecular Biosciences, Australian Infectious Disease Research Centre, The University of Queensland, Brisbane, Australia
| | - Brian M Forde
- Faculty of Medicine, UQ Centre for Clinical Research, The University of Queensland, Brisbane, Australia
| | - Benjamin L Schulz
- School of Chemistry and Molecular Biosciences, Australian Infectious Disease Research Centre, The University of Queensland, Brisbane, Australia
| | - Nicholas P West
- School of Chemistry and Molecular Biosciences, Australian Infectious Disease Research Centre, The University of Queensland, Brisbane, Australia.
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2
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Huang J, Jiang T, Li J, Qie J, Cheng X, Wang Y, Zhou T, Liu J, Han H, Yao K, Yu L. Biomimetic Corneal Stroma for Scarless Corneal Wound Healing via Structural Restoration and Microenvironment Modulation. Adv Healthc Mater 2024; 13:e2302889. [PMID: 37988231 DOI: 10.1002/adhm.202302889] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2023] [Revised: 10/31/2023] [Indexed: 11/23/2023]
Abstract
Corneal injury-induced stromal scarring causes the most common subtype of corneal blindness, and there is an unmet need to promote scarless corneal wound healing. Herein, a biomimetic corneal stroma with immunomodulatory properties is bioengineered for scarless corneal defect repair. First, a fully defined serum-free system is established to derive stromal keratocytes (hAESC-SKs) from a current Good Manufacturing Practice (cGMP)-grade human amniotic epithelial stem cells (hAESCs), and RNA-seq is used to validate the phenotypic transition. Moreover, hAESC-SKs are shown to possess robust immunomodulatory properties in addition to the keratocyte phenotype. Inspired by the corneal stromal extracellular matrix (ECM), a photocurable gelatin-based hydrogel is fabricated to serve as a scaffold for hAESC-SKs for bioengineering of a biomimetic corneal stroma. The rabbit corneal defect model is used to confirm that this biomimetic corneal stroma rapidly restores the corneal structure, and effectively reshapes the tissue microenvironment via proteoglycan secretion to promote transparency and inhibition of the inflammatory cascade to alleviate fibrosis, which synergistically reduces scar formation by ≈75% in addition to promoting wound healing. Overall, the strategy proposed here provides a promising solution for scarless corneal defect repair.
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Affiliation(s)
- Jianan Huang
- Eye Center, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang Provincial Key Laboratory of Ophthalmology, Zhejiang Provincial Clinical Research Center for Eye Diseases, Zhejiang Provincial Engineering Institute on Eye Diseases, Hangzhou, 310009, P. R. China
- MOE Laboratory of Biosystems Homeostasis & Protection & College of Life Sciences-iCell Biotechnology Regenerative Biomedicine Laboratory, College of Life Sciences, Zhejiang University, Hangzhou, 310058, P. R. China
| | - Tuoying Jiang
- MOE Laboratory of Biosystems Homeostasis & Protection & College of Life Sciences-iCell Biotechnology Regenerative Biomedicine Laboratory, College of Life Sciences, Zhejiang University, Hangzhou, 310058, P. R. China
| | - Jinying Li
- MOE Laboratory of Biosystems Homeostasis & Protection & College of Life Sciences-iCell Biotechnology Regenerative Biomedicine Laboratory, College of Life Sciences, Zhejiang University, Hangzhou, 310058, P. R. China
- College of Traditional Chinese Medicine and Health Industry, Lishui University, Lishui, 323000, P. R. China
| | - Jiqiao Qie
- Eye Center, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang Provincial Key Laboratory of Ophthalmology, Zhejiang Provincial Clinical Research Center for Eye Diseases, Zhejiang Provincial Engineering Institute on Eye Diseases, Hangzhou, 310009, P. R. China
| | - Xiaoyu Cheng
- Eye Center, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang Provincial Key Laboratory of Ophthalmology, Zhejiang Provincial Clinical Research Center for Eye Diseases, Zhejiang Provincial Engineering Institute on Eye Diseases, Hangzhou, 310009, P. R. China
| | - Yiyao Wang
- Eye Center, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang Provincial Key Laboratory of Ophthalmology, Zhejiang Provincial Clinical Research Center for Eye Diseases, Zhejiang Provincial Engineering Institute on Eye Diseases, Hangzhou, 310009, P. R. China
| | - Tinglian Zhou
- Eye Center, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang Provincial Key Laboratory of Ophthalmology, Zhejiang Provincial Clinical Research Center for Eye Diseases, Zhejiang Provincial Engineering Institute on Eye Diseases, Hangzhou, 310009, P. R. China
| | - Jia Liu
- MOE Laboratory of Biosystems Homeostasis & Protection & College of Life Sciences-iCell Biotechnology Regenerative Biomedicine Laboratory, College of Life Sciences, Zhejiang University, Hangzhou, 310058, P. R. China
| | - Haijie Han
- Eye Center, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang Provincial Key Laboratory of Ophthalmology, Zhejiang Provincial Clinical Research Center for Eye Diseases, Zhejiang Provincial Engineering Institute on Eye Diseases, Hangzhou, 310009, P. R. China
- State Key Laboratory of Trauma, Burn and Combined Injury, Third Military Medical University, Chongqing, 400038, P. R. China
| | - Ke Yao
- Eye Center, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang Provincial Key Laboratory of Ophthalmology, Zhejiang Provincial Clinical Research Center for Eye Diseases, Zhejiang Provincial Engineering Institute on Eye Diseases, Hangzhou, 310009, P. R. China
| | - Luyang Yu
- MOE Laboratory of Biosystems Homeostasis & Protection & College of Life Sciences-iCell Biotechnology Regenerative Biomedicine Laboratory, College of Life Sciences, Zhejiang University, Hangzhou, 310058, P. R. China
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3
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Huynh KM, Wong ACY, Wu B, Horschman M, Zhao H, Brooks JD. Sprr2f protects against renal injury by decreasing the level of reactive oxygen species in female mice. Am J Physiol Renal Physiol 2020; 319:F876-F884. [PMID: 33017192 DOI: 10.1152/ajprenal.00318.2020] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Renal injury leads to chronic kidney disease, with which women are not only more likely to be diagnosed than men but have poorer outcomes as well. We have previously shown that expression of small proline-rich region 2f (Sprr2f), a member of the small proline-rich region (Sprr) gene family, is increased several hundredfold after renal injury using a unilateral ureteral obstruction (UUO) mouse model. To better understand the role of Sprr2f in renal injury, we generated a Sprr2f knockout (Sprr2f-KO) mouse model using CRISPR-Cas9 technology. Sprr2f-KO female mice showed greater renal damage after UUO compared with wild-type (Sprr2f-WT) animals, as evidenced by higher hydroxyproline levels and denser collagen staining, indicating a protective role of Sprr2f during renal injury. Gene expression profiling by RNA sequencing identified 162 genes whose expression levels were significantly different between day 0 and day 5 after UUO in Sprr2f-KO mice. Of the 162 genes, 121 genes were upregulated after UUO and enriched with those involved in oxidation-reduction, a phenomenon not observed in Sprr2f-WT animals, suggesting a protective role of Sprr2f in UUO through defense against oxidative damage. Consistently, bilateral ischemia-reperfusion injury resulted in higher serum blood urea nitrogen levels and higher tissue reactive oxygen species in Sprr2f-KO compared with Sprr2f-WT female mice. Moreover, cultured renal epithelial cells from Sprr2f-KO female mice showed lower viability after oxidative damage induced by menadione compared with Sprr2f-WT cells that could be rescued by supplementation with reduced glutathione, suggesting that Sprr2f induction after renal damage acts as a defense against reactive oxygen species.
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Affiliation(s)
- Kieu My Huynh
- Department of Urology, School of Medicine, Stanford University, Stanford, California
| | - Anny Chuu-Yun Wong
- Department of Urology, School of Medicine, Stanford University, Stanford, California
| | - Bo Wu
- Department of Urology, School of Medicine, Stanford University, Stanford, California
| | - Marc Horschman
- Department of Urology, School of Medicine, Stanford University, Stanford, California
| | - Hongjuan Zhao
- Department of Urology, School of Medicine, Stanford University, Stanford, California
| | - James D Brooks
- Department of Urology, School of Medicine, Stanford University, Stanford, California
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4
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Su M, Hu X, Lin J, Zhang L, Sun W, Zhang J, Tian Y, Qiu W. Identification of Candidate Genes Involved in Renal Ischemia/Reperfusion Injury. DNA Cell Biol 2019; 38:256-262. [PMID: 30668132 PMCID: PMC6434600 DOI: 10.1089/dna.2018.4551] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Renal ischemia/reperfusion injury (IRI) is a main risk factor for the occurrence of delayed graft function or primary graft nonfunction of kidney transplantation. However, it lacks ideal molecular markers for indicating IRI in kidney transplantation. The present study is to explore novel candidate genes involved in renal IRI. Experimental renal IRI mouse models were constructed, and the differentially expressed genes were screened using a microarray assay. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were performed. The expression of genes was detected using real-time qPCR assay. Western blotting and immunohistochemistry staining assays were performed for protein determination. We identified that renal IRI induced the upregulation of SPRR2F, SPRR1A, MMP-10, and long noncoding RNA (lncRNA) Malat1 in kidney tissues for 479.3-, 4.98-, 238.1-, and 3.79-fold, respectively. The expression of miR-139-5p in kidney tissues of IRI-treated mice was decreased to 40.4% compared with the sham-operated mice. These genes are associated with keratinocyte differentiation, regeneration and repair of kidney tissues, extracellular matrix degradation and remodeling, inflammation, and cell proliferation in renal IRI. Identification of novel biomarkers involved in renal IRI may provide evidences for the diagnosis and treatment of renal IRI.
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Affiliation(s)
- Ming Su
- 1 Department of Clinical Laboratory, Peking University People's Hospital, Beijing, People's Republic of China
| | - Xinyi Hu
- 2 Department of Urology, Beijing Friendship Hospital, Capital Medical University, Beijing, People's Republic of China
| | - Jun Lin
- 2 Department of Urology, Beijing Friendship Hospital, Capital Medical University, Beijing, People's Republic of China
| | - Lei Zhang
- 2 Department of Urology, Beijing Friendship Hospital, Capital Medical University, Beijing, People's Republic of China
| | - Wen Sun
- 2 Department of Urology, Beijing Friendship Hospital, Capital Medical University, Beijing, People's Republic of China
| | - Jian Zhang
- 2 Department of Urology, Beijing Friendship Hospital, Capital Medical University, Beijing, People's Republic of China
| | - Ye Tian
- 2 Department of Urology, Beijing Friendship Hospital, Capital Medical University, Beijing, People's Republic of China
| | - Wei Qiu
- 2 Department of Urology, Beijing Friendship Hospital, Capital Medical University, Beijing, People's Republic of China
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5
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Oh IY, de Guzman Strong C. The Molecular Revolution in Cutaneous Biology: EDC and Locus Control. J Invest Dermatol 2017; 137:e101-e104. [PMID: 28411839 DOI: 10.1016/j.jid.2016.03.046] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2015] [Revised: 02/10/2016] [Accepted: 03/02/2016] [Indexed: 12/12/2022]
Abstract
The epidermal differentiation complex (EDC) locus consists of a cluster of genes important for the terminal differentiation of the epidermis. While early studies identified the functional importance of individual EDC genes, the recognition of the EDC genes as a cluster with its shared biology, homology, and physical linkage was pivotal to later studies that investigated the transcriptional regulation of the locus. Evolutionary conservation of the EDC and the transcriptional activation during epidermal differentiation suggested a cis-regulatory mechanism via conserved noncoding elements or enhancers. This line of pursuit led to the identification of CNE 923, an epidermal-specific enhancer that was found to mediate chromatin remodeling of the EDC in an AP-1 dependent manner. These genomic studies, as well as the advent of high-throughput sequencing and genome engineering techniques, have paved the way for future investigation into enhancer-mediated regulatory networks in cutaneous biology.
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Affiliation(s)
- Inez Y Oh
- Department of Internal Medicine, Division of Dermatology, Center for Pharmacogenomics, Center for the Study of Itch, Washington University School of Medicine, St. Louis, Missouri, USA
| | - Cristina de Guzman Strong
- Department of Internal Medicine, Division of Dermatology, Center for Pharmacogenomics, Center for the Study of Itch, Washington University School of Medicine, St. Louis, Missouri, USA.
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6
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Suzuki A, Watanabe H, Mizutani T, Sato T, Ohta Y, Iguchi T. Global Gene Expression in Mouse Vaginae Exposed to Diethylstilbestrol at Different Ages. Exp Biol Med (Maywood) 2016; 231:632-40. [PMID: 16636312 DOI: 10.1177/153537020623100518] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Estrogens regulate proliferation and differentiation of cells in target organs such as the female reproductive tract. In mature mice, estrogens stimulate cell proliferation, whereas ovariectomy results in atrophy of the female reproductive tract. In contrast, perinatal exposure to estrogens, including diethylstilbestrol (DES), induces persistent, ovary-independent vaginal stratification and cervico-vaginal tumors later in life. These effects are due to altered cell fate following DES exposure during a critical developmental period. The detailed mechanisms underlying the reversible and irreversible cell proliferation in vaginae induced by DES at different ages has not been clarified. Therefore, we examined differences in gene expression pattern using DNA microarray analysis in mouse vaginae 6 hrs after a single injection of 2 μg DES per gram of body weight, and proliferation of vaginal epithelial and stromal cells 24 hrs after the injection at postnatal days (PNDs) 0, 5, 20, and 70. After DES stimulation, vaginal epithelial and stromal cells showed cell proliferation at PNDs 20 and 70, and at PNDs 0 and 5, respectively. DNA microarray analysis exhibited 54 DES-induced genes and 9 DES-repressed genes in vaginae at PND 0, whereas more than 200 DES-induced genes were found in vaginae at PNDs 5 and 20, and 350 genes at PND 70. Clustering analysis of DES-induced genes in the vaginae at different ages revealed that genes induced by DES at PND 5 were closer to the adult type than that of PND 0. Genes related to keratinocyte differentiation, such as Gadd45α, p21, 14–3–3 sigma, small proline-rich protein 2f (Sprr2f), and Krupple-like factor 4 (Klf4), were induced by DES. The number of DES-induced genes during the critical period, PND 0, was smaller than those found after the critical period. These results give insight toward understanding the molecular mechanisms underlying the critical period in mouse vaginae.
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Affiliation(s)
- Atsuko Suzuki
- The United Graduate School of Veterinary Science, Yamaguchi University, 1677-1, Yoshida, Yamaguchi, 753-8515, Japan
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7
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Jiang Y, Xie M, Chen W, Talbot R, Maddox JF, Faraut T, Wu C, Muzny DM, Li Y, Zhang W, Stanton JA, Brauning R, Barris WC, Hourlier T, Aken BL, Searle SMJ, Adelson DL, Bian C, Cam GR, Chen Y, Cheng S, DeSilva U, Dixen K, Dong Y, Fan G, Franklin IR, Fu S, Guan R, Highland MA, Holder ME, Huang G, Ingham AB, Jhangiani SN, Kalra D, Kovar CL, Lee SL, Liu W, Liu X, Lu C, Lv T, Mathew T, McWilliam S, Menzies M, Pan S, Robelin D, Servin B, Townley D, Wang W, Wei B, White SN, Yang X, Ye C, Yue Y, Zeng P, Zhou Q, Hansen JB, Kristensen K, Gibbs RA, Flicek P, Warkup CC, Jones HE, Oddy VH, Nicholas FW, McEwan JC, Kijas J, Wang J, Worley KC, Archibald AL, Cockett N, Xu X, Wang W, Dalrymple BP. The sheep genome illuminates biology of the rumen and lipid metabolism. Science 2014; 344:1168-1173. [PMID: 24904168 DOI: 10.1126/science.1252806] [Citation(s) in RCA: 333] [Impact Index Per Article: 30.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Sheep (Ovis aries) are a major source of meat, milk, and fiber in the form of wool and represent a distinct class of animals that have a specialized digestive organ, the rumen, that carries out the initial digestion of plant material. We have developed and analyzed a high-quality reference sheep genome and transcriptomes from 40 different tissues. We identified highly expressed genes encoding keratin cross-linking proteins associated with rumen evolution. We also identified genes involved in lipid metabolism that had been amplified and/or had altered tissue expression patterns. This may be in response to changes in the barrier lipids of the skin, an interaction between lipid metabolism and wool synthesis, and an increased role of volatile fatty acids in ruminants compared with nonruminant animals.
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Affiliation(s)
- Yu Jiang
- State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.,CSIRO Animal Food and Health Sciences, St Lucia, QLD 4067, Australia.,College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
| | - Min Xie
- BGI-Shenzhen, Shenzhen 518083, China
| | | | - Richard Talbot
- Ediburgh Genomics, University of Edinburgh, Easter Bush, Midlothian EH 25 9RG, UK
| | - Jillian F Maddox
- Department of Veterinary Science, University of Melbourne, Victoria 3010, Australia
| | - Thomas Faraut
- INRA, Laboratoire de Génétique Cellulaire, UMR 444, Castanet-Tolosan F-31326, France
| | - Chunhua Wu
- Utah State University, Logan, UT 84322-1435-1435, USA
| | - Donna M Muzny
- Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA
| | | | - Wenguang Zhang
- State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.,Inner Mongolia Agricultural University, Hohhot 010018, China.,Institute of ATCG, Nei Mongol Bio-Information, Hohhot, China
| | - Jo-Ann Stanton
- Department of Anatomy, University of Otago, Dunedin 9054, New Zealand
| | - Rudiger Brauning
- AgResearch, Invermay Agricultural Centre, Mosgiel 9053, New Zealand
| | - Wesley C Barris
- CSIRO Animal Food and Health Sciences, St Lucia, QLD 4067, Australia
| | - Thibaut Hourlier
- Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.,European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdom
| | - Bronwen L Aken
- Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.,European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdom
| | - Stephen M J Searle
- Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK
| | - David L Adelson
- CSIRO Animal Food and Health Sciences, St Lucia, QLD 4067, Australia
| | - Chao Bian
- BGI-Shenzhen, Shenzhen 518083, China
| | - Graham R Cam
- CSIRO Animal Food and Health Sciences, St Lucia, QLD 4067, Australia
| | - Yulin Chen
- College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
| | | | - Udaya DeSilva
- CSIRO Animal Food and Health Sciences, St Lucia, QLD 4067, Australia
| | - Karen Dixen
- Department of Biology, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark
| | - Yang Dong
- State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China
| | | | - Ian R Franklin
- CSIRO Animal Food and Health Sciences, St Lucia, QLD 4067, Australia
| | - Shaoyin Fu
- Inner Mongolia Agricultural University, Hohhot 010018, China
| | - Rui Guan
- BGI-Shenzhen, Shenzhen 518083, China
| | - Margaret A Highland
- USDA-ARS Animal Disease Research Unit, Pullman, WA 99164 USA.,Department of Veterinary Microbiology & Pathology, Washington State University, Pullman, WA 99164 USA
| | - Michael E Holder
- Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA
| | | | - Aaron B Ingham
- CSIRO Animal Food and Health Sciences, St Lucia, QLD 4067, Australia
| | - Shalini N Jhangiani
- Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA
| | - Divya Kalra
- Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA
| | - Christie L Kovar
- Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA
| | - Sandra L Lee
- Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA
| | | | - Xin Liu
- BGI-Shenzhen, Shenzhen 518083, China
| | | | - Tian Lv
- BGI-Shenzhen, Shenzhen 518083, China
| | - Tittu Mathew
- Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA
| | - Sean McWilliam
- CSIRO Animal Food and Health Sciences, St Lucia, QLD 4067, Australia
| | - Moira Menzies
- CSIRO Animal Food and Health Sciences, St Lucia, QLD 4067, Australia
| | | | - David Robelin
- INRA, Laboratoire de Génétique Cellulaire, UMR 444, Castanet-Tolosan F-31326, France
| | - Bertrand Servin
- INRA, Laboratoire de Génétique Cellulaire, UMR 444, Castanet-Tolosan F-31326, France
| | - David Townley
- CSIRO Animal Food and Health Sciences, St Lucia, QLD 4067, Australia
| | | | - Bin Wei
- BGI-Shenzhen, Shenzhen 518083, China.,Maize Research Institute, Sichuan Agricultural University, Chengdu 611130, China
| | - Stephen N White
- USDA-ARS Animal Disease Research Unit, Pullman, WA 99164 USA.,Department of Veterinary Microbiology & Pathology, Washington State University, Pullman, WA 99164 USA
| | | | - Chen Ye
- BGI-Shenzhen, Shenzhen 518083, China
| | - Yaojing Yue
- Lanzhou Institute of Husbandry and Pharmaceutical Science, Lanzhou,730050,China
| | - Peng Zeng
- BGI-Shenzhen, Shenzhen 518083, China
| | - Qing Zhou
- BGI-Shenzhen, Shenzhen 518083, China
| | - Jacob B Hansen
- Department of Biology, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark
| | - Karsten Kristensen
- Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark
| | - Richard A Gibbs
- Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA
| | - Paul Flicek
- European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdom
| | | | - Huw E Jones
- Biosciences KTN, The Roslin Institute, Easter Bush, Midlothian, EH25 9RG, UK
| | - V Hutton Oddy
- School of Environmental and Rural Science, University of New England, Armidale, NSW 2351, Australia
| | - Frank W Nicholas
- Faculty of Veterinary Science, University of Sydney, NSW 2006, Australia
| | - John C McEwan
- AgResearch, Invermay Agricultural Centre, Mosgiel 9053, New Zealand
| | - James Kijas
- CSIRO Animal Food and Health Sciences, St Lucia, QLD 4067, Australia
| | - Jun Wang
- BGI-Shenzhen, Shenzhen 518083, China.,Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark.,Princess Al Jawhara Center of Excellence in the Research of Hereditary Disorders, King Abdulaziz University, Jeddah 21589, Saudi Arabia.,Macau University of Science and Technology, Macau 999078, China
| | - Kim C Worley
- Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA
| | - Alan L Archibald
- The Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush, Midlothian EH 25 9RG, UK
| | | | - Xun Xu
- BGI-Shenzhen, Shenzhen 518083, China
| | - Wen Wang
- State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China
| | - Brian P Dalrymple
- CSIRO Animal Food and Health Sciences, St Lucia, QLD 4067, Australia
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8
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Carregaro F, Stefanini ACB, Henrique T, Tajara EH. Study of small proline-rich proteins (SPRRs) in health and disease: a review of the literature. Arch Dermatol Res 2013; 305:857-66. [DOI: 10.1007/s00403-013-1415-9] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2013] [Revised: 09/11/2013] [Accepted: 09/16/2013] [Indexed: 12/26/2022]
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9
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Lee HA, Kim HR, Lee YJ, Lee SJ, Kim WJ, Han SS, Yang SR, Woo HM, Na S, Song H, Hong SH. Immunolocalisation and oestrogen regulation of small proline-rich protein 2a protein in the mouse uterus. Reprod Fertil Dev 2013; 26:682-9. [PMID: 23735658 DOI: 10.1071/rd12408] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2012] [Accepted: 04/21/2013] [Indexed: 11/23/2022] Open
Abstract
Small proline-rich protein 2a (Sprr2a) is one of the structural components of the cornified keratinocyte cell envelope that contributes to form a protective barrier in the skin against dehydration and environmental stress. Interestingly, Sprr2a mRNA is detected in the mouse uterus and is regulated by 17β-oestradiol (E2). In the present study, we investigated the effects of E2 and oestrogenic compounds on the regulation and localisation of Sprr2a protein in the mouse uterus. Immunohistochemical staining revealed that Sprr2a protein is detected only in the adult uterus, and not in the ovary, oviduct or testis. We also demonstrated that Sprr2a protein is tightly regulated by E2 in the mouse uterus and exclusively detected in luminal and glandular epithelial cells. Furthermore, Sprr2a is dose-dependently induced by oestrogenic compounds such as bisphenol A and 4-tert-octylphenol. Collectively, our studies suggest that Sprr2a protein may have a unique function in physiological events in the mouse uterus and can be used as an indicator to detect compounds with oestrogenic activity in the mouse uterus.
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Affiliation(s)
- Hyang-Ah Lee
- Department of Obstertics and Gynecology, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea
| | - Hye-Ryun Kim
- Department of Biomedical Science, CHA University, Seoul 135-081, South Korea
| | - Young Jin Lee
- Center for Biomedical Engineering and Technology and Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
| | - Seung-Joon Lee
- Department of Internal Medicine, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea
| | - Woo Jin Kim
- Department of Internal Medicine, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea
| | - Seon-Sook Han
- Department of Internal Medicine, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea
| | - Se-Ran Yang
- Department of Thoracic and Cardiovascular Surgery, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea
| | - Heung-Myong Woo
- College of Veterinary Medicine, Kangwon National Univeristy, Chuncheon 200-701, South Korea
| | - Sunghun Na
- Department of Obstertics and Gynecology, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea
| | - Haengseok Song
- Department of Biomedical Science, CHA University, Seoul 135-081, South Korea
| | - Seok-Ho Hong
- Department of Internal Medicine, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea
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10
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Liu S, Kam WR, Ding J, Hatton MP, Sullivan DA. Effect of growth factors on the proliferation and gene expression of human meibomian gland epithelial cells. Invest Ophthalmol Vis Sci 2013; 54:2541-50. [PMID: 23493293 DOI: 10.1167/iovs.12-11221] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
PURPOSE We hypothesize that growth factors, including epidermal growth factor (EGF) and bovine pituitary extract (BPE), induce proliferation, but not differentiation (e.g., lipid accumulation), of human meibomian gland epithelial cells. We also hypothesize that these actions involve a significant upregulation of genes linked to cell cycle processes, and a significant downregulation of genes associated with differentiation. Our objective was to test these hypotheses. METHODS Immortalized human meibomian gland and conjunctival epithelial cells were cultured for varying time periods in the presence or absence of EGF, BPE, EGF + BPE, or serum, followed by cell counting, neutral lipid staining, or RNA isolation for molecular biological procedures. RESULTS Our studies show that growth factors stimulate a significant, time-dependent proliferation of human meibomian gland epithelial cells. These effects are associated with a significant upregulation of genes linked to cell cycle, DNA replication, ribosomes, and translation, and a significant decrease in those related to cell differentiation, tissue development, lipid metabolic processes, and peroxisome proliferator-activated receptor signaling. Serum-induced differentiation, but not growth factor-related proliferation, elicits a pronounced lipid accumulation in human meibomian gland epithelial cells. This lipogenic response is unique, and is not duplicated by human conjunctival epithelial cells. CONCLUSIONS Our results demonstrate that EGF and BPE stimulate human meibomian gland epithelial cells to proliferate. Further, our findings show that action is associated with an upregulation of cell cycle and translation ontologies, and a downregulation of genetic pathways linked to differentiation and lipid biosynthesis.
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Affiliation(s)
- Shaohui Liu
- Schepens Eye Research Institute, Boston, Massachusetts 02114, USA
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11
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Fang K, Zhang S, Glawe J, Grisham MB, Kevil CG. Temporal genome expression profile analysis during t-cell-mediated colitis: identification of novel targets and pathways. Inflamm Bowel Dis 2012; 18:1411-23. [PMID: 22179924 PMCID: PMC4413946 DOI: 10.1002/ibd.22842] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/18/2011] [Accepted: 11/03/2011] [Indexed: 01/30/2023]
Abstract
BACKGROUND T cells critically regulate inflammatory bowel disease (IBD), with T-cell-dependent experimental colitis models gaining favor in identifying potential pathogenic mechanisms; yet limited understanding of specific pathogenic molecules or pathways still exists. METHODS In this study we sought to identify changes in whole genome expression profiles using the CD4CD45Rbhi T-cell transfer colitis model compared to genome expression differences from Crohn's disease (CD) tissue specimens. Colon tissue was used for histopathological and genome expression profiling analysis at 0, 2, 4, or 6 weeks after adoptive T-cell transfer. RESULTS We identified 1775 genes that were significantly altered during disease progression, with 361 being progressively downregulated and 341 progressively upregulated. Gene expression changes were validated by quantitative real-time polymerase chain reaction (qRT-PCR), confirming genome expression analysis data. Differentially expressed genes were clearly related to inflammation/immune responses but also strongly associated with metabolic, chemokine signaling, Jak-STAT signaling, and angiogenesis pathways. Ingenuity network analysis revealed 25 unique network associations that were associated with functions such as antigen presentation, cell morphology, cell-to-cell signaling and interaction, as well as nervous system development and function. Moreover, many of these genes and pathways were similarly identified in CD specimens. CONCLUSIONS These findings reveal novel, complex, and dynamic changes in gene expression that may provide useful targets for future therapeutic approaches.
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Affiliation(s)
- Kai Fang
- Department of Pathology, Louisiana State University Health Science Center, Shreveport, LA, 71103, USA
| | - Songlin Zhang
- Department of Pathology, Louisiana State University Health Science Center, Shreveport, LA, 71103, USA
| | - John Glawe
- Department of Pathology, Louisiana State University Health Science Center, Shreveport, LA, 71103, USA
| | - Matthew B. Grisham
- Molecular and Cellular Physiology, Louisiana State University Health Science Center, Shreveport, LA, 71103, USA
| | - Christopher G Kevil
- Department of Pathology, Louisiana State University Health Science Center, Shreveport, LA, 71103, USA,Molecular and Cellular Physiology, Louisiana State University Health Science Center, Shreveport, LA, 71103, USA,Corresponding author: Christopher G. Kevil, Ph.D., Department of Pathology, LSU Health Science Center-Shreveport, 1501 Kings Highway, Shreveport, LA 71103, Phone: (318) 675-4694, Fax: (318) 675-7662,
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12
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Liu S, Richards SM, Lo K, Hatton M, Fay A, Sullivan DA. Changes in gene expression in human meibomian gland dysfunction. Invest Ophthalmol Vis Sci 2011; 52:2727-40. [PMID: 21372006 PMCID: PMC3088560 DOI: 10.1167/iovs.10-6482] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2010] [Revised: 12/15/2010] [Accepted: 03/01/2011] [Indexed: 12/14/2022] Open
Abstract
PURPOSE Meibomian gland dysfunction (MGD) may be the leading cause of dry eye syndrome throughout the world. However, the precise mechanism(s) underlying the pathogenesis of this disease is unclear. This study was conducted to identify meibomian gland genes that may promote the development and/or progression of human MGD. METHODS Lid tissues were obtained from male and female MGD patients and age-matched controls after eyelid surgeries (e.g., to correct entropion or ectropion). Meibomian glands were isolated and processed for RNA extraction and the analysis of gene expression. RESULTS The results show that MGD is associated with significant alterations in the expression of almost 400 genes in the human meibomian gland. The levels of 197 transcripts, including those encoding various small proline-rich proteins and S100 calcium-binding proteins, are significantly increased, whereas the expression of 194 genes, such as claudin 3 and cell adhesion molecule 1, is significantly decreased. These changes, which cannot be accounted for by sex differences, are accompanied by alterations in many gene ontologies (e.g., keratinization, cell cycle, and DNA repair). The findings also show that the human meibomian gland contains several highly expressed genes that are distinct from those in an adjacent tissue (i.e., conjunctival epithelium). CONCLUSIONS The results demonstrate that MGD is accompanied by multiple changes in gene expression in the meibomian gland. The nature of these alterations, including the upregulation of genes encoding small proline-rich proteins and S100 calcium-binding proteins, suggest that keratinization plays an important role in the pathogenesis of MGD.
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Affiliation(s)
- Shaohui Liu
- From the Schepens Eye Research Institute
- the Department of Ophthalmology, and
| | - Stephen M. Richards
- From the Schepens Eye Research Institute
- the Department of Ophthalmology, and
| | - Kristine Lo
- the Department of Ophthalmology, and
- the Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts; and
| | - Mark Hatton
- From the Schepens Eye Research Institute
- the Department of Ophthalmology, and
- Ophthalmic Consultants of Boston, Boston, Massachusetts
| | - Aaron Fay
- the Department of Ophthalmology, and
- the Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts; and
| | - David A. Sullivan
- From the Schepens Eye Research Institute
- the Department of Ophthalmology, and
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Schäfer M, Dütsch S, auf dem Keller U, Navid F, Schwarz A, Johnson DA, Johnson JA, Werner S. Nrf2 establishes a glutathione-mediated gradient of UVB cytoprotection in the epidermis. Genes Dev 2010; 24:1045-58. [PMID: 20478997 DOI: 10.1101/gad.568810] [Citation(s) in RCA: 132] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Ultraviolet (UV) B irradiation can severely damage the skin and even induce tumorigenesis. It exerts its effects by direct DNA modification and by formation of reactive oxygen species (ROS). We developed a strategy to genetically activate target gene expression of the transcription factor NF-E2-related factor 2 (Nrf2) in keratinocytes in vivo based on expression of a constitutively active Nrf2 mutant. Activation of Nrf2 target genes strongly reduced UVB cytotoxicity through enhancement of ROS detoxification. Remarkably, the protective effect was extended to neighboring cells. Using different combinations of genetically modified mice, we demonstrate that Nrf2 activates the production, recycling, and release of glutathione and cysteine by suprabasal keratinocytes, resulting in protection of basal cells in a paracrine, glutathione/cysteine-dependent manner. Most importantly, we found that endogenous Nrf2 controls selective protection of suprabasal keratinocytes from UVB-induced apoptosis through activation of cytoprotective genes. This finding explains the preferential UVB-induced apoptosis of basal cells, which is important for elimination of mutated stem cells as well as for preservation of skin integrity. Taken together, our results identify Nrf2 as a key regulator in the UV response of the skin.
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Affiliation(s)
- Matthias Schäfer
- Department of Biology, Institute of Cell Biology, ETH Zurich, Zurich, Switzerland.
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14
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Contreras CM, Akbay EA, Gallardo TD, Haynie JM, Sharma S, Tagao O, Bardeesy N, Takahashi M, Settleman J, Wong KK, Castrillon DH. Lkb1 inactivation is sufficient to drive endometrial cancers that are aggressive yet highly responsive to mTOR inhibitor monotherapy. Dis Model Mech 2010; 3:181-93. [PMID: 20142330 DOI: 10.1242/dmm.004440] [Citation(s) in RCA: 94] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Endometrial cancer--the most common malignancy of the female reproductive tract--arises from the specialized epithelial cells that line the inner surface of the uterus. Although significant advances have been made in our understanding of this disease in recent years, one significant limitation has been the lack of a diverse genetic toolkit for the generation of mouse models. We identified a novel endometrial-specific gene, Sprr2f, and developed a Sprr2f-Cre transgene for conditional gene targeting within endometrial epithelium. We then used this tool to generate a completely penetrant Lkb1 (also known as Stk11)-based mouse model of invasive endometrial cancer. Strikingly, female mice with homozygous endometrial Lkb1 inactivation did not harbor discrete endometrial neoplasms, but instead underwent diffuse malignant transformation of their entire endometrium with rapid extrauterine spread and death, suggesting that Lkb1 inactivation was sufficient to promote the development of invasive endometrial cancer. Mice with heterozygous endometrial Lkb1 inactivation only rarely developed tumors, which were focal and arose with much longer latency, arguing against the idea--suggested by some prior studies--that Lkb1 is a haploinsufficient tumor suppressor. Lastly, the finding that endometrial cancer cell lines were especially sensitive to the mTOR (mammalian target of rapamycin) inhibitor rapamycin prompted us to test its efficacy against Lkb1-driven endometrial cancers. Rapamycin monotherapy not only greatly slowed disease progression, but also led to striking regression of pre-existing tumors. These studies demonstrate that Lkb1 is a uniquely potent endometrial tumor suppressor, but also suggest that the clinical responses of some types of invasive cancers to mTOR inhibitors may be linked to Lkb1 status.
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Affiliation(s)
- Cristina M Contreras
- Department of Pathology, UT Southwestern Medical Center, UT Southwestern Medical Center, Dallas, TX 75390, USA
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15
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Chen J, Chen Y, Capizzi S, Yang M, Deng L, Bledsoe SB, Evan AP, Tischfield JA, Sahota A. 2,8-dihydroxyadenine nephrolithiasis induces developmental stage-specific alterations in gene expression in mouse kidney. Urology 2009; 75:914-22. [PMID: 20035974 DOI: 10.1016/j.urology.2009.10.031] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2009] [Revised: 10/06/2009] [Accepted: 10/15/2009] [Indexed: 11/24/2022]
Abstract
OBJECTIVES To identify factors that may be crucial for the initiation and progression of stone-induced injury in the developing mouse kidney by a prospective observational study using microarray analysis. Kidney stone diseases are common in premature infants, but the underlying molecular and cellular mechanisms are not fully defined. METHODS Mice with adenine phosphoribosyltransferase deficiency develop 2,8-dihydroxyadenine (DHA) nephrolithiasis. The gene expression changes between Aprt(-/-) and Aprt(+/+) kidneys from newborn and adult mice were compared using Affymetrix gene chips. Targets of interest were further analyzed by quantitative real-time polymerase chain reaction and immunohistochemistry. RESULTS We identified a set of genes that were differentially expressed in the developing kidney in response to DHA-induced injury. In 1-week-old Aprt(-/-) mice, the expression of Sprr2f and Clu was highly augmented and that of Egf was significantly decreased. We also observed that maturation-related gene expression changes were delayed in developing Aprt(-/-) kidneys, and immature Aprt(-/-) kidneys contained large numbers of intercalated cells that were blocked from terminal differentiation. CONCLUSIONS This study presents a comprehensive picture of the transcriptional changes induced by DHA stone injury in the developing mouse kidney. Our findings help explain growth impairment in kidneys subject to injury during the early stages of development.
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Affiliation(s)
- Jianmin Chen
- Department of Genetics, Rutgers University, Piscataway, New Jersey 08854, USA
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16
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Lee HJ, Pazin DE, Kahlon RS, Correa SM, Albrecht KH. Novel markers of early ovarian pre-granulosa cells are expressed in an Sry-like pattern. Dev Dyn 2009; 238:812-25. [PMID: 19301398 DOI: 10.1002/dvdy.21902] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Mammalian gonad differentiation involves sexually dimorphic cell-fate decisions within the bipotential gonadal primordia. Testis differentiation is initiated by a center-to-poles wave of Sry expression that induces supporting cell precursors (SCPs) to become Sertoli rather than granulosa cells. The initiation of ovary differentiation is less well understood. We identified two novel SCP markers, 1700106J16Rik and Sprr2d, whose expression is ovary-biased during early gonad development, and altered in Wnt4, Sf1, Wt1, and Fog2 mutant gonads. In XX and XY gonads, both genes were up-regulated at approximately E11 in a center-to-poles wave, and then rapidly down-regulated in XY gonads in a center-to-poles wave, which is reminiscent of Sry expression in XY gonads. Our data suggest that 1700106J16Rik and Sprr2d may have important roles in early gonad development, and are consistent with the hypothesis that ovarian SCP differentiation occurs in a center-to-poles wave with similar timing to that of testicular SCP differentiation.
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Affiliation(s)
- Hyunjoo J Lee
- Department of Medicine, Genetics Program, Boston University School of Medicine, Boston, Massachusetts 02118, USA
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17
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Fischer DF, Backendorf C. Identification of regulatory elements by gene family footprinting and in vivo analysis. ADVANCES IN BIOCHEMICAL ENGINEERING/BIOTECHNOLOGY 2007; 104:37-64. [PMID: 17290818 DOI: 10.1007/10_027] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/11/2023]
Abstract
Gene families of recently duplicated but subsequently diverged genes provide an unique opportunity for comparative analysis of regulatory elements. We have studied the human SPRR gene family of small proline rich proteins involved in barrier function of stratified squamous epithelia. These genes are all expressed in normal human keratinocytes, but respond differently to environmental insults. Comparisons of the functional promoter regions allows the rapid identification of both conserved and of novel regulatory elements that appeared after gene duplication. Competitive electrophoretic mobility shift assays can be used to confirm their presence. Here we show the power of gene family footprinting by the identification of two novel elements in the SPRR3 promoter, not present in SPRR1A and SPRR2A. One of these elements binds a protein similar to GAAP-1, a pro-apoptotic activator of IRF-1 and p53. In vivo analysis shows that this element functions as an inhibitor of SPRR3 transcription. The second novel element functions as an activator of promoter activity and is characterized by its A/T rich sequence. The latter interacting protein indeed binds through contacts in the minor groove, and strikingly, depends on the presence of calcium for DNA interaction.
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Affiliation(s)
- David F Fischer
- Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands
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18
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Tan YF, Sun XY, Li FX, Tang S, Piao YS, Wang YL. Gene expression pattern and hormonal regulation of small proline-rich protein 2 family members in the female mouse reproductive system during the estrous cycle and pregnancy. ACTA ACUST UNITED AC 2006; 46:641-55. [PMID: 17169311 DOI: 10.1051/rnd:2006037] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2006] [Accepted: 07/07/2006] [Indexed: 01/15/2023]
Abstract
Small proline-rich proteins (SPRR) are known to construct the cornified cell envelope (CE) in the stratified squamous epithelial cell. Their functions in the simple epithelium such as the uterine epithelium are not clear hitherto. In the present study, the mRNA expression patterns of sprr2 family members in the mouse uterus and vagina during the estrous cycle and pregnancy as well as their regulation by steroids were investigated. Using semi-quantitative RT-PCR, it was revealed that the transcripts of sprr2b, 2e and 2g genes were up-regulated in the proestrous and estrous uteri, and sprr2d was up-regulated only in the estrous uterus. In the vagina, transcription of sprr2a, 2b, 2d, 2e and 2k genes were up-regulated at the metestrous stage. Northern blot analysis demonstrated that the overall expression of sprr2 was highly up-regulated in the estrous uterus and the metestrous vagina. During pregnancy, the sprr2 mRNA in the uterus was sharply repressed from day 3 postcoitus on, and began to be induced around labor time. In situ hybridization showed that the sprr2 transcripts were localized in uterine luminal and glandular epithelial cells as well as vaginal stratified epithelial cells. In ovariectomized mice, the expression of sprr2a, 2d, 2e and 2f genes in the uterus were induced by estrogen, and the effect of estrogen on sprr2d and 2e expression could be partly abolished by progesterone. The data indicate that the sprr2 genes have unique regulation patterns in different reproductive tissues under different physiological conditions, and the encoded proteins might play diverse functions in the female reproductive system.
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Affiliation(s)
- Yin-fei Tan
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China
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19
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Sibug RM, Datson N, Tijssen AMI, Morsink M, de Koning J, de Kloet ER, Helmerhorst FM. Effects of urinary and recombinant gonadotrophins on gene expression profiles during the murine peri-implantation period. Hum Reprod 2006; 22:75-82. [PMID: 17053000 DOI: 10.1093/humrep/del363] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
BACKGROUND Controlled ovarian stimulation (COS) with urinary gonadotrophins but not recombinant gonadotrophins, adversely affect the implantation process. In this study, we investigated the effects of urinary and recombinant gonadotrophins on gene expression profiles at implantation sites during the mouse peri-implantation period and the possible molecular mechanisms involved in the detrimental effects of urinary gonadotrophins using microarray technology. METHODS Adult female CD1 mice were treated with (i) urinary human FSH (hFSH) and urinary HCG, (ii) recombinant hFSH and recombinant human LH or (iii) saline. Gene expression profiling with GeneChip mouse genome 430 2.0 arrays, containing 45 101 probe sets, was performed using implantation sites on embryonic day 5. Data were statistically analysed using Significance Analysis of Microarrays. Ten genes from the microarray analysis were selected for validation using quantitative RT-PCR (qRT-PCR). A parallel group of pregnant mice was allowed to give birth to study the effect of gonadotrophins on resorption. RESULTS Urinary gonadotrophins differentially up-regulated the expression of 30 genes, increased resorption and reduced litter size, whereas recombinant gonadotrophins did not. Nine of the 10 genes were confirmed by qRT-PCR. CONCLUSIONS Urinary gonadotrophins, but not recombinant gonadotrophins, exerted differential effects on gene expression during the murine peri-implantation period. These findings might contribute to improve protocols for COS, leading to higher successful pregnancy rates.
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Affiliation(s)
- R M Sibug
- Division of Medical Pharmacology, Leiden Amsterdam Center for Drug Research/Leiden University Medical Center, The Netherlands
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20
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Demetris AJ, Fontes P, Lunz JG, Specht S, Murase N, Marcos A. Wound healing in the biliary tree of liver allografts. Cell Transplant 2006; 15 Suppl 1:S57-65. [PMID: 16826796 DOI: 10.3727/000000006783982386] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
An increasing need for liver transplantation requires evaluation and triage of organs harvested from "extended criteria" donors. Although there is currently no widely accepted definition, most would agree that "extended criteria" includes organs donated by individuals that are old (>65 years), obese, infected with HBV or HCV, non-heart beating (NHBD), or had an unstable blood pressure before harvesting or the organ experienced a long cold ischemic time. These organs carry a statistical risk of dysfunction early after transplantation, but in the majority of recipients, hepatic parenchymal function recovers. Later, however, a small but significant percentage of extended criteria donors develop biliary strictures within several months after transplantation. The strictures occur primarily because of preservation injury that leads to "ischemic cholangitis" or deep wounding of the bile duct wall. Subsequent partial wound healing and wound contraction, but failed restitution of the biliary epithelial cell (BEC) lining, result in biliary tract strictures that cause progressive biliary fibrosis, increased morbidity, and decreased organ half-life. Better understanding of the pathophysiologic mechanisms that lead to biliary strictures in extended criteria donors provides an ideal proving ground for regenerative medicine; it also can provide insights into other diseases, such as extrahepatic biliary atresia and primary sclerosing cholangitis, that likely share certain pathogenic mechanisms. Possible points of therapeutic intervention include limiting cold and warm ischemic times, donor and/or donor organ treatment, ex vivo, to minimize the ischemic/preservation injury, maximize blood flow after transplantation, promote BEC wound healing, and limit myofibroblasts activation and proliferation in the bile duct wall. The pathobiology of biliary wound healing and therapeutic potential of interleukin-6 (IL-6) are highlighted.
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Affiliation(s)
- A J Demetris
- Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.
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Demetris AJ, Lunz JG, Specht S, Nozaki I. Biliary wound healing, ductular reactions, and IL-6/gp130 signaling in the development of liver disease. World J Gastroenterol 2006; 12:3512-22. [PMID: 16773708 PMCID: PMC4087567 DOI: 10.3748/wjg.v12.i22.3512] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Basic and translational wound healing research in the biliary tree lag significantly behind similar studies on the skin and gastrointestinal tract. This is at least partly attributable to lack of easy access to the biliary tract for study. But clinical relevance, more interest in biliary epithelial cell (BEC) pathophysiology, and widespread availability of BEC cultures are factors reversing this trend. In the extra-hepatic biliary tree, ineffectual wound healing, scarring and stricture development are pressing issues. In the smallest intra-hepatic bile ducts either impaired BEC proliferation or an exuberant response can contribute to liver disease. Chronic inflammation and persistent wound healing reactions in large and small bile ducts often lead to liver cancer. General concepts of wound healing as they apply to the biliary tract, importance of cellular processes dependent on IL-6/gp130/STAT3 signaling pathways, unanswered questions, and future directions are discussed.
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Affiliation(s)
- A-J Demetris
- The Thomas E. Starzl Transplantation Institute, Department of Pathology, Division of Transplantation, University of Pittsburgh Medical Center, UPMC-Montefiore E-741, 200 Lothrop Street, Pittsburgh, PA 15213-2582, USA.
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22
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Hawthorne SK, Goodarzi G, Bagarova J, Gallant KE, Busanelli RR, Olend WJ, Kleene KC. Comparative genomics of the sperm mitochondria-associated cysteine-rich protein gene. Genomics 2006; 87:382-91. [PMID: 16325371 DOI: 10.1016/j.ygeno.2005.09.010] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2005] [Revised: 08/28/2005] [Accepted: 09/20/2005] [Indexed: 10/25/2022]
Abstract
The sperm mitochondrial cysteine-rich protein (SMCP) is a rapidly evolving cysteine- and proline-rich protein that is localized in the mitochondrial capsule and enhances sperm motility. The sequences of the SMCP protein, gene, and mRNA in a variety of mammals have been compared to understand their evolution and regulation. SMCP can now be reliably identified by its tripartite structure including a short amino-terminal segment; a central segment containing short tandem repeats rich in cysteine, proline, glutamine, and lysine; and a C-terminal segment containing no repeats, few cysteines, and a C-terminal lysine. The SMCP gene is located in the epidermal differentiation complex (EDC), a large gene cluster that functions in forming epithelial barriers. Similarities in chromosomal location, molecular function, intron-exon structure, and protein organization argue that SMCP originated from an EDC gene and acquired spermatogenic cell-specific transcriptional and translational regulation and a novel cellular function in sperm motility. The SMCP 5' UTR and 3' UTR contain conserved elements and uORFs that may function in cytoplasmic regulation of gene expression, and the levels of SMCP mRNA in human are much lower than in other mammals, a feature of male-biased expression. The evolution of SMCP has been accompanied by changes in the sequence, number, and length of repeat units, including three alleles in dogs. The major proteins associated with the mitochondrial capsule, SMCP and phospholipid hydroperoxide glutathione peroxidase, provide outstanding examples of changes in cellular function driven by selective pressures on sperm motility, an important determinant of male reproductive success.
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Affiliation(s)
- Sabrina K Hawthorne
- Department of Biology, University of Massachusetts at Boston, 100 Morrissey Boulevard, Boston, MA 02125, USA
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Baden HP, Champliaud MF, Sundberg JP, Viel A. Targeted deletion of the sciellin gene resulted in normal development and maturation. Genesis 2005; 42:219-28. [PMID: 16028229 DOI: 10.1002/gene.20133] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Sciellin, together with other precursor proteins, was cross-linked by transglutaminase 1 to form the cornified envelope, an essential component of the physical barrier of the epidermis and stratified squamous epithelia. To more fully understand the function of sciellin in cornified envelope formation, we generated sciellin null mice. The mice appeared normal in their development and maturation and there were no structural features that distinguished them from littermate controls. Isolated cornified envelopes appeared normal in structure and were not more fragile to mechanical stress. There was no evidence of decreased barrier function or altered expression of other cornified envelope components. Transgenic mice expressing the repeat domain appeared to have a normal phenotype, like the null, and did not alter endogenous sciellin expression. We conclude that sciellin null mice had no structural anomalies and the transgenic mice did not act as a dominant-negative mutation.
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Affiliation(s)
- Howard P Baden
- Department of Dermatology, Cutaneous Biology Research Center, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02129, USA.
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Backendorf C, de Wit J, van Oosten M, Stout GJ, Mitchell JR, Borgstein AM, van der Horst GT, de Gruijl FR, Brouwer J, Mullenders LHF, Hoeijmakers JHJ. Repair characteristics and differentiation propensity of long-term cultures of epidermal keratinocytes derived from normal and NER-deficient mice. DNA Repair (Amst) 2005; 4:1325-36. [PMID: 16182615 DOI: 10.1016/j.dnarep.2005.07.011] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2005] [Revised: 07/20/2005] [Accepted: 07/21/2005] [Indexed: 01/15/2023]
Abstract
Epidermal keratinocytes constitute the most relevant cellular system in terms of DNA damage because of their continuous exposure to UV light and genotoxic chemicals from the environment. Here, we describe the establishment of long-term keratinocyte cultures from the skin of wild-type and nucleotide excision repair (NER) deficient mouse mutants. The use of media with a lowered calcium concentration and the inclusion of keratinocyte growth factor (KGF) permitted repeated passaging of the cultures and resulted in the generation of stable cell lines that proliferated efficiently. The cells retained their normal ability to engage into terminal differentiation when triggered with high calcium concentrations or after suspension in semi-solid medium. The cultures reflected the cellular characteristics (i.e. repair and transcription profiles) of the Xpa(-/-), Xpc(-/-), Csb(-/-) and Xpd(TTD) mouse models from which they were derived. For instance, in line with earlier in vivo results, Xpd(TTD) keratinocytes were disturbed in their ability to terminally differentiate in vitro. This was concluded from a delay in calcium-induced stratification and by reduced transcription of both early (keratin 10) and late (loricrin) terminal differentiation marker genes. UDS measurements in wild-type cells committed to terminal differentiation did not reveal any reduction in global DNA repair that could be indicative of differentiation associated repair (DAR) as found in neurons. UV sensitivity data revealed that in keratinocytes global genome repair contributes more to cell survival than previously concluded from fibroblast studies. It is inferred that these fully controllable in vitro cultures will be a valuable tool to assess critical parameters of genome care-taking systems in cell proliferation and differentiation.
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Affiliation(s)
- Claude Backendorf
- Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Einsteinweg 55, 2333 CC Leiden, The Netherlands.
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25
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Jackson B, Tilli CMLJ, Hardman MJ, Avilion AA, MacLeod MC, Ashcroft GS, Byrne C. Late cornified envelope family in differentiating epithelia--response to calcium and ultraviolet irradiation. J Invest Dermatol 2005; 124:1062-70. [PMID: 15854049 DOI: 10.1111/j.0022-202x.2005.23699.x] [Citation(s) in RCA: 130] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
The late cornified envelope (LCE) gene cluster within the epidermal differentiation complex on human chromosome one (mouse chromosome three) contains multiple conserved genes encoding stratum-corneum proteins. Within the LCE cluster, genes form "groups" based on chromosomal position and protein homology. We link a recently accepted nomenclature for the LCE cluster (formerly XP5, small proline-rich-like, late-envelope protein genes) to gene structure, groupings, and chromosomal organization, and carry out a pan-cluster quantitative expression analysis in a variety of tissues and environmental conditions. This analysis shows that (i) the cluster organizes into two "skin" expressing groups and a third group with low-level, tissue-specific expression patterns in all barrier-forming epithelia tested, including internal epithelia; (ii) LCE genes respond "group-wise" to environmental stimuli such as calcium levels and ultraviolet (UV) light, highlighting the functional significance of groups; (iii) in response to UV stimulation there is massive upregulation of a single, normally quiescent, non-skin LCE gene; and (iv) heterogeneity occurs between individuals with one individual lacking expression of an LCE skin gene without overt skin disease, suggesting LCE genes affect subtle attributes of skin function. This quantitative and pan-cluster expression analysis suggests that LCE groups have distinct functions and that within groups regulatory diversification permits specific responsiveness to environmental challenge.
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Affiliation(s)
- Benjamin Jackson
- Faculty of Life Sciences, University of Manchester, Manchester, UK
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26
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Zimmermann N, Doepker MP, Witte DP, Stringer KF, Fulkerson PC, Pope SM, Brandt EB, Mishra A, King NE, Nikolaidis NM, Wills-Karp M, Finkelman FD, Rothenberg ME. Expression and regulation of small proline-rich protein 2 in allergic inflammation. Am J Respir Cell Mol Biol 2005; 32:428-35. [PMID: 15731505 DOI: 10.1165/rcmb.2004-0269oc] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Asthma is a complex inflammatory pulmonary disorder that is on the rise despite intense ongoing research. We aimed to elucidate novel pathways involved in the pathogenesis of asthma. Employing asthma models induced by different allergens (ovalbumin and Aspergillus fumigatus), we uncovered the involvement of two members of the small proline-rich protein (SPRR) family, SPRR2a and SPRR2b, known to be involved in epithelial differentiation but not allergic disease. In situ hybridization revealed induction of SPRR2 signal in a subset of bronchial epithelial cells and mononuclear cells associated with inflammation after allergen challenge. Allergen-induced SPRR2 mRNA accumulation in the lung occurred in a time-dependent manner, with peak expression 10-96 h after a second ovalbumin challenge. Transgenic overexpression of interleukin (IL)-13 in the lungs resulted in a marked increase of SPRR2 expression, and allergen-induced SPRR2 expression was significantly decreased in IL-13-deficient mice. Studies in gene-targeted mice revealed that allergen-induced SPRR2 was dependent upon STAT6. Finally, we aimed to determine if the induction of SPRR2 by allergen was tissue specific. Notably, SPRR2 was markedly increased in the small intestine after induction of allergic gastrointestinal inflammation. Thus, SPRR2 is an allergen- and IL-13-induced gene in experimental allergic responses that may be involved in disease pathophysiology.
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Affiliation(s)
- Nives Zimmermann
- Division of Allergy and Immunology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA
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27
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Martin N, Patel S, Segre JA. Long-range comparison of human and mouse Sprr loci to identify conserved noncoding sequences involved in coordinate regulation. Genome Res 2005; 14:2430-8. [PMID: 15574822 PMCID: PMC534667 DOI: 10.1101/gr.2709404] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Mammalian epidermis provides a permeability barrier between an organism and its environment. Under homeostatic conditions, epidermal cells produce structural proteins, which are cross-linked in an orderly fashion to form a cornified envelope (CE). However, under genetic or environmental stress, specific genes are induced to rapidly build a temporary barrier. Small proline-rich (SPRR) proteins are the primary constituents of the CE. Under stress the entire family of 14 Sprr genes is upregulated. The Sprr genes are clustered within the larger epidermal differentiation complex on mouse chromosome 3, human chromosome 1q21. The clustering of the Sprr genes and their upregulation under stress suggest that these genes may be coordinately regulated. To identify enhancer elements that regulate this stress response activation of the Sprr locus, we utilized bioinformatic tools and classical biochemical dissection. Long-range comparative sequence analysis identified conserved noncoding sequences (CNSs). Clusters of epidermal-specific DNaseI-hypersensitive sites (HSs) mapped to specific CNSs. Increased prevalence of these HSs in barrier-deficient epidermis provides in vivo evidence of the regulation of the Sprr locus by these conserved sequences. Individual components of these HSs were cloned, and one was shown to have strong enhancer activity specific to conditions when the Sprr genes are coordinately upregulated.
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Affiliation(s)
- Natalia Martin
- National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA
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28
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Rundhaug JE, Hawkins KA, Pavone A, Gaddis S, Kil H, Klein RD, Berton TR, McCauley E, Johnson DG, Lubet RA, Fischer SM, Aldaz CM. SAGE profiling of UV-induced mouse skin squamous cell carcinomas, comparison with acute UV irradiation effects. Mol Carcinog 2005; 42:40-52. [PMID: 15547921 DOI: 10.1002/mc.20064] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Ultraviolet (UV) irradiation is the primary environmental insult responsible for the development of most common skin cancers. To better understand the multiple molecular events that contribute to the development of UV-induced skin cancer, in a first study, serial analysis of gene expression (SAGE) was used to compare the global gene expression profiles of normal SKH-1 mice epidermis with that of UV-induced squamous cell carcinomas (SCCs) from SKH-1 mice. More than 200 genes were found to be differentially expressed in SCCs compared to normal skin (P < 0.0005 level of significance). As expected, genes related to epidermal proliferation and differentiation were deregulated in SCCs relative to normal skin. However, various novel genes, not previously associated with skin carcinogenesis, were also identified as deregulated in SCCs. Northern blot analyses on various selected genes validated the SAGE findings: caspase-14 (reduced 8.5-fold in SCCs); cathepsins D and S (reduced 3-fold and increased 11.3-fold, respectively, in SCCs); decorin, glutathione S-transferase omega-1, hypoxia-inducible factor 1 alpha, insulin-like growth factor binding protein-7, and matrix metalloproteinase-13 (increased 18-, 12-, 12-, 18.3-, and 11-folds, respectively, in SCCs). Chemokine (C-C motif), ligand 27 (CCL27), which was found downregulated 12.7-fold in SCCs by SAGE, was also observed to be strongly downregulated 6-24 h after a single and multiple UV treatments. In a second independent study we compared the expression profile of UV-irradiated versus sham-treated SKH-1 epidermis. Interestingly, numerous genes determined to be deregulated 8 h after a single UV dose were also deregulated in SCCs. For instance, genes whose expression was upregulated both after acute UV-treated skin and SCCs included keratins 6 and 16, small proline-rich proteins, and S100 calcium binding protein A9. Studies like those described here do not only provide insights into genes and pathways involved in skin carcinogenesis but also allow us to identify early UV irradiation deregulated surrogate biomarkers of potential use in chemoprevention studies.
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Affiliation(s)
- Joyce E Rundhaug
- Department of Carcinogenesis, The University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957, USA
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29
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Troy TC, Turksen K. Commitment of embryonic stem cells to an epidermal cell fate and differentiation in vitro. Dev Dyn 2005; 232:293-300. [PMID: 15614782 DOI: 10.1002/dvdy.20223] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Abstract
The epidermis develops from a stem cell population in the surface ectoderm that feeds a single vertical terminal differentiation pathway. To date, however, the limited capacity for the isolation or purification of epidermal stem or precursor cells has hampered studies on early commitment and differentiation events. We have developed a two-step culture scheme in which pluripotent mouse embryonic stem (ES) cells are induced first to a surface ectoderm phenotype and then are positively selected for putative epidermal stem cells. We show that the earliest stages of epidermal development follow an ordered sequence that is similar to that observed in vivo (expression of keratin 8, keratin 19, keratin 17, and keratin 14), suggesting that ES cell-derived surface ectoderm-like cells can be induced to follow the epidermal developmental pathway. At a low frequency, keratin 14-positive early epidermal cells progressed to keratin 1-positive and terminally differentiated cells producing a cornified envelope. This culturing protocol provides an invaluable system in which to study both the mechanisms that direct stem cells along the epidermal pathway as well as those that influence their subsequent epidermal differentiation.
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Affiliation(s)
- Tammy-Claire Troy
- Development Program, Ottawa Health Research Institute, 725 Parkdale Avenue, Ottawa, Ontario, K1Y 4E9, Canada
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30
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Nozaki I, Lunz JG, Specht S, Stolz DB, Taguchi K, Subbotin VM, Murase N, Demetris AJ. Small proline-rich proteins 2 are noncoordinately upregulated by IL-6/STAT3 signaling after bile duct ligation. J Transl Med 2005; 85:109-23. [PMID: 15558059 DOI: 10.1038/labinvest.3700213] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Small proline-rich proteins 2 (SPRR2) are coordinately expressed with other epidermal differential complex (EDC) genes in the skin. They function as crosslinking proteins that form bridges between other proteins that comprise the cornified cell envelope, which is the major barrier against the environment. IL-6 is invariably produced at sites of biliary tract injury and IL-6-deficient (IL-6(-/-)) mice show impaired barrier function after bile duct ligation (BDL). Screening microarray analysis identified noncoordinate expression of SPRR2 as a candidate gene that is: (a) expressed in biliary epithelial cells (BEC); (b) IL-6 responsive; and (c) potentially related to biliary barrier function. Therefore, we studied in detail the regulation of BEC SPRR2A expression, in vitro; and tested the hypothesis that if BEC SPRR2 expression contributes to biliary barrier function, it should be increased after BDL in IL-6-wild type (IL-6(+/+)) mice and not in IL-6(-/-) mice. In vitro studies confirmed that IL-6/gp130-signaling, mediated primarily by signal transducer and activator of transcription 3 (STAT3), stimulated noncoordinate BEC SPRR2 expression. In vivo, noncoordinate upregulation of BEC SPRR2 expression after BDL was seen in the IL-6(+/+) mice and was unrelated to squamous metaplasia. IL-6(-/-) mice showed deficient BEC SPRR2 expression after BDL associated with impaired barrier function, as evidenced by smaller diameters of obstructed ducts, decreased bile volume, and an inability to form 'white bile' compared to IL-6(+/+) mice at 12 weeks after BDL. IL-6 replacement therapy reversed the barrier defect in IL-6(-/-) mice after BDL, coincident with recovery of SPRR2A expression. SPRR2 in diseased mouse and human liver localized subjacent to the apical plasma membrane of BEC lining bile ducts, but was more diffusely expressed throughout the cytoplasm of cholangioles. In conclusion, BEC IL-6/gp130/STAT3 signaling noncoordinately upregulates BEC SPRR2 that appears to contribute to modification of the biliary barrier under conditions of stress.
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Affiliation(s)
- Isao Nozaki
- Thomas E Starzl Transplantation Institute, Divisions of Transplantation, University of Pittsburgh Medical Center, Pittsburgh, PA, USA
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31
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Huang WW, Yin Y, Bi Q, Chiang TC, Garner N, Vuoristo J, McLachlan JA, Ma L. Developmental diethylstilbestrol exposure alters genetic pathways of uterine cytodifferentiation. Mol Endocrinol 2004; 19:669-82. [PMID: 15591538 DOI: 10.1210/me.2004-0155] [Citation(s) in RCA: 60] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
The formation of a simple columnar epithelium in the uterus is essential for implantation. Perturbation of this developmental process by exogenous estrogen, such as diethylstilbestrol (DES), results in uterine metaplasia that contributes to infertility. The cellular and molecular mechanism underlying this transformation event is not well understood. Here we use a combination of global gene expression analysis and a knockout mouse model to delineate genetic pathways affected by DES. Global gene expression profiling experiment revealed that neonatal DES treatment alters uterine cell fate, particularly in the luminal epithelium by inducing abnormal differentiation, characterized by the induction of stratified epithelial markers including members of the small proline-rich protein family and epidermal keratins. We show that Msx2, a homeodomain transcription factor, functions downstream of DES and is required for the proper expression of several genes in the uterine epithelium including Wnt7a, PLAP, and K2.16. Finally, Msx2-/- uteri were found to exhibit abnormal water trafficking upon DES exposure, demonstrating the importance of Msx2 in tissue responsiveness to estrogen exposure. Together, these results indicate that developmental exposure to DES can perturb normal uterine development by affecting genetic pathways governing uterine differentiation.
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Affiliation(s)
- Wei-Wei Huang
- Tulane/Xavier Center for Bioenvironmental Research, Tulane University Medical Center, New Orleans, Louisiana 70112, USA
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32
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Knight PA, Pemberton AD, Robertson KA, Roy DJ, Wright SH, Miller HRP. Expression profiling reveals novel innate and inflammatory responses in the jejunal epithelial compartment during infection with Trichinella spiralis. Infect Immun 2004; 72:6076-86. [PMID: 15385512 PMCID: PMC517597 DOI: 10.1128/iai.72.10.6076-6086.2004] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Infection with intestinal nematodes induces profound pathological changes to the gut that are associated with eventual parasite expulsion. We have applied expression profiling as an initial screening process with oligonucleotide microarrays (Affymetrix MG-U74AV2 gene chips) and time course kinetics to investigate gene transcription triggered by the intraepithelial nematode Trichinella spiralis in jejunal epithelium from BALB/c mice. Of the 4,114 genes detected, 2,617 were present in all uninfected and T. spiralis-infected replicates, 8% of which were notably upregulated, whereas 12% were downregulated at the time of worm expulsion (day 14 postinfection). Upregulation of goblet cell mucin gene transcripts intestinal mucin gene 3 (MUC3), calcium chloride channel 5 (CLCA5), and goblet cell gene 4 (GOB4) is consistent with enhanced production and alteration of mucus, whereas a 60- to 70-fold upregulation of transcripts for mast cell proteases 1 and 2 (MCPT-1 and -2) is consistent with intraepithelial mucosal mast cell recruitment. Importantly, there was novel expression of sialyltransferase 4C (SIAT4C), small proline-rich protein 2A (SPRR2A), and resistin-like molecule beta (RELMbeta) on day 14 postinfection. In contrast, DNase I and regenerating protein 3 (REG3) transcripts were substantially downregulated. Time course analyses revealed early (within 48 h of infection) induction of Siat4c, Sprr2A, and Relmbeta and later (within 120 h) induction of Mcpt-1 and -2. The findings demonstrate early innate responses and later inflammatory changes within the epithelium. The early epithelial responses may be associated both with repair (Sprr2A) and with the development of innate immunity (Siat4c and Relmbeta).
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Affiliation(s)
- Pamela A Knight
- Division of Veterinary Clinical Studies, University of Edinburgh, Roslin, Midlothian, United Kingdom.
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33
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Steinert PM, Parry DAD, Marekov LN. Trichohyalin mechanically strengthens the hair follicle: multiple cross-bridging roles in the inner root shealth. J Biol Chem 2003; 278:41409-19. [PMID: 12853460 DOI: 10.1074/jbc.m302037200] [Citation(s) in RCA: 93] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Trichohyalin is expressed in specialized epithelia that are unusually mechanically strong, such as the inner root sheath cells of the hair follicle. We have previously shown that trichohyalin is sequentially subjected to post-synthetic modifications by peptidylarginine deaminases, which convert many of its arginines to citrullines, and by transglutaminases, which introduce intra- and interprotein chain cross-links. Here we have characterized in detail the proteins to which it becomes cross-linked in vivo in the inner root sheath of the mouse hair follicle. We suggest that it has three principal roles. First, it serves as an interfilamentous matrix protein by becoming cross-linked both to itself and to the head and tail end domains of the inner root sheath keratin intermediate filament chains. A new antibody reveals that arginines of the tail domains of the keratins are modified to citrullines before cross-linking, which clarifies previous studies. Second, trichohyalin serves as a cross-bridging reinforcement protein of the cornified cell envelope of the inner root sheath cells by becoming cross-linked to several known or novel barrier proteins, including involucrin, small proline-rich proteins, repetin, and epiplakin. Third, it coordinates linkage between the keratin filaments and cell envelope to form a seamless continuum. Together, our new data document that trichohyalin is a multi-functional cross-bridging protein that functions in the inner root sheath and perhaps in other specialized epithelial tissues by conferring to and coordinating mechanical strength between their peripheral cell envelope barrier structures and their cytoplasmic keratin filament networks.
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Affiliation(s)
- Peter M Steinert
- Laboratory of Skin Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892-8023, USA
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34
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Bierie B, Nozawa M, Renou JP, Shillingford JM, Morgan F, Oka T, Taketo MM, Cardiff RD, Miyoshi K, Wagner KU, Robinson GW, Hennighausen L. Activation of beta-catenin in prostate epithelium induces hyperplasias and squamous transdifferentiation. Oncogene 2003; 22:3875-87. [PMID: 12813461 DOI: 10.1038/sj.onc.1206426] [Citation(s) in RCA: 101] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The Wnt/beta-catenin signaling pathway is critical for normal mammalian development, the specification of epidermal cells and neoplastic transformation of intestinal epithelium. However, precise molecular information regarding cell-specific responses to beta-catenin signaling has been limited. This question was addressed using a mouse model in which exon 3 of the beta-catenin gene was deleted in several cell types with loxP-mediated recombination utilizing a Cre transgene under control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). The stabilization of beta-catenin in prostate epithelium resulted in hyperplasias and extensive transdifferentiation into epidermal-like structures, which expressed keratins 1 and 6, filaggrin, loricrin and involucrin. The cell-specific loss of NKCC1 protein and reduced nuclear Stat5a is further suggestive of a loss of prostate epithelial characteristics. In addition to the prostate, hyperplasias and squamous metaplasias were detected in epithelia of the epididymis, vas deferens, coagulating gland, preputial gland and salivary gland. However, and in contrast to a recent study, no lesions reminiscent of high-grade prostate intraepithelial neoplasia were detected. Since beta-catenin was activated in several cell types and impinged upon the viability of these mice, it was not possible to evaluate the cumulative effect over more than 3 months. To assess long-term consequences of beta-catenin activation, mutant and control prostate tissues were transplanted into the mammary fat pads of wild-type males. Notably, squamous metaplasias, intra-acinous hyperplasia and possible neoplastic transformation were observed after a total of 18 weeks of beta-catenin stimulation. This suggests that the transdifferentiation into squamous metaplasias is an early response of endoderm-derived cells to beta-catenin, and that the development of intra-acinous hyperplasias or neoplastic foci is a later event.
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Affiliation(s)
- Brian Bierie
- Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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35
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Turksen K, Troy TC. Overexpression of the calcium sensing receptor accelerates epidermal differentiation and permeability barrier formation in vivo. Mech Dev 2003; 120:733-44. [PMID: 12834872 DOI: 10.1016/s0925-4773(03)00045-5] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
The calcium sensing receptor (CaSR) has emerged as an important mediator of a wide range of Ca(2+)-dependent physiological responses (Ca(2+) signaling) in various tissues. To explore the role of CaSR in the epidermis, we utilised the keratin 14 promoter to express CaSR cDNA constitutively in the basal cells of the stratified squamous epithelium of transgenic mice. Analysis of the transgenic mice revealed that a sensitized response to CaSR signaling accelerates the epidermal differentiation program with the precocious formation of the epidermal permeability barrier (EPB) during development and an accelerated hair growth at birth. Our observations indicate that overexpression of CaSR in the undifferentiated basal cells leads to changes in the differentiation program of the transgenic epidermis, including the stimulation of keratins 1 and 6 as well as the overexpression of several markers of terminal differentiation such as filaggrin, loricrin and involucrin. Our data suggest that the observed modifications in the differentiation pathway are a consequence of a CaSR-induced enhancement of Ca(2+) signaling involving cross-talk with other signaling pathways (e.g. EGF and Wnt/Ca(2+)). These studies provide new insights into the role of CaSR in epidermal differentiation including EPB development and hair follicle morphogenesis.
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Affiliation(s)
- Kursad Turksen
- Ottawa Health Research Institute, 725 Parkdale Ave., Ontario, K1Y 4E9 Ottawa, Canada.
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36
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Pearton DJ, Dale BA, Presland RB. Functional analysis of the profilaggrin N-terminal peptide: identification of domains that regulate nuclear and cytoplasmic distribution. J Invest Dermatol 2002; 119:661-9. [PMID: 12230510 DOI: 10.1046/j.1523-1747.2002.01831.x] [Citation(s) in RCA: 68] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Profilaggrin is expressed in the differentiating granular layer of epidermis and other stratified epithelia, where it forms a major component of cytoplasmic keratohyalin granules. It consists of two distinct domains, an N-terminal S100-like Ca2+- binding domain containing two EF-hands and multiple filaggrin units that aggregate keratin filaments in the stratum corneum. Here, we report structure-function studies of the N-terminal peptide from mouse, human, and rat profilaggrin. The profilaggrin N- terminal peptides of all species contain two S100-like EF-hands, bipartite nuclear localization sequences, and proprotein convertase cleavage sites. The nuclear localization signals in human and mouse profilaggrin were shown to be functional by transfection of epithelial cells and depended on the absence of filaggrin sequences. The nuclear localization of the processed (free) N-terminal peptide of human profilaggrin is consistent with immunolocalization findings in normal human skin and in parakeratotic skin disorders, which exhibit nuclear staining of granular and/or cornified layers. The mouse profilaggrin N-terminus undergoes proteolytic processing in two steps, first releasing an N-terminal peptide containing some filaggrin sequence and finally the free N-terminus of 28-30 kDa; these peptides have cytoplasmic and nuclear distributions, respectively, when expressed in transfected cells. The N-terminal processing may occur prior to or simultaneously with the proteolytic processing of the polyfilaggrin domain. The nuclear accumulation of the profilaggrin N-terminal peptide in epidermis and in transfected cells strongly suggests a calcium-dependent nuclear function for the profilaggrin N-terminus during epidermal terminal differentia tion when the free N-terminus is released from profilaggrin by specific proteolysis.
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Affiliation(s)
- David J Pearton
- Department of Oral Biology, University of Washington, Seattle, Washington 98195-7132, USA
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37
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Holland P, Willis C, Kanaly S, Glaccum M, Warren A, Charrier K, Murison J, Derry J, Virca G, Bird T, Peschon J. RIP4 is an ankyrin repeat-containing kinase essential for keratinocyte differentiation. Curr Biol 2002; 12:1424-8. [PMID: 12194825 DOI: 10.1016/s0960-9822(02)01075-8] [Citation(s) in RCA: 85] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
The epidermis is a stratified, continually renewing epithelium dependent on a balance among cell proliferation, differentiation, and death for homeostasis. In normal epidermis, a mitotically active basal layer gives rise to terminally differentiating keratinocytes that migrate outward and are ultimately sloughed from the skin surface as enucleated squames. Although many proteins are known to function in maintaining epidermal homeostasis, the molecular coordination of these events is poorly understood. RIP4 is a novel RIP (receptor-interacting protein) family kinase with ankyrin repeats cloned from a keratinocyte cDNA library. RIP4 deficiency in mice results in perinatal lethality associated with abnormal epidermal differentiation. The phenotype of RIP4(-/-) mice in part resembles that of mice lacking IKKalpha, a component of a complex that regulates NF-kappaB. Despite the similar keratinocyte defects in RIP4- and IKKalpha-deficient mice, these kinases function in distinct pathways. RIP4 functions cell autonomously within the keratinocyte lineage. Unlike IKKalpha, RIP4-deficient skin fails to fully differentiate when grafted onto a normal host. Instead, abnormal hair follicle development and epidermal dysplasia, indicative of progression into a more pathologic state, are observed. Thus, RIP4 is a critical component of a novel pathway that controls keratinocyte differentiation.
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Affiliation(s)
- Pamela Holland
- Immunex Corporation, 51 University Street, Seattle, WA 98101, USA.
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38
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Turksen K, Troy TC. Permeability barrier dysfunction in transgenic mice overexpressing claudin 6. Development 2002; 129:1775-84. [PMID: 11923212 DOI: 10.1242/dev.129.7.1775] [Citation(s) in RCA: 158] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
A defective epidermal permeability barrier (EPB) in premature birth remains a leading cause of neonatal death as a result of its associated complications, which include poor temperature stability, infection by micro-organisms through the skin, and the outflow of water. Despite its importance in survival, the mechanisms involved in the formation and maintenance of the EPB are not well understood. To address the possibility that claudins, a new superfamily of tight junctional molecules, are involved, we engineered transgenic mice with claudin 6 (Cldn6) overexpressed via the involucrin (Inv) promoter. Interestingly, the Inv-Cldn6 transgenic animals die within 2 days of birth, apparently due to the lack of an intact EPB as evidenced by increased water loss and the penetration of X-gal through the skin. Barrier dysfunction was manifested biochemically by the aberrant expression of late epidermal differentiation markers, including K1, filaggrin, loricrin, transglutaminase 3, involucrin, repetin, members of the SPRR family and the transcriptional regulator Klf4. The overall claudin profile of the epidermis was also modified. Our data suggest that repetin and SPRR1A and 2A are downregulated in response to the downregulation of Klf4 in the transgenic animals, which would contribute to decreased protein crossbridging leading to fragile, defective cornified envelopes. These results provide new insights into the role of claudin 6 in epithelial differentiation and EPB formation. In addition, the epidermal phenotype of these transgenic mice, which is very reminiscent of that in pre-term infant skin, suggest that they will be an important and novel model for studies on human premature EPB-related morbidity.
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Affiliation(s)
- Kursad Turksen
- Ottawa Health Research Institute, Ottawa Hospital, Ottawa, Ontario, Canada.
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39
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Jarnik M, de Viragh PA, Schärer E, Bundman D, Simon MN, Roop DR, Steven AC. Quasi-normal cornified cell envelopes in loricrin knockout mice imply the existence of a loricrin backup system. J Invest Dermatol 2002; 118:102-9. [PMID: 11851882 DOI: 10.1046/j.0022-202x.2001.01661.x] [Citation(s) in RCA: 39] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
The cornified cell envelope, a lipoprotein layer that assembles at the surface of terminally differentiated keratinocytes, is a resilient structure on account of covalent crosslinking of its constituent proteins, principally loricrin, which accounts for up to 60%-80% of total protein. Despite the importance of the cell envelope as a protective barrier, knocking out the loricrin gene in mice results in only mild syndromes. We have investigated the epidermis and forestomach epithelium of these mice by electron microscopy. In both tissues, corneocytes have normal-looking cell envelopes, despite the absence of loricrin, which was confirmed by immunolabeling, and the absence of the distinctive loricrin-containing keratohyalin granules (L-granules). Isolated cell envelopes were normal in thickness (approximately 15 nm) and mass per unit area (approximately 7.3 kDa per nm2); however, metal shadowing revealed an altered substructure on their cytoplasmic surface. Their amino acid compositions indicate altered protein compositions. Analysis of these data implies that the epidermal cell envelopes have elevated levels of the small proline-rich proteins, and cell envelopes of both kinds contain other protein(s) that, like loricrin, are rich in glycine and serine. These observations imply that, in the absence of loricrin, the mechanisms that govern cell envelope assembly function normally but employ different building-blocks.
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Affiliation(s)
- Michal Jarnik
- Laboratory of Structural Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892-8025, USA
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40
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Itin PH, Sarasin A, Pittelkow MR. Trichothiodystrophy: update on the sulfur-deficient brittle hair syndromes. J Am Acad Dermatol 2001; 44:891-920; quiz 921-4. [PMID: 11369901 DOI: 10.1067/mjd.2001.114294] [Citation(s) in RCA: 147] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Trichothiodystrophy (TTD) refers to a heterogeneous group of autosomal recessive disorders that share the distinctive features of short, brittle hair and an abnormally low sulfur content. Within the spectrum of the TTD syndromes are numerous interrelated neuroectodermal disorders. The TTD syndromes show defective synthesis of high-sulfur matrix proteins. Abnormalities in excision repair of ultraviolet (UV)-damaged DNA are recognized in about half of the patients. Three distinct autosomal recessive syndromes are associated with nucleotide excision repair (NER) defects: the photosensitive form of TTD, xeroderma pigmentosum, and Cockayne syndrome. The unifying feature of these conditions is exaggerated sensitivity to sunlight and UV radiation. In contrast to patients with xeroderma pigmentosum, no increase of skin cancers in patients with TTD has been observed. Genetically, 3 complementation groups have been characterized among photosensitive patients with TTD. Most patients exhibit mutations on the two alleles of the XPD gene. Rarely, mutated XPB gene or an unidentified TTD-A gene may result in TTD. In UV-sensitive TTD, the TFIIH transcription factor containing XPB and XPD helicase activities necessary for both transcription initiation and DNA repair is damaged. Beyond deficiency in the NER pathway, it is hypothesized that basal transcription may be altered leading to decreased transcription of specific genes. Depressed RNA synthesis may account for some clinical features, such as growth retardation, neurologic abnormalities, and brittle hair and nails. Therefore the attenuated expression of some proteins in differentiated cells is most likely explained by a mechanism distinct from DNA repair deficiency. The first transgenic mouse models for NER deficiencies have been generated. The TTD mouse as well as related cell models will provide important tools to understand the complex relationships between defects in DNA repair, low-sulfur hair shaft disorders, and the genotype-phenotype correlates for this constellation of inherited disorders, including the lack of predisposition to cancer in patients with TTD.
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Affiliation(s)
- P H Itin
- Department of Dermatology, University of Basel and Kantonsspital Aarau, Switzerland
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41
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Cabral A, Voskamp P, Cleton-Jansen AM, South A, Nizetic D, Backendorf C. Structural organization and regulation of the small proline-rich family of cornified envelope precursors suggest a role in adaptive barrier function. J Biol Chem 2001; 276:19231-7. [PMID: 11279051 DOI: 10.1074/jbc.m100336200] [Citation(s) in RCA: 128] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The protective barrier provided by stratified squamous epithelia relies on the cornified cell envelope (CE), a structure synthesized at late stages of keratinocyte differentiation. It is composed of structural proteins, including involucrin, loricrin, and the small proline-rich (SPRR) proteins, all encoded by genes localized at human chromosome 1q21. The genetic characterization of the SPRR locus reveals that the various members of this multigene family can be classified into two distinct groups with separate evolutionary histories. Whereas group 1 genes have diverged in protein structure and are composed of three different classes (SPRR1 (2x), SPRR3, and SPRR4), an active process of gene conversion has counteracted diversification of the protein sequences of group 2 genes (SPRR2 class, seven genes). Contrasting with this homogenization process, all individual members of the SPRR gene family show specific in vivo and in vitro expression patterns and react selectively to UV irradiation. Apparently, creation of regulatory rather than structural diversity has been the driving force behind the evolution of the SPRR gene family. Differential regulation of highly homologous genes underlines the importance of SPRR protein dosage in providing optimal barrier function to different epithelia, while allowing adaptation to diverse external insults.
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Affiliation(s)
- A Cabral
- Department of Molecular Genetics, Leiden Institute of Chemistry, P. O. Box 9502, 2300 RA Leiden, The Netherlands
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42
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Medico E, Gambarotta G, Gentile A, Comoglio PM, Soriano P. A gene trap vector system for identifying transcriptionally responsive genes. Nat Biotechnol 2001; 19:579-82. [PMID: 11385465 DOI: 10.1038/89343] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
We present a method for fast and efficient trapping of genes whose transcription is regulated by exogenous stimuli. We constructed a promoterless retroviral vector transducing a green fluorescent protein-nitroreductase (GFNR) fusion protein downstream from a splice acceptor site. Flow cytometric analysis of the infected population allows identification and sorting of cells in which the trap is integrated downstream from an active promoter. Conversely, the nitroreductase (NTR) moiety allows pharmacological selection against constitutive GFNR expression. Using hepatocyte growth factor (HGF) stimulation of liver cells combined with either positive or negative selection, we recovered cell populations carrying traps in induced or suppressed genes, respectively. Several distinct responsive clones were isolated, and regulated expression of the trapped gene was confirmed at the RNA level. Positive and negative selection can be calibrated to recover traps in genes showing different levels of basal expression or transcriptional regulation. The flexibility and efficiency of the GFNR-based trap screening procedure make it suitable for wide surveys of transcriptionally regulated genes.
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Affiliation(s)
- E Medico
- Institute for Cancer Research and Treatment, University of Torino School of Medicine, 10060 Candiolo, Italy.
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43
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Presland RB, Dale BA. Epithelial structural proteins of the skin and oral cavity: function in health and disease. CRITICAL REVIEWS IN ORAL BIOLOGY AND MEDICINE : AN OFFICIAL PUBLICATION OF THE AMERICAN ASSOCIATION OF ORAL BIOLOGISTS 2001; 11:383-408. [PMID: 11132762 DOI: 10.1177/10454411000110040101] [Citation(s) in RCA: 297] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Epithelial tissues function to protect the organism from physical, chemical, and microbial damage and are essential for survival. To perform this role, epithelial keratinocytes undergo a well-defined differentiation program that results in the expression of structural proteins which maintain the integrity of epithelial tissues and function as a protective barrier. This review focuses on structural proteins of the epidermis and oral mucosa. Keratin proteins comprise the predominant cytoskeletal component of these epithelia. Keratin filaments are attached to the plasma membrane via desmosomes, and together these structural components form a three-dimensional array within the cytoplasm of epithelial cells and tissues. Desmosomes contain two types of transmembrane proteins, the desmogleins and desmocollins, that are members of the cadherin family. The desmosomal cadherins are linked to the keratin cytoskeleton via several cytoplasmic plaque proteins, including desmoplakin and plakoglobin (gamma-catenin). Epidermal and oral keratinocytes express additional differentiation markers, including filaggrin and trichohyalin, that associate with the keratin cytoskeleton during terminal differentiation, and proteins such as loricrin, small proline-rich proteins, and involucrin, that are cross-linked into the cornified envelope by transglutaminase enzymes. The importance of these cellular structures is highlighted by the large numbers of genetic and acquired (autoimmune) human disorders that involve mutations in, or autoantibodies to, keratins and desmosomal and cornified envelope proteins. While much progress has been made in the identification of the structural proteins and enzymes involved in epithelial differentiation, regulation of this process is less clear. Both calcium and retinoids influence epithelial differentiation by altering the transcription of target genes and by regulating activity of enzymes critical in epithelial differentiation, such as transglutaminases, proteinases, and protein kinases. These studies have furthered our understanding of how epithelial tissue and cell integrity is maintained and provide a basis for the future treatment of skin and oral disorders by gene therapy and other novel therapeutics.
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Affiliation(s)
- R B Presland
- Department of Oral Biology, University of Washington, Seattle 98195, USA.
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44
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Stern LE, Erwin CR, Falcone RA, Huang FS, Kemp CJ, Williams JL, Warner BW. cDNA microarray analysis of adapting bowel after intestinal resection. J Pediatr Surg 2001; 36:190-5. [PMID: 11150463 DOI: 10.1053/jpsu.2001.20050] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
BACKGROUND/PURPOSE Studies of the genetic regulation of various physiologic processes have been hampered by methodologies that are limited to the analysis of individual genes. The advent of cDNA microarray technology has permitted the simultaneous screening of numerous genes for alterations in expression. In this study, cDNA microarrays were used to evaluate gene expression changes during the intestinal adaptive response to massive small bowel resection (SBR). METHODS Male ICR mice (n = 20) underwent either a 50% SBR or sham operation and then were given either orogastric epidermal growth factor (EGF, 50 microg/kg/d) or saline. After 3 days, cDNA microarray analysis was performed on mRNA extracted from the remnant ileum. RESULTS From over 8,700 different genes, the array identified 27 genes that were altered 2-fold or greater after SBR. Small proline-rich protein 2 (sprr2), the gene with the greatest expression change (4.9-fold), was further upregulated by EGF. This gene has never been characterized in the intestine or described in intestinal adaptation. CONCLUSIONS cDNA microarray analysis showed enhanced expression of sprr2, a gene not previously known to be involved in the physiology of adaptation after SBR. This technology provides a more rapid and efficient means of dissecting the complex genetic regulation of gut adaptation.
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Affiliation(s)
- L E Stern
- Division of Pediatric Surgery, Children's Hospital Medical Center, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, OH 45229-3039, USA
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Koch PJ, de Viragh PA, Scharer E, Bundman D, Longley MA, Bickenbach J, Kawachi Y, Suga Y, Zhou Z, Huber M, Hohl D, Kartasova T, Jarnik M, Steven AC, Roop DR. Lessons from loricrin-deficient mice: compensatory mechanisms maintaining skin barrier function in the absence of a major cornified envelope protein. J Cell Biol 2000; 151:389-400. [PMID: 11038185 PMCID: PMC2192642 DOI: 10.1083/jcb.151.2.389] [Citation(s) in RCA: 217] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
The epidermal cornified cell envelope (CE) is a complex protein-lipid composite that replaces the plasma membrane of terminally differentiated keratinocytes. This lamellar structure is essential for the barrier function of the skin and has the ability to prevent the loss of water and ions and to protect from environmental hazards. The major protein of the epidermal CE is loricrin, contributing approximately 70% by mass. We have generated mice that are deficient for this protein. These mice showed a delay in the formation of the skin barrier in embryonic development. At birth, homozygous mutant mice weighed less than control littermates and showed skin abnormalities, such as congenital erythroderma with a shiny, translucent skin. Tape stripping experiments suggested that the stratum corneum stability was reduced in newborn Lor(-/-) mice compared with wild-type controls. Isolated mutant CEs were more easily fragmented by sonication in vitro, indicating a greater susceptibility to mechanical stress. Nevertheless, we did not detect impaired epidermal barrier function in these mice. Surprisingly, the skin phenotype disappeared 4-5 d after birth. At least one of the compensatory mechanisms preventing a more severe skin phenotype in newborn Lor(-/-) mice is an increase in the expression of other CE components, such as SPRRP2D and SPRRP2H, members of the family of "small proline rich proteins", and repetin, a member of the "fused gene" subgroup of the S100 gene family.
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Affiliation(s)
- P J Koch
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
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De Heller-Milev M, Huber M, Panizzon R, Hohl D. Expression of small proline rich proteins in neoplastic and inflammatory skin diseases. Br J Dermatol 2000; 143:733-40. [PMID: 11069449 DOI: 10.1046/j.1365-2133.2000.03768.x] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
BACKGROUND The formation of the cornified cell envelope (CE) during the late stages of epidermal differentiation is essential for epidermal barrier function and protects the body against environmental attack and water loss. Formation of the CE involves the replacement of the plasma membrane by cross-linkage of precursor proteins such as involucrin, small proline rich proteins (SPRR) and loricrin. In normal epidermis, SPRR1 is restricted to appendages, SPRR2 is also expressed in interfollicular areas, while SPRR3 is completely absent; the latter is most abundant in oral epithelium. This differential expression indicates an important part for SPRRs in specific barrier requirements, and reflects their importance in the biomechanical properties of the CE. OBJECTIVES We report here on the expression of SPRR1, SPRR2 and SPRR3 in a wide range of cutaneous neoplastic and inflammatory diseases. METHODS We used immunohistochemistry; in addition, Northern blot analysis of malignant tumours was performed. RESULTS Increased suprabasal expression of SPRR1 and SPRR2, but no SPRR3 expression, was noted in inflammatory dermatoses with orthokeratotic and parakeratotic squamous differentiation. By contrast, differentiating epidermal tumours such as Bowen's disease, keratoacanthoma and squamous cell carcinoma expressed SPRR3. CONCLUSIONS As SPRRs were originally cloned on the basis of their expression in ultraviolet light-irradiated keratinocytes, the expression of SPRR3 in actinic lesions is of interest, and might serve as a diagnostic tool.
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Affiliation(s)
- M De Heller-Milev
- Department of Dermatology, CHUV/DHURDV, CH-1011 Lausanne, Switzerland
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Lee CH, Marekov LN, Kim S, Brahim JS, Park MH, Steinert PM. Small proline-rich protein 1 is the major component of the cell envelope of normal human oral keratinocytes. FEBS Lett 2000; 477:268-72. [PMID: 10908733 DOI: 10.1016/s0014-5793(00)01806-8] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Oral keratinocytes of buccal and gingival tissues undergo a terminal differentiation program to form a protective epithelial barrier as non-keratinized or parakeratinized stratified cells. We have examined the protein composition of cell envelopes (CEs) from normal human buccal and gingival tissues as well as keratinocytes from normal human gingival cells grown in culture. Biochemical and sequencing analyses reveal that the CEs contain 60-70% small proline-rich protein 1a/b (SPR1a/b), together with smaller amounts of involucrin, annexin I and several other known CE proteins. The data imply a specialized role for SPR1 proteins in the unique barrier function requirements of oral epithelia.
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Affiliation(s)
- C H Lee
- Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, MD 20892-7252, USA
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Abstract
Involucrin, loricrin and the small proline-rich proteins (SPRRs) are precursors of the cornified envelope of terminally differentiated keratinocytes. The genes for these proteins are closely linked on mouse chromosome 3. Each of the proteins is encoded by a single exon and is largely composed of a segment of short tandem repeats. No size polymorphism of either loricrin or the SPRRs was observed. In contrast, involucrin was found in at least eight polymorphic forms of different size with molecular weights ranging from 51 to 82kDa. Two classes of involucrin alleles were identified. Size polymorphism of involucrin has resulted from the recent expansion of the segment of repeats in one class of alleles, but not in the other. In expanding alleles, repeats were added at a precise location within the segment of repeats, in a 5'-to-3' direction. A study of a large number of allele-specific markers, located on both sides of the site of repeat addition, revealed no evidence for recombination between any of the alleles examined. Expansion of the segment of repeats of the gene for mouse involucrin must result from an intra-allelic process controlled by a cis-acting element, active in one class of alleles, and inactive in the other.
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Affiliation(s)
- B Delhomme
- CNRS-UPR 2228. Régulation de la Transcription et Maladies Génétiques, Université René Descartes, UFR Biomédicale, 45 rue des Saints-Pères, 75270 cedex 06, Paris, France
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Peters JM, Lee SS, Li W, Ward JM, Gavrilova O, Everett C, Reitman ML, Hudson LD, Gonzalez FJ. Growth, adipose, brain, and skin alterations resulting from targeted disruption of the mouse peroxisome proliferator-activated receptor beta(delta). Mol Cell Biol 2000; 20:5119-28. [PMID: 10866668 PMCID: PMC85961 DOI: 10.1128/mcb.20.14.5119-5128.2000] [Citation(s) in RCA: 540] [Impact Index Per Article: 21.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
To determine the physiological roles of peroxisome proliferator-activated receptor beta (PPARbeta), null mice were constructed by targeted disruption of the ligand binding domain of the murine PPARbeta gene. Homozygous PPARbeta-null term fetuses were smaller than controls, and this phenotype persisted postnatally. Gonadal adipose stores were smaller, and constitutive mRNA levels of CD36 were higher, in PPARbeta-null mice than in controls. In the brain, myelination of the corpus callosum was altered in PPARbeta-null mice. PPARbeta was not required for induction of mRNAs involved in epidermal differentiation induced by O-tetradecanoylphorbol-13-acetate (TPA). The hyperplastic response observed in the epidermis after TPA application was significantly greater in the PPARbeta-null mice than in controls. Inflammation induced by TPA in the skin was lower in wild-type mice fed sulindac than in similarly treated PPARbeta-null mice. These results are the first to provide in vivo evidence of significant roles for PPARbeta in development, myelination of the corpus callosum, lipid metabolism, and epidermal cell proliferation.
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Affiliation(s)
- J M Peters
- Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
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Candi E, Paci M, Oddi S, Paradisi A, Guerrieri P, Melino G. Ordered structure acquisition by the N- and C-terminal domains of the small proline-rich 3 protein. J Cell Biochem 2000; 77:179-85. [PMID: 10723085 DOI: 10.1002/(sici)1097-4644(20000501)77:2<179::aid-jcb2>3.0.co;2-h] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
The cell envelope (CE) is a vital structure for barrier function in terminally differentiated dead stratified squamous epithelia. It is assembled by transglutaminase (TGase) cross-linking of several proteins, including hSPR3 in certain specialized epithelia normally subjected to mechanical trauma. Biochemical studies show that hSPR3 serves as a complete substrate for TGase1, TGase2, and TGase3. Multiple adjacent glutamines and lysines of only head-and-tail domain sequences are used by each enzyme for cross-linking. Structural data suggest that the hSPR3 central repeats, as well as hSPR1 and hSPR 2, are highly flexible and mobile; thus, the TGases might not be able to recognize the residues localized on the repeats as adequate substrate. To investigate this hypothesis further and to complete the structural investigation of hSPR3, we performed circular dichroism (CD) studies on peptides corresponding to the N- and C-terminal domain. CD spectra have also been carried out in the presence of different concentrations of the structure-promoting agent cosolvent trifluoroethanol (TFE), which mimics a partial hydrophobic environment found in vivo in or next to the membrane. In fact, this agent increases the dielectric constant of water proportionally, depending on its concentration, and confers structuring properties to the solution, to peptides and proteins that have a structuring propensity. The results indicate that in both the N-terminal and C-terminal, peptides acquire a more ordered structure as a function of the TFE concentration in water. This ability of both N- and C-terminal domain to acquire a more stable ordered conformation might be relevant for SPR3 to act as substrate of TGases. Indeed, only the N- and C-terminus is cross-linked by TGase1 and 3.
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Affiliation(s)
- E Candi
- Laboratory of Biochemistry, Istituto Dermopatico dell'Immacolata, Department of Experimental Medicine, University of Tor Vergata, 00133 Rome, Italy
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