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Role of syntaxin3 an apical polarity protein in poorly polarized keratinocytes: regulation of asymmetric barrier formations in the skin epidermis

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Abstract

The skin epidermis exhibits an asymmetric structure composed of multilayered keratinocytes and those in the outer layers form two-way physical barriers, cornified cell envelope (CCE), and tight junctions (TJs). While undifferentiated keratinocytes in the basal layer continuously deliver daughter cells outward, which undergo successive differentiation with losing their polarized characteristics, they retain the expression of several polarity proteins. In the present study, we revealed that the t-SNARE protein syntaxin3, a critical element for the formation of the apical compartment in simple epithelial cells, is required to confer the ability to organize the physical barriers on “poorly polarized” keratinocytes in epidermal outer layers. HaCaT keratinocytes with genetic ablation of syntaxin3 readily succumbed to hydrogen peroxide-induced cell death. Additionally, they lost the ability to organize TJ and CCE structures, accompanied by notable downregulation of transglutaminase1 and caspase14 (a cornification regulator) expression. These syntaxin3-knockout cells appeared to restore oxidative stress tolerance and functional TJ formation ability, in response to the inducible re-expression of exogenous syntaxin3. While plausible mechanisms underlying these phenomena remain unclear, syntaxin3, an apical polarity protein in the simple epithelia, has emerged as a potentially crucial element for barrier formation in poorly polarized keratinocytes in polarized epidermal tissue.

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Data availability

The data that support the finding of this study are available from the corresponding author upon request.

References

  • Ali NJA, Dias Gomes M, Bauer R, Brodesser S, Niemann C, Iden S (2016) Essential role of polarity protein Par3 for epidermal homeostasis through regulation of barrier function, Keratinocyte Differentiation, and Stem Cell Maintenance. J Invest Dermatol 136:2406–2416

    Article  CAS  PubMed  Google Scholar 

  • Aono S, Hirai Y (2008) Phosphorylation of claudin-4 is required for tight junction formation in a human keratinocyte cell line. Exp Cell Res 314:3326–3339

    Article  CAS  PubMed  Google Scholar 

  • Bennett MK, Garcia-Arraras JE, Elferink LA, Peterson K, Fleming AM, Hazuka CD, Scheller RH (1993) The syntaxin family of vesicular transport receptors. Cell 74:863–873

    Article  CAS  PubMed  Google Scholar 

  • Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE (1988) Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 106:761–771

    Article  CAS  PubMed  Google Scholar 

  • Brunger AT (2005) Structure and function of SNARE and SNARE-interacting proteins. Q Rev Biophys 38:1–47

    Article  CAS  PubMed  Google Scholar 

  • Candi E, Schmidt R, Melino G (2005) The cornified envelope: a model of cell death in the skin. Nat Rev Mol Cell Biol 6:328–340

    Article  CAS  PubMed  Google Scholar 

  • Costanzo A, Fausti F, Spallone G, Moretti F, Narcisi A, Botti E (2015) Programmed cell death in the skin. Int J Dev Biol 59:73–78

    Article  CAS  PubMed  Google Scholar 

  • Denda M, Fujiwara S, Hibino T (2006) Expression of voltage-gated calcium channel subunit alpha1C in epidermal keratinocytes and effects of agonist and antagonists of the channel on skin barrier homeostasis. Exp Dermatol 15:455–460

    Article  CAS  PubMed  Google Scholar 

  • El-Chami C, Haslam IS, Steward MC (2020) O’Neill CA Author correction: organic osmolytes preserve the function of the developing tight junction in ultraviolet B-irradiated rat epidermal keratinocytes. Sci Rep 10(1):8639. https://doi.org/10.1038/s41598-020-65508-w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • El-Chami C, Haslam IS, Steward MC, O’Neill CA (2018) Organic osmolytes preserve the function of the developing tight junction in ultraviolet B-irradiated rat epidermal keratinocytes. Sci Rep 8:018–22533

    Article  Google Scholar 

  • Elsholz F, Harteneck C, Muller W, Friedland K (2014) Calcium–a central regulator of keratinocyte differentiation in health and disease. Eur J Dermatol 24:650–661

    Article  CAS  PubMed  Google Scholar 

  • Giovannone AJ, Winterstein C, Bhattaram P, Reales E, Low SH, Baggs JE, Xu M, Lalli MA, Hogenesch JB, Weimbs T (2018) Soluble syntaxin 3 functions as a transcriptional regulator. J Biol Chem 293:5478–5491

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gossen M, Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89:5547–5551

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Heiskala M, Peterson PA, Yang Y (2001) The roles of claudin superfamily proteins in paracellular transport. Traffic (copenhagen, Denmark) 2:93–98

    Article  CAS  PubMed  Google Scholar 

  • Helfrich I, Schmitz A, Zigrino P, Michels C, Haase I, le Bivic A, Leitges M, Niessen CM (2007) Role of aPKC isoforms and their binding partners Par3 and Par6 in epidermal barrier formation. J Invest Dermatol 127:782–791

    Article  CAS  PubMed  Google Scholar 

  • Hirai Y, Nelson CM, Yamazaki K, Takebe K, Przybylo J, Madden B, Radisky DC (2007) Non-classical export of epimorphin and its adhesion to {alpha}v-integrin in regulation of epithelial morphogenesis. J Cell Sci 120:2032–2043

    Article  CAS  PubMed  Google Scholar 

  • Hirai Y, Nose A, Kobayashi S, Takeichi M (1989) Expression and role of E- and P-cadherin adhesion molecules in embryonic histogenesis. II. Skin morphogenesis. Development (cambridge, England) 105:271–277

    Article  CAS  PubMed  Google Scholar 

  • Hitomi K (2005) Transglutaminases in skin epidermis. Eur J Dermatol 15:313–319

    CAS  PubMed  Google Scholar 

  • Hori H, Kotani A, Abe J, Matsuguchi S, Hirai Y (2022) Extracellular epimorphin impairs expression and processing of profilaggrin in HaCaT keratinocytes. Cytotechnology

  • Iizuka M, Sasaki K, Hirai Y, Shindo K, Konno S, Itou H, Ohshima S, Horie Y, Watanabe S (2007) Morphogenic protein epimorphin protects intestinal epithelial cells from oxidative stress by the activation of EGF receptor and MEK/ERK, PI3 kinase/Akt signals. Am J Physiol Gastrointest Liver Physiol 292:G39-52

    Article  CAS  PubMed  Google Scholar 

  • Ishida-Yamamoto A, Kishibe M, Murakami M, Honma M, Takahashi H, Iizuka H (2012) Lamellar granule secretion starts before the establishment of tight junction barrier for paracellular tracers in mammalian epidermis. PLoS ONE 7:6

    Article  Google Scholar 

  • Ishida-Yamamoto A, Tanaka H, Nakane H, Takahashi H, Hashimoto Y, Iizuka H (1999) Programmed cell death in normal epidermis and loricrin keratoderma. Multiple functions of profilaggrin in keratinization. The Journal of investigative dermatology Symposium proceedings / the Society for Investigative Dermatology, Inc Eur Soc Dermatol Res 4:145–149

  • Kadono N, Miyazaki T, Okugawa Y, Nakajima K, Hirai Y (2012) The impact of extracellular syntaxin4 on HaCaT keratinocyte behavior. Biochem Biophys Res Commun 417:1200–1205

    Article  CAS  PubMed  Google Scholar 

  • Kim SY, Bae CD (1998) Calpain inhibitors reduce the cornified cell envelope formation by inhibiting proteolytic processing of transglutaminase 1. Exp Mol Med 30:257–262

    Article  CAS  PubMed  Google Scholar 

  • Kirschner N, Brandner JM (2012) Barriers and more: functions of tight junction proteins in the skin. Ann N Y Acad Sci 1257:158–166

    Article  CAS  PubMed  Google Scholar 

  • Lanzafame M, Botta E, Teson M, Fortugno P, Zambruno G, Stefanini M (2015) Orioli D Reference genes for gene expression analysis in proliferating and differentiating human keratinocytes. Exp Dermatol 24(4):314–316. https://doi.org/10.1111/exd.12657

    Article  CAS  PubMed  Google Scholar 

  • Lechler T, Fuchs E (2005) Asymmetric cell divisions promote stratification and differentiation of mammalian skin. Nature 437:275–280

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lehmann B (1997) HaCaT cell line as a model system for vitamin D3 metabolism in human skin. J Invest Dermatol 108:78–82

    Article  CAS  PubMed  Google Scholar 

  • Liu H, Tuchinda P, Fishelevich R, Harberts E, Gaspari AA (2014) Human in vitro skin organ culture as a model system for evaluating DNA repair. J Dermatol Sci 74:236–241

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Madison KC (2003) Barrier function of the skin: “la raison d’être” of the epidermis. J Invest Dermatol 121:231–241

    Article  CAS  PubMed  Google Scholar 

  • Micallef L, Belaubre F, Pinon A, Jayat-Vignoles C, Delage C, Charveron M, Simon A (2009) Effects of extracellular calcium on the growth-differentiation switch in immortalized keratinocyte HaCaT cells compared with normal human keratinocytes. Exp Dermatol 18:143–151

    Article  CAS  PubMed  Google Scholar 

  • Michels C, Aghdam SY, Niessen CM (2009) Cadherin-mediated regulation of tight junctions in stratifying epithelia. Ann New York Acad Sci

    Book  Google Scholar 

  • Miyazaki T, Kadono N, Konishi Y, Hagiwara N, Maekubo K, Hirai Y (2013) Effluent syntaxin3 from dying cells affords protection against apoptosis in epidermal keratinocytes. Exp Dermatol 22:845–847

    Article  CAS  PubMed  Google Scholar 

  • Morita K, Miyachi Y (2003) Tight junctions in the skin. J Dermatol Sci 31:81–89

    Article  PubMed  Google Scholar 

  • Muroyama A, Lechler T (2012) Polarity and stratification of the epidermis. Semin Cell Dev Biol 23:890–896

    Article  PubMed  PubMed Central  Google Scholar 

  • Natsuga K (2014) Epidermal barriers. Cold Spring Harbor Perspectives in Medicine 4

  • O’Neill CA, Garrod D (2011) Tight junction proteins and the epidermis. Exp Dermatol 20:88–91

    Article  CAS  PubMed  Google Scholar 

  • Okugawa Y, Hirai Y (2008) Overexpression of extracellular epimorphin leads to impaired epidermal differentiation in HaCaT keratinocytes. J Invest Dermatol 128:1884–1893

    Article  CAS  PubMed  Google Scholar 

  • Okugawa Y, Hirai Y (2013) Extracellular epimorphin modulates epidermal differentiation signals mediated by epidermal growth factor receptor. J Dermatol Sci 69:236–242

    Article  CAS  PubMed  Google Scholar 

  • Parenteau NL, Bilbo P, Nolte CJ, Mason VS, Rosenberg M (1992) The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function. Cytotechnology 9:163–171

    Article  CAS  PubMed  Google Scholar 

  • Pistritto G, Jost M, Srinivasula SM, Baffa R, Poyet JL, Kari C, Lazebnik Y, Rodeck U, Alnemri ES (2002) Expression and transcriptional regulation of caspase-14 in simple and complex epithelia. Cell Death Differ 9:995–1006

    Article  CAS  PubMed  Google Scholar 

  • Proksch E, Brandner JM, Jensen JM (2008) The skin: an indispensable barrier. Exp Dermatol 17:1063–1072

    Article  PubMed  Google Scholar 

  • Risselada HJ, Grubmüller H (2012) How SNARE molecules mediate membrane fusion: recent insights from molecular simulations. Curr Opin Struct Biol 22:187–196

    Article  CAS  PubMed  Google Scholar 

  • Sanchez E, Gonzalez EA, Moreno DS, Cardenas RA, Ramos MA, Davalos AJ, Manllo J, Rodarte AI, Petrova Y, Moreira DC, Chavez MA, Tortoriello A, Lara A, Gutierrez BA, Burns AR, Heidelberger R, Adachi R (2019) Syntaxin 3, but not syntaxin 4, is required for mast cell-regulated exocytosis, where it plays a primary role mediating compound exocytosis. J Biol Chem 294:3012–3023

    Article  CAS  PubMed  Google Scholar 

  • Sapra B, Jindal M, Tiwary AK (2012) Tight junctions in skin: new perspectives. Ther Deliv 3:1297–1327

    Article  CAS  PubMed  Google Scholar 

  • Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Seo MD, Kang TJ, Lee CH, Lee AY, Noh M (2012) HaCaT keratinocytes and primary epidermal keratinocytes have different transcriptional profiles of cornified envelope-associated genes to T helper cell cytokines. Biomol Ther 20:171–176

    Article  CAS  Google Scholar 

  • Sharma N, Low SH, Misra S, Pallavi B, Weimbs T (2006) Apical targeting of syntaxin 3 is essential for epithelial cell polarity. J Cell Biol 173:937–948

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shono M, Yoshioka R, Chatani Y, Hirai Y (2013) Ectopic expression of syntaxin3 affects behaviors of B16 melanoma by controlling actin dynamics. Cell Struct Funct 38:97–107

    Article  CAS  PubMed  Google Scholar 

  • Simpson CL, Patel DM, Green KJ (2011) Deconstructing the skin: cytoarchitectural determinants of epidermal morphogenesis. Nat Rev Mol Cell Biol 12:565–580

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tanaka F, Uda M, Hirose Y, Hirai Y (2020) Restoration of calcium-induced differentiation potential and tight junction formation in HaCaT keratinocytes by functional attenuation of overexpressed high mobility group box-1 protein. Cytotechnology 72:165–174

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Torkko JM, Manninen A, Schuck S, Simons K (2008) Depletion of apical transport proteins perturbs epithelial cyst formation and ciliogenesis. J Cell Sci 121:1193–1203

    Article  CAS  PubMed  Google Scholar 

  • Van Itallie CM, Anderson JM (2014) Architecture of tight junctions and principles of molecular composition. Semin Cell Dev Biol 36:157–165

    Article  PubMed  Google Scholar 

  • Volksdorf T, Heilmann J, Eming SA, Schawjinski K, Zorn-Kruppa M, Ueck C, Vidal YSS, Windhorst S, Jücker M, Moll I, Brandner JM (2017) Tight junction proteins claudin-1 and occludin are important for cutaneous wound healing. Am J Pathol 187:1301–1312

    Article  CAS  PubMed  Google Scholar 

  • Wang W, Lin C, Lu D, Ning Z, Cox T, Melvin D, Wang X, Bradley A, Liu P (2008) Chromosomal transposition of PiggyBac in mouse embryonic stem cells. Proc Natl Acad Sci USA 105:9290–9295

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yano T, Kanoh H, Tamura A, Tsukita S (2017) Apical cytoskeletons and junctional complexes as a combined system in epithelial cell sheets. Ann N Y Acad Sci 1:32–43

    Article  Google Scholar 

  • Yokouchi M, Atsugi T, Logtestijn MV, Tanaka RJ, Kajimura M, Suematsu M, Furuse M, Amagai M, Kubo A (2016) Epidermal cell turnover across tight junctions based on Kelvin’s tetrakaidecahedron cell shape. Elife 29:19593

    Article  Google Scholar 

  • Yoon TY, Munson M (2018) SNARE complex assembly and disassembly. Current Biology: CB 28:R397–R401

    Article  CAS  PubMed  Google Scholar 

  • Yoshida K, Yokouchi M, Nagao K, Ishii K, Amagai M, Kubo A (2013) Functional tight junction barrier localizes in the second layer of the stratum granulosum of human epidermis. J Dermatol Sci 71:89–99

    Article  PubMed  Google Scholar 

  • Yuki T, Hachiya A, Kusaka A, Sriwiriyanont P, Visscher MO, Morita K, Muto M, Miyachi Y, Sugiyama Y, Inoue S (2011) Characterization of tight junctions and their disruption by UVB in human epidermis and cultured keratinocytes. J Invest Dermatol 131:744–752

    Article  CAS  PubMed  Google Scholar 

  • Zihni C, Mills C, Matter K, Balda MS (2016) Tight junctions: from simple barriers to multifunctional molecular gates. Nat Rev Mol Cell Biol 17:564–580

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Drs. Motomu Manabe and Akira Nagafuchi for HaCaT keratinocytes and the ZO-1 antibodies. We are grateful to all members of the Hirai laboratory for helpful discussions.

Funding

Part of this work was supported by the Kwansei Gakuin University Special Research Fund (to YH).

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Authors and Affiliations

  1. Department of Biomedical Sciences, Graduate School of Science and Technology, Kwansei Gakuin University, Gakuen-Uegahara, Sanda, 669-1330, Japan

    Kaho Hayashi, Sae Nozaki, Kanako Tokushima, Fumika Tanaka & Yohei Hirai

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  1. Kaho Hayashi
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  2. Sae Nozaki
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  3. Kanako Tokushima
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  4. Fumika Tanaka
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  5. Yohei Hirai
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Contributions

K.H. mainly performed experiments; K.T. analyzed NHEK cells; F.T. performed epimorphin study; S.N. validated data; YH analyzed NHEK cells, supervised the study, and wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Yohei Hirai.

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Experiments using mice were approved by the Committee for Animal Experimentation at the Kwansei Gakuin University (approval number: 2020–27).

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Hayashi, K., Nozaki, S., Tokushima, K. et al. Role of syntaxin3 an apical polarity protein in poorly polarized keratinocytes: regulation of asymmetric barrier formations in the skin epidermis. Cell Tissue Res 393, 523–535 (2023). https://doi.org/10.1007/s00441-023-03798-y

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