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ORAI1 Calcium Channel Orchestrates Skin Homeostasis PNAS PLUS

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ORAI1 Calcium Channel Orchestrates Skin Homeostasis PNAS PLUS ORAI1 calcium channel orchestrates skin homeostasis PNAS PLUS Matthieu Vandenberghea,1, Maylis Raphaëla,1,V’yacheslav Lehen’kyia,1, Dmitri Gordienkoa,2, Ryan Hastieb, Thierry Oddosc, Anjana Raob, Patrick G. Hoganb,3, Roman Skrymaa,3, and Natalia Prevarskayaa,3,4 aInstitut National de la Santé et de la Recherche Médicale U1003, Equipe Labellisée par la Ligue Nationale Contre le Cancer, Laboratory of Excellence Ion Channel Science and Therapeutics, Université des Sciences et Technologies de Lille, 59650 Villeneuve d’Ascq, France; bLa Jolla Institute for Allergy and Immunology, La Jolla, CA 92037; and cPharmacology Department, Johnson and Johnson Santé Beauté France, Campus de Maigremont, 27100 Val de Reuil, France Edited* by Michael J. Berridge, The Babraham Institute, Cambridge, United Kingdom, and approved October 25, 2013 (received for review June 4, 2013) To achieve and maintain skin architecture and homeostasis, processes of skin homeostasis. In the present study, using both keratinocytes must intricately balance growth, differentiation, and human primary keratinocytes and the keratinocytes obtained from −/− polarized motility known to be governed by calcium. Orai1 is a pore orai1 mice, we found a previously undescribed role of Orai1 in subunit of a store-operated Ca2+ channel that is a major molecular epidermal physiology. Indeed, in contrast to its expected pro- counterpart for Ca2+ influx in nonexcitable cells. To elucidate the differentiative role, we show that Orai1 constitutively inhibits ter- physiological significance of Orai1 in skin, we studied its functions minal keratinocyte differentiation and is indispensable for the in epidermis of mice, with targeted disruption of the orai1 gene, physiological control of proliferation and migration of basal ker- human skin sections, and primary keratinocytes. We demonstrate atinocytes. We demonstrate that Orai1 protein is mainly confined that Orai1 protein is mainly confined to the basal layer of epidermis to the basal layer of the epidermis where it plays a critical role in where it plays a critical role to control keratinocyte proliferation and the control of keratinocyte proliferation and polarized motility by polarized motility. Orai1 loss of function alters keratinocyte differen- enhancing focal adhesion turnover via the EGFR-PKCβ-Calpain- tiation both in vitro and in vivo. Exploring underlying mechanisms, focal adhesion kinase (FAK) pathway. Orai1 loss of function we show that the activation of Orai1-mediated calcium entry leads to decreases keratinocyte proliferation and inhibits directional mi- enhancing focal adhesion turnover via a PKCβ-Calpain-focal adhesion gration, thereby accelerating the expression of differentiation- kinase pathway. Our findings provide insight into the functions of regulating genes. Finally, Orai1 loss of function alters the skin the Orai1 channel in the maintenance of skin homeostasis. homeostasis in an in vivo mice model, confirming our findings obtained on primary keratinocytes. calcium signaling | cell migration | orai1 KO mice Results he involvement of calcium-dependent mechanisms in the Orai1 Protein Is Mostly Expressed in Stratum Basale and Diminishes Tinduction and regulation of keratinocyte proliferation, mi- During Differentiation. Firstly, we have studied the expression of gration, and differentiation is now well established (1–3). Kera- Orai1 protein in human skin sections (Fig. 1). Immunohisto- tinocytes are arranged in highly organized, specialized layers chemical studies showed that the Orai1 protein is mostly according to their functions and the programmed life cycle. expressed in the stratum basale of human epidermis, with a slight Proliferating keratinocytes comprise the stratum basale. Basal- presence in upper layers of the skin (Fig. 1A). The same ten- cell proliferation is appreciably higher and inversely correlated dency was observed for the STIM1 protein, which was also with the calcium gradient in the skin, reflecting the importance of calcium signaling in differentiation (3). As a result of pro- Significance liferation, keratinocytes leave the stratum basale, moving toward the exterior with the onset of differentiation in the stratum spi- The epidermis of the skin is composed of keratinocytes that are nosum. Differentiation is completed in the stratum granulosum, organized in several layers. Basal cells divide and produce cells thereby constituting the enucleated stratum corneum, which moving outwards the epidermis while undergoing the process CELL BIOLOGY plays the major role as a permeability barrier (1). Besides dif- of terminal differentiation, crucial for the barrier function of the ferentiation and proliferation, the balance of which determines skin. Calcium is an indispensible ion for differentiation, and cal- the epidermis physiology, the polarized motility of keratinocytes cium channels are of primary importance. Unexpectedly, we follows the same vertical pathway, suggesting its crucial impor- discovered that the Orai1 calcium channel is mainly expressed in tance for skin homeostasis (4). the basal layer, functioning to negatively control differentiation. For years, calcium has been considered as a potent inducer of The Orai1 channel supplies calcium to sustain proliferation and, keratinocyte differentiation; for this reason, calcium channels in particular, to drive migration of keratinocytes, both processes have been suggested to be indispensable in its promotion. Of them, being the feature of basal keratinocytes. store-operated calcium channels (SOCs) are a major mechanism 2+ – of Ca entry in nonexcitable cells (5 7). A molecular candidate Author contributions: V.L. and N.P. designed research; M.V., M.R., V.L., D.G., and R.H. for SOC termed Orai1 has been identified and characterized (8– performed research; M.R., R.H., T.O., A.R., and P.G.H. contributed new reagents/analytic 12). Numerous studies have demonstrated that Orai1 mediates tools; M.V., M.R., V.L., T.O., A.R., P.G.H., R.S., and N.P. analyzed data; and V.L., P.G.H., R.S., calcium release-activated currents and SOC in a large variety of and N.P. wrote the paper. cells and is involved in a wide range of cell functions, including The authors declare no conflict of interest. endothelial cell proliferation (13), lymphocyte proliferation (14), *This Direct Submission article had a prearranged editor. and mast cell activation (15), as well as skeletal muscle development Freely available online through the PNAS open access option. and a contractile function (16). However, the role of Orai1 in skin 1M.V., M.R., and V.L. contributed equally to this work. physiology remains poorly understood. The phenotypic features of 2 orai1−/− Second address: Laboratory of Molecular Pharmacology and Biophysics of Cell Signalling, the homozygous mice have been recently shown as sporadic State Key Laboratory of Molecular and Cell Biology, O. O. Bogomoletz Institute of Phys- hair loss, resembling the cyclical alopecia, thinner epidermis with iology, Kiev 01024, Ukraine. lower cell density, and narrower follicles (17), which indicates the 3P.G.H., R.S., and N.P. contributed equally to this work. important role of the Orai1 channel in skin homeostasis. Although 4To whom correspondence should be addressed. E-mail: Natacha.Prevarskaya@univ-lille1. the first findings on the role of Orai1 in differentiation and migra- fr. tion of isolated keratinocytes have very recently appeared (18, 19), This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. they do not reflect the complex role of this channel in the overall 1073/pnas.1310394110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1310394110 PNAS | Published online November 25, 2013 | E4839–E4848 Downloaded by guest on October 2, 2021 (e.g., IVL, KRT1 and -10), intermediate (e.g., TGM1), and late (e.g., Filaggrin, Loricrin), was studied. To confirm the immunohistochemical studies, we have studied the expression of Orai1, STIM1, and the late differentiation marker FLG in human primary keratinocytes (hPKs) using real- time quantitative PCR (Fig. 1B). Quantification of the mRNA transcripts from three independent experiments showing the expression levels of Orai1, STIM1, and FLG in hPK kept in 0.07 mM Ca2+ [undifferentiated hPK (UhPK)] or hPK cells treated with 1.7 mM Ca2+ during 1, 3, or 6 d [differentiated hPK (DhPK)] is indicated in Fig. 1B. The expression data for Orai1, STIM1, and FLG confirmed those of immunohistochemical studies indicating the down-regulation of these proteins during the differentiation process in contrast to the FLG, whose ex- pression increases during differentiation. To study the role of Orai1 in calcium-induced differentiation of hPK, we used a so-called Ca2+-switch protocol. The aim was to see whether the knockdown of Orai1 protein may influence the onset of keratinocyte differentiation. Thus, the cells are pre- transfected with the Orai1-specific siRNA tested preliminarily as shown in Fig. S1A. After 24 h, 1.7 mM Ca2+ is added to UhPK to trigger differentiation for an additional 24 h. The control hPK cells kept in 0.07 mM Ca2+ and control hPK cells with the in- duced Ca2+ switch were transfected with siCtrl. The pretrans- fection of hPK cells with siOrai1 significantly induced the ex- pression of differentiation markers such as cytokeratin 1 (KRT1), IVL, keratinocyte transglutaminase (TGM1), and even FLG, which was superior to the extent of Ca2+ switch itself (Fig. 1C). These data strongly indicate the important role of Orai1 in the process of keratinocyte differentiation. Because
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