Expression Level of Orai3 Correlates with Aging-Related Changes in Mechanical Stimulation-Induced Calcium Signalling in Keratinocytes

Epidermis shows morphological and functional alterations with aging: the number of layers of stratum corneum increases, the skin surface becomes dry and flaky, and keratohyalin granules, which produce free amino acids and cornified envelope, decrease.1 Barrier homeostasis of stratum corneum becomes less robust with aging, and recovery after tape stripping becomes slower.2 The calcium gradient in epidermis plays a crucial role in epidermal barrier homeostasis.3 In epidermis of healthy, young subjects, Ca2+ concentration is higher in the uppermost layer.4 We reported that the calcium gradient in epidermis was disrupted with aging.5 Thus, alteration of the epidermal calcium gradient might be a cause of morphological and functional aging of the epidermis. The STIM1 (stromal interaction molecule 1)-Orai1 (ORAI calcium release-activated calcium modulator 1) system mediates calcium signalling. Activation of STIM1 by depletion of Ca2+ from endoplasmic reticulum leads to formation of a highly selective calcium channel with Orai1 in plasma membrane.6 Loss of Orai1 function alters keratinocyte differentiation, and knockdown of Orai1 or STIM1 delays wound healing.7 However, the roles of Orai2 and Orai3 are unknown.8 We speculated that changes in expression or function of STIM or Orai family members might be related to aged epidermal phenotypes. Here, we focused on Orai3, based on our observation that it is differentially expressed in keratinocytes from young and aged subjects. Are changes of calcium dynamics that are associated with aging-related skin phenotypes related to changes in expression of Orai3 in keratinocytes? To examine aging-related changes in calcium dynamics, we applied hydraulic pressure stimulation to cultured normal human epidermal keratinocytes from subjects of various ages as described previously,9 and imaged changes of [Ca2+]i in single cells with the fluorescent dye Fura-2 AM. We used hydraulic stress because the level of stress could be easily and precisely regulated. Expression of Orai mRNAs was measured by PCR, and the putative role of Orai3 in keratinocytes was confirmed by knockdown with siRNA. Localization of Orai3 in human skin sections was examined immunohistochemically. Methods are described in the supplement. We observed [Ca2+]i profiles after application of hydraulic pressure to keratinocytes from 11 subjects, aged 22 to 67 years. Differentiated keratinocytes in the presence of 1.8 mmol/L calcium were used, as they are more sensitive than undifferentiated cells to mechanical stimuli.9 Figure 1a and b shows representative time courses of [Ca2+]i in stimulated single cells from young (24-year-old) and aged (63-year-old) subjects, respectively, and Figure 1c and d shows representative fluorescence images. Peak [Ca2+]i (at 35 seconds) was similar in cells from young and old subjects. However, recovery of [Ca2+]i to baseline was slower in cells from aged subjects. The half-bandwidth (time duration sliced at half the Δ ratio; see Figure 1A,B) at age 63 was longer than that at age 24. There was a significant correlation between age and half-bandwidth (Figure 1E). Recovery of [Ca2+]i from hydraulic stress-induced elevation tended to be delayed in keratinocytes from older subjects. We then evaluated mRNA levels of calcium pump ATPases and STIM-Orai family members in differentiated keratinocytes from subjects aged 19 to 70 (n=14, average 42.7 years). STIM and Orai play important roles in calcium dynamics, and their coordinated function mediates cytosolic and endoplasmic reticulum Ca2+ homeostasis and generation of both transient and long-term Ca2+ signals.6 The correlation coefficient (r) between each mRNA level and age or half-bandwidth is shown in Table S1. Among mRNAs examined, only Orai3 mRNA showed a significant correlation with both age and half-bandwidth (Figure 2A,B). Orai3 expression was doubled in keratinocytes from subjects in their 60s, compared with subjects in their 20s. Next, we investigated the effect of Orai knockdown with targeted siRNA in keratinocytes from aged subjects. The results of real-time quantitative PCR are shown in Figure 2C. mRNA was extracted from transfected cells 4 days after siRNA treatment (ie 2 days after differentiation induction; the same time as calcium imaging). Compared with negative control siRNA-treated cells, Orai siRNA-treated cells showed approximately 80-90% decrease in the targeted mRNA. We then examined the profile of [Ca2+]i in Orai-knockdown cells exposed to hydraulic stress. Representative profiles are shown in Figure 2E. Knockdown of Orai1 or Orai2 did not significantly change the half-bandwidth or peak value of fura-2 ratio (data not shown). Compared with controls, Orai3-knockdown keratinocytes from subjects aged 56, 63 and 64 years showed significantly decreased half-bandwidth (Figure 2E), corresponding to faster recovery from hydraulic stress. Expression levels of Orai1 and Orai2 were unchanged in Orai3-knockdown cells. Orai3 knockdown in keratinocytes from young subjects had no effect on half-bandwidth (ages 0, 24; data not shown). We also examined mRNA expression of differentiation marker proteins (involucrin, loricrin, filaggrin, keratin 1, transglutaminase 1) in the Orai3-knockdown cells used in Figure 2E and found no significant difference between control and knockdown cells at the time of calcium imaging (data not shown). Immunohistochemical examination showed that Orai3 was expressed in the uppermost (granular) layer of human epidermis (Figure S1). Skin sections from donors aged 19 to 64 years (N=8) all showed similar expression patterns. Orai3 mRNA expression in differentiated human keratinocytes increased with increasing age of donors, and the increase corresponded well to slower recovery of [Ca2+]i from hydraulic stress-induced elevation in keratinocytes of older donors. This delay in recovery was reduced by knockdown of Orai3 with siRNA. Thus, increased Orai3 expression might be associated with aging-related phenotypes of human skin. Further, as expression of Orai3 was observed throughout the epidermis, including the granular layer, Orai3 might be associated with sensing of external stimuli, as well as epidermal permeability barrier homeostasis. However, Orai3 expression in human skin samples did not appear to be age dependent, so Orai3 expressed in intact skin might be functionally different from that of cultured keratinocytes. Mechanistic studies of aging-related changes in keratinocyte responses to calcium channel agonists/antagonists, together with in vivo calcium imaging, might open up new avenues in dermatological therapy. MD and SD designed the study and wrote the manuscript; KT, JK, SD and MG performed the experiments. The authors have no conflict of interest to declare. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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