Calcineurin Activation and Hair Development Figure 1. Molecular characteristics of a novel variant of CnAß (CnAß-FK). (A) CnA expression in PHK cells and mouse brain. PHK cells were treated with 1 mM ALLM or incubated with 1 mM Ca2+ for 24 h. (B) Expression of CnAß in the dermis and epidermis of rat. An atypical CnAß with a molecular mass of 48 kDa was observed in the dermis and PHK cells. ß -actin expression (Actin) was used as the control. (C) Comparison of the primary structure of CnAß-FK with other splicing variants of CnAß. CnAß-FK lacks an autoinhibitory domain. The gray box in variant 2 represents amino acid sequences translated from mRNAs of the intron. (D) Comparison of the nucleotide sequence of the 39 terminus of CnAß-FK cDNA with that of variant 1. Blue box, cDNA derived from exons; red box, cDNA derived from introns. *, stop codon. (E) Ectopic expression of CnAß-FK in HEK293 cells. PHK cells lysate was loaded as a positive control. (F) Relative CnAß activity in brain lysate and each cell line. HEK 293 cells were transfected with pcDNA3.1 plasmid containing CnAß-FK cDNA and harvested 24 h later. Cn phosphatase activity was assayed in the presence or absence of 1 mM Ca2+ or 1 mM cyclosporin A (CsA). Phosphatase activity of each sample was standardized with that in the presence of Ca2+ but not CsA. Purified CnA from bovine brain was used as a positive control. n = 5 each sample. *P,0.01. (G) Subcellular distribution of NFATc2 in PHK cells and HEK293 cells. Cells were transfected with a GFP-NFATc2 plasmid and incubated for 24 h. PHK cells were then treated with 11R-VIVIT (1 mM) for 6 h or stimulated with Ca2+ (1 mM) for 15 min. Note that GFP signals were seen not only in the cytoplasm but also in the nucleus of control PHK cells. In HEK293 cells, under basal conditions, GFP signals were seen only in the cytoplasm. Scale bar, 10 mm. doi:10.1371/journal.pone.0017685.g001 PHK and 293 cells overexpressing CnAß-FK (Fig. 1E & 1F). CnAß-FK was stably expressed in the cells (Fig. 1E). Cn activity of a brain lysate and of purified Cn was Ca2+-dependent, whereas PHK cells had high Cn activity under low Ca2+ conditions (Fig. 1F). In cells overexpressing CnAß-FK, moreover, the activity was weakly sensitive to Ca2+ (Fig. 1F). CnAß-FK activity was inhibited by cyclosporin A (CsA), a potent inhibitor of Cn. The subcellular distribution of NFAT was examined by optical imaging in PHK and HEK293 cells that had been transfected with GFP-NFATc2 cDNA. In HEK293 cells lacking CnAß-FK, NFAT was located in the cytoplasm (Fig. 1G). In intact PHK cells, on the other hand, NFAT was observed in both the nucleus and cytoplasm (Fig. 1G). In PHK cells treated with the membrane- permeable specific NFAT inhibitor, 11R-VIVIT, which inhibits nuclear translocation of NFAT by blocking the NFAT-Cn interaction [25,26], NFAT was observed in the cytoplasm but not in the nucleus (Fig. 1G). In contrast, PHK cells stimulated with Ca2+ showed translocation of NFAT to the nucleus (Fig. 1G). Moreover, the pattern of distribution of endogenous NFATc2 was observed in PHK cells under a low Ca2+ (40 mM) concentration. Endogenous NFATc2 was observed in both the nucleus and cytoplasm (Fig. S3). Our observations suggest that Cn has high activity in PHK cells regardless of intracellular Ca2+ levels and induces NFAT translocation into the nucleus. Localization of CnAß–FK mRNA in Rat Skin Immunohistochemical analysis using CnAß antibodies showed that CnAß was strongly expressed in both epidermal and follicular keratinocytes of the rat skin (Fig. S4). CnAßwas also observed in the cells of the outer root sheath (ORS) and the inner root sheath (IRS) but was undetectable in other cells (Fig. S4). However, the immunohistochemical analysis does not distinguish signals from typical CnAß (,60 kDa) and CnAß-FK. Therefore, we performed an in situ hybridization (ISH) using a specific anti-sense probe to CnAß-FK mRNA to verify the distribution of CnAß-FK mRNA in rat skin (Fig. 2A). During the late phase of the anagen stage (P28), in which almost follicular keratinocytes are mature, CnAß-FK mRNA was strongly expressed in follicular keratinocytes of the hair shaft and matrix (Fig. 2B). It was also strongly expressed in IRS cells but was not expressed in ORS cells or the dermal papillae of hair follicles (Fig. 2B and 3D). Strong mRNA expression was also seen in cells of the sebaceous gland (SG) (Fig. 2D). In contrast, no CnAß-FK mRNA was detected in either bulge stem cells or epidermal keratinocytes (Fig. 2D). Interestingly, at an early phase of the anagen stage (P25) and at the transition from telogen to anagen stages (P24), when immature follicular keratinocytes in hair germ (HG) are proliferating, CnAß-FK mRNA was not expressed in the keratinocytes of the HG (Fig. S5A–D). At these stages, high levels of mRNA expression were only detected in the SG (Fig. S5A–D). Moreover, at the catagen and telogen stages, when follicular keratinocytes were absent, CnAß-FK mRNA expression was restricted to SG cells (Fig. 3B, C, E and F). CnAß-FK was also expressed in human hair follicle (Fig. S6). These results agree with those from the western blotting analysis shown in Fig. 1B, and suggest that CnAß-FK may be predominantly expressed in mature follicular keratinocytes and SG cells. If CnAß-FK is constitutively active as shown in Fig. 1, then it is possible that some NFATc2 might be localized in the nuclei of mature follicular keratinocytes and SG cells. To investigate this hypothesis, we determined the subcellular distribution of NFATc2 at each hair cycle stage in rat skin. At the transition from the catagen to telogen stages (P49), NFATc2 was present in the nuclei of cells of the SG (arrowheads in Fig. 3I), whereas it was expressed in the cytoplasm but not the nuclei of dermal papilla cells and follicular keratinocytes (Fig. 3I). Nuclear localization of NFATc2 was also observed in SG cells during the late phase of the anagen stage (arrowheads in Fig. 3H). Moreover, NFATc2 was strongly expressed in the nuclei of mature follicular keratinocytes at the late phase of the anagen stage (Fig. 3G, J–L). NFATc1 was specifically expressed in bulge stem cells (Fig. 3H) as previously reported [5]. At P24 and P25, NFATc2 was observed in the cytoplasm of proliferating follicular keratinocytes in which CnAß- FK was not expressed (compare Fig. S5A and C with Fig. S5E and F). These results suggest that CnAß-FK localized in mature keratinocytes and SG cells, and that a consequence of this localization was the translocation of NFATc2 in the cell nuclei. Effect of NFAT Inhibition on Hair Growth in Nude Mice Cyclosporin A (CsA) and FK506 are widely used immunosup- pressants; both have the well-known side effect of causing hypertrichosis [1]. The Cn/NFAT pathway is involved in the mechanism of this side effect [1,3]. We gave a topical application of an ointment containing 11R-VIVIT to the vertex of nude mice on seven successive days from postnatal day 28 (late phase of the anagen stage). Significant hair growth was observed in the area treated with ointment containing 11R-VIVIT (Figure 4B). Histo- logical analysis of skin specimens from control nude mice revealed dystrophic follicles containing fragmented hair shafts that did not emerge from the hair follicles, and showed little birefringence of the cuticle (Fig. 4C). In contrast, treatment with 11R-VIVIT resulted in relatively normal follicles containing well-differentiated straight hair shafts that emerged from hair follicles and reached the surface of the skin, and showed clear birefringence of the cuticle (Fig. 4C). Moreover, the number of hair follicles containing birefringent hair was significantly increased in 11R-VIVIT-treated mice compared to the control mice (Fig. 4D). These results suggest that inhibition of CN/NFAT signaling pathway enhanced hair growth. Involvement of Cyclin G2 in Cell Cycle Arrest in Follicular Keratinocytes As shown in Figure 1G, NFATc2 is a target of CnAß-FK. NFATc2 has been shown to regulate the hair cycle by determining the level of PLoS ONE | www.plosone.org 5 March 2011 | Volume 6 | Issue 3 | e17685