the signaling pathway in follicular keratinocytes remained unclear. In the present study, we cloned a novel splicing variant of CnAß (CnAß–FK) in follicular keratinocytes. CnAß–FK lacked the autoinhibitory domain, resulting in a constitutively active form, and was localized in the nucleus of mature follicular keratinocytes along with NFATc2. In contrast, CnAß–FK was not expressed in immature keratinocytes of the hair germ. These results suggest that constant activation of the CN/NFAT signaling pathway in mature keratinocytes is involved in the regulation of hair growth. Normal hair growth requires a balance between keratinocyte growth and differentiation in the hair follicle [2]. CnAß–FK may be involved in inhibition of follicular keratinocyte growth and the induction of differentiation of the cells. One of the most common side effects of treatment with the immunosuppressant drug CsA is hypertrichosis; this condition affects 50–80% of transplant recipients treated with the drug [28]. In hair follicle stem cells, inhibition of the Cn/ NFATc1 pathway by CsA induced the proliferation of stem cells [5]. Overall, the results of previous studies and those described here suggest that CsA inhibits both the CnAß–FK/NFATc2 pathway in follicular keratinocytes and the Cn/NFATc1 pathway in follicular stem cells, resulting in excessive hair growth. CnAß-FK is encoded by an intron-retaining mRNA and is predominantly expressed in follicular keratinocytes. Under ordinary intracellular conditions, intron-containing pre-mature mRNAs are confined within the nucleus, or are degraded by the cellular nonsense-mediated decay system when transported to the cytoplasm; both of these mechanisms result in restriction of their translation [29,30]. However, several recent studies have indicated the existence of a mechanism for export of pre-mature mRNAs from the nucleus [31,32], and also suggested that these mRNAs may play physiologically important roles in addition to being involved in pathological conditions [33–35]. Our results here provide the first evidence that an intron-containing pre-mature mRNA has a physiological function in the regulation of hair follicle development. Although the mechanisms of pre-mRNA nuclear export have been investigated, the details remain unclear [36,37]. Further study is needed to determine the effects of intron 11 and 12 of CnAß on mRNA stability and on expression of CnAß-FK. In the present study, we also found that CnAß-FK was strongly expressed in cells of the sebaceous gland. There have been no previous reports that Cn might have a function in the sebaceous gland. It would be interesting to investigate the physiological functions of CnAß-FK in the sebaceous gland. Another extension of research on this variant would be to investigate the mechanisms of alternative splicing and posttran- scriptional modification. Central dogma states that Cn activity in mammals is tightly regulated by Ca2+ through CnB and calmodulin binding. In response to changes in intracellular Ca2+ levels, the autoinhibitory domain of CnA is removed and the catalytic site is exposed [38]. We showed previously that the autoinhibitory domain of CnA is cleaved by calpain and that Cn becomes the constitutively active form during neurodegeneration [15,23]. This pattern of cleavage is also observed in patients with diseased myocardium [39], suggesting that Cn changes the constitutively active form in some pathological conditions. However, the present study demonstrated that CnAß-FK was a normally occurring variant and was the constitutively active form with a physiological function in mature follicular keratinocytes. Moreover, a splicing variant form of CnAß (variant 2 in Fig. 1C) has been cloned and is also encoded by an intron-retaining mRNA [40]. In variant 2 of CnAß, the autoinhibitory domain has been replaced with a unique C- terminal region generated by the translation of intron 12 (41 amino acids); consequently, variant 2 is also constitutively active Calcineurin Activation and Hair Development [41]. A recent study showed that variant 2 is highly expressed in proliferating myoblasts and regenerates skeletal muscle fibers [41]. Together, these findings indicate that constitutively active variants of Cn may play important roles in the regulation of physiological functions such as cell proliferation and differentiation in various organs and tissues. The present study showed that CsA blocked the Cn activity of CnAß-FK. This result agrees with that of truncated CnA by calpain [15,23]. In contrast, variant 2 of CnAß has reduced sensitivity to the action of immmunosuppressants [41]. Variant 2 of CnAß has a unique C-terminal region generated by the translation of intronic sequences. The alternative C-terminal domain in variant 2 of CnAß may interfere with the binding of the immunosuppressant- immunophilin complex to Cn and prevent its inactivation. The side effects of immunosuppressants are seen in hair follicles and SG as hypertrichosis and hyperplasia but not in skeletal muscle. Differences in sensitivity to immunosuppressants between CnAß-FK and variant 2 of CnAß might determine the nature of any side effects of immunosuppressant treatment. We showed here that cyclin G2 is a novel target of Cn/NFATc2 in follicular keratinocytes. Cyclin G2 is an atypical cyclin involved in cell cycle arrest and apoptosis [19,27]. Previous studies identified the cyclin-dependent kinase inhibitors p21waf/cip1 and p27kip1 as targets of Cn/NFATc2 in follicular keratinocytes [4,16]. Both proteins inhibit proliferation and induce differentiation of keratinocytes [4]. Overall, these observations suggest that activation of the Cn/NFATc2 signaling pathway is involved in the induction of the expression of some signaling molecules that inhibit proliferation and induce terminal differentiation in keratinocytes. Therefore, immunosuppressants may induce prolif- eration of follicular keratinocytes and block terminal differentia- tion in these cells. In conclusion, the data we present here demonstrate a novel signaling cascade involving CnAß-FK, a constitutively active variant of CnA, that controls the growth of follicular keratinocytes through the regulation of expression of cell-cycle regulatory genes such as cyclin G2 and p21waf/cip1. Supporting Information Figure S1 Full-length nucleotide sequence of human CnAß-FK cDNA. (A) The underline indicates the ATG initiation codon and the double underline indicates the stop codon. Black, cDNA derived from exons; blue, cDNA derived from introns; red, poly A tail. (B) Comparison of 39 sequence derived from exon 11 and 12 in CnAß variants. In CnAß-FK cDNA, two sequential stop codons have been inserted at the 39 end of the cDNA derived from exon 11 (arrow). (TIF) Figure S2 Comparison of the alignment of amino acid sequences among each variant of CnAß. The thin bar indicates the calmodulin-binding site and the thick bar indicates the autoinhibitory domain (AID). Dashes indicate missing amino acids. CnAß-FK and variant 2 have no autoinhibitory domain. (TIF) Figure S3 Localization of NFATc2 in PHK cells. Endog- enous NFATc2 was observed in both cytoplasm and nucleus of the cells. Scale Bar, 50 mm. (TIF) Figure S4 Expression of CnAß in male rat skin at postnatal day 28. (A) Endogenous CnAß in hair follicle at a PLoS ONE | www.plosone.org 10 March 2011 | Volume 6 | Issue 3 | e17685