keratinocytes or of the mechanism of Cn-dependent regulation of the growth of hair follicles. Interestingly, it has been pointed out that Cn is functionally active and activates NFATc2-dependent gene transcription in follicular keratinocytes, resulting in inhibition of the hair cycle [4]. Overall, the molecular mechanism of Cn activation in follicular keratinocytes seems to differ from that in other tissues such as neurons and T cells. In the present study, we show that a novel splicing variant of CnAß(CnAßin follicular keratinocytes, CnAß-FK) is predominantly expressed in follicular keratinocytes. CnAß-FK was deficient in the autoinhibitory domain and was therefore active independently of Ca2+ levels. To clarify the roles of CnAß-FK in the hair cycle, we sought to identify the cell cycle regulators whose expression was regulated by CnAß-FK/NFATc2. We found that cyclin G2, an atypical cyclin that correlates with cell cycle arrest, was regulated by CnAß-FK/ NFATc2 [18,19]. Our findings provide the first indication of the role of functionally active Cn in follicular keratinocytes. Materials and Methods Cell Culture PHK16-0b (PHK) cells established from human keratinocytes, HEK293 cells and HeLa cells were provided by the Health Science Research Resources Bank (Osaka, Japan). PHK cells were cultured in the serum-free medium EpiLife in the presence of 60 mM CaCl2 (Cascade Biologics), supplemented with EDGS (growth supplement, S-012-5, Cascade Biologics), penicillin, streptomycin, and amphotericin B (Nacalai tesque, Japan). At 80% confluence, cells were trypsinized (Trypsin/EDTA, Toyobo) and subcultured onto culture plates coated with human Type-1 collagen (Coating Matrix Kit, Cascade Biologics). HEK 293 cells and HeLa cells were cultured in D-MEM (Gibco) with fetal bovine serum (10%) and antibiotics. Cloning of a Splicing Variant of CnAßCnAß-FK) A cDNA library was constructed from PHK cells by Takara Bio (Ohtsu, Japan). Briefly, cDNAs were synthesized from PHK cells using an Oligo(dT)-Anchor primer (Takara Bio) and subcloned into the EcoRI/XhoI sites of pBluescript II SK(+) plasmids. The full nucleotide sequence of CnAß-FK was determined by PCR and nested-PCR using the primers listed in Tables S1-A and -B. PCR products were cloned into pCR-Blunt II-TOPO (Invitrogen) and analyzed using the listed sequencing primers (Table S1-a and -b). For further determination of the C-terminus, 39-RACE (Rapid Amplification of cDNA Ends) was performed with the 39 Full RACE Core Set (Takara Bio) (Table S1-c). mRNAs were isolated from PHK cells using the FastTrack 2.0 kit (Invitrogen) according to manufacturer’s protocol. Details of the primer sequences and cloning process are given in Table S1. PCR was performed using the KOD FX DNA polymerase (Toyobo). Calcineurin Phosphatase Assay Calcineurin activity was determined by a calcineurin phospha- tase assay that measures dephosphorylation of p-nitrophenol phosphate (pNPP). HEK293 cells were transfected with pcDNA3.1 plasmid (Invitrogen) containing human CnAß-FK cDNA. After 24 h, the cells were harvested and lysed in a buffer comprised of 20 mM Tris-HCl (pH 7.2), 1 mM EGTA, 100 mM sodium chloride, 1% Triton X-100, and Protease Inhibitor Cocktail (Roche). Mouse brain and PHK cells were lysed in the same buffer. After centrifugation at 65,000 g, each supernatant (1 mg of total protein) was immunoprecipitated using 10 mg of polyclonal anti-CnAß antibodies (Chemicon). Immunoprecipitat- ed samples were incubated on 96-well microplates in a Cn assay Calcineurin Activation and Hair Development buffer (50 mM Tris-HCl (pH 7.2), 1 mg/ml CaM, 1% BSA. 1 mM okadaic acid) in the presence or absence of 2 mM CaCl2, 2 mM EGTA or 1 mM CsA at 37uC for 15 min. After adding 1 mg/ml pNPP, the samples were further incubated for 15 min. The reaction was stopped by adding sodium hydroxide (final concentration, 300 mM), then the absorbance at 405 nm was measured with a microplate reader. Purified bovine CaN was purchased from Upstate (14-390). In each sample, the data were standardized against Ca2+ stimulation data. Animal Studies The effect of 11R-VIVIT on hair growth in vivo was investigated in male nude mice aged 28 days (BALB/c Slc-nu, Shimizu Laboratory Supplies, Kyoto). For drug application, 11R-VIVIT (10 mg) was dissolved in 1 g hydrophilic ointment (Merck). Twenty-five mg of the ointment containing 250 mg of 11R-VIVIT was applied to the skins of the mice once per day. After application of the ointment for seven days, the animals were killed, and the skins excised and immediately fixed with 4% paraformaldehyde (PFA) at 4uC overnight. As a control, mice were treated as above, but with ointment lacking 11R-VIVIT. To investigate the effect of CsA, Wistar rats aged 24 days (Shimizu Laboratory Supplies) were intraperitoneally injected with 100 mg/kg CsA on 3 successive days. The animals were then killed and the skins then excised and used for immunohistochem- istry. As a control, rats were injected with vehicle only. All procedures were approved by the Ethics Review Committee for Animal Experimentation of our institute (OKU-2009192). In Situ Hybridization In situ hybridization was carried out as described previously [20]. Briefly, probes for detecting rat CnAß-FK mRNA were prepared by first cloning the full sequence of intron 11 of CnAß- FK cDNA (Figure 2A) into pCR-Blunt II-TOPO (Invitrogen). The plasmid was linearized and transcribed with SP6 or T7 RNA polymerase. Digoxigenin-UTP-labeled RNA probes were gener- ated from the DNA template using a DIG RNA labeling kit (Roche). Fresh frozen sections (6 mm thickness) were prepared on silane-coated glass microscope slides and immediately fixed with 4% paraformaldehyde (PFA) and 0.1% glutaraldehyde (GA) in 0.1 M phosphate buffer (PB, pH 7.4) for 15 min. Prior to hybridization, RNA probes were denatured at 80uC for 5 min. Hybridization was performed at 60uC for 16 h with a digoxigenin- labeled probe (1 mg/ml) in hybridization buffer (50% formamide, 26SSC, 1% SDS). After a series of wash steps, the single-stranded RNA probes were removed with RNase A (10 mg/ml) at 37uC for 30 min. The sections were incubated with alkaline phosphatase- conjugated anti-DIG antibody and hybridization signals were detected by an NBT/BCIP colorimetric reaction according to manufacturer’s recommendation (Roche). For specificity control, a sense probe was used. Immunofluorescent Staining Skin sections from male Wistar rats were immunofluorescently stained as follows. Excised skin samples were immediately frozen in Optimal Cutting Temperature compound (Sakura Finetek, Japan) and sequentially sectioned at a thickness of 10 mm. The sections were fixed with 4% PFA in 0.1 M phosphate buffer (pH 7.4) for 15 min and then incubated with 5% normal goat serum (Abcam) in blocking buffer (16PBS, 0.3% Triton X-100) at room temperature for 1 h. After washing in PBS, the sections were incubated with primary antibodies in dilution buffer (16 PBS, 10% BSA, 0.3% Triton X-100) at 4uC overnight. The sections were incubated with secondary antibodies in dilution buffer at PLoS ONE | www.plosone.org 2 March 2011 | Volume 6 | Issue 3 | e17685