Expression of a constitutively active calcineurin encoded by an intron-retaining mRNA in follicular keratinocytes

Abstract

Hair growth is a highly regulated cyclical process. Immunosuppressive immunophilin ligands such as cyclosporin A (CsA) and FK506 are known as potent hair growth modulatory agents in rodents and humans that induce active hair growth and inhibit hair follicle regression. The immunosuppressive effectiveness of these drugs has been generally attributed to inhibition of T cell activation through well-characterized pathways. Specifically, CsA and FK506 bind to intracellular proteins, principally cyclophilin A and FKBP12, respectively, and thereby inhibit the phosphatase calcineurin (Cn). The calcineurin (Cn)/NFAT pathway has an important, but poorly understood, role in the regulation of hair follicle development. Here we show that a novel-splicing variant of calcineurin Aß CnAß-FK, which is encoded by an intron-retaining mRNA and is deficient in the autoinhibitory domain, is predominantly expressed in mature follicular keratinocytes but not in the proliferating keratinocytes of rodents. CnAß-FK was weakly sensitive to Ca(2+) and dephosphorylated NFATc2 under low Ca(2+) levels in keratinocytes. Inhibition of Cn/NFAT induced hair growth in nude mice. Cyclin G2 was identified as a novel target of the Cn/NFATc2 pathway and its expression in follicular keratinocytes was reduced by inhibition of Cn/NFAT. Overexpression of cyclin G2 arrested the cell cycle in follicular keratinocytes in vitro and the Cn inhibitor, cyclosporin A, inhibited nuclear localization of NFATc2, resulting in decreased cyclin G2 expression in follicular keratinocytes of rats in vivo. We therefore suggest that the calcineurin/NFAT pathway has a unique regulatory role in hair follicle 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 µM 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 3′ 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 µM 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 µm.

Figure 2. Localization of CnAß -FK mRNA in rat skin.

(A) Schema of sense and anti-sense probes used for in situ hybridization (ISH). The probes correspond to the full sequence of intron 11 and are specific for CnAß-FK mRNA. Sense probes were used as the negative control. (B and C) Expression of CnAß-FK mRNA in rat skin. Skin sections from the parietal region were prepared from male Wistar rat (postnatal day 28, corresponding to the late phase of the second anagen). (D) Higher magnification micrograph to show the mRNA expression in epidermais and SG. Epi, epidermis; SG, sebaceous gland. Scale bars in B and C, 1 mm; in D, 50 µm.

Figure 3. CnAß-FK mRNA and NFATc2 Localizations in rat skin.

(A) Time-scale of the hair cycle in male Wister rats. Each phase was determined using criteria described previously . The intensity of the gray shading indicates the rate of proliferation of follicular keratinocytes: white

Figure 4. Effects of 11R-VIVIT on hair growth induction in nude mice.

(A) The vertex region (blue circle) was chosen as the treatment spot with 11R-VIVIT. (B) Macroscopic and microscopic assessment of hair growth induction. Upper panels, control mouse treated with ointment only. Lower panels, 11R-VIVIT-treated mouse. Right panels, longitudinal sections stained with H. E. Scale bar, 1 mm. (C) Histological features of the skin of control and 11R-VIVIT-treated mice. i–iv, control mice; v–viii, 11R-VIVIT-treated mice. Longitudinal (i, ii, v and vi) and transverse (iii, iv, vii and viii) sections stained with H.E. (i, iii, v and vii), and the same sections assessed for birefringent hair shafts using a polarizing microscope (ii, iv, vi and viii). Scale bars in ii and v, 500 µm; in iv and vii, 200 µm. (D) Hair growth in control (−) and 11R-VIVIT-treated (+) mice was quantified using a polarizing microscope by counting the number of hair follicles containing birefringent hair shafts. n = 10 each. *P<0.0005.

Figure 5. Cyclin G2 is a target molecule of Cn/NFATc2 in follicular keratinocytes.

(A) The microarray analysis identified 24 genes that showed down-regulated expression in PHK cells treated with either 11R-VIVIT or CsA; ccng2 (cyclin G2) expression showed the greatest decrease after Cn/NFAT inhibition. (B) Time-dependent changes in cyclin G2 and p21^waf/cip1 expression in PHK cells treated with 11R-VIVIT. (C) Quantitative analysis of the changes in expression changes of cyclin G2 and p21^waf/cip1 in PHK cells after 11R-VIVIT treatment. n = 5 for each sample group. * P<0.01, ^† P<0.05. (D) Effect of cyclin G2 overexpression on the proliferation of PHK cells. Cells were infected with recombinant adenoviruses carrying cyclin G2 and lacZ at an MOI of 100. Overexpression of cyclin G2 significantly inhibited cell proliferation compared with LacZ-infected cells (*P<0.01). (E) Expression of cyclin G2 in rat hair follicles at postnatal days 9 (first anagen stage) and 49 (second telogen stage). Arrowheads, differentiated keratinocytes in a distal part of the hair shaft; arrows, proliferating keratinocytes in the proximal hair shaft. DP, dermal papillae; HG, hair germ; SG, sebaceous gland. Scale Bar = 50 µm.