Constitutive activation of Stat3 in mouse epidermis is linked to hair deficiency and cytoskeletal network damage.

Abstract

The hair follicle (HF) is a complicated mini-organ comprised of multiple layers that form three major structures: the hair shaft, the inner root sheath and the outer root sheath. The HF undergoes continuous lifelong cycles of rapid growth (anagen), regression (catagen) and rest (telogen). During the anagen stage, keratinocyte stem cells in the bulge region proliferate and subsequently differentiate into several lineages of cells to form new HF 1. HF cycling and hair growth are regulated in a myriad of ways via cytokines, growth factors, receptors and transcription factors 1, 2. Uncontrolled signalling pathways resulting in overexpression and underexpression of HF proteins can cause severe hair growth abnormalities including hair loss 3, 4. Signal transducers and activators of transcription (Stats) are a family of cytoplasmic proteins that participate in the cellular responses involved in various physiological functions including apoptosis, cell cycle regulation and tumor angiogenesis 5. Upon activation, phosphorylated Stats dimerize and translocate to the nucleus where they modulate the expression of target genes. Constitutive activation of Stats is associated with a number of epithelial cancers 5. Studies focusing on Stat3 within skin showed that Stat3 has a critical role in the development of tumors induced by chemical exposure or ultraviolet B radiation 6, 7. Studies also revealed that Stat3-deficient mice develop diffuse alopecia along with impaired wound healing and keratinocytes derived from these mice display impaired growth factor-dependent migration which contributes to defects in wound healing and hair cycling (8,S1,S2). Furthermore, Stat3 is required for the onset of anagen (S1,S2) and it regulates the behaviour of keratinocyte stem cells and other progenitor cells in the bulge region (S3). The cytokine leptin controls HF cycling by stimulating Stat3 activation to induce anagen 9, and hepatocyte growth factor stimulates Stat3 activation and HF development (S1,S2). Together, the data suggest strict regulation of Stat3 is critical to maintain HF cycling and growth. The objective of this study was to identify genes involved in HF formation that are regulated by Stat3. Global gene expression patterns in the epidermis of transgenic mice (BK5.Stat3C) with keratinocytes expressing a constitutively active form of Stat3 (S4) were compared with the patterns in the epidermis of wild-type mice using mouse whole-genome microarray analysis. Of approximately 130 differentially expressed genes detected by microarray, approximately 70 genes were significantly upregulated in epidermis by constitutive activation of Stat3. Thirty-three of these genes were HF-related genes (Table S1), including trichohyalin, hair keratins and keratin-associated proteins that are expressed in the hair shaft and inner root sheath as essential structural components (S5). Aberrant expression of these genes can cause severe hair structure and growth defects (S6). For example, trichohyalin has been found to be associated with alopecia (S7). Interestingly, 26 of the 33 hair follicle genes are located on mouse chromosomes 11 and 16 (Figure S1), indicating that Stat3 regulates distinct subsets of genes expressed in the hair shaft and inner root sheath. To confirm the microarray results, five genes whose expression was upregulated in the epidermis of BK5.Stat3C mice were chosen for quantitative RT-PCR analysis. Consistent with the microarray data, the expression of Tchh, Krtap16-8, Krtap6-1, Krt33a and Krt25d was significantly increased in the epidermis of BK5.Stat3C mice compared with wild-type mice (Fig. 1a–e). In addition, Krtap16-1, which was not identified by microarray analysis but is localized on mouse chromosome 16, was highly expressed in the epidermis of BK5.Stat3C mice in comparison with wild-type mice(Fig. 1f). This raises the possibility that Stat3 regulates groups of HF genes clustered on mouse chromosome 16 and 11. Expression of Cyclin D1 and Bcl-xL, known target genes of Stat3, was also increased in the epidermis of BK5.Stat3C mice compared with that of wild-type mice (Fig. 1g,h). Of 63 significantly downregulated genes, 26 genes encode cytoskeletal proteins including myosin-related proteins, indicating that Stat3 may exert negative effects on the maintenance of keratinocyte shape and motility by destabilizing cytoskeletal proteins (Table S2). To confirm the results, two genes whose expression was downregulated in the epidermis of BK5.Stat3C mice were chosen for quantitative RT-PCR analysis. The expression of Tnnt3 and Myh1 was significantly decreased in the epidermis of BK5.Stat3C mice compared with that of wild-type mice (Fig. 1i,j). The abnormal expression of HF and cytoskeletal genes could explain the appearance of hair loss in BK5.Stat3C mice (Fig. 2a). BK5.Stat3C mice showed continuous hair loss through all hair cycles beginning with the onset of first hair growth. Scanning electron microscopy analysis of dorsal skin from BK5.Stat3C mice showed a marked reduction in hair fibres compared with wild-type mice (Fig. 2b). Furthermore, transmission electron microscopy analysis of cross-sections of BK5.Stat3C HFs showed that BK5.Stat3C HFs exhibit structural disorganization characterized by diffused demarcation of the boundaries between HF layers, especially in the hair cuticle and inner root sheath, whereas wild-type mice display well-formed HF layers (Fig. 2b,c), implying that disruption of HF and cytoskeletal gene regulation by constitutive expression of Stat3 contributes to defects in HF growth/cycling. Studies have shown that Stat3 plays a role in hair growth control, and thereby, it could contribute to hair disorders such as alopecia. This report provides evidence that demonstrates Stat3 regulates sets of HF and cytoskeletal genes that are important for HF cycling. Constitutive Stat3 activation results in impaired hair growth and formation which is the result of aberrant expression of HF and cytoskeletal genes induced by Stat3. Further investigation will help characterize the mechanism of HF differentiation and development and can lead to the development of new therapeutics for hair disorders. This work was supported by the National Cancer Institute Grant CA76520 (J. DiGiovanni), the institutional fund from the Edinburg Regional Academic Health Center, University of Texas Health Science Center at San Antonio (D.J. Kim). HL and DJK performed the research. JD and DJK designed the research study. HL, MK, PKR, JD and DJK analysed the data. HL, LDM and DJK wrote the manuscript. None of the authors have a conflict of interest with regard to this manuscript. Table S1. Thirty three hair follicle genes upregulated by constitutive activation of Stat3. Table S2. Twenty six cytoskeletal genes down-regulated by constitutive activation of Stat3. Figure S1. a) Genomic map of hair follicle genes on mouse chromosome 16 C3.3. b) Genomic map of hair follicle genes on mouse chromosome 11 D. 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