Interleukin 6 and STAT 3 regulate p63 isoform expression in keratinocytes during regeneration
Year: 2015
Type: article
Abstract: Epidermal stem cells express several transcriptional modulators required to maintain their undifferentiated and slow-cycling characteristics. One of these transcriptional modulators is p63, the oldest conserved homolog of the tumor suppressor, p53. p63 activity is vital to skin development, homeostasis and wound healing. Regardless of genetic background, p63-null mice have no epidermis or epithelial appendages, and severe limb/craniofacial defects. Two different isoforms of p63 have been described and yet, the exact contribution of each isoform (TAp63 and ΔNp63) to skin's functions is unknown because of conflicting results from numerous mouse models and human disease states 1. It is fairly well accepted that TAp63, expressed in the uncommitted surface ectoderm, induces proliferation and inhibits terminal differentiation 2, while ΔNp63 isoforms are expressed after the ectoderm has committed to stratification 2. Our previous work has demonstrated how the IL-6/STAT3 signalling pathway promotes wound-induced hair follicle neogenesis (WIHN) 3. We reasoned that as p63 is required for normal epidermal functions, including hair follicle formation 1, it may also be necessary during WIHN. A link between IL-6/STAT3 and the p63 isoforms, TAp63 or ΔNp63, has not been described in skin. Does p63 play a role in wound-induced hair neogenesis and is p63 expression in keratinocytes modulated by IL-6/Stat3 signalling pathway? We examined the role of p63 during WIHN using wild-type mice (C57BL/6) and investigated the impact of IL-6/STAT3 signalling on p63 isoform expression using mice and cultured human keratinocytes. All animal protocols were approved by the Johns Hopkins University Animal Care and Use Committee. A role for p63 during WIHN has not been described. We hypothesized that WIHN would be decreased in the absence of p63. Following our WIHN assay protocol, 1 cm2 full-thickness wounds on the backs of wild-type (C57BL/6) 21-day-old male and female mice were created as previously described 3. A single intra-dermal injection of pan-p63 siRNA was introduced into the healing wound, 7 days after wounding (WD7) but before complete re-epithelialization, was sufficient to inhibit p63 expression by 72% in vivo (Fig. 1a). This single injection of pan-p63 siRNA reduced the number of regenerated hair follicles ~2.5-fold in wild-type mice compared with control injections (n = 3; P = 0.07) (Fig 1b). These data suggest that p63 is partially necessary for WIHN in mice. Our prior studies illustrated that IL-6/STAT3 signalling is necessary during WIHN; IL-6 increased WIHN, whereas inhibition of Stat3 blocked WIHN (Fig. 2a) 3. Therefore, we next investigated whether the IL-6/STAT3 signalling pathway regulates p63 function in human keratinocytes. We hypothesized that IL-6/STAT3 signalling would maintain a basal keratinocyte phenotype, consistent with the described role of p63 in stem cell maintenance, through induction of TAp63 and inhibition of ΔNp63. In vitro, TAp63 mRNA expression was significantly increased in normal human epidermal keratinocytes (NHEK) with IL-6 and inhibited by cucurbitacin I, a specific pharmacological JAK/STAT3 inhibitor, in a dose-dependent fashion (Fig. 2b). It is plausible that the level of induction from IL-6 alone is modest since cell culture systems already have significant p-STAT3 stimulation. Abundant levels of ΔNp63 were also detected in monolayer keratinocyte cell culture. IL-6 also significantly increased TAp63 protein expression in C57BL/6 mice (Fig. 2c). Less consistently, ΔNp63 protein levels decreased with IL-6. Most impressively of all, cucurbitacin I significantly decreased TAp63 and increased ΔNp63 protein expression in vivo (Fig. 2d). The molecular switch in p63 isoform expression is critical to epidermal stratification (Koster et al., 2004). In previous work, we show that IL-6 inhibits stratification in vivo and epidermal differentiation marker, keratin 1 (KRT1) by 4-fold (graph inset, Fig. 2e) 3. In this study, we examined whether or not IL-6's mediated changes in KRT1 expression are dependent on TAp63. In the presence of IL-6, TAp63-specific siRNA sufficiently blocks TAp63 mRNA expression in NHEKs (Fig. S1). KRT1 mRNA expression was significantly increased in NHEKs with TAp63-specific siRNA compared to non-targeting scrambled siRNA (Fig. 2e). Together, these data suggest that TAp63 plays a key role in mediating IL-6's effects in keratinocytes. p63 mutations cause ectodermal dysplasia characterized by faulty hair development 1. Increasing TAp63 expression may represent one mechanism whereby IL-6 maintains a basilar keratinocyte phenotype, inhibits keratinocyte differentiation and promotes WIHN 3. TAp63-deficient mice develop skin ulcerations, have significantly delayed wound healing and have accelerated ageing 4. Additionally, ectopic overexpression of TAp63 decreases terminal differentiation 5. These studies are consistent with our findings. Moreover, our data suggests a novel function for IL-6/P-STAT3 activation influencing p63 isoform expression in healing wounds, which may contribute to WIHN. Unlike ΔNp63, the promoter region of the TAp63 isoform does not contain a putative STAT3 response element; however, multiple NFκB binding sites are present 6. STAT3 and NFκB can interact after IL-6 stimulation and regulate gene transcription at the κB sites within promoters 7-9. It is conceivable that TAp63 expression is regulated by this STAT3/NFκB mechanism. In sum, these findings underscore the important crosstalk between immune and developmental signalling pathways during wound healing and regeneration. Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases, part of the National Institutes of Health, under Award Number F32AR062932 to AMN and R01AR064297 to LAG. This work was also supported by the Department of Defense, Armed Forces Institute of Regenerative Medicine, Extremities Regeneration (AFIRM2-ER11), Northrop Grumman Electronic Systems and Alliance for Veterans Support, Inc. (Veteran/Amputee Skin Regeneration Program Initiative) as well as the Thomas Provost, MD Young Faculty Development Fund of Johns Hopkins Dermatology to LAG. AMN and LAG designed the studies, analysed and interpreted the results and co-wrote the paper. In addition, ASK and TSR assisted AMN in performing laboratory experiments. All members discussed results and helped formulate ongoing conduct of the project. All animal protocols are approved by the Johns Hopkins University Animal Care and Use Committee. The authors have declared no conflicting interests. Figure S1. TAp63-specific siRNA decreased expression of target gene by 65% in NHEK with no effect on dNp63 Data S1. Supplemental Methods and Materials Data S2. Supplementary References Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Source: Experimental Dermatology
DOI: 10.1111/exd.12896
Institutions: Johns Hopkins University, Johns Hopkins Medicine
Language: English
Topic: Hair Growth and Disorders
Subfield: Urology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2173623840