The Role of Yes-Associated Protein (YAP) in Skin Regeneration

Mechanical force is increasingly recognized to be a crucial player in stem cell regulation during normal tissue homeostasis and in cancer development, with Yes-associated protein (YAP) rapidly taking the centre stage as key mechanoprotein. It is critical to unravel the molecular mechanisms underlying the interplay between mechanical force and cell signalling events in normal tissue homeostasis to better understand cancer development, and for the development of therapeutics. My PhD project elucidated that epidermal YAP activity drives β-catenin activation to promote keratinocyte proliferation in mouse skin in vivo (Chapter 3) via mechanotransduction pathways regulated by ROCK, the major mediator of RhoA/C, and elevated collagen-mediated dermal stiffness (Chapter 4). Furthermore, I found evidence that YAP also promotes epidermal Hedgehog signalling via β-catenin activation in mouse skin in vivo (Chapter 5). Interestingly, I observed increased YAP, β-catenin and ROCK activity and dermal fibrosis in mouse and human squamous cell carcinoma (SCC), and in human BCC (Chapter 4, 5). Lastly, I contributed to the development of a simple method to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, Ki67 labelled proliferating cells, Nile Red labelled sebocytes, and DAPI nuclear labelling at the single cell resolution in 3D (Chapter 6).These findings constitute a significant conceptual advance over the established paradigm that YAP purely acts as a mechanosensor in tissue homeostasis, and hence a responder to changes in extracellular matrix stiffness. My work reveals that YAP acts as a central mechanoprotein and also drives tissue mechanics through regulatory interactions with β-catenin, ROCK and Hedgehog signalling pathways. This has important implications for our understanding of normal tissue regeneration and the development of cancers or other regenerative disease displaying increased YAP activity

Positive regulatory interactions between YAP and Hedgehog signalling in skin homeostasis and BCC development in mouse skin in vivo

Skin is a highly plastic tissue that undergoes tissue turnover throughout life, but also in response to injury. YAP and Hedgehog signalling play a central role in the control of epidermal stem/progenitor cells in the skin during embryonic development, in postnatal tissue homeostasis and in skin carcinogenesis. However, the genetic contexts in which they act to control tissue homeostasis remain mostly unresolved. We provide compelling evidence that epidermal YAP and Hedgehog/GLI2 signalling undergo positive regulatory interactions in the control of normal epidermal homeostasis and in basal cell carcinoma (BCC) development, which in the large majority of cases is caused by aberrant Hedgehog signalling activity. We report increased nuclear YAP and GLI2 activity in the epidermis and BCCs of K14-CreER/Rosa-SmoM2 transgenic mouse skin, accompanied with increased ROCK signalling and ECM remodelling. Furthermore, we found that epidermal YAP activity drives GLI2 nuclear accumulation in the skin of YAP2-5SA-ΔC mice, which depends on epidermal β-catenin activation. Lastly, we found prominent nuclear activity of GLI2, YAP and β-catenin, concomitant with increased ROCK signalling and stromal fibrosis in human BCC. Our work provides novel insights into the molecular mechanisms underlying the interplay between cell signalling events and mechanical force in normal tissue homeostasis in vivo, that could potentially be perturbed in BCC development

Activated ROCK-signalling, increased dermal fibroblast numbers, and dermal fibrosis in the skin of K14-CreER/Rosa-SmoM2 transgenic mice.

<p>(A-G) Immunofluorescence staining and area coverage analysis of dorsal skin tamoxifen- and vehicle-treated K14-CreER/Rosa-SmoM2 transgenic littermate mice detecting Fsp1 (A & B) and Vimentin (C), Phalloidin (G), DIAPH3 (H), Thr696-phosphorylated MYPT1 (I & J), Thr18/Ser19-phosphorylated MLC2 (K & L). (D) Masson’s trichrome histological staining of sections through the dorsal neck skin of tamoxifen- and vehicle-treated K14-CreER/Rosa-SmoM2 mice. (E & J) Dual two-photon SHG and monochromatic transmission (Trans; grayscale in merge) images showing collagen (white in single channel, magenta in merged) in tamoxifen- and vehicle-treated K14-CreER/Rosa-SmoM2 skin sections. Area coverage analysis (5 fields/sample from three mice per genotype) of SHG is quantified. Basement membranes and hair follicles are demarcated with dashed lines. DAPI, 4, 6-diamidino-2-phenylindole. Scale bars = 20 μm.</p

Human BCCs exhibit nuclear YAP and β-catenin in association with ROCK signalling activation and increased ECM collagen deposition.

<p>Representative images of immunohistochemical staining (brown) of Gli2 (A), YAP (B), Thr696-phosphorylated MYPT (C) and β-catenin (D) in normal and human BCCs skin samples. (E) Masson’s trichrome histological staining. IHC, Immunohistochemistry. Scale bars = 20 <b>μ</b>m.</p

GLI2 activation in the skin of YAP2-5SA-ΔC transgenic mice.

<p>(A) Immunofluorescence staining of dorsal skin sections of YAP2-5SA-ΔC transgenic and wildtype mice detecting GLI2 (green) and YAP (red). Quantification of % YAP-GLI2 co-positive (arrowheads—B), % GLI2 positive (C) and % YAP (D) positive nuclei in the skin sections of YAP2-5SA-ΔC transgenic and wildtype mice. Basement membranes are demarcated with dashed lines. DAPI, 4, 6-diamidino-2-phenylindole. Scale bars = 20 μm.</p

A model outlining the cross-regulatory interactions between epidermal YAP, ROCK, β-catenin and Hh signalling.

<p>(A) Epidermal SmoM2 activates YAP, ROCK signalling and dermal fibroblasts in the dorsal skin of K14-CreER/Rosa-SmoM2 transgenic mice (based on Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.g002" target="_blank">2</a>). (B) Epidermal YAP activates GLI2 mediated by β-catenin activation in the dorsal skin of YAP2-5SA-ΔC transgenic mice (based on Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.g004" target="_blank">4</a>). (C) A model outlining the proposed regulatory interactions between epidermal YAP, Hedgehog and ROCK-dependent mechanosignalling to balance skin regeneration based on our findings (red arrows) and on cited studies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref025" target="_blank">25</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref027" target="_blank">27</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref029" target="_blank">29</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref034" target="_blank">34</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref039" target="_blank">39</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref040" target="_blank">40</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref045" target="_blank">45</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref047" target="_blank">47</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref048" target="_blank">48</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref063" target="_blank">63</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref064" target="_blank">64</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref065" target="_blank">65</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref066" target="_blank">66</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref069" target="_blank">69</a>].</p

YAP activation in the skin of K14-CreER/Rosa-SmoM2 transgenic mice.

<p>H&E histological staining (A) and Immunofluorescence (B) staining of dorsal skin sections of tamoxifen- and vehicle-treated K14-CreER/Rosa-SmoM2 transgenic mice detecting GLI2 and YAP. Quantification of % GLI2-YAP co-positive (C), % GLI2 positive (D) and % YAP positive nuclei (E). (F) qPCR quantification of mRNA levels of <i>Thbs</i>, <i>Ctgf</i>, <i>Inhba</i> and <i>Gli2</i> genes relative to <i>18S</i> in lysates extracted from the dorsal skin of tamoxifen (control) and vehicle-treated K14-CreER/Rosa-SmoM2 transgenic mice. Basement membranes are demarcated with dashed lines. DAPI, 4, 6-diamidino-2-phenylindole. Scale bars = 20 μm.</p