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, 108 (6), 2270-5

Yes-associated Protein (YAP) Transcriptional Coactivator Functions in Balancing Growth and Differentiation in Skin

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Yes-associated Protein (YAP) Transcriptional Coactivator Functions in Balancing Growth and Differentiation in Skin

Haiying Zhang et al. Proc Natl Acad Sci U S A.

Abstract

In mammals, skin begins as a single-layered epithelium, which, through a series of signals, either stratifies and differentiates to become epidermis or invaginates downward to make hair follicles (HFs). To achieve and maintain proper tissue architecture, keratinocytes must intricately balance growth and differentiation. Here, we uncover a critical and hitherto unappreciated role for Yes-associated protein (YAP), an evolutionarily conserved transcriptional coactivator with potent oncogenic potential. We show that YAP is highly expressed and nuclear in single-layered basal epidermal progenitors. Notably, nuclear YAP progressively declines with age and correlates with proliferative potential of epidermal progenitors. Shortly after initiation of HF morphogenesis, YAP translocates to the cytoplasm of differentiating cells. Through genetic analysis, we demonstrate a role for YAP in maintaining basal epidermal progenitors and regulating HF morphogenesis. YAP overexpression causes hair placodes to evaginate into epidermis rather than invaginate into dermis. YAP also expands basal epidermal progenitors, promotes proliferation, and inhibits terminal differentiation. In vitro gain-and-loss of function studies show that primary mouse keratinocytes (MKs) accelerate proliferation, suppress differentiation, and inhibit apoptosis when YAP is activated and reverse these features when YAP is inhibited. Finally, we identify Cyr61 as a target of YAP in MKs and demonstrate a requirement for TEA domain (TEAD) transcriptional factors to comediate YAP functions in MKs.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Expression of YAP during embryonic skin development. (AC) Immunofluorescence microscopy of frozen back-skin sections labeled with pan-YAP Ab. (D and E) Progressive decline in nuclear YAP correlates with an age-related reduction in the proliferative potential of basal epidermal progenitors. EdU was administered 4 h before tissue processing and quantifications (n = 8). Primary Abs are color-coded according to their secondary Abs, and nuclei are counterstained with DAPI (blue). (Scale bar, 10 μm.) β4, β4 integrin; B, basal; Der, dermis; DP, dermal papilla; Epi, epidermis; K5, keratin 5 (a pan-marker of basal keratinocytes); SB, suprabasal. Dotted lines denote the dermoepidermal border.
Fig. 2.
Fig. 2.
Elevated nuclear YAP results in hyperthickening of interfollicular epidermis and evagination of HFs. (A) Dox-induced Tg pups were distinguished from WT by their lack of a milk spot (*) and open eyes and mouth. (B) H&E staining reveals hyperthickening of Tg interfollicular epidermis accompanied by thinner stratum corneum. (C) HF evaginations in Tg skin are evident by alkaline phosphatase (AP) enzymatic activity (black) in DP. (D) Semithin 1-μm sections of P0 back-skins were stained with toluidine blue, and basal cell density was quantified (as discussed in the text). (E and F) Ultrastructure reveals aberrant cellular morphology in the basal layer and evaginated HFs in Tg mice. B, basal; Der, dermis; DP, dermal papilla; Epi, epidermis; Gr, granular layer; Me, melanin granules (denoted with arrows in F, prevalent in hair shaft and its progenitor Mx cells); Sc, stratum corneum; Sp, spinous layer. (Scale bar, 10 μm.)
Fig. 3.
Fig. 3.
YAP(S127A) induction leads to an expansion of basal-like layers, increased proliferation, and diminished terminal differentiation. (A–C) Immunofluorescence (IF) reveals enhanced proliferation in P0 YAP Tg epidermis. Note enhanced Ki67- and pH3-positive cells mostly in the basal layer of the epidermis and evaginating HFs but also in some suprabasal cells (arrows in expanded boxed areas). Note also the expanded layers of basal markers K5 and p63 in YAP Tg skin. (D–F) IF shows aberrant terminal differentiation in YAP Tg epidermis. B, basal; Der, dermis; Epi, epidermis; K1, keratin 1; Lor, loricrin; SB, suprabasal. Dotted lines denote the dermoepidermal border. (Scale bar,10 μm.)
Fig. 4.
Fig. 4.
YAP(S127A) induction in MKs in vitro increases proliferation potential and inhibits apoptosis and differentiation. Dox-induced WT and K14-rtTA/TRE-YAP(S127A) Tg MKs were assayed 13 d and 1 d later, respectively, for differences in efficiency to form colonies (A, n = 3) and apoptosis (B, n = 3). Images are phase-contrast. (C) Calcium-induced activation of terminal differentiation genes is suppressed by YAP(S127A) induction in MKs. mRNAs were isolated 24 h after the switch from low to high Ca+, and real-time RT-PCR assays were performed (n = 3). F, feeders. *, statistical significance at the level of P < 0.05.
Fig. 5.
Fig. 5.
Loss of YAP leads to impaired growth, enhanced cell death, and precocious differentiation of epidermal MKs. Cultured WT and/or K14rtTA/TRE-YAP(S127A) Tg MKs were infected with lentivirus carrying either Yap shRNA or scramble shRNA as a control. Infected MKs were selected with puromycin (2 μg/mL) 2 d after infection and harvested 5 d later. (A) WB analysis reveals efficient KD of YAP expression (β-actin control). (B and C) Differences in cellular morphology (phase-contrast microscopy) and cell growth on Yap KD. Note that the abnormalities can be rescued by activating YAP(S127A). (D) Apoptosis assays. (E) Precocious induction of late-differentiation genes by Yap KD in MKs cultured in low-Ca2+ medium. mRNAs were isolated from FACS-purified MKs 5 d after infection with lentivirus carrying either Yap shRNAs or scramble shRNAs as well as red fluorescent protein (RFP) and were analyzed with real-time RT-PCR (n = 3 in CE). *, statistical significance at the level of P < 0.05.
Fig. 6.
Fig. 6.
Identification of a target of YAP and of a role for TEADs in mediating YAP function in MKs. (A) Real-time RT-PCR shows that Cyr61 expression is elevated by increased nuclear YAP (ind., induced) in basal epidermal MKs in vivo and in vitro and is repressed by Yap KD (n = 3). (B) Growth defects conferred by Cyr61 KD in MKs. MKs were analyzed 5 d after Cyr61 KD by lentiviral shRNAs (n = 3). (C) ChIP of YAP and IgG (negative control) at the Cyr61 promoter (n = 2). (D and E) Growth defects caused by Tead KD in WT (uninduced, −Dox) and YAP(S127A) overexpressing (induced, +Dox) MKs (n = 3). (F) Effects of Tead KD on Cyr61 expression (n = 3). *, statistical significance at the level of P < 0.05.

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