Bone Morphogenetic Protein Signaling Suppresses Wound-Induced Skin Repair by Inhibiting Keratinocyte Proliferation and Migration Christopher J

ORIGINAL ARTICLE

Bone Morphogenetic Protein Signaling Suppresses Wound-Induced Skin Repair by Inhibiting Keratinocyte Proliferation and Migration Christopher J. Lewis1,2,AndreiN.Mardaryev1, Krzysztof Poterlowicz1, Tatyana Y. Sharova3, Ahmar Aziz3, David T. Sharpe2, Natalia V. Botchkareva1 and Andrey A. Sharov3

Bone morphogenetic protein (BMP) signaling plays a key role in the control of skin development and postnatal remodeling by regulating keratinocyte proliferation, differentiation, and apoptosis. To study the role of BMPs in wound-induced epidermal repair, we used transgenic mice overexpressing the BMP downstream component Smad1 under the control of a K14 promoter as an in vivo model, as well as ex vivo and in vitro assays. K14- caSmad1 mice (transgenic mice overexpressing a constitutively active form of Smad1 under K14 promoter) exhibited retarded wound healing associated with signiﬁcant inhibition of proliferation and increased apoptosis in healing wound epithelium. Furthermore, microarray and quantitative real-time reverse-transcriptase–PCR (qRT– PCR) analyses revealed decreased expression of a number of cytoskeletal/cell motility–associated genes including wound-associated keratins (Krt16, Krt17)andMyosin VA (Myo5a), in the epidermis of K14-caSmad1 mice versus wild-type (WT) controls during wound healing. BMP treatment signiﬁcantly inhibited keratinocyte migration ex vivo, and primary keratinocytes of K14-caSmad1 mice showed retarded migration compared with WT controls. Finally, small interfering RNA (siRNA)–mediated silencing of BMPR-1B in primary mouse keratinocytes accelerated cell migration and was associated with increased expression of Krt16, Krt17, and Myo5a compared with controls. Thus, this study demonstrates that BMPs inhibit keratinocyte proliferation, cytoskeletal organization, and migration in regenerating skin epithelium during wound healing, and raises a possibility for using BMP antagonists for the management of chronic wounds. Journal of Investigative Dermatology (2014) 134, 827–837; doi:10.1038/jid.2013.419; published online 7 November 2013

INTRODUCTION BMP signaling is activated by binding of BMP ligands to Bone morphogenetic proteins (BMPs) are members of the type I and type II serine–threonine kinase receptors (BMPRs) transforming growth factor-b (TGF-b)superfamily,playingkey followed by activation of the BMP-Smad and/or BMP– rolesinthecontrolofskindevelopmentandpostnatal Mitogen-Activated Protein Kinase pathways (Miyazono et al., remodeling by regulating cell proliferation, differentiation, 2010). BMP signaling activity is controlled by endogenous and apoptosis (Botchkarev and Sharov, 2004; Miyazono antagonists, including Noggin (Walsh et al., 2010), which et al., 2010; Walsh et al., 2010). preferentially binds BMP-2 and BMP-4 with very high afﬁnities, preventing their interactions with receptors (Zimmerman et al., 1996; Krause et al., 2011). 1Centre for Skin Sciences, University of Bradford, Bradford, West Yorkshire, During skin and hair follicle (HF) development, BMPs UK; 2Plastic Surgery and Burns Research Unit, University of Bradford, inhibit HF initiation (Botchkarev et al., 1999; Jamora et al., 3 Bradford, West Yorkshire, UK and Department of Dermatology, 2003). In adult HFs, BMP signaling is involved in the control Boston University, Boston, Massachusetts, USA of telogen–anagen transition by maintaining bulge stem cells Correspondence: Natalia V. Botchkareva, Centre for Skin Sciences, University of Bradford, Richmond Road, Bradford, West Yorkshire, BD7 1DP, UK. in a quiescent state through the binding of BMP-6 to BMPR-1A E-mail: [email protected] or Andrey A. Sharov, Department of (Blanpain et al., 2004), and suppressing Wnt/b-catenin and Dermatology, Boston University, 609 Albany Street, Boston, Massachusetts, Hedgehog signaling pathways in the stem cell niche USA. E-mail: [email protected] (Botchkarev et al., 2001; Andl et al., 2004; Ming Kwan Abbreviations: BMP, bone morphogenetic protein; BMPR, bone et al., 2004; Yuhki et al., 2004; Zhang et al., 2006; Kobielak morphogenetic protein receptor; DAPI, 4’,6-diamidino-2-phenylindole; HF, hair follicle; K14-caSmad1, transgenic mice overexpressing a constitutively et al., 2007; Plikus et al., 2008; Kandyba et al., 2013). active form of Smad1 under K14 promoter; Krt, keratin; Myo5a, Myosin VA; In anagen HFs, BMP signaling inhibits proliferation and PMEK, primary mouse epidermal keratinocyte; qRT–PCR, quantitative real- promotes differentiation of hair matrix keratinocytes into time reverse-transcriptase–PCR; siRNA, small interfering RNA; TG, transgenic; hair shaft and inner root sheath lineages (Kulessa et al., TGF, transforming growth factor; WT, wild type 2000; Kobielak et al., 2003; Andl et al., 2004; Ming Received 19 April 2013; revised 2 September 2013; accepted 16 September 2013; accepted article preview online 14 October 2013; published online Kwan et al., 2004; Yuhki et al., 2004; Sharov et al., 2006), 7 November 2013 as well as regulating melanogenesis (Sharov et al., 2005; Yaar

& 2014 The Society for Investigative Dermatology www.jidonline.org 827 CJ Lewis et al. Role of BMP Signaling in Skin Repair

9 Bmp-2

6 BMPR-1A BMPR-1B pSmad-1/5/8

mRNA 3 * *

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60 Bmp-4 BMPR-1A BMPR-1B pSmad-1/5/8 40 WE

mRNA 20 * 0 * Day 3 wound Day Tel d3 d5 HF HF WE WE Bmp-7 12 BMPR-1A BMPR-1B pSmad-1/5/8 8

mRNA WE 4 ** HF HF SG 0 5 wound Day Tel d3 d5 WE WE BMPR-1A BMPR-1B pSmad-1/5/8 45 Noggin

mRNA 15 ** 7 wound Day 0 ** Tel d3 d5

Figure 1. Expression of bone morphogenetic protein (Bmp) pathway components during skin healing. (a) Quantitative real-time reverse-transcriptase–PCR (qRT–PCR): signiﬁcant decreases of Bmp-2, Bmp-4, Bmp-7,andNoggin transcripts on day 3 (d3) and day 5 (d5) after wounding. (b) Immunoﬂuorescence: in telogen (Tel) skin, BMPR-1A expression is restricted to the hair follicle (HF) bulge (arrowhead); BMPR-1B is seen in the basal (arrows) and suprabasal epidermal layers (arrowheads); pSmad-1/5/8 is expressed in the basal (arrows) and more prominently in suprabasal epidermal layers (arrowheads). On days 3, 5, and 7 after wounding, BMPR-1A expression is low in HF bulges near the wound (arrows) but remains strongly expressed in those further from the wound (inset, arrowhead); there is strong expression of BMPR-1B and pSmad-1/5/8 in the wound epithelial tongue and the adjacent unwounded epidermis (arrows). Mean±SD, *Po0.01, **Po0.0001, Student’s t-test. SG, sebaceous gland; WE, wound epithelium. Bar ¼ 100 mm.

et al.,2006;Parket al., 2009; Singh et al., 2012). In addition, wound, forming a layer of hyperproliferative epithelium BMPs contribute to the regulation of epidermal differen- (Falanga, 1993; Taylor et al., 2000; Li et al., 2007; Schafer tiation by activating BMPR-1B that is expressed in the supra- and Werner, 2007). Stem cells residing in the HF infundi- basal epidermis (Hwang et al., 2001; Sharov et al., 2003; Yu bulum and bulge adjacent to the wound become inherently et al., 2010). involved in the processes of epithelial regeneration, giving rise Wound healing is a dynamic process of overlapping phases to daughter cells that migrate to the sites of injury and assist in of inﬂammation, proliferation, extracellular matrix formation, skin repair (Ito et al., 2005; Cotsarelis, 2006; Levy et al., 2007; re-epithelialization, and remodeling (Li et al., 2007; Schafer Ito and Cotsarelis, 2008; Langton et al., 2008; Hsu et al., and Werner, 2007; Barrientos et al., 2008; Shaw and Martin, 2011; Kasper et al., 2011; Wong and Reiter, 2011). Similar to 2009). These stages are characterized by coordinated intrinsic the HF, sweat gland stem cells also contribute to wound repair cellular responses in keratinocytes, ﬁbroblasts, neutrophils, (Lu et al., 2012). macrophages, and endothelial cells regulated by an orchestra Different members of the TGF-b superfamily also play of mediators including platelet-derived growth factor, tumor important yet distinct roles in the control of wound healing: necrosis factor-a, IGF-1, epidermal growth factor, TGF-a, TGF-b signaling inhibits skin repair, at least in part by vascular endothelial growth factor, and platelet factor-IV suppressing keratinocyte migration, whereas activin signaling (Falanga, 1993; Li et al., 2007; Schafer and Werner, 2007). stimulates the wound healing response and promotes kerati- Skin injury triggers immediate stress responses in epidermal nocyte proliferation (Ashcroft et al., 1999; Munz et al., 1999; keratinocytes that begin to proliferate and migrate toward the Wankell et al., 2001; Hosokawa et al., 2005). Several

828 Journal of Investigative Dermatology (2014), Volume 134 CJ Lewis et al. Role of BMP Signaling in Skin Repair

Kpnl ATG Notl EVE Flag expressing K14- hKeratin 14 Flag caSmad1 SV40pA plasmid caSmad1 WT promoter Flag

WT K14-caSmad1 Actin Smad1 Smad1

Day 3 Day 5 Day 7 WT

Day 3 Day 5 Day 7

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WE WE 350 400 WE 2 WT 350 WT 300 K14-caSmad1 300 250 K14-caSmad1 250 200 200 ** 150 150 WE ** ** 100 * 100 * GT WE 50 * 50 * K14-caSmad1 WE Epithelial tongue length, µ m Wound epithelial area, µ m Wound 0 0 Day 3 Day 5 Day 7 Day 3 Day 5 Day 7

Figure 2. Histomorphological analysis of wound epithelium in K14-caSmad1 (transgenic mice overexpressing a constitutively active form of Smad1 under K14 promoter) and wild-type (WT) mice. (a) Transgenic construct used to generate K14-caSmad1 mice. (b) K14-caSmad1 mice show markedly increased Smad1 expression in the epidermis and hair follicles (HFs) versus control mice as detected by anti-Smad1 antibody. (c) Western blot confirmation of FLAG-tag expression in dorsal skin of transgenic K14-caSmad1 mice versus controls. (d) Representative images of macroscopic wound appearance and (e) wound histology in K14- caSmad1 and WT mice at 3, 5, and 7 days after wounding. (f) Significantly reduced area of wound epithelium in K14-Smad1 mice on days 3, 5, and 7 after wounding versus WT controls. (g) Significantly reduced wound epithelial tongue length in K14-caSmad1 mice on days 3, 5, and 7 after wounding (mean±SD, *Po0.01, **Po0.0001, Student’s t-test). GT, granulation tissue; hKeratin, human keratin; WE, wound epithelium. Bar ¼ 100 mm. indications suggest that BMP signaling can also be involved in RESULTS the regulation of skin repair. BMP-6 expression has been BMP pathway components show differential expression patterns shown to be induced in response to wounding in both during wound healing keratinocytes and dermal fibroblasts; in addition, high BMP- To understand the role of the BMP/Smad pathway in wound 6 levels have been found in chronic wounds (Kaiser et al., repair, the expressions of BMP ligands (BMP-2, BMP-4, 1998). Overexpression of BMP-6 in mouse epidermis results in BMP-7), their receptors (BMPR-1A, BMPR-1B), intracellular delayed re-epithelialization (Kaiser et al., 1998). It is also component of the BMP pathway phosphorylated-Smad-1/5/8 known that BMP-2 and BMP-4 exert negative effects on (p-Smad-1/5/8), and NOGGIN were examined at different keratinocyte proliferation (Sharov et al., 2006; Ahmed et al., stages after application of wounds to mouse skin containing all 2011). However, the expression of distinct BMP signaling hair follicles at the telogen (resting) stage of the hair cycle. components and the effects of BMPs and their antagonist Quantitative real-time reverse-transcriptase–PCR (qRT–PCR) Noggin in skin healing after injury remain to be explored. analysis revealed significant (Po0.01) decreases in the expres- In this paper, we provide evidence that BMP-2, BMP-4, sion of Bmp-2, Bmp-4, Bmp-7,andNoggin (Po0.0001) BMP-7, BMPRs, and antagonist Noggin exhibit a dynamic transcripts on days 3 and 5 after skin injury compared with expression pattern during wound healing. By using K14- unwounded skin (Figure 1a). caSmad1 mice (transgenic mice overexpressing a constitu- Immunofluorescent analysis showed that BMPR-1A expres- tively active form of Smad1 under K14 promoter) and small sion was restricted to the HF bulge in telogen skin (Figure 1b interfering RNA (siRNA)–mediated silencing of keratinocyte and Supplementary Figure S1a online) (Botchkarev et al., BMPRs in vitro, we demonstrate that BMPs inhibit keratino- 2001; Kobielak et al., 2003). BMPR-1B expression was cyte proliferation, cytoskeletal organization, and migration found in the basal and suprabasal layers of the epidermis, induced by BMPR-1B in regenerating skin epithelium during whereas it was not detected in the HF (Yu et al., 2010) wound healing, and raise a possibility for using BMP antago- (Figure 1b; data not shown; Supplementary Figure S1a online). nists for the management of chronic wounds. The expression of pSmad-1/5/8 was seen throughout the

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epidermis, being more prominent in the suprabasal layers in epithelium at days 3, 5, and 7 versus time-matched controls telogen skin (Figure 1b and Supplementary Figure S1a online). (Figure 3c and d, Supplementary Figure S1f online). During wound healing, BMPR-1A expression was progres- Because injury-induced repair is associated with profound sively decreased followed by its disappearance from the HFs changes in cytoskeletal organization, we also examined the immediately adjacent to the wound bed on days 5 and 7 after expressions of keratin 16 (KRT16) and keratin 17 (KRT17), injury, whereas it remained present in the bulge of HFs distant whose expression is induced in response to wounding from the wound (Figure 1b and Supplementary Figure S1a (Paladini et al., 1996; Coulombe, 1997; Patel et al., 2006). online). In contrast, there was prominent expression of In contrast to KRT14 whose expression was not changed in the BMPR-1B and p-Smad-1/5/8 in the wound epithelial tongue wound epithelium of K14-caSmad1 versus WT mice and in the adjacent unwounded epidermis, HFs, and dermal (Supplementary Figure S1g online), KRT16 and KRT17 expres- cells (Figure 1b and Supplementary Figure S1a online). sions were dramatically reduced in K14-caSmad1 mouse wounds compared with WT controls at days 3 and 5 after Overexpression of Smad1 in the epidermis compromises wounding (Figure 3e and f and Supplementary Figure S1h, I wound healing online). Analysis of keratinocyte morphology revealed that the To elucidate a role for BMP signaling in skin healing, K14- epithelial tongue of WT mice contained more elongated caSmad1 transgenic (TG) mice overexpressing a constitutively keratinocytes (Figure 3e and f), an important characteristic of active form of Smad1 as a key component of the ‘‘canonical’’ actively migrating cells in the wound epithelial tongue BMP pathway were employed. K14-caSmad1 mice were (Driscoll et al., 2012; Meyer et al., 2012; Allard and generated using a TG construct containing human K14 Mogilner, 2013). In contrast, epithelial cells in K14-caSmad1 promoter, FLAG-tagged human complementary DNA encod- mice lost this flattened appearance and showed a more ing phospho-mimetic activated Smad1 in which the C-term- cuboidal shape (Figure 3e and f, Supplementary Figure S1h, inal SVS phosphorylation sites (S463 and S465) were mutated i online). This suggested that the delayed wound healing in into EVE (Fuentealba et al., 2007), and human growth K14-caSmad1 mice may also be caused by impaired kerati- hormone poly-A sequence (Sharov et al., 2009) (Figure 2a). nocyte migration. K14-caSmad1 mice were viable, fertile, and showed relatively normal skin and HF development (Supplementary Figure S1b Global microarray analysis reveals changes in expression of online). K14-caSmad1 mice showed markedly increased cytoskeletal and cell migration–associated genes in the epidermis Smad1 expression in both the epidermis and HFs versus of K14-caSmad1 mice corresponding wild-type (WT) mice (Figure 2b and Supple- To define the genetic program regulated by Smad1 in epider- mentary Figure S1c online). TG genotype was confirmed by mal keratinocytes in the context of the mechanisms underlying western blot detection of FLAG-tag expression in dorsal skin alterations in the wound healing in K14-caSmad1 mice, global samples (Figure 2c). microarray analyses of the epidermal keratinocytes isolated Macroscopically, wound healing in K14-caSmad1 mice was from telogen skin on day 20 after birth (P20) from WT and delayed compared with WT controls, with visibly larger skin K14-caSmad1 was performed, as described previously (Fessing wounds at time-matched points (Figure 2d). Histomorpholo- et al., 2010; Mardaryev et al., 2011). Microarray data were gical analysis of skin wounds confirmed that the areas covered validated by qRT–PCR analyses of RNA samples isolated from by hyperproliferative epithelium and the epithelial tongue the epidermis of unwounded telogen skin, or from the wound length in K14-caSmad1 mice were significantly smaller at days epithelium obtained 3 or 5 days after wound infliction 3, 5, and 7 after wounding than that of controls (Figure 2e–g (Mardaryev et al., 2011). and Supplementary Figure S1d online). Bioinformatic analyses of the microarray data revealed 2- fold and higher changes in expression of 1,600 genes in the K14-caSmad1 mice show altered proliferation/apoptosis and epidermis of K14caSmad1 mice compared with WT controls changes in cytoskeletal organization in the wound epithelium (Figure 4a and Supplementary Tables S1 and S2 online). These To ascertain whether changes in the dynamics of epithelial genes belonged to different functional categories and encoded regeneration observed in the K14-caSmad1 mice were asso- distinct adhesion/extracellular matrix molecules, cell cycle/ ciated with altered keratinocyte proliferation and/or apoptosis, apoptosis regulators, cytoskeletal/cell motility markers, meta- a quantitative analysis of Ki-67 þ cells and cells positive for bolic enzymes, signaling/transcription regulators, and so on active caspase 3 was performed. In telogen skin of K14- (Figure 4a and Supplementary Tables S1 and S2 online). caSmad1 mice, the epidermis showed significantly fewer Ki- Among these functional categories, significant enrichment 67 þ keratinocytes (Figure 3a and b and Supplementary (Po0.01) was found for the genes that encode cytoskeletal/ Figure S1eonline) than in the controls. During healing, there cell motility–associated markers, and qRT–PCR validation was no difference in K14-caSmad1 wound epithelial prolif- showed significant downregulation in the expression of the eration at day 3, but there was a significantly lower proportion selected epidermal keratins (Krt1, Krt10, Krt16, Krt17)inthe of Ki-67 þ keratinocytes in K14-caSmad1 wound epithelium wound epithelium of K14-caSmad1 mice compared with WT on days 5 and 7 (Figure 3a and b and Supplementary controls (Figure 4b). Figure S1e online) after wounding compared with time- Furthermore, transcripts for MYOSIN VA (Myo5a), an actin- matched controls. In contrast, K14-caSmad1 mice displayed dependent protein required for cell motility (Cao et al., 2004; a higher proportion of active caspase-3 þ cells in the wound Sloane and Vartanian, 2007; Lan et al., 2010), as well as for

830 Journal of Investigative Dermatology (2014), Volume 134 CJ Lewis et al. Role of BMP Signaling in Skin Repair

Keratin 16 Ki67 Day 3 Day 5 Unwounded Day 5 80 WT 70 K14-caSmad1 60 GT 50 Wild type Wild type * ** 40 30 % Kl67+ cells ** 20 GT 10 0 Telogen Day 3 Day 5 Day 7 K14-caSmad1 K14-caSmad1

Active caspase 3 Keratin 17 Day 3 Day 5 25 ** WT 20 K14-caSmad1 GT

15 Wild type Wild type GT ** 10 **

% Caspase 3+ cells 5 GT 0 GT Day 3 Day 5 Day 7 GT K14-caSmad1

K14-caSmad1 GT

Figure 3. Quantitative analysis of proliferation and apoptosis and assessment of Keratin 16 and Keratin 17 expression in the wound epithelium of K14-caSmad1 (transgenic mice overexpressing a constitutively active form of Smad1 under K14 promoter) and wild-type (WT) mice. (a) Proliferative Ki-67 þ cells are seen in the basal layer of telogen skin and in the wound epithelium on day 5 after injury (arrowheads). (b) Significant reduction in Ki-67 þ cells in K14-caSmad1 telogen skin on days 5 and 7 after wounding versus WT. (c) Apoptotic active caspase 3 þ cells are seen in the wound epithelium at days 3 and 5 after wounding; inset illustrates cell-specific staining. (d) Significant increase in active caspase 3 þ cells in K14-caSmad1 wound epithelium at days 3, 5, and 7 after wounding versus WT (mean±SD, *Po0.01, **Po0.001, Student’s t-test). (e, f) Reduced expression of (e) Keratin 16 and (f) Keratin 17 in K14-caSmad1 wounds and keratinocytes were cuboidal (arrowheads), whereas those in WT mice were elongated (arrows). GT, granulation tissue. Bar ¼ 100 mm. other cell motility-associated genes, such as Ablim2 and significantly increased keratinocyte migration compared with Tubb6 (encoding actin-binding LIM protein family, member controls, suggesting that antagonism of BMP signaling accel- 2 and tubulin b6, respectively), were significantly more erated cell migration in this model (Figure 5a and b). strongly downregulated in K14-caSmad1 mice in response to Modulation of BMP activity in the keratinocytes also led to wounding than in controls (Figure 4b). These data suggested changes in cell morphology, which was determined by Alexa that excess of BMP/Smad1 signaling in epidermal keratino- Fluor 488-phalloidin staining detecting the endogenous actin cytes inhibits wound healing, at least in part, via alterations in filament network (Lengsfeld et al., 1974; Wulf et al., 1979; cytoskeletal organization and cell migration. Meyer et al., 2012). In the control group, the majority of migrating keratinocytes displayed an elongated appearance BMP inhibitory effects on keratinocyte migration are mediated with actin fibers seen across the cell body (Figure 5c), whereas by BMPR-1B the majority of cells migrating from BMP-4/7-treated explants To elucidate the effects of BMP signaling on keratinocyte had acquired a spherical shape (Figure 5c), and did not show a migration, ex vivo skin explants (Mazzalupo et al., 2002) were defined actin fiber network. Notably more polarized cell shapes treated with BMP-4/7, Noggin, or their combination, and cell were observed in NOGGIN-treated samples (Figure 5c), which migratory area from the explants was measured at different might reflect their accelerated migration (Figure 5b). Cell time points. Keratinocyte migration was significantly retarded morphology of the keratinocytes cotreated with BMP-4/7 and by BMP-4/7 treatment at both day 5 and day 7 (Po0.05) NOGGINwassimilartothatseenincontrolcells(Figure5c). compared with control explants (Figure 5a and b). NOGGIN In addition, the inhibitory effects of BMP on cell migration negated this BMP-induced inhibition of migration when were studied using transwell assay with primary mouse explants were exposed to both treatments and restored epidermal keratinocytes (PMEKs) as previously described keratinocyte movement back to that seen in controls (Yin et al., 2005; Merlo et al., 2009). BMP-4/7 significantly (Figure 5a and b). Interestingly, NOGGIN used alone slowed (Po0.001) PMEK migration compared with control

www.jidonline.org 831 CJ Lewis et al. Role of BMP Signaling in Skin Repair

Downregulated (633) Upregulated (697) Transport 2.25% Transport 4.2% Adhesion/extracellular matrix 3.14% Translation 1.35% Translation 0.33% Adhesion/extracellular matrix 2.88% Transcription regulators 7.86% Cell cycle/apoptosis 4.04% Transcription regulators 10.73% Cell cycle/apoptosis 6.19% Cytoskeleton/differentiation, cell motility 11.15% Cytoskeleton/differentaition, cell motility 8.41% Signaling 23.05% Signaling 15.04%

Metabolism 18.71% Metabolism 16.81% RNA processing 2.84% RNA processing 7.19% Proteolysis 6.89% Proteolysis 8.52% Nuclear and chromatin assembly 5.31% Nuclear and chromatin assembly 9.29% Protein folding 1.42% Protein folding 0.88% Others 11.98%

Krt1 Krt16 Krt17 16 80 10 WT WT WT K14-caSmad1 9 8 K14-caSmad1 12 K14-caSmad1 60 7 6 8 40 5 *

mRNA 4 mRNA mRNA 3 4 20 * 2 ** ** ** 1 0 0 0 Tel d3 d5 Tel d3 d5 Tel d3 d5

Ablim2 Tubb6 14 Myo5a 50 25 WT WT WT 12 20 40 K14-caSmad1 K14-caSmad1 10 K14-caSmad1 8 30 15 6

mRNA 10 mRNA 20 mRNA 4 * 10 * 5 ** 2 * ** 0 0 0 Tel d3 d5 Tel d3 d5 Tel d3 d5

Figure 4. Global gene expression proﬁling of analyses of the epidermal keratinocytes isolated from telogen skin of wild-type (WT) and K14-caSmad1 (transgenic mice overexpressing a constitutively active form of Smad1 under K14 promoter) mice. (a) Microarray analysis of the global gene expression in the keratinocytes K14-caSmad1 versus WT: functional assignments of the genes with altered expression. (b) K14-caSmad1 mice displayed a signiﬁcant decrease in Krt1, Krt16, and Krt17 expression (upper panel) and Myo5a, Ablim2,andTubb6 (lower panel) in response to wounding versus WT (mean±SD, *Po0.01, **Po0.0001, Student’s t-test). d3, day 3; d5, day 5; Tel, telogen.

keratinocytes, whereas it was negated when BMP was DISCUSSION co-administered with Noggin (Figure 5d). Noggin alone In this study, we investigated the effects of BMP signaling on significantly (Po0.04) accelerated migration compared with epidermal keratinocytes during skin repair. Using TG mice controls (Figure 5d). Furthermore, cell migration was signifi- that overexpress Smad1 in basal epidermal keratinocytes, as cantly (Po0.0001) inhibited in the keratinocytes obtained well as ex vivo and in vitro models, we have illustrated that from K14-caSmad1 versus WT mice (Figure 5e). BMP signaling slows wound healing by suppressing keratino- To further define the role that individual BMP receptors play cyte proliferation and increasing apoptosis in the wound in the regulation of keratinocyte migration, PMEKs were epithelium, as well as by attenuating keratinocyte migration, transfected with siRNA to BMPR-1A or BMPR-1B and pro- an effect that is mediated, at least in part, via BMPR-1B. These cessed for transwell plate expression assay. BMPR-1B silen- data are consistent with previous observations that BMP cing accelerated (Po0.01) PMEK migration compared with signaling negates the facets of proliferation and migration controls (Figure 5f); however, no effect was seen with BMPR- seen in wound repair (Kaiser et al., 1998; Sharov et al., 2006; 1A silencing. In order to delineate which genes may be Ahmed et al., 2011). responsible for BMPR-1B-mediated acceleration in migration, The expression pattern of BMP receptors that we observed transfected PMEKs were processed for qRT–PCR. Following in telogen skin was consistent with that previously described, confirmation of significant (Po0.001) Bmpr-1B silencing by with BMPR-1A localized to the HF bulge (Blanpain and Fuchs, Bmpr-1B siRNA (Figure 5g), we found increased expression of 2006) and BMPR-1B in the suprabasal epidermis (Hwang Myo5a (Po0.02), Krt16 (Po0.01), and Krt17 (Po0.001) in et al., 2001; Sharov et al., 2003; Yu et al., 2010), where they BMPR-1B-silenced keratinocytes compared with controls. This were colocalized with the downstream signaling component suggests that BMP signaling can cause delayed re-epitheliali- pSmad-1/5/8. The prominent expression of BMPR-1A was zation, at least in part, by inhibition of keratinocyte cytoske- consistent with its role in maintaining stem cell quiescence letal organization and migration, the effects that are mediated, in conjunction with BMP ligands (Blanpain et al., 2004; at least in part, by BMPR-1B. Blanpain and Fuchs, 2006; Zhang et al., 2006; Kobielak

832 Journal of Investigative Dermatology (2014), Volume 134 CJ Lewis et al. Role of BMP Signaling in Skin Repair

Day 3 Day 5 Day 7 2 0.06 * Control BMP-4/7 NOGGIN BMP-4/7 + NOGGIN 0.05 0.04 0.03 ** 0.02 * Explant Explant Explant Explant 0.01 *

Area of migration, mm 0

Control BMP-4/7 NOGGIN BMP-4/7+ NOGGIN

Control BMP-4/7 NOGGIN BMP-4/7 + NOGGIN

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0 PMEKs/microscopic field 0 PMEKs/microscopic field WT K14- Cntr Bmpr-1A Bmpr-1B Relative mRNA expression Bmpr-1B Krt16 Krt17 Myo5a Cntr caSmad1 siRNA siRNA siRNA BMP-4/7 NOGGIN BMP-4/7

+ NOGGIN

Figure 5. Bone morphogenetic protein (BMP) pathway modulation alters keratinocyte migration and morphology. (a, b) Skin explant model. (b) Bmp-4/7 inhibited migration at days 5 and 7 (*Po0.05). Noggin alone increased migration at days 5 (**Po0.01) and 7 (*Po0.05). (c) Phalloidin staining of actin filament networks; control keratinocytes were elongated with actin fibers across the cell body; Bmp-4/7-treated keratinocytes were spherical and lacked defined actin fibers (arrowheads); Noggin increased cell polarity and actin formation. (d–f) Transwell assay: Bmp-4/7 inhibited migration (**Po0.001); Noggin (*Po0.05) increased migration; (e) significant delay in primary mouse epidermal keratinocyte (PMEK) migration obtained from K14-caSmad1 mice (transgenic mice overexpressing a constitutively active form of Smad1 under K14 promoter) (***Po0.0001); Bmpr-1B knockdown accelerated (*Po0.01) PMEK movement (f). Cntr, control. (g) Quantitative real-time reverse-transcriptase–PCR (qRT–PCR) confirmation of Bmpr-1B silencing (***Po0.001); Bmpr-1B small interfering RNA (siRNA) upregulated Myo5a (*Po0.02), Krt16 (**Po0.01), and Krt17 (***Po0.001) transcripts. Bar ¼ 100 mm; mean±SD, Student’s t-test. et al., 2007; Fuchs, 2008) indeed, the localized downre- During wound healing, the expression pattern of BMPR-1B gulation of bulge BMPR-1A expression in those HFs immedia- also show cytoplasmic staining in the epithelial tongue tely adjacent to the wound, together with reduced levels of keratinocytes, as opposed to that expected on the cell mem- BMP ligands and Smads, suggested a local suppression of the brane, which was seen in unwounded epidermis. Intracellular BMP axis in response to wounding (Mathura et al., 2000). localization of both BMPRs has been described in human Thus, our data suggest that downregulation of both BMP osteoblasts (Singhatanadgit et al., 2008), with BMPR-1B in ligands and BMPR-1A may facilitate an increase in HF stem particular found in the perinuclear region, suggesting that in cell activity, which accompanies wound healing (Botchkarev the absence of an appropriate ligand, BMPR-1B undergoes et al., 1999; Hwang et al., 2001; Kobielak et al., 2003; Ito internalization or represents newly synthesized receptors et al., 2005; Cotsarelis, 2006; Zhang et al., 2006; Ito and before their transport to the cell surface (Singhatanadgit Cotsarelis, 2008; Plikus et al., 2008; Wong and Reiter, 2011). et al., 2008) to play roles in wound repair. The extensive expression of BMPR-1B and pSmad-1/5/8 in The K14-caSmad1 mouse model used in this study provided the wound epithelium implicates an involvement of BMP in vivo evidence that constitutive BMP signal activation signaling in the control of skin repair. Within developing skin, delays wound healing. These data are consistent with BMPR-1B is involved in the control of cell differentiation of previous studies showing that other components of the suprabasal epidermal layers (Botchkarev and Sharov, 2004; TGF-b signaling pathways, such as Smad2/3 and Smad4, Pardali et al., 2005; Plikus et al., 2008; Fessing et al., 2010). negatively regulate skin repair (Ashcroft et al., 1999;

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Flanders et al., 2003; Hosokawa et al.,2005;Tomikawaet al., proliferation and migration in epidermal keratinocytes during 2012; Yang et al., 2012). Our data provide evidence wound healing, this analysis will help in the development of that Smad1, a crucial downstream regulator of the BMP antagonists for the management of chronic wounds. BMP-Smad pathway (Botchkarev and Sharov, 2004), slows wound healing by attenuating keratinocyte proliferation MATERIALS AND METHODS and migration and augmenting wound epithelial apoptosis. Animals and tissue collection It has previously been shown that activation of BMP Animal studies were performed under protocols approved by Boston signaling slows keratinocyte proliferation (Drozdoff et al., University (Boston, MA) and Home Office Project License (UK). K14- 1994; Kaiser et al., 1998; D’Souza et al., 2001; McDonnell caSmad1 mice were generated on FVB background using a TG et al., 2001; Park and Morasso, 2002; Ahmed et al., 2011), construct containing human K14 promoter, FLAG-tagged human whereas overexpression of the BMP antagonist NOGGIN complementary DNA encoding phospho-mimetic activated Smad1 results in epidermal hyperplasia due to hyperproliferation (EVE; provided by E De Robertis, Howard Hughes Medical Institute, (Sharov et al., 2009) and suppressed apoptosis (Sharov et al., University of California, Los Angeles, CA) (Fuentealba et al.,2007), 2003). Thus, our study suggests that Smad1 has an important and human growth hormone poly-A sequence (Sharov et al.,2009). function in regulating epidermal homeostasis during skin Skin samples were collected from dorsal skin of newborn mice, as repair. well as from P20 mice with skin wounds (days 0, 3, 5, and 7 after Our data also assert that BMP signaling, and in particular wounding), as previously described (Kaiser et al., 1998; Mardaryev BMPR-1B, negatively regulates keratinocyte migration during et al., 2011; Wong et al., 2011). In each experiment, at least four to wound repair. Microarray analyses reveal downregulated five mice of each strain per time point were used for analyses in both expression of a number of genes encoding distinct cytoskeletal experimental and control groups. proteins (Krt1,Krt10,Krt16,Krt17) and cell motility markers (Myo5a, Ablim2, and Tubb6) in the epidermis of K14- Immunohistochemistry and western blotting caSmad1 mice versus WT controls. In K14-caSmad1 mice, Formalin-fixed cryosections (10 mM) were incubated with primary the epithelial tongue was shorter and contained few antibody (Supplementary Table S3 online) overnight followed by polarized migratory keratinocytes (Kurosaka and Kashina, application of the corresponding Alexa-546- or Alexa-555-labeled 2008), suggesting that Smad1 activation slows migration. antibodies (Invitrogen, Paisley, UK) for 45 minutes at 37 1C. To detect Furthermore, BMPR-1B knockdown in keratinocytes caused endogenous and transgenic expression of Smad1 in K14-caSmad1 increased expression of Krt16 and Krt17 that are typically and WT mice, rabbit polyclonal anti-Smad1 antibody (Abcam; Cam- upregulated following wounding and play roles in the bridge, UK, Supplementary Table S3 online) was used. Cell nuclei cytoplasmic keratin network reorganization (Paladini et al., were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; Vec- 1996; McGowan and Coulombe, 1998; Tomic-Canic et al., tor Biolabs, Peterborough, UK). Image analysis was performed using a 1998; Wawersik and Coulombe, 2000; Wawersik et al., 2001; fluorescent microscope in combination with DS-C1 digital camera Mazzalupo et al., 2003; Hosokawa et al., 2005; Patel et al., and ACT-2U image analysis software (Nikon, Amsterdam, 2006; Moll et al., 2008). The Netherlands). Western blot analysis of total tissue proteins Consistent with these data, cultured BMP-treated keratino- obtained from the extracts of full-thickness skin of K14-caSmad1 cytes lacked the defined cytoplasmic actin filament network and WT mice was performed using mouse mAb EPR4759 against required for movement, whereas Noggin-treated keratinocytes DKK1 (FLAG) epitope (Origene, Rockville, MD), as previously displayed an accelerated migration and showed elongated described (Sharov et al., 2006). shape with endogenous actin filaments visible (Kurosaka and Kashina, 2008; Fletcher and Mullins, 2010; Firat-Karalar and Microarray and qRT–PCR analysis Welch, 2011; Reymann et al., 2012; Allard and Mogilner, For microarray analysis, total RNA was isolated from primary 2013). In addition, BMP signaling shows a negative effect on epidermal keratinocytes of P20 WT and TG mice using RNeasy kit the expression of Myo5a, an actin-dependent molecular motor (Qiagen, Manchester, UK), and processed for one-round RNA involved in cell motility and metastasis (Wang et al., 1996; amplification using RiboAmp RNA Amplification Kit (Molecular Cao et al., 2004; Eppinga et al., 2008; Kurosaka and Kashina, Devices, Sunnyvale, CA). Gene expression array analysis was 2008; Lan et al., 2010). These data are consistent with performed by Mogene LLC (St Louis, MO) using 44K Whole Mouse previous results showing the inhibitory effects of the TGF-b Genome 60-mer oligo-microarray (manufactured by Agilent family on cell migration during skin repair (Tsuboi et al., 1992; Technologies, Santa Clara, CA). Functional annotation of the over- Hosokawa et al.,2005),aswellaswithdatademonstrating represented and underrepresented genes was performed as described that Smad2 (Hosokawa et al., 2005), Smad3 (Ashcroft et al., before (Sharov et al., 2009; Fessing et al., 2010) using the NIA Array 1999; Flanders et al., 2003), and Smad4 (Yang et al., 2012) Analysis software (http://lgsun.grc.nia.nih.gov/ANOVA/), and inhibit cell movement in other models. enrichment of the genes in different functional categories was However, additional studies including chromatin immuno- assessed by using hypergeometric or Fisher’s exact tests. Microarray precipitation–sequencing and reporter assay analyses are data have been deposited to the Gene Expression Omnibus. For qRT– required to define the complete set of the downstream target PCR, total RNA was isolated from snap-frozen samples of full- genes that are regulated by the BMP/Smad pathway in thickness wounds using TRIzol (Invitrogen) (Mardaryev et al.,2010) keratinocytes during wound healing. Together with the results followed by conversion into complementary DNA using Reverse of this study, demonstrating that BMPs directly inhibit cell Transcription System (Promega, Southampton, UK). PCR primers were

834 Journal of Investigative Dermatology (2014), Volume 134 CJ Lewis et al. Role of BMP Signaling in Skin Repair

designed with Beacon Designer software (Premier Biosoft, Palo Alto, experiment) was counted and compared. Statistical analysis was CA; Supplementary Table S4 online). qRT–PCR was performed performed using unpaired Student’s t-test; differences were deemed on MyiQ single-color real-time PCR detection system (Bio-Rad, significant if Po0.05. Hertfordshire, UK) using SYBR Green master mix (Applied Biosys- tems, Paislay, UK). Differences between samples and controls were calculated using the Genex database software (Bio-Rad) based on the CONFLICT OF INTEREST DD Ct ( Ct) equitation method and normalized to Gapdh. Data from The authors state no conflict of interest. triplicates were pooled and statistical analysis was performed using unpaired Student’s t-test. ACKNOWLEDGMENTS Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases, part of the National Institutes Quantitative wound histomorphometry of Health, under award number K01AR056771, AR049778 to AAS. The Wound samples (n ¼ 8–10 from each strain) were processed for content is solely the responsibility of the authors and does not necessarily hematoxylin and eosin staining (Sharov et al., 2006), and analyzed represent the official views of the National Institutes of Health. This study was using VisiCam (VWR International, Leicestershire, UK) software. The also supported in part by the Medical Research Council UK via grant MR/ 2 K011324/1 to NVB, and by charitable donations to the Bradford Plastic Surgery epithelial tongue area (mm ) and length (mm) were measured and and Burns Research Unit. We thank Dr Eddy M De Robertis (Howard Hughes compared at time-matched intervals. To assess cell proliferation and Medical Institute, University of California, Los Angeles, CA) for plasmid apoptosis, the number of Ki-67 þ ,caspase-3þ , and DAPI þ cells encoding caSmad1 cDNA. was counted along the basal layer of the wound epithelial tongue or intact epidermis at time-matched intervals using ImageJ software SUPPLEMENTARY MATERIAL (National Institutes of Health, Bethesda, MD) as previously described Supplementary material is linked to the online version of the paper at http:// (Mardaryev et al., 2011). Statistical analysis was performed using www.nature.com/jid unpaired Student’s t-test; differences were deemed significant if Po0.05. REFERENCES Ahmed MI, Mardaryev AN, Lewis CJ et al. (2011) MicroRNA-21 is an Ex vivo skin explant migration assay important downstream component of BMP signalling in epidermal Explant migration assay was performed as previously described keratinocytes. J Cell Sci 124:3399–404 (Mazzalupo et al., 2002). Explants were treated daily with 1 mgml 1 Allard J, Mogilner A (2013) Traveling waves in actin dynamics and cell motility. Curr Opin Cell Biol 25:107–15 recombinant BMP-4/7 (R&D Systems, Minneapolis, MN), 600 ng ml 1 Andl T, Ahn K, Kairo A et al. (2004) Epithelial BMPR1a regulates differentiation NOGGIN (R&D Systems), 1 mgml 1 of BMP-4/7 and 600 ng ml 1 of and proliferation in postnatal hair follicles and is essential for tooth NOGGIN combined, or BSA; all treatments were performed in development. Development 131:2257–68 triplicate. Photomicrographs were taken every 48 hours using a light Ashcroft GS, Yang X, Glick AB et al. (1999) Mice lacking Smad3 show microscope (Leitz Labovert, Wetzlar, Germany). Image analysis was accelerated wound healing and an impaired local inflammatory response. performed using the Visicam software (VWR International); the Nat Cell Biol 1:260–6 migratory area (mm2) of PMEKs from the skin explant was measured Barrientos S, Stojadinovic O, Golinko MS et al. (2008) Growth factors and at each time point. Following fixation, skin discs were removed and the cytokines in wound healing. Wound Repair Regen 16:585–601 remaining keratinocytes were stained with Alexa Fluor 488-phalloidin Blanpain C, Fuchs E (2006) Epidermal stem cells of the skin. Annu Rev Cell Dev Biol 22:339–73 antibody (Invitrogen). Blanpain C, Lowry WE, Geoghegan A et al. (2004) Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell Cell culture and transwell migration assay niche. Cell 118:635–48 PMEKs were prepared from newborn mice as described previously Botchkarev VA, Botchkareva NV, Nakamura M et al. (2001) NOGGIN is (Lichti et al., 2008) and were grown in EMEM (Lonza, UK) required for induction of hair follicle growth phase in postnatal skin. supplemented with 4% chelated fetal bovine serum (Gibco, Paislay, FASEB J 15:2205–14 UK). Transwell assay was performed as previously described (Yin Botchkarev VA, Botchkareva NV, Roth W et al. (1999) NOGGIN is a et al., 2005; Merlo et al., 2009). Following attachment, PMEKs were mesenchymally derived stimulator of hair-follicle induction. Nat Cell Biol 1:158–64 either transfected with mouse smartpool siRNAs directed against Botchkarev VA, Sharov AA (2004) BMP signaling in the control of skin BMPR-1A or BMPR-1B and a nontargeting control (Thermo Scientific, development and hair follicle growth. Differentiation 72:512–26 Loughborough, UK) using Lipofectamine RNAimax (Invitrogen) 1 Cao TT, Chang W, Masters SE et al. (2004) Myosin-Va binds to and according to the manufacturers’ protocol, or treated with 1 mgml mechanochemically couples microtubules to actin filaments. Mole Biol recombinant BMP-4/7 (R&D Systems), 600 ng ml 1 NOGGIN (R&D Cell 15:151–61 1 1 Systems), 1 mgml of BMP-4/7 and 600 ng ml of NOGGIN Cotsarelis G (2006) Epithelial stem cells: a folliculocentric view. J Invest combined, or BSA; all transfection and treatments were performed Dermatol 126:1459–68 in duplicate (Ahmed et al., 2011). PMEKs were allowed to migrate Coulombe PA (1997) Towards a molecular definition of keratinocyte activation over 48 hours through the insert membrane, after which cells that after acute injury to stratified epithelia. Biochem Biophys Res Commun 236:231–8 adherent to the top surface of the membrane were removed with a D’Souza SJ, Pajak A, Balazsi K et al. (2001) Ca2 þ and BMP-6 signaling cotton swab; cells that had migrated to the bottom surface were regulate E2F during epidermal keratinocyte differentiation. JBiolChem formalin fixed and counterstained with DAPI (Vector Labs). The 276:23531–8 number of DAPI þ nuclei of migrated PMEKs per microscopic field Driscoll MK, McCann C, Kopace R et al. (2012) Cell shape dynamics: from (10 randomly selected fields/transwell from two transwells per waves to migration. PLoS Comput Biol 8:e1002392

www.jidonline.org 835 CJ Lewis et al. Role of BMP Signaling in Skin Repair

Drozdoff V, Wall NA, Pledger WJ (1994) Expression and growth inhibitory Langton AK, Herrick SE, Headon DJ (2008) An extended epidermal response effect of decapentaplegic Vg-related protein 6: evidence for a regulatory heals cutaneous wounds in the absence of a hair follicle stem cell role in keratinocyte differentiation. Proc Natl Acad Sci USA 91:5528–32 contribution. J Invest Dermatol 128:1311–8 Eppinga RD, Peng IF, Lin JL et al. (2008) Opposite effects of overexpressed Lengsfeld AM, Low I, Wieland T et al. (1974) Interaction of phalloidin with myosin Va or heavy meromyosin Va on vesicle distribution, cytoskeleton actin. Proc Natl Acad Sci USA 71:2803–7 organization, and cell motility in nonmuscle cells. Cell Motil Cytoskeleton Levy V, Lindon C, Zheng Y et al. (2007) Epidermal stem cells arise from the 65:197–215 hair follicle after wounding. FASEB J 21:1358–66 Falanga V (1993) Growth factors and wound healing. JDermatolSurgOncol Li J, Chen J, Kirsner R (2007) Pathophysiology of acute wound healing. Clin 19:711–4 Dermatol 25:9–18 Fessing MY, Atoyan R, Shander B et al. (2010) BMP signaling induces cell-type- Lichti U, Anders J, Yuspa SH (2008) Isolation and short-term culture of primary specific changes in gene expression programs of human keratinocytes and keratinocytes, hair follicle populations and dermal cells from newborn fibroblasts. J Invest Dermatol 130:398–404 mice and keratinocytes from adult mice for in vitro analysis and for Firat-Karalar EN, Welch MD (2011) New mechanisms and functions of actin grafting to immunodeficient mice. Nat Protoc 3:799–810 nucleation. Curr Opin Cell Biol 23:4–13 Lu CP, Polak L, Rocha AS et al. (2012) Identification of stem cell populations in Flanders KC, Major CD, Arabshahi A et al. (2003) Interference with transform- sweat glands and ducts reveals roles in homeostasis and wound repair. ing growth factor-beta/ Smad3 signaling results in accelerated healing of Cell 150:136–50 wounds in previously irradiated skin. Am J Pathol 163:2247–57 Mardaryev AN, Ahmed MI, Vlahov NV et al. (2010) Micro-RNA-31 controls Fletcher DA, Mullins RD (2010) Cell mechanics and the cytoskeleton. Nature hair cycle-associated changes in gene expression programs of the skin and 463:485–92 hair follicle. FASEB J 24:3869–81 Fuchs E (2008) Skin stem cells: rising to the surface. JCellBiol180:273–84 Mardaryev AN, Meier N, Poterlowicz K et al. (2011) Lhx2 differentially Fuentealba LC, Eivers E, Ikeda A et al. (2007) Integrating patterning signals: regulates Sox9, Tcf4 and Lgr5 in hair follicle stem cells to promote Wnt/GSK3 regulates the duration of the BMP/Smad1 signal. Cell epidermal regeneration after injury. Development 138:4843–52 131:980–93 Mathura JR Jr., Jafari N, Chang JT et al. (2000) Bone morphogenetic proteins-2 Hosokawa R, Urata MM, Ito Y et al. (2005) Functional significance of Smad2 in and -4: negative growth regulators in adult retinal pigmented epithelium. regulating basal keratinocyte migration during wound healing. J Invest Invest Ophthalmol Vis Sci 41:592–600 Dermatol 125:1302–9 Mazzalupo S, Wawersik MJ, Coulombe PA (2002) An ex vivo assay to assess Hsu YC, Pasolli HA, Fuchs E (2011) Dynamics between stem cells, niche, and the potential of skin keratinocytes for wound epithelialization. J Invest progeny in the hair follicle. Cell 144:92–105 Dermatol 118:866–70 Hwang EA, Lee HB, Tark KC (2001) Comparison of bone morphogenetic Mazzalupo S, Wong P, Martin P et al. (2003) Role for keratins 6 and 17 during protein receptors expression in the fetal and adult skin. Yonsei Med J wound closure in embryonic mouse skin. Dev Dyn 226:356–65 42:581–6 McDonnell MA, Law BK, Serra R et al. (2001) Antagonistic effects of TGFbeta1 Ito M, Cotsarelis G (2008) Is the hair follicle necessary for normal wound and BMP-6 on skin keratinocyte differentiation. Exp Cell Res 263:265–73 healing? J Invest Dermatol 128:1059–61 McGowan KM, Coulombe PA (1998) Onset of keratin 17 expression coincides Ito M, Liu Y, Yang Z et al. (2005) Stem cells in the hair follicle bulge contribute with the definition of major epithelial lineages during skin development. to wound repair but not to homeostasis of the epidermis. Nat Med JCellBiol143:469–86 11:1351–4 Merlo S, Frasca G, Canonico PL et al. (2009) Differential involvement of Jamora C, DasGupta R, Kocieniewski P et al. (2003) Links between signal estrogen receptor alpha and estrogen receptor beta in the healing transduction, transcription and adhesion in epithelial bud development. promoting effect of estrogen in human keratinocytes. J Endocrinol Nature 422:317–22 200:189–97 Kaiser S, Schirmacher P, Philipp A et al. (1998) Induction of bone morpho- Meyer M, Muller AK, Yang J et al. (2012) FGF receptors 1 and 2 are key genetic protein-6 in skin wounds. Delayed reepitheliazation and scar regulators of keratinocyte migration in vitro and in wounded skin. J Cell formation in BMP-6 overexpressing transgenic mice. J Invest Dermatol Sci 125:5690–701 111:1145–52 Ming Kwan K, Li AG, Wang XJ et al. (2004) Essential roles of BMPR-IA Kandyba E, Leung Y, Chen YB et al. (2013) Competitive balance of intrabulge signaling in differentiation and growth of hair follicles and in skin BMP/Wnt signaling reveals a robust gene network ruling stem cell tumorigenesis. Genesis 39:10–25 homeostasis and cyclic activation. Proc Natl Acad Sci USA 110:1351–6 Miyazono K, Kamiya Y, Morikawa M (2010) Bone morphogenetic protein Kasper M, Jaks V, Are A et al. (2011) Wounding enhances epidermal receptors and signal transduction. J Biochem 147:35–51 tumorigenesis by recruiting hair follicle keratinocytes. Proc Natl Acad Moll R, Divo M, Langbein L (2008) The human keratins: biology and Sci USA 108:4099–104 pathology. Histochem Cell Biol 129:705–33 Kobielak K, Pasolli HA, Alonso L et al. (2003) Defining BMP functions in the Munz B, Smola H, Engelhardt F et al. (1999) Overexpression of activin A in the hair follicle by conditional ablation of BMP receptor IA. JCellBiol skin of transgenic mice reveals new activities of activin in epidermal 163:609–23 morphogenesis, dermal fibrosis and wound repair. EMBO J 18:5205–15 Kobielak K, Stokes N, de la Cruz J et al. (2007) Loss of a quiescent niche but Paladini RD, Takahashi K, Bravo NS et al. (1996) Onset of re-epithelialization not follicle stem cells in the absence of bone morphogenetic protein after skin injury correlates with a reorganization of keratin filaments in signaling. Proc Natl Acad Sci USA 104:10063–8 wound edge keratinocytes: defining a potential role for keratin 16. JCell Krause C, Guzman A, Knaus P (2011) NOGGIN. Int J Biochem Cell Biol 43: Biol 132:381–97 478–81 Pardali K, Kowanetz M, Heldin CH et al. (2005) Smad pathway-specific Kulessa H, Turk G, Hogan BL (2000) Inhibition of BMP signaling affects growth transcriptional regulation of the cell cycle inhibitor p21(WAF1/Cip1). J and differentiation in the anagen hair follicle. EMBO J 19:6664–74 Cell Physiol 204:260–72 Kurosaka S, Kashina A (2008) Cell biology of embryonic migration. Birth Park GT, Morasso MI (2002) Bone morphogenetic protein-2 (BMP-2) transacti- Defects Res C Embryo Today 84:102–22 vates Dlx3 through Smad1 and Smad4: alternative mode for Dlx3 Lan L, Han H, Zuo H et al. (2010) Upregulation of myosin Va by induction in mouse keratinocytes. Nucleic Acids Res 30:515–22 Snail is involved in cancer cell migration and metastasis. Int J Cancer Park HY, Wu C, Yaar M et al. (2009) Role of BMP-4 and its signaling pathways 126:53–64 in cultured human melanocytes. Int J Cell Biol 2009:750482

836 Journal of Investigative Dermatology (2014), Volume 134 CJ Lewis et al. Role of BMP Signaling in Skin Repair

Patel GK, Wilson CH, Harding KG et al. (2006) Numerous keratinocyte Tsuboi R, Sato C, Shi CM et al. (1992) Stimulation of keratinocyte migration by subtypes involved in wound re-epithelialization. J Invest Dermatol growth factors. J Dermatol 19:652–3 126:497–502 Walsh DW, Godson C, Brazil DP et al. (2010) Extracellular BMP-antagonist Plikus MV, Mayer JA, de la Cruz D et al. (2008) Cyclic dermal BMP signalling regulation in development and disease: tied up in knots. Trends Cell Biol regulates stem cell activation during hair regeneration. Nature 451: 20:244–56 340–4 Wang FS, Wolenski JS, Cheney RE et al. (1996) Function of myosin-V in Reymann AC, Boujemaa-Paterski R, Martiel JL et al. (2012) Actin network ﬁlopodial extension of neuronal growth cones. Science 273:660–3 architecture can determine myosin motor activity. Science 336:1310–4 Wankell M, Munz B, Hu¨bner G et al. (2001) Impaired wound healing in Schafer M, Werner S (2007) Transcriptional control of wound repair. Annu Rev transgenic mice overexpressing the activin antagonist follistatin in the Cell Dev Biol 23:69–92 epidermis. EMBO J 20:5361–72 Sharov AA, Fessing M, Atoyan R et al. (2005) Bone morphogenetic Wawersik M, Coulombe PA (2000) Forced expression of keratin 16 alters the protein (BMP) signaling controls hair pigmentation by means of cross- adhesion, differentiation, and migration of mouse skin keratinocytes. Mol talk with the melanocortin receptor-1 pathway. Proc Natl Acad Sci U S A Biol Cell 11:3315–27 102:93–8 Wawersik MJ, Mazzalupo S, Nguyen D et al. (2001) Increased levels of keratin 16 alter epithelialization potential of mouse skin keratinocytes in vivo and Sharov AA, Mardaryev AN, Sharova TY et al. (2009) Bone morphogenetic ex vivo. Mol Biol Cell 12:3439–50 protein antagonist noggin promotes skin tumorigenesis via stimulation of the Wnt and Shh signaling pathways. Am J Pathol 175:1303–14 Wong SY, Reiter JF (2011) Wounding mobilizes hair follicle stem cells to form tumors. Proc Natl Acad Sci USA 108:4093–8 Sharov AA, Sharova TY, Mardaryev AN et al. (2006) Bone morphogenetic protein signaling regulates the size of hair follicles and modulates the Wong VW, Sorkin M, Glotzbach JP et al. (2011) Surgical approaches to create expression of cell cycle-associated genes. Proc Natl Acad Sci USA murine models of human wound healing. J Biomed Biotechnol 103:18166–71 2011:969618 Sharov AA, Weiner L, Sharova TY et al. (2003) NOGGIN overexpression Wulf E, Deboben A, Bautz FA et al. (1979) Fluorescent phallotoxin, a tool for inhibits eyelid opening by altering epidermal apoptosis and differentia- the visualization of cellular actin. Proc Natl Acad Sci USA 76:4498–502 tion. EMBO J 22:2992–3003 Yaar M, Wu C, Park HY et al. (2006) Bone morphogenetic protein-4, a novel modulator of melanogenesis. JBiolChem281:25307–14 Shaw TJ, Martin P (2009) Wound repair at a glance. JCellSci122:3209–13 Yang L, Li W, Wang S et al. (2012) Smad4 disruption accelerates keratinocyte Singh SK, Abbas WA, Tobin DJ (2012) Bone morphogenetic proteins reepithelialization in murine cutaneous wound repair. Histochem Cell differentially regulate pigmentation in human skin cells. J Cell Sci 125: Biol 138:573–82 4306–19 Yin T, Getsios S, Caldelari R et al. (2005) Plakoglobin suppresses keratinocyte Singhatanadgit W, Mordan N, Salih V et al. (2008) Changes in bone motility through both cell-cell adhesion-dependent and -independent morphogenetic protein receptor-IB localisation regulate osteogenic mechanisms. Proc Natl Acad Sci USA 102:5420–5 responses of human bone cells to bone morphogenetic protein-2. Int J Biochem Cell Biol 40:2854–64 Yu X, Espinoza-Lewis RA, Sun C et al. (2010) Overexpression of constitutively active BMP-receptor-IB in mouse skin causes an ichthyosis-vulgaris-like Sloane JA, Vartanian TK (2007) Myosin Va controls oligodendrocyte morpho- disease. Cell Tissue Res 342:401–10 genesis and myelination. J Neurosci 27:11366–75 Yuhki M, Yamada M, Kawano M et al. (2004) BMPR1A signaling is necessary Taylor G, Lehrer MS, Jensen PJ et al. (2000) Involvement of follicular stem for hair follicle cycling and hair shaft differentiation in mice. Develop- cells in forming not only the follicle but also the epidermis. Cell 102: ment 131:1825–33 451–61 Zhang J, He XC, Tong WG et al. (2006) Bone morphogenetic protein signaling Tomic-Canic M, Komine M, Freedberg IM et al. (1998) Epidermal signal inhibits hair follicle anagen induction by restricting epithelial stem/ transduction and transcription factor activation in activated keratinocytes. progenitor cell activation and expansion. Stem Cells 24:2826–39 J Dermatol Sci 17:167–81 Zimmerman LB, De Jesus-Escobar JM, Harland RM (1996) The Spemann Tomikawa K, Yamamoto T, Shiomi N et al. (2012) Smad2 decelerates re- organizer signal noggin binds and inactivates bone morphogenetic epithelialization during gingival wound healing. J Dent Res 91:764–70 protein 4. Cell 86:599–606