Overexpression of CD109 in the Epidermis Differentially Regulates ALK1 Versus ALK5 Signaling and Modulates Extracellular Matrix Synthesis in the Skin  Joshua Vorstenbosch, Christopher M. Nguyen, Shufeng Zhou, You Jung Seo, Aya Siblini, Kenneth W. Finnson, Albane A. Bizet, Simon D. Tran, Anie Philip  Journal of Investigative Dermatology  Volume 137, Issue 3, Pages 641-649 (March 2017) DOI: 10.1016/j.jid.2016.09.039 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Overexpression of CD109 in the epidermis does not affect dermal architecture but alters ALK5 versus ALK1 expression and favors signaling through the Smad1/5 pathway over the Smad2/3 pathway. (a) Hematoxylin and eosin and Masson Trichrome staining of WT and CD109 transgenic mouse skin show similar epidermal and dermal features (left 2 panels). Scale bar = 50 μm). Immunohistochemistry of skin (a, right 5 panels) and (b) immunoblot analyses of skin protein extract (right panel) and densitometric analysis of immunoblot data (b, left panel) show increased ALK1 expression, decreased ALK5 expression, increased pSmad1/5, and decreased phospho-Smad2 (pmad2) and phosphorylated Smad3 (pSmad3) in CD109 transgenic mice compared with wild-type littermates. (c) CD109 transgenic mouse skin shows reduced TGF-β1 levels compared with WT littermate skin, as detected by immunohistochemistry (left panel). Scale bar = 50 μm). Cultured CD109 transgenic mouse keratinocytes also show reduced TGF-β1 levels compared with cultured WT littermate keratinocytes as detected by real-time PCR (c, right panel). ∗P < 0.05, ∗∗P < 0.005. H&E, hematoxylin and eosin; TG, transgenic; TGF-β, transforming growth factor-β; WT, wild type. Journal of Investigative Dermatology 2017 137, 641-649DOI: (10.1016/j.jid.2016.09.039) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Primary cultures of keratinocytes from CD109 transgenic mice show increased Smad1/5 signaling and decreased Smad2/3 signaling when compared with those from wild-type littermates, whereas no differences in those signaling pathways were observed between primary cultures of skin fibroblasts from transgenic mice and wild-type littermates. Keratinocytes and fibroblasts were isolated from CD109 transgenic and wild-type littermate mice skin, and cell lysates were prepared from primary cultures and analyzed by Western blot. Transgenic keratinocytes display (a) increased ALK1 and ALK5 expression, (c) increased Smad1/5 and decreased Smad2/3 phosphorylation in response to 15 pmol/L TGF-β treatment for 30 and 60 minutes, and (e) increased CD109, and decreased collagen I and fibronectin expression, compared to WT keratinocytes. ∗P < 0.05, ∗∗P < 0.005. In contrast, fibroblasts from transgenic mice skin exhibit similar levels of (b) ALK1 and ALK5 expression, (d) Smad1/5 and Smad2/3 phosphorylation, and (f) CD109, collagen I and fibronectin expression, as fibroblasts from wild-type mice skin. M, mol/L; min, minutes; P, phosphorylated; TG, transgenic; TGF-β, transforming growth factor-β; WT, wild type. Journal of Investigative Dermatology 2017 137, 641-649DOI: (10.1016/j.jid.2016.09.039) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Treatment with media conditioned by CD109 transgenic keratinocytes or co-culturing with CD109 transgenic epidermal explants decreases extracellular matrix expression in primary murine fibroblasts. Skin fibroblasts prepared from wild-type mice were (a, b) treated with conditioned media collected from transgenic and wild-type keratinocytes or (c, d) co-cultured with epidermal explants prepared from wild-type or transgenic skin. Western blot analysis of fibroblast cell lysates shows that (a, b) treatment with conditioned media from transgenic keratinocytes and (c, d) co-culture with transgenic epidermal explants, when compared with treatment with wild-type keratinocyte conditioned media or co-culture with wild-type epidermal explants, respectively, resulted in decreased α-smooth muscle actin, CTGF, and fibronectin expression in primary mouse skin fibroblasts. ∗P < 0.05, ∗∗P < 0.005. a-SMA, α-smooth muscle actin; TG, transgenic; WT, wild type. Journal of Investigative Dermatology 2017 137, 641-649DOI: (10.1016/j.jid.2016.09.039) Copyright © 2016 The Authors Terms and Conditions

Figure 4 CD109 interacts and co-localizes with ALK1 in mouse keratinocytes. (a) Immunofluorescent staining of primary cultures of keratinocytes isolated from transgenic mice shows co-localization of CD109 and ALK1. Cells cultured in chamber slides were fixed and incubated with anti-CD109 or anti-ALK1 or both antibodies overnight at 4 °C and then with the appropriate secondary antibodies conjugated with FITC and Cy3, respectively. The nuclei were stained with DAPI. Scale bar = 50 μm. Inset, the enlarged area highlights ALK1 staining (left, red), CD109 staining (middle, green), and co-localization ALK1 and CD109 (right, yellow). (b) Immunofluorescent staining of transgenic mice skin shows co-localization of CD109 and ALK1 in the mouse epidermis. Top panel shows ALK1 (red, left), CD109 (green, middle), and co-localization of ALK1 and CD109 (yellow, right) in the epidermis of skin obtained from mice. Scale bar = 20 μm. Arrows indicate positively stained areas. Inset, the enlarged area highlights the co-localization of ALK1 and CD109 staining. The bottom panel highlights that ALK1 (red) is detected in the epidermis/keratinocytes as it co-localizes with the keratinocyte marker cytokeratin (PanCK, green). The bottom left panel shows the detection of ALK1 (red) in the dermis as it co-localizes with the fibroblast marker α-smooth muscle actin (aSMA, green). Inset, the enlarged area highlights the ALK1/α-smooth muscle actin staining (yellow). The nuclei were stained with DAPI (blue). (c) Co-immunoprecipitation in primary mouse keratinocytes shows that ALK1 interacts with CD109. Protein extracts of keratinocytes prepared from wild-type and transgenic mice were immunoprecipitated with anti-ALK1 or control IgG at 4 °C, followed by precipitation with protein A–Sepharose and immunoblotting with anti-CD109 antibody (left). Keratinocyte extracts immunoblotted with CD109 antibody without immunoprecipitation is also shown (right). der, dermis; epi, epidermis; IP, immunoprecipitation; TG, transgenic; WT, wild type. Journal of Investigative Dermatology 2017 137, 641-649DOI: (10.1016/j.jid.2016.09.039) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Schematic diagram demonstrating role of CD109 in regulating ALK1-Smad1/5 and ALK5-Smad2/3 signaling and ECM synthesis in the skin. CD109 directly interacts with ALK5 and ALK1 and inhibits Smad2/3 activation while enhancing the Smad1/5 activation, leading to alterations in keratinocyte/fibroblast paracrine interactions and a decrease in ECM production. Journal of Investigative Dermatology 2017 137, 641-649DOI: (10.1016/j.jid.2016.09.039) Copyright © 2016 The Authors Terms and Conditions