Bone morphogenetic protein signaling suppresses wound-induced skin repair by inhibiting keratinocyte proliferation and migration

Abstract

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 (transgenic mice overexpressing a constitutively active form of Smad1 under K14 promoter) mice 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) and Myosin 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.

Figure 1. Expression of Bmp pathway components during skin healing

(a) qRT-PCR: significant decreases of Bmp-2, Bmp-4, Bmp-7 (*p<0.01) and noggin (**p<0.001) transcripts on days 3 and 5 post-wounding; (b) Immunofluorescence: In telogen skin, Bmpr-1A expression is restricted to the 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 post-wounding, Bmpr-1A expression is low in HF bulges near the wound (arrowed) 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, *p<0.01, **p<0.0001, Student’s t-test). HF – hair follicle, SG – sebaceous gland, WE – wound epithelium, scale bar 100µm.

Figure 2. Histomorphological analysis of wound epithelium in K14-caSmad1 and WT mice

(a) Transgenic construct used to generate K14-caSmad1 mice; (b) K14-caSmad1 mice show markedly increased Smad1 expression in the epidermis and 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 3, 5, and 7 days post-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 post-wounding (mean ± SD, *p<0.01, **p<0.0001, Student’s t-test). GT – granulation tissue, WE – wound epithelium, scale bar 100µm.

Figure 3. Quantitative analysis of proliferation and apoptosis and assessment of Keratin 16 and Keratin 17 expression in the wound epithelium of K14-caSmad1 and WT mice

(a) Proliferative Ki-67+ 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+ cells in K14-caSmad1 telogen skin, on day 5 and 7 after wounding versus WT; (c) Apoptotic active caspase 3+ cells are seen in the wound epithelium at days 3 and 5 post-wounding – inset illustrates cell-specific staining; (d) significant increase in active caspase 3+ cells in K14-caSmad1 wound epithelium at days 3, 5 and 7 post-wounding versus WT (mean ± SD, *p<0.01, **p<0.0001, Student’s t-test); (e–f) reduced expression of keratin-16 and (e) keratin-17 expression (f) in K14-caSmad1 wounds and keratinocytes were cuboidal (arrowheads), while those in WT mice were elongated (arrows). GT – granulation tissue, scale bar 100µm.

Figure 4. Global gene expression profiling of analyses of the epidermal keratinocytes isolated from telogen skin of WT and K14-caSmad1

(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 Keratin - 1, 16 and -17 expression (upper panel) and Myo5a, Ablim2, Tubb6 (lower panel) in response to wounding versus WT (mean ± SD, *p<0.01, **p<0.0001, Student’s t-test).

Figure 5. BMP pathway modulation alters keratinocyte migration and morphology

(a–b) Skin explant model; (b) Bmp-4/7 inhibited migration at days 5 (*p<0.05) and 7 (*p<0.05); Noggin alone increased migration at days 5 (**p<0.01) and 7 (*p<0.05); (c) Phalloidin staining of actin filament networks; control keratinocytes were elongated with actin fibres across the cell body; Bmp-4/7-treated keratinocytes were spherical and lacked defined actin fibres (arrowheads); Noggin increased cell polarity and actin formation; (d–f) transwell assay: Bmp-4/7 inhibited migration (**p<0.001); Noggin (*p<0.05) increased migration; (d); significant delay in PMEK migration obtained from K14-caSmad1 mice (***p<0.0001); Bmpr-1B knockdown accelerated (*p<0.01) PMEK movement (f); (g) qRT-PCR confirmation of Bmpr-1B silencing (***p<0.001); Bmpr-1B siRNA up-regulated Myo5a (*p<0.02), Krt16 (**p<0.01) and Krt17 (***p<0.001) transcripts. PMEK – primary mouse epidermal keratinocyte, scale bar 100µm, mean±SD, Student’s t-test.