CD9 Is Critical for Cutaneous Wound Healing through JNK Signaling Jiaping Zhang, Jianda Dong, Hua Gu, Sidney Yu, Xiaohu Zhang, Yulin Gou, Wenming Xu, Andrew Burd, Lin Huang, Kenji Miyado, Yuesheng Huang, Hsiao Chang Chan  Journal of Investigative Dermatology  Volume 132, Issue 1, Pages 226-236 (January 2012) DOI: 10.1038/jid.2011.268 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Downregulation of CD9 in epidermis during wound healing in wild-type mice. Immunofluorescence staining of CD9 in normal unwounded skin (day 0), day 3, day 5, day 7, and day 10 wound sections obtained from wild-type mice showing downregulation of CD9 in migrating epidermis during normal wound re-epithelialization. Wounds were close to re-epithelialization on day 7 and fully re-epithelialized on day 10. K10 served as an internal control as it is known to be downregulated in migrating epidermis during wound healing (Mathay et al., 2008; n=6 wounds). Bar=100μm. GT, granulation tissue; WM, wound margin. Narrow-dotted line: interface between epidermis and dermis or leading edge of migrating epidermis (day 5). Wide-dotted line: wound margin. Journal of Investigative Dermatology 2012 132, 226-236DOI: (10.1038/jid.2011.268) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Delayed wound healing and impaired re-epithelialization in CD9-null mice. (a, b) Macroscopic (a) and histological (b) comparison of wound healing in CD9-null (knockout, KO) mice with wild-type (WT) controls at indicated time points. Arrows indicate the leading edges of epidermis. Bar=200μm. (c–e) Summary of averaged wound gape (c), dermal gape (d), and length of migrating epidermis (e) over the course of healing. Results are mean±SEM, n=8–12 wounds; *P<0.01, #P<0.05 compared with WT controls; (f, g) keratinocyte proliferating cell nuclear antigen (PCNA) staining (f) and the percentage of positive cells (g) at wound edge on day 5 post wounding. Bar=250μm, n=8. (h) Western blot results of PCNA expression in day 5 wounds, n=3. Journal of Investigative Dermatology 2012 132, 226-236DOI: (10.1038/jid.2011.268) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Hyper-activation of C-JUN NH2 terminal kinase (JNK) in CD9-null wounds. (a) Representative western blot results showing increased JNK phosphorylation on day 3 post wounding in knockout (KO) mice, with no alteration to its protein level. (b, c) Representative western blot results (b) and densitometric analysis (c, n=3) of JNK and phospho-JNK (p-JNK) expression in normal skin and wounds from day 3 to day 7. *P<0.01 compared with wild type (WT) controls; (d–g) p-JNK staining in normal skin (d, e) and wounds on day 5 (f, g) from WT (d, f) and KO (e, g) mice. Arrows indicate migrating epidermis. Bar=100μm, n=6 wounds. ERK, extracellular signal-regulated kinase; p-ERK, phospho-ERK. Journal of Investigative Dermatology 2012 132, 226-236DOI: (10.1038/jid.2011.268) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Abnormal elevation of matrix metalloproteinase (MMP)-9 in CD9-null wounds. (a) Western blot results showing abnormally elevated expression of MMP-9 in knockout (KO) wounds compared with wild-type (WT) controls (n=3). (b) Results of gelatin zymograms showing abnormally elevated activity of MMP-9 in KO wounds compared with WT controls. C, cleaved form; M, intermediate active form; P, precursor. (c) Abnormally increased mRNA level of MMP-9 in day 3 wounds from KO mice compared with WT controls (n=3). (d) Immunostaining results showing a stronger signal of MMP-9 in migrating epidermis on day 5 post wounding in KO mice than that in WT controls. GT, granulation tissue; WM, wound margin. Bar=50μm, n=6 wounds. *P<0.01, #P<0.05 compared with WT controls. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Journal of Investigative Dermatology 2012 132, 226-236DOI: (10.1038/jid.2011.268) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Excessive degradation of type IV collagen in CD9-null wounds. (a) Immunostaining results of type IV collagen showing reduced signal at basement membrane on day 5 and day 7 post wounding in knockout (KO) mice compared with wild-type (WT) controls. Arrows indicate the expression of type IV collagen in basement membrane. Bar=50μm, n=5 wounds. (b) Western blot results showing a significant reduction of type IV collagen (160kDa) associated with an increase in its degraded fragments (40kDa) on day 5 and day 7 post wounding in KO mice compared with WT controls. *P<0.01, n=3. (c) Western blot results showing suppressed degradation of type IV collagen by matrix metalloproteinase (MMP)-9 inhibitor in day 7 KO wounds, as compared with vehicle controls. *P<0.01, n=3. PBS, phosphate-buffered saline. Journal of Investigative Dermatology 2012 132, 226-236DOI: (10.1038/jid.2011.268) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 C-JUN NH2 terminal kinase (JNK) suppression rescued healing delay in CD9-null mice. (a, b) Representative results (a) and densitometric analysis (b, n=3) showing suppressed expressions or activities of phospho-JNK and matrix metalloproteinase (MMP)-9 in day 5 wounds by SP600125 (JNKi) compared with vehicle control (DMSO). (c) Reduced MMP-9 in CD9-deficient keratinocytes in an in vitro wound by SP600125 (20μm). Arrows indicate a positive staining of cytoplasmic MMP-9. Bar=25μm. (d) Reduced degradation of type IV collagen by SP600125 in day 5 wounds. (e) Accelerated healing by SP600125 treatment. (f, g) Representative sections (f, bar=200μm) and summary of wound gape and migrating epidermis length (g, results are mean±SEM, n=10 wounds) confirming improved re-epithelialization by SP600125 on day 7 post wounding. *P<0.01, #P<0.05. Journal of Investigative Dermatology 2012 132, 226-236DOI: (10.1038/jid.2011.268) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions