The Abnormal Architecture of Healed Diabetic Ulcers Is the Result of FAK Degradation by Calpain 1  Wei Liu, Kun Ma, Sun Hyung Kwon, Ravi Garg, Yoda R. Patta, Toshihiro Fujiwara, Geoffrey C. Gurtner  Journal of Investigative Dermatology  Volume 137, Issue 5, Pages 1155-1165 (May 2017) DOI: 10.1016/j.jid.2016.11.039 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Downregulation of FAK under a high glucose condition in fibroblasts. Western blot of total-FAK (T-FAK), phosphorylated-FAK (p-FAK) (Y397), and β-actin (a) and quantification of the expression of T-FAK and p-FAK (Y397) normalized by β-actin (b) in human keratino cytes and fibroblasts that were cultured in different concentrations of glucose (5.5 mM,12 mM, and 25 mM) for 7 days. Under a high glucose (25 mM) condition, T-FAK and p-FAK expression was downregulated in fibroblasts (*25 mM group vs. 5.5 mM group; P = 0.0073 for T-FAK/β-actin, P = 0.0149 for p-FAK/β-actin). (c) ΔΔCt (sample/β-actin) of three different FAK mRNA splice variants (FAK-V1, FAK-V2, and FAK-V3) of human fibroblasts that were cultured in different concentrations of glucose (5.5 mM, 12 mM, and 25 mM) for 7 days. Error bars: mean ± SD. n = 3/cell strain; 3 cell strains were used for each cell type of fibroblasts and keratinocytes. FAK, focal adhesion kinase; SD, standard deviation. Journal of Investigative Dermatology 2017 137, 1155-1165DOI: (10.1016/j.jid.2016.11.039) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Degradation of FAK under a high glucose condition by calpain 1 in fibroblasts. Western blot (a) and quantification (b) of the expression of FAK, calpain 1 (calp 1), calpain 2 (calp 2) in human fibroblasts cultured under different concentrations of glucose (Glu 5.5 mM, 12 mM, and 25 mM) or mannose (Man 25 mM) for 7 days. Mannose was used as an osmotic control. Under a high glucose culture condition, FAK degradation was observed and calpain 1 level was increased. *In (b), 25 mM Glu versus 25 mM Man, P < 0.001. (c) BOC-CMAC assay on human fibroblasts cultured with normal glucose medium (5.5 mM) for 7 days. Blue fluorescence suggests calpain activation. Scale bar =10 μm. (d) Quantification of calpain activity in human fibroblasts cultured with normal or high glucose medium (5.5 mM and 25 mM) with or without the calpain inhibitor PD150606 for 7 days. Ctrl Normal Glu: group of 5.5 mM glucose; Ctrl High Glu: group of 25 mM glucose; PD Normal Glu: group of 5.5 mM glucose and 25 μg/ml calpain inhibitor PD150606; PD High Glu: group of 25 mM glucose and 25 μg/ml calpain inhibitor PD150606. Calpain activity in the Ctrl High Glu group had more than 2-folds increase compared with the Ctrl Normal Glu group (9.55 vs. 4.01), and PD150606 completely inhibits calpain activity in either treatment groups (4.01 vs. 0.01; 9.55 vs. 0.02). *in (d), Ctrl High Glu versus Ctrl Normal Glu, P < 0.001; ▲: PD High Glu versus Ctrl High Glu, P < 0.001; ●: PD Normal Glu versus Ctrl Normal Glu, P < 0.001. Error bars: mean ± SD. n = 3/cell strain; 3 cell strains of fibroblasts were used. BOC-CMAC, 7-amino-4-chloromethylcoumarin, t-BOC-l-leucyl-l-methionine amide; FAK, focal adhesion kinase; SD, standard deviation. Journal of Investigative Dermatology 2017 137, 1155-1165DOI: (10.1016/j.jid.2016.11.039) Copyright © 2017 The Authors Terms and Conditions

Figure 3 AGE downregulates FAK expression but can be reversed by calpain inhibition. (a) Western blot of total-FAK, collagen I, phospho-NF-κB p65, total NF-κB, phospho-ERK, total ERK and α-tubulin and quantification of the expression of total FAK, collagen I, phospho-NF-κB p65, and phospho-ERK in human fibroblasts cultured under 5.5 mM and 25 mM glucose condition with or without 0.5 μM FPS-ZM1 (an inhibitor for RAGE activation) for 7 days. *Group 2 versus group 1, P < 0.05; ▴: Group 4 versus group 2, P < 0.05. (b) Western blot of total-FAK, phospho-AKT, total-AKT, phospho-ERK, total-ERK, phospho-P38, and total-P38 and quantification of the expression of total-FAK, phospho-AKT, phospho-ERK, and phospho-P38 in human fibroblasts cultured with 40 μM BSA or AGE-BSA for 7 days. *AGE-BSA 40 μM versus BSA 40 μM, P < 0.05. (c) Zymogram of calpain 1/2 in human fibroblasts cultured in medium containing different concentrations (25 μg/ml, 50 μg/ml, 100 μg/ml, and 200 μg/ml) of BSA and AGE-BSA for 48 hours. The AGE-BSA-treated group demonstrated an active form of calpain (a truncated form after activation) with lower molecular bands, which indicate calpain activation (boxed area). (d, e) Western blot of total-FAK and β-actin in human fibroblasts cultured under 5.5 mM glucose condition and treated with 40 μM BSA or AGE-BSA for 7 days. Error bars: mean ± SD. *P < 0.001 versus BSA group. n = 3/cell strain; 3 cell strains of fibroblasts were used. AGE, advance glycation end product; AKT, acutely transforming retrovirus AKT8 in rodent T-cell lymphoma; BSA, bovine serum albumin; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; FPS-ZM1, N-benzyl-4-chloro-N-cyclohexylbenzamide; RAGE, receptor for AGE; SD, standard deviation. Journal of Investigative Dermatology 2017 137, 1155-1165DOI: (10.1016/j.jid.2016.11.039) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Defective wound healing and accumulation of FAK and calpain 1 in a splinted excisional db/db skin wound model. H&E staining (a) and dermal thickness (b) of wound sites in wild-type and db/db mice on day 21 after wounding. Db/db mice showed a significant defect in dermal healing after reepithelialization. *P < 0.01, n = 8. (c) Young’s modulus (E) of wounds in wild-type and db/db mice groups at 8 weeks after wounding. *P < 0.01 versus wild type, n = 8. (d) Immunofluorescence staining of FAK in wild-type and db/db mice on day 21 after wounding, scale bar = 100 μm. Arrow: Abnormal aggregation of FAK. Magnified images of the boxed areas in (d) were shown in (e) correspondingly. (e) Immunofluorescence staining of FAK (green), calpain 1 (red), and cell nuclei (blue) in wild-type and db/db mice on day 21 after wounding. Scale bar = 2 μm. Error bars: mean ± SD. FAK, focal adhesion kinase; H&E, hematoxylin and eosin; SD, standard deviation. Journal of Investigative Dermatology 2017 137, 1155-1165DOI: (10.1016/j.jid.2016.11.039) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Improved wound healing in db/db mice by PD150606. H&E staining (a) and dermal thickness (b) of wound sites in DMSO control and PD150606-treated groups in db/db mice on day 21 after wounding. Scale bar = 100 μm. *P = 0.005 versus PD150606, n = 14. (c) Immunofluorescence staining of FAK (green) and cell nuclei (blue) in control and -reated groups in db/db mice on day 21 after wounding. Scale bar = 100 μm. (d) Masson’s trichrome staining of control and PD150606-treated groups in db/db on day 21 after wounding. Scale bar = 100 μm. Blue: collagen; red: cytoplasm and muscle; black: cell nuclei. PD150606 significantly increased dermal thickness, FAK expression in the dermis, and collagen deposition. (e). Blue color density per unit area in the dermal area representing collagen expression in Masson’s trichrome staining images. Error bars: mean ± SD. *P = 0.0014, n = 14. FAK, focal adhesion kinase; H&E, hematoxylin and eosin; SD, standard deviation. Journal of Investigative Dermatology 2017 137, 1155-1165DOI: (10.1016/j.jid.2016.11.039) Copyright © 2017 The Authors Terms and Conditions

Figure 6 PD150606 significantly improves diabetic wound stiffness. Representative true stress-true strain curve (a) and Young’s modulus (b) of the control and PD150606-treated groups in db/db mice on day 60 after wounding. PD150606 treatment significantly improved skin wound stiffness in db/db mice excisional wounds. *P = 0.002, n = 8, for the control group and n = 10 for the PD150606 group. (c) Schematic description of calpain-mediated FAK downregulation in diabetic wound dermis. Error bars: mean ± SD. AGE, advance glycation end product; FAK, focal adhesion kinase; SD, standard deviation. Journal of Investigative Dermatology 2017 137, 1155-1165DOI: (10.1016/j.jid.2016.11.039) Copyright © 2017 The Authors Terms and Conditions