Functions of Rhomboid Family Protease RHBDL2 and Thrombomodulin in Wound Healing Tsung-Lin Cheng, Yu-Ting Wu, Hung-Yu Lin, Fu-Chih Hsu, Shi-Kai Liu,

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Functions of Rhomboid Family Protease RHBDL2 and Thrombomodulin in Wound Healing Tsung-Lin Cheng, Yu-Ting Wu, Hung-Yu Lin, Fu-Chih Hsu, Shi-Kai Liu, Bi-Ing Chang, Wei-Sheng Chen, Chao-Han Lai, Guey-Yueh Shi, Hua-Lin Wu Journal of Investigative Dermatology Volume 131, Issue 12, Pages 2486-2494 (December 2011) DOI: 10.1038/jid.2011.230 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Thrombomodulin (TM) is cleaved by rhomboid-like-2 (RHBDL2) to release soluble TM (sTM) from keratinocytes. (a) Illustration of the protein domain structure of TM and recombinant TM (rTM). Arrow indicates the predicted cutting site of RHBDL2. Dotted line indicates the antibody region against TM (D-3). Purified rTMD23 and rTMD123 were analyzed by Coomassie blue staining (b) and western blotting (c). (d) Western blot analysis of TM in HaCaT lysates, purified rTMD123 (20ng), and sTM in the serum-free conditioned medium (CM) collected at the indicated time points. (e) Western blotting and quantitative representation of the sTM after treatment with 3,4-dichloroisocoumarin (DCI) for 24hours. (f) Western blotting and quantitative representation of the sTM from HaCaT and stable transfected cells (green fluorescent protein (GFP), RHBDL2-GFP, and shRHBDL2). *P<0.05, **P<0.01, ***P<0.001. aa, amino acid; GST, glutathione S-transferase. Journal of Investigative Dermatology 2011 131, 2486-2494DOI: (10.1038/jid.2011.230) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Injury-induced rhomboid-like-2 (RHBDL2) upregulation and thrombomodulin (TM) ectodomain shedding in HaCaT cells were inhibited by RNase. (a) Western blot analysis of RHBDL2 expression in cell lysates (left) and soluble TM (sTM, right) in the conditioned medium of HaCaT after scratch wounding. (b) Quantitative representation of the results of a. Western blot analysis of normal HaCaT cocultured with UV-irradiated keratinocytes (UVR cells) (c) and scratch-wounded cells (d) that were pretreated with DNase (4Uml−1) or RNase (20μgml−1). (e, f) Quantitative representation of the results of c and d. The number of scratch wounds is five. Data are from three independent experiments. *P<0.05, **P<0.01, ***P<0.001 versus untreated controls. GST, glutathione S-transferase; Lys, lysate; Med, medium. Journal of Investigative Dermatology 2011 131, 2486-2494DOI: (10.1038/jid.2011.230) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Scratch-wound-induced thrombomodulin (TM) ectodomain shedding in HaCaT cells inhibited by serine protease inhibitor 3,4-dichloroisocoumarin (DCI) and rhomboid-like-2 (RHBDL2) short hairpin RNA (shRNA). (a) The soluble TM (sTM) in concentrated conditioned medium and RHBDL2 in cell lysates of HaCaT cells after scratch wounding and treatment with DCI (left panel) or transient transfection with RHBDL2 shRNA (right panel) were analyzed by western blotting. (b) Quantitative representation of the results of a. The number of scratches is five. Data are from three independent experiments. ***P<0.001. For all western blot analyses, one representative experiment of three is shown. GST, glutathione S-transferase; Lys, lysate; Med, medium. Journal of Investigative Dermatology 2011 131, 2486-2494DOI: (10.1038/jid.2011.230) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Short hairpin RNA rhomboid-like-2 (shRHBDL2)-derived downregulation of RHBDL2 caused the inhibition of thrombomodulin (TM) shedding, wound recovery, cell migration, and proliferation in HaCaT. (a) Western blot analysis of TM shedding and RHBDL2 expression in stably transfected cells (green fluorescent protein (GFP) and shRHBDL2). (b) Scratch-wound healing assay of stably transfected cells cultured in complete medium and observed by time-lapse recording. (c) Quantitative representation of the results of b. Data were calculated on the basis of the experiments with three different stable clones. (d) Transwell migration assays for HaCaT, GFP, and shRHBDL2 stable clones. (e) Cell growth was monitored by cell counts using a hemocytometer. Bar=200μm. Data are from three independent experiments. ***P<0.001. GST, glutathione S-transferase; Lys, lysate; Med, medium; sTM, soluble TM. Journal of Investigative Dermatology 2011 131, 2486-2494DOI: (10.1038/jid.2011.230) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Role of rhomboid-like-2 (RHBDL2) in thrombomodulin (TM) shedding and wound healing. Conditioned medium (CM) from scratch-wounded HaCaT cells or human rTMD123 enhances scratch-wound healing. (a) The CM from scratch-wounded HaCaT enhanced wound healing (left 2). The anti-TM antibody (Ab) blocked the effect of the CM (left 3). The CM from scratch-wounded short hairpin RNA rhomboid-like-2 (shRHBDL2) cells did not promote wound healing (left 4). Purified rTMD123 (20nM) could promote wound healing in HaCaT cells (right). Bar=200μm. (b) Quantitative representation of a. Data are from three independent experiments. ***P<0.001. Journal of Investigative Dermatology 2011 131, 2486-2494DOI: (10.1038/jid.2011.230) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Cutaneous wounding in mice induces rhomboid-like-2 (RHBDL2) expression and soluble thrombomodulin (sTM) release, which is involved in wound healing. (a) The lysates of wound-edge cells were harvested at the indicated time points and subjected to western blot analysis. (b) The conditioned medium from wounded culture skin was harvested for western blot analysis after 2 days. (c) Macroscopic observation of wound healing in mice that were subcutaneously injected with vehicle (Lipovenoes), 3,4-dichloroisocoumarin (DCI, 1mM), or DCI plus rTMD123 at the margin of the wound. Bar=4mm. (d) Quantitative representation of c. rTMD123 treatment group compared with DCI group; *P<0.05 (n=5). (e) Schematic model of RHBDL2 and sTM promotion of wound healing. Ab, antibody; GST, glutathione S-transferase; TM, thrombomodulin. Journal of Investigative Dermatology 2011 131, 2486-2494DOI: (10.1038/jid.2011.230) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions