Discoidin Domain Receptor 2–microRNA 196a–Mediated Negative Feedback against Excess Type I Collagen Expression Is Impaired in Scleroderma Dermal Fibroblasts

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Discoidin Domain Receptor 2–microRNA 196a–Mediated Negative Feedback against Excess Type I Collagen Expression Is Impaired in Scleroderma Dermal Fibroblasts Katsunari Makino, Masatoshi Jinnin, Jun Aoi, Ayaka Hirano, Ikko Kajihara, Takamitsu Makino, Keisuke Sakai, Satoshi Fukushima, Yuji Inoue, Hironobu Ihn Journal of Investigative Dermatology Volume 133, Issue 1, Pages 110-119 (January 2013) DOI: 10.1038/jid.2012.252 Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 The discoidin domain receptor 2 (DDR2) expression in normal and systemic sclerosis (SSc) skin. (a) The mRNA levels of DDR1 and DDR2 in skin tissues from five controls, five limited cutaneous SSc (lcSSc) patients, and five diffuse cutaneous SSc (dcSSc) patients. Horizontal lines show means. *P<0.05 compared with values in control skin (1.0). (b) The mRNA levels of DDR1 and DDR2 in normal and SSc-cultured fibroblasts (n=5). (c) Lysates from normal and SSc-cultured dermal fibroblasts were subjected to immunoblotting with antibody against DDR2 or β-actin. Three representative results for five normal and SSc fibroblasts are shown. (d) Lysates were obtained from cultured normal and SSc fibroblasts at passages 5, 10, and 15 (p5, p10, and p15), and subjected to immunoblotting (n=3). (e, f) Hematoxylin and eosin (HE) and DDR2 staining of (e) normal and SSc skin or (f) mouse skin treated with phosphate-buffered saline (PBS) or bleomycin (n=3). DDR2 was stained brown. Arrowheads indicate dermal fibroblasts. Bar=100μm. Journal of Investigative Dermatology 2013 133, 110-119DOI: (10.1038/jid.2012.252) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Decreased discoidin domain receptor 2 (DDR2) synthesis is induced by stimulation with transforming growth factor-β (TGF-β). (a) Normal and systemic sclerosis (SSc) fibroblasts were incubated for 3, 9, and 12hours after treatment with 2.5μgml−1 actinomycin D. DDR2 mRNA expression was analyzed by real-time PCR and normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH; n=3). The values in untreated fibroblasts were set at 100%. (b, c) Normal and SSc fibroblasts were incubated in the presence or absence of 2ngml−1 TGF-β1 for 24hours. (b) DDR2 mRNA levels were determined by real-time PCR (n=5). (c) Protein was analyzed by immunoblotting. (d, e) Normal and SSc fibroblasts were transfected with control or TGF-β1 small interfering RNA (siRNA) for 96hours. (d) The mRNA levels of TGF-β1 and collagen were quantified by real-time PCR (n=5). (e) The expression of DDR2 was analyzed by immunoblotting. *P<0.05 compared with controls. Journal of Investigative Dermatology 2013 133, 110-119DOI: (10.1038/jid.2012.252) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 The effect of discoidin domain receptor 2 (DDR2) on the expression of type I collagen. (a) DDR2 was immunoprecipitated and the phosphorylated DDR2 was detected by immunoblotting using 4G10 antibody. EGF-stimulated A431 cell lysates were used as a positive control (n=3). (b, c) Normal (b) and systemic sclerosis (SSc; c) fibroblasts were transfected with the indicated small interfering RNA (siRNA). Lysates were subjected to immunoblotting. Three representative results for five normal and SSc fibroblasts are shown (left). The relative amounts of α2(I) collagen transcripts were determined by real-time PCR (n=5, right). (d, e) Normal fibroblasts were treated with increasing amounts (0, 2, and 5μl) of virus-containing medium with a (d) control or (e) DDR2 expression vector. Protein was analyzed by immunoblotting (n=3). (f) SSc fibroblasts were transduced with the control vector or DDR2 vector for 96hours, and then the cell lysates were subjected to immunoblotting. Journal of Investigative Dermatology 2013 133, 110-119DOI: (10.1038/jid.2012.252) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Regulation of microRNA-196a (miR-196a) by discoidin domain receptor 2 (DDR2). (a, b) Fibroblasts were transfected with control or DDR2 small interfering RNA (siRNA). Cell lysates were subjected to immunoblotting. (a) A representative result from five independent experiments is shown. (b) The mRNA levels of transforming growth factor-β1 (TGF-β1), TGF-β receptor I (TGFBR1), TGF-β receptor II (TGFBR2), endoglin (ENG), thrombospondin-1 (TSP-1), integrin β3 (ITGB3), and integrin β5 (ITGB5) were determined by real-time PCR (n=5). (c, d) Fibroblasts were treated with control vector or DDR2 expression vector. Lysates were subjected to immunoblotting. (c) A representative result from five independent experiments is shown. (d) The mRNA levels of indicated molecules were determined by real-time PCR (n=3). (e, f) Fibroblasts were transfected with (e) siRNAs or (f) expression vectors. The miR-196a levels were determined by real-time PCR (n=5). *P<0.05 compared with cells treated with controls (1.0). Journal of Investigative Dermatology 2013 133, 110-119DOI: (10.1038/jid.2012.252) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The microRNA-196a (miR-196a) expression in normal and systemic sclerosis (SSc) fibroblasts. (a, b) Normal (a) and SSc (b) fibroblasts were transfected with control or miR-196a mimic in the presence or absence of miScript Target protector for 96hours. Lysates were subjected to immunoblotting. Type I procollagen protein levels quantified by scanning densitometry and corrected for β-actin levels in the same samples are shown (n=3, right). (c) Relative miR-196a expressions in tissues from normal and SSc skin (n=4). (d) Relative miR-196a expressions in normal and SSc-cultured dermal fibroblasts (n=4). (e) Normal fibroblasts were stimulated with 2ngml−1 transforming growth factor-β1 (TGF-β1) for 24hours. Relative miR-196a expressions were determined by real-time PCR (n=4). (f) SSc fibroblasts were transfected with control small interfering RNA (siRNA) or TGF-β1 siRNA for 96hours. Then, relative miR-196a levels were determined by PCR (n=4). *P<0.05 compared with controls. Journal of Investigative Dermatology 2013 133, 110-119DOI: (10.1038/jid.2012.252) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 A hypothetical model of the role of discoidin domain receptor 2 (DDR2) in systemic sclerosis (SSc) fibroblasts. DDR2 has a negative effect on microRNA-196a (miR-196a) expression and an inducible effect on collagen expression in normal fibroblasts. In SSc fibroblasts, autocrine transforming growth factor-β (TGF-β) stimulation induces collagen expression, and also downregulates DDR2 expression as a negative feedback mechanism to protect against excess collagen expression. However, the DDR2–miR-196a–mediated negative feedback is impaired: the constitutive downregulation of miR-196a by TGF-β causes an unresponsiveness of the miRNA to DDR2, leading to tissue fibrosis. Journal of Investigative Dermatology 2013 133, 110-119DOI: (10.1038/jid.2012.252) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions