The Downregulation of microRNA let-7a Contributes to the Excessive Expression of Type I Collagen in Systemic and Localized Scleroderma This information is current as of October 1, 2021. Katsunari Makino, Masatoshi Jinnin, Ayaka Hirano, Keitaro Yamane, Mitsuhiko Eto, Takamitsu Kusano, Noritoshi Honda, Ikko Kajihara, Takamitsu Makino, Keisuke Sakai, Shinichi Masuguchi, Satoshi Fukushima and Hironobu Ihn J Immunol published online 15 March 2013 Downloaded from http://www.jimmunol.org/content/early/2013/03/15/jimmun ol.1200822 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 1, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 15, 2013, doi:10.4049/jimmunol.1200822 The Journal of Immunology The Downregulation of microRNA let-7a Contributes to the Excessive Expression of Type I Collagen in Systemic and Localized Scleroderma Katsunari Makino, Masatoshi Jinnin, Ayaka Hirano, Keitaro Yamane, Mitsuhiko Eto, Takamitsu Kusano, Noritoshi Honda, Ikko Kajihara, Takamitsu Makino, Keisuke Sakai, Shinichi Masuguchi, Satoshi Fukushima, and Hironobu Ihn Systemic and localized scleroderma (SSc and LSc) is characterized by excessive deposition of collagen and tissue fibrosis in the skin. Although they have fundamental common characteristics including autoimmunity, little is known about the exact mechanism that mediates the excessive collagen expression in these disorders. In the current study, we tried to evaluate the possibility that micro- RNAs (miRNAs) play some roles in the pathogenesis of fibrosis seen in these diseases. miRNA expression patterns were evaluated by Downloaded from miRNA array analysis, real-time PCR, and in situ hybridization. The function of miRNAs in dermal fibroblasts was assessed us- ing miRNA inhibitors, precursors, or protectors. In the mouse model of bleomycin-induced dermal sclerosis, the overexpression of miRNAs was performed by i.p. miRNA injection. We demonstrated let-7a expression was downregulated in SSc and LSc skin both in vivo and in vitro, compared with normal or keloid skin. The inhibition or overexpression of let-7a in human or mouse skin fibroblasts affected the protein expression of type I collagen or luciferase activity of collagen 39-untranslated region. Also, we found let-7a was detectable and quantitative in the serum and investigated serum let-7a levels in patients with SSc or LSc. let-7a http://www.jimmunol.org/ concentration was significantly decreased in these patients, especially in LSc patients. Moreover, we revealed that the intermittent overexpression of let-7a in the skin by i.p. miRNA injection improved the skin fibrosis induced by bleomycin in mice. Investigation of more detailed mechanisms of miRNA-mediated regulation of collagen expression may lead to new therapeutic approaches against SSc and LSc. The Journal of Immunology, 2013, 190: 000–000. ystemic scleroderma (SSc) is a connective tissue disorder In contrast, keloids are also characterized by cutaneous fibrosis that results in fibrosis of the skin and internal organs. that invade adjacent healthy tissue. However, keloid is not an auto- S Localized scleroderma (LSc) also manifests tissue fibrosis immune disease, and it is still controversial whether the collagen by guest on October 1, 2021 limited to the skin and s.c. tissue, occasionally involving the production by keloid fibroblasts is also upregulated (12–15). Thus, muscular tissues beneath the cutaneous lesions (1, 2), but the different factors are likely to mediate the tissue fibrosis in SSc/LSc presence of Raynaud’s phenomenon, acrosclerosis, and involve- and keloid, but the exact mechanism involved in each fibrotic ment of internal organs differentiates SSc from LSc (3). Abnormal condition is still unknown. collagen metabolism (4–8) and autoimmunity (9, 10) are consid- Recently, microRNAs (miRNAs) have attracted attention for its ered to be fundamental common characteristics of SSc and LSc, involvement in the pathogenesis of various human diseases such as and especially, excess collagen production by dermal fibroblasts immunological disorders, cancers, and metabolic disorders (16–18). is thought to be caused by intrinsic activation of TGF-b signaling miRNAs are small noncoding RNAs that bind to complementary in both diseases (5, 11). sequences in the 39-untranslated regions (UTRs) of target mRNAs, resulting in inhibiting their translation into protein. Because .1000 Department of Dermatology and Plastic Surgery, Faculty of Life Sciences, Kuma- of miRNAs have been identified, which corresponds to 1–5% of all moto University, Kumamoto 860-8556, Japan genes in the human genome, miRNAs are thought to be the most Received for publication March 14, 2012. Accepted for publication February 13, abundant class of regulators (19). However, little is known about the 2013. role of miRNAs in the pathogenesis of SSc and LSc. In the current This work was supported in part by a grant for scientific research from the Japanese study, we tried to evaluate the possibility that miRNAs also play some Ministry of Education, Science, Sports, and Culture, by project research on intrac- parts in the collagen metabolism and skin fibrosis of SSc/LSc. table diseases from the Japanese Ministry of Health, Labor, and Welfare, and by a research grant from Shiseido. The sequences presented in this article have been submitted to the Gene Expression Materials and Methods Omnibus microarray data repository (http://www.ncbi.nlm.nih.gov/geo/) under acces- Patient materials sion number GSE43469. Serum samples were obtained from 39 SSc patients in their first visit. These Address correspondence and reprint requests to Dr. Masatoshi Jinnin, Department of Dermatology and Plastic Surgery, Faculty of Life Sciences, Kumamoto University, patients were grouped according to the classification system proposed by 1-1-1 Honjo, Kumamoto 860-8556, Japan. E-mail address: [email protected] LeRoy et al. (20): 20 patients had diffuse cutaneous SSc (dcSSc) and 19 had limited cutaneous SSc (lcSSc), as described previously (21). Similarly, Abbreviations used in this article: Ct, threshold cycle; dcSSc, diffuse cutaneous 32 patients with LSc were classified into following three subgroups, as systemic scleroderma; DM, dermatomyositis; GM, generalized morphea; lcSSc, lim- ited cutaneous systemic scleroderma; LS, linear scleroderma; LSc, localized sclero- described previously (22, 23): 19 patients with morphea (one or a few derma; miRNA, microRNA; SLE, systemic lupus erythematosus; SSc, systemic circumscribed sclerotic plaque), 8 with linear scleroderma (LS, with linear scleroderma; UTR, untranslated region. distribution), and 5 with generalized morphea (GM, four or more lesions . 3 cm in diameter and involvement of two or more areas of the body out Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 of seven areas). When patients had both morphea and LS, they were diag- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200822 2 let-7a EXPRESSION IN SSc AND LSc nosed as having GM (22). We also included serum samples from other Cell lysis and immunoblotting rheumatic diseases: 8 patients with systemic lupus erythematosus (SLE) and 8 with dermatomyositis (DM). Control serum samples were obtained from Human or mouse dermal fibroblasts were washed with PBS twice and lysed 17 healthy age-/sex-matched volunteers. Skin specimens were derived from in lysis buffer (BioSource International). Aliquots of cell lysates were involved skin of 7 SSc, 7 LSc, and 5 keloid patients. These skin samples and separated by electrophoresis on 10% SDS-PAGE and transferred to poly- seven control skins were collected and fixed in formaldehyde immediately vinylidene difluoride membranes, which were blocked in blocking One P after resections. buffer (Nacalai Tesque) for 1 h and incubated overnight at 4˚C with primary Clinical and laboratory data reported in this study were obtained at the Ab for type I collagen–UNLB (Southern Biotechnology Associates). The time of serum sampling. Institutional review board approval and written membranes were washed with TBS and 0.1% TBST, probed with HRP- informed consent were obtained according to the Declaration of Helsinki. conjugated secondary Ab for 1 h, and then washed with TBST again. The detection was performed using ECL system (Thermo Scientific), according Cell cultures to the manufacturer’s recommendations. As a loading control, the same membrane was stripped and reprobed with an Ab against b-actin (Santa Human dermal fibroblasts were obtained by skin biopsy from the affected Cruz Biotechnology). areas of five SSc patients, LSc patients, and healthy donors. All biopsies were performed with institutional review board approval and written in- In situ hybridization formed consent according to the Declaration