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ABSTRACT: Background: Dermatomyositis (DM) is characterized by skin manifestations accompanying and preceding muscle weakness. Gottron's papules, one of the skin manifestations, are of great diagnostic value because they are specific to DM. However, the pathogenesis of Gottron's papules remains unclear. Objectives: We investigated the expression pattern of miRNAs in Gottron's papules of DM patients and evaluated the possibility that miRNAs play a role in its pathogenesis. Materials and methods: miRNAs were extracted from skin tissues and sera of patients with DM, clinically amyopathic DM (CADM) and healthy controls. To identify pathogenic miRNAs, we performed miRNA PCR array analysis. The results were confirmed by in situ hybridization, immunohistochemistry, immunoblotting and transient transfection of siRNAs or miRNA inhibitors. Results: PCR array analysis using tissue miRNAs demonstrated the miR-223 level was markedly decreased in Gottron's papules of DM and CADM in vivo, but not in psoriasis skin. The protein expression of PKCɛ, a predicted target of miR-223, was increased in DM/CADM skin. The transfection of a specific inhibitor of miR-223 in keratinocytes led to up-regulation of the PKCɛ protein, and resulted in increased cell proliferation. On the other hand, cell numbers were significantly decreased when cells were transfected with siRNA for PKCɛ. The serum miR-223 concentration was decreased in DM/PM patients, particularly in CADM patients, compared with healthy controls. Conclusions: A decreased miR-223 expression and the subsequently increased PKCɛ levels may therefore play a key role in the pathogenesis of Gottron's papules.
European journal of dermatology : EJD. 04/2013;
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Katsunari Makino,
Masatoshi Jinnin,
Ayaka Hirano, Keitaro Yamane,
Mitsuhiko Eto,
Takamitsu Kusano,
Noritoshi Honda,
Ikko Kajihara,
Takamitsu Makino,
Keisuke Sakai,
Shinichi Masuguchi,
Satoshi Fukushima,
Hironobu Ihn
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ABSTRACT: 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 microRNAs (miRNAs) play some roles in the pathogenesis of fibrosis seen in these diseases. miRNA expression patterns were evaluated by miRNA array analysis, real-time PCR, and in situ hybridization. The function of miRNAs in dermal fibroblasts was assessed using 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 3'-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 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 03/2013; · 5.79 Impact Factor
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ABSTRACT: BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. There is an urgent need to develop new therapeutic approaches against skin fibrosis. Although intravenous immunoglobulin (IVIG) may be one of the promising treatments, the mechanisms by which IVIG improves the fibrosis of SSc remain unknown. OBJECTIVES: To compare the cytokine profile in the sera and skin of SSc patients before and after IVIG administration, and try to clarify the mechanism of the effect of IVIG. METHODS: Each three patients received 5-day administration of IVIG, or the same dose of physiologic saline for placebo. Cytokine levels were determined by ELISA array, immunostaining, and real-time PCR. RESULTS: Cytokine array revealed that the serum levels of IFN-γ and IL-12, representative Th1 cytokines, were increased by IVIG treatment, but not by placebo. The percentage of IFN-γ- and IL-12-positive cell number/CD4-positive T lymphocyte cell number was also significantly increased by IVIG in SSc skin. Furthermore, mRNA expression of IFN-γ and IL-12 in SSc skin tissue was significantly up-regulated after IVIG treatment. CONCLUSION: The expression of Th1 cytokine is reported to be decreased in SSc. Our study suggested IVIG recovered the suppressed levels of Th1 cytokines, and that the treatment improves skin fibrosis by correcting the Th1/Th2 balance. In order to facilitate the clinical use of IVIG for SSc, it is necessary to perform a larger study in the future.
Journal of dermatological science 09/2012; · 3.71 Impact Factor
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Mitsuhiko Etoh,
Masatoshi Jinnin,
Katsunari Makino, Keitaro Yamane,
Wakana Nakayama,
Jun Aoi,
Noritoshi Honda,
Ikko Kajihara,
Takamitsu Makino,
Satoshi Fukushima,
Hironobu Ihn
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ABSTRACT: Localized scleroderma (LSc), a connective tissue disorder restricted to the skin and subcutaneous tissue, is characterized by skin fibrosis due to an excessive deposition of types I collagen. The mechanism of such fibrosis is still unknown, but epigenetics may play some roles in the excessive collagen expression. In the present study, we investigated the mechanism of fibrosis seen in LSc, focusing on microRNA (miRNA). miRNA expression was determined by PCR array, real-time PCR, and in situ hybridization. The function of miRNA was evaluated using specific inhibitor. Immunoblotting was performed to detect α2(I) collagen protein. PCR array analysis using tissue miRNA demonstrated miR-7 level was significantly decreased in LSc skin as well as keloid tissue compared to normal skin in vivo. In situ hybridization also showed miR-7 expression in dermal fibroblasts was decreased in LSc dermis. The transfection of specific inhibitor for miR-7 into cultured normal dermal fibroblasts resulted in the up-regulation of α2(I) collagen protein in vitro. Also, the serum levels of miR-7 were significantly decreased in LSc patients compared with healthy controls, but serum miR-29a levels not. Systemic or local down-regulation of miR-7 may contribute to the pathogenesis of LSc via the overexpression of α2(I) collagen, and serum miR-7 may be useful as a disease marker. Investigation of the regulatory mechanisms of LSc by miRNA may lead to new treatments by the transfection into the lesional skin of this disease.
Archives for Dermatological Research 09/2012; · 2.28 Impact Factor
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Keitaro Yamane,
Masatoshi Jinnin,
Tomomi Etoh,
Yuki Kobayashi,
Naoki Shimozono,
Satoshi Fukushima,
Shinichi Masuguchi,
Keishi Maruo,
Yuji Inoue,
Tsuyoshi Ishihara,
Jun Aoi,
Yuichi Oike,
Hironobu Ihn
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ABSTRACT: Squamous cell carcinoma (SCC) is one of the most common skin cancers. Because its potential to recur and metastasize leads to a poor prognosis and significant mortality, it is necessary to develop new early diagnostic tools and new therapeutic approaches. In this study, we found protein levels of ERK1 and ERK2 were increased in SCC cell lines without changing mRNA levels and that ERK1/2 mediates abnormal cell proliferation in these cells. Then, mechanisms underlying the overexpression of ERK1/2 in SCC were investigated focusing on microRNA. We found that miR-214 is the regulator of ERK1, whereas ERK2 is regulated by miR-124 and miR-214. Expression of miR-124 and miR-214 was significantly down-regulated in SCC in vitro and in vivo. Treatment with 5-aza-deoxycytidine and trichostatin A synergistically recovered the miR-124/-214 down-regulation in SCC cell line. However, bisulphite sequencing revealed the methylation status of miR-124/-214 promoter was not increased in the SCC cell line and tumor tissue. Taken together, the down-regulation of miR-124/-214 in SCC is most likely caused, at least in part, by hypermethylation of other promoter regions rather than the miR-124/-214 promoter. Supplementation of these microRNAs in the SCC cell line reduced the abnormal cell proliferation by normalizing ERK1/2 levels. Additionally, serum concentration of miR-124 was correlated with miR-124 expression levels in the tumor tissues and inversely correlated with tumor progression. On the other hand, miR-214 was not detected in the serum. Investigation of the regulatory mechanisms of keratinocyte proliferation by microRNA may lead to develop new biomarkers and treatments using microRNA.
Journal of Molecular Medicine 07/2012; · 4.67 Impact Factor
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Takaomi Sing,
Masatoshi Jinnin, Keitaro Yamane,
Norihito Honda,
Kastunari Makino,
Ikko Kajihara,
Takamitsu Makino,
Keisuke Sakai,
Shinichi Masuguchi,
Satoshi Fukushima,
Hironobu Ihn
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ABSTRACT: microRNAs (miRNAs) play a part in various cellular activities. However, the role of miRNA in SSc is not fully understood. This study investigated the expression and role of miR-92a in SSc patients and evaluated the possibility that miR-92a is involved in the pathogenesis of this disease.
Serum samples were obtained from 61 SSc patients. mRNAs were purified from serum and levels of miR-92a and miR-135 were measured with quantitative real-time PCR. miR-92a expression in dermal fibroblasts was also determined by quantitative real-time PCR. Immunoblotting was performed to detect MMP-1 protein.
The median serum levels of miR-92a, not miR-135, were significantly higher in SSc patients than normal subjects. The constitutive up-regulated miR-92a expression was also found in cultured dermal fibroblasts from SSc skin, which was decreased by the transfection with siRNA of TGF-β. Furthermore, the forced overexpression of miR-92a in normal dermal fibroblasts using miR-92a mimic resulted in the down-regulation of MMP-1 expression.
The increase of miR-92a in SSc may be due to the stimulation of intrinsic TGF-β activation seen in this disease. There is also a possibility that MMP-1 is the target of miR-92a and that increased miR-92a expression therefore plays a role in excessive collagen accumulation in SSc via the down-regulation of MMP-1. Clarifying the role of miRNAs in SSc may result in a better understanding of this disease and the development of new therapeutic approaches.
Rheumatology (Oxford, England) 06/2012; 51(9):1550-6. · 4.24 Impact Factor
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Taiji Nakashima,
Masatoshi Jinnin, Keitaro Yamane,
Noritoshi Honda,
Ikko Kajihara,
Takamitsu Makino,
Shinichi Masuguchi,
Satoshi Fukushima,
Yoshinobu Okamoto,
Minoru Hasegawa,
Manabu Fujimoto,
Hironobu Ihn
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ABSTRACT: Among IL-17 families, IL-17A and IL-17F share amino acid sequence similarity and bind to IL-17R type A. IL-17 signaling is implicated in the pathogenesis of various autoimmune diseases, but its role in the regulatory mechanism of extracellular matrix expression and its contribution to the phenotype of systemic sclerosis (SSc) both remain to be elucidated. This study revealed that IL-17A expression was significantly increased in the involved skin and sera of SSc patients, whereas the IL-17F levels did not increase. In contrast, the expression of IL-17R type A in SSc fibroblasts significantly decreased in comparison with that in normal fibroblasts, due to the intrinsic TGF-β1 activation in these cell types. Moreover, IL-17A, not IL-17F, reduced the protein expression of α1(I) collagen and connective tissue growth factor. miR-129-5p, one of the downregulated microRNAs in SSc fibroblasts, increased due to IL-17A and mediated the α1(I) collagen reduction. These results suggest that IL-17A signaling, not IL-17F, has an antifibrogenic effect via the upregulation of miR-129-5p and the downregulation of connective tissue growth factor and α1(I) collagen. IL-17A signaling is suppressed due to the downregulation of the receptor by the intrinsic activation of TGF-β1 in SSc fibroblasts, which may amplify the increased collagen accumulation and fibrosis characteristic of SSc. Increased IL-17A levels in the sera and involved skin of SSc may be due to negative feedback. Clarifying the novel regulatory mechanisms of fibrosis by the cytokine network consisting of TGF-β and IL-17A may lead to a new therapeutic approach for this disease.
The Journal of Immunology 03/2012; 188(8):3573-83. · 5.79 Impact Factor
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Ikko Kajihara,
Masatoshi Jinnin, Keitaro Yamane,
Takamitsu Makino,
Noritoshi Honda,
Toshikatsu Igata,
Shinichi Masuguchi,
Satoshi Fukushima,
Yoshinobu Okamoto,
Minoru Hasegawa,
Manabu Fujimoto,
Hironobu Ihn
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ABSTRACT: The aim of the present study was to determine the expression and role of thrombospondin-2 (TSP-2) in systemic sclerosis (SSc). Both TSP-2 mRNA levels and protein synthesis in cell lysates were significantly lower in cultured SSc fibroblasts than in normal fibroblasts; however, the TSP-2 protein that accumulated in the conditioned medium of SSc fibroblasts was up-regulated, compared with that of normal fibroblasts, because of an increase in the half-life of the protein. In vivo serum TSP-2 levels were higher in SSc patients than in healthy control subjects, and SSc patients with elevated serum TSP-2 levels tended to have pitting scars and/or ulcers. TSP-2 knockdown resulted in the down-regulation of type I collagen expression and the up-regulation of miR-7, one of the miRNAs with an inhibitory effect on collagen expression. Expression levels of miR-7 were also up-regulated in SSc dermal fibroblasts both in vivo and in vitro. Taken together, these findings suggest that the increased extracellular TSP-2 deposition in SSc fibroblasts may contribute to tissue fibrosis by inducing collagen expression. Down-regulation of intracellular TSP-2 synthesis and the subsequent miR-7 up-regulation in SSc fibroblasts may be due to a negative feedback mechanism that prevents increased extracellular TSP-2 deposition and/or tissue fibrosis. Thus, TSP-2 may play an important role in the maintenance of fibrosis and angiopathy in patients with SSc.
American Journal Of Pathology 12/2011; 180(2):703-14. · 4.89 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) are post-transcriptional regulators that bind to complementary sequences in the 3' UTRs of mRNAs, leading to gene silencing, and their serum levels can be useful biomarkers for diagnosis, prognosis and therapeutic value in various diseases. Although miRNAs are thought to be involved in the pathogenesis of human diseases, little is known about miRNAs in psoriasis. Recently, psoriasis has attracted attention for its characteristics as a Th17 disease; the expression of IL-17 is increased in lesional skin and serum. We hypothesized that miRNAs contribute to the mechanism underlying the overexpression of IL-17. Therefore, serum levels of miR-1266, a putative regulator of IL-17A, in psoriasis patients were determined with the expectation that miR-1266 levels may be decreased in these patients, which may result in induction of IL-17. However, real-time PCR demonstrated that serum miR-1266 levels were considerably higher in psoriasis patients than in healthy control subjects. Furthermore, miR-1266 levels showed weak inverse correlations with Psoriasis Area Severity Index scores and body surface areas of involved skin. Taken together, serum miR-1266 may have potential for a new disease marker. miR-1266 is not likely to regulate IL-17A expression directly, but may be involved in the pathogenesis of psoriasis by regulating other target molecules.
European journal of dermatology: EJD 11/2011; 22(1):68-71. · 2.53 Impact Factor
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ABSTRACT: IL-13, a T helper type 2 cytokine, is reported to be increased in the tissue of patients with atopic dermatitis (AD). In addition, chronic lichenified plaques in AD show thickened epidermis and dermis. We hypothesized that IL-13 is involved in tissue remodeling by altering the expression of matrix metalloproteinases (MMPs). In this study, we examined the MMP-related genes targeted by IL-13 in human dermal fibroblasts using a complementary DNA microarray. We focused on the MMP-13 gene, which was identified as one of the MMPs suppressed by IL-13. IL-13 downregulated both MMP-13 protein and mRNA expression. IL-13 suppressed MMP-13 expression more effectively in the presence of protein kinase C (PKC)-δ inhibitor, whereas IL-13 upregulated MMP-13 in the presence of inhibitors of phosphoinositide 3-kinase (PI3K)/Akt pathway or Akt3-specific small interfering RNA. Our results suggest that MMP-13 expression is negatively controlled by PI3K/Akt3 and positively regulated by PKC-δ in the presence of IL-13. Taken together, these findings indicate that IL-13 may induce the formation of thickened dermis in AD by decreasing collagen degradation. Blockade of IL-13 signaling cascades in AD patients may be a new therapeutic approach.
Journal of Investigative Dermatology 03/2011; 131(3):655-61. · 6.31 Impact Factor
