Hironobu Ihn

Pathogenesis of fibrosis: Role of TGF-β and CTGF

Transforming growth factor (TGF)-beta regulates diverse biologic activities including cell growth, cell death or apoptosis, cell differentiation, and extracellular matrix (ECM) synthesis. TGF-beta is believed to be a key mediator of tissue fibrosis as a consequence of ECM accumulation in pathologic states such as systemic sclerosis. TGF-beta is known to induce the expression of ECM proteins in mesenchymal cells, and to stimulate the production of protease inhibitors that prevent enzymatic breakdown of the ECM. Connective tissue growth factor (CTGF), which is induced by TGF-beta, has been reported to mediate stimulatory actions of TGF-beta ECM synthesis. This review focuses on the possible role of TGF-beta and CTGF in the pathogenesis of fibrosis.

Impaired Smad7-Smurf-mediated negative regulation of TGF-β signaling in scleroderma fibroblasts

The principal effect of TGF-beta1 on mesenchymal cells is its stimulation of ECM synthesis. Previous reports indicated the significance of the autocrine TGF-beta loop in the pathogenesis of scleroderma. In this study, we focused on Smad7 and Smurfs, principal molecules in the negative regulation of TGF-beta signaling, to further understand the autocrine TGF-beta loop in scleroderma. Scleroderma fibroblasts exhibited increased Smad7 levels compared with normal fibroblasts in vivo and in vitro. Smad7 constitutively formed a complex with the TGF-beta receptors, and the inhibitory effect of Smad7 on the promoter activity of human alpha2(I) collagen and 3TP-lux was completely impaired in scleroderma fibroblasts. Furthermore, the protein stability of TGF-beta receptor type I was significantly increased in scleroderma fibroblasts compared with normal fibroblasts. There was no significant difference in Smurf1 and Smurf2 levels between normal and scleroderma fibroblasts, and the transiently overexpressed Smurf1 and/or Smurf2 did not affect TGF-beta receptor type I protein levels in scleroderma fibroblasts. These results indicate that the impaired Smad7-Smurf-mediated inhibitory effect on TGF-beta signaling might contribute to maintaining the autocrine TGF-beta loop in scleroderma fibroblasts. To our knowledge, this is the first report of a disturbed negative regulation of TGF-beta signaling in fibrotic disorders.

Increased Expression of Integrin αvβ3 Contributes to the Establishment of Autocrine TGF-β Signaling in Scleroderma Fibroblasts

The constitutive secretion of latent TGF-β by many cell types in culture suggests that extracellular mechanisms to control the activity of this potent cytokine are important in the pathogenesis of the diseases in which this cytokine may be involved, including fibrotic disorders. In this study, we focused on the αvβ3 integrin, which is recently demonstrated to function as an active receptor for latent TGF-β1 through its interaction with latency-associated peptide-β1, and investigated the involvement of this integrin in the pathogenesis of scleroderma. Scleroderma fibroblasts exhibited increased αvβ3 expression compared with normal fibroblasts in vivo and in vitro. In scleroderma fibroblasts, ERK pathway was constitutively activated and such abnormality induced the up-regulation of αvβ3. Transient overexpression of αvβ3 in normal fibroblasts induced the increase in the promoter activity of human α2(I) collagen gene and the decrease in that of human MMP-1 gene. These effects of αvβ3 were almost completely abolished by the treatment with anti-TGF-β Ab or TGF-β1 antisense oligonucleotide. Furthermore, the addition of anti-αvβ3 Ab reversed the expression of type I procollagen protein and MMP-1 protein, the promoter activity of human α2(I) collagen gene, and the myofibroblastic phenotype in scleroderma fibroblasts. These results suggest that the up-regulated expression of αvβ3 contributes to the establishment of autocrine TGF-β loop in scleroderma fibroblasts, and this integrin is a potent target for the treatment of scleroderma.

The circulating microRNA-221 level in patients with malignant melanoma as a new tumor marker

BACKGROUND MicroRNA-221 (miR-221) is known to be abnormally expressed in malignant melanoma (MM) cells, and it favors the induction of the malignant phenotype through down-modulation of p27Kip1/CDKN1B and the c-KIT receptor. This suggests that the serum level of miR-221 might increase in patients with MM and thus could be used as a new tumor marker. OBJECTIVE To evaluate the possibility that the serum miR-221 level can be a marker of MM. METHODS Serum samples were obtained from 94 MM patients and 20 healthy controls. MicroRNAs were purified from serum, and miR-221 levels were measured by quantitative real-time polymerase chain reaction. RESULTS Circulating miR-221 was detectable and could be quantified in serum samples. MM patients had significantly higher miR-221 levels than healthy controls. Among the MM patients, the miR-221 levels were significantly increased in patients with stage I-IV MM compared to those with MM in situ, and the levels were correlated with tumor thickness. Moreover, a longitudinal study revealed a tendency for the miR-221 levels to decrease after surgical removal of the primary tumor, and to increase again at recurrence. CONCLUSIONS Serum levels of miR-221 were significantly increased in MM patients and may be useful not only for the diagnosis of MM, but also for the differentiating MM in situ from stage I-IV MM, and for evaluating tumor progression and monitoring patients during the follow-up period. In addition, considering that the serum levels of miR-221 were correlated with tumor thickness, miR-221 might also be useful as a prognostic marker for patients with MM.

A randomized double-blind trial of intravenous immunoglobulin for pemphigus

BACKGROUND Pemphigus is a rare life-threatening intractable autoimmune blistering disease caused by IgG autoantibodies to desmogleins. It has been difficult to conduct a double-blind clinical study for pemphigus partly because, in a placebo group, appropriate treatment often must be provided when the disease flares. OBJECTIVE A multicenter, randomized, placebo-controlled, double-blind trial was conducted to investigate the therapeutic effect of a single cycle of high-dose intravenous immunoglobulin (400, 200, or 0 mg/kg/d) administered over 5 consecutive days in patients relatively resistant to systemic steroids. METHODS We evaluated efficacy with time to escape from the protocol as a novel primary end point, and pemphigus activity score, antidesmoglein enzyme-linked immunosorbent assay scores, and safety as secondary end points. RESULTS We enrolled 61 patients with pemphigus vulgaris or pemphigus foliaceus who did not respond to prednisolone (> or =20 mg/d). Time to escape from the protocol was significantly prolonged in the 400-mg group compared with the placebo group (P < .001), and a dose-response relationship among the 3 treatment groups was observed (P < .001). Disease activity and enzyme-linked immunosorbent assay scores were significantly lower in the 400-mg group than in the other groups (P < .05 on day 43, P < .01 on day 85). There was no significant difference in the safety end point among the 3 treatment groups. LIMITATION Prednisolone at 20 mg/d or more may not be high enough to define steroid resistance. CONCLUSION Intravenous immunoglobulin (400 mg/kg/d for 5 d) in a single cycle is an effective and safe treatment for patients with pemphigus who are relatively resistant to systemic steroids. Time to escape from the protocol is a useful indicator for evaluation in randomized, placebo-controlled, double-blind studies of rare and serious diseases.

Impaired IL-17 Signaling Pathway Contributes to the Increased Collagen Expression in Scleroderma Fibroblasts

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.

Tenascin-C upregulation by transforming growth factor-β in human dermal fibroblasts involves Smad3, Sp1, and Ets1

In cultured human dermal fibroblasts, transforming growth factor (TGF)-β induced the mRNA expression of tenascin-C (TN-C). The molecular mechanism(s) underlying this process is not presently understood. In this study, we performed serial 5′ deletion and a transient transfection analysis to define a region in the TN-C promoter mediating the inducible responsiveness to TGF-β. This region contains an atypical nucleotide recognition element for the Smad family of transcriptional regulators. A DNA affinity precipitation assay revealed that Smad2/Smad3 bound to this site in a transient and specific manner. Overexpression of Smad3 or Smad4 activated the TN-C promoter activity and superinduced the TN-C promoter activity stimulated by TGF-β. Moreover, simultaneous cotransfection of Smad3 and Smad4 activated the TN-C promoter activity in a synergistic manner. Mutation of the Smad-binding sites, the Ets-binding sites, or Sp1/3-binding sites in the TN-C promoter abrogated the TGF-β/Smad-inducible promoter activity. Immunoprecipitation analysis revealed that Smad3, Sp1, and Ets1 form a transcriptionally active complex. Furthermore, the interaction between Smads and CBP/p300 in TGF-β signaling was confirmed. These findings demonstrate the existence of a novel, functional binding element in the proximal region of the TN-C promoter mediating responsiveness to TGF-β involving Smad3/4, Sp1, Ets1, and CBP/p300.

TGF-β–Mediated Downregulation of MicroRNA-196a Contributes to the Constitutive Upregulated Type I Collagen Expression in Scleroderma Dermal Fibroblasts

Previous reports indicated the significance of the TGF-β signaling in the pathogenesis of systemic sclerosis. We tried to evaluate the possibility that microRNAs (miRNAs) play a part in the type I collagen upregulation seen in normal fibroblasts stimulated with exogenous TGF-β and systemic sclerosis (SSc) fibroblasts. miRNA expression profile was evaluated by miRNA PCR array and real-time PCR. The protein expression of type I collagen was determined by immunoblotting. In vivo detection of miRNA in paraffin section was performed by in situ hybridization. Several miRNAs were found to be downregulated in both TGF-β–stimulated normal fibroblasts and SSc fibroblasts compared with normal fibroblasts by PCR array. Among them, miR-196a expression was decreased in SSc both in vivo and in vitro by real-time PCR or in situ hybridization. In SSc fibroblasts, miR-196a expression was normalized by TGF-β small interfering RNA. miR-196a inhibitor leads to the overexpression of type I collagen in normal fibroblasts, whereas overexpression of the miRNA resulted in the downregulation of type I collagen in SSc fibroblasts. In addition, miR-196a was detectable and quantitative in the serum of SSc patients. Patients with lower serum miR-196a levels had significantly higher ratio of diffuse cutaneous SSc:limited cutaneous SSc, higher modified Rodnan total skin thickness score, and higher prevalence of pitting scars than those without. miR-196a may play some roles in the pathogenesis of SSc. Investigation of the regulatory mechanisms of type I collagen expression by miR-196a may lead to new treatments using miRNA.

Characterization of monocyte/macrophage subsets in the skin and peripheral blood derived from patients with systemic sclerosis

IntroductionRecent accumulating evidence indicates a crucial involvement of macrophage lineage in the pathogenesis of systemic sclerosis (SSc). To analyze the assembly of the monocyte/macrophage population, we evaluated the expression of CD163 and CD204 and various activated macrophage markers, in the inflammatory cells of the skin and in the peripheral blood mononuclear cells (PBMCs) derived from patients with SSc.MethodsSkin biopsy specimens from 6 healthy controls and 10 SSc patients (7 limited cutaneous SSc and 3 diffuse cutaneous SSc) were analyzed by immunohistochemistry using monoclonal antibody against CD68 (pan-macrophage marker), CD163 and CD204. Surface and/or intracellular protein expression of CD14 (marker for monocyte lineage), CD163 and CD204 was analysed by flow cytometry in PBMCs from 16 healthy controls and 41 SSc patients (26 limited cutaneous SSc and 15 diffuse cutaneous SSc). Statistical analysis was carried out using Mann-Whitney U test for comparison of means.ResultsIn the skin from SSc patients, the number of CD163+ cells or CD204+ cells between the collagen fibers was significantly larger than that in healthy controls. Flow cytometry showed that the population of CD14+ cells was significantly greater in PBMCs from SSc patients than that in healthy controls. Further analysis of CD14+ cells in SSc patients revealed higher expression of CD163 and the presence of two unique peaks in the CD204 histogram. Additionally, we found that the CD163+ cells belong to CD14brightCD204+ population.ConclusionsThis is the first report indicating CD163+ or CD204+ activated macrophages may be one of the potential fibrogenic regulators in the SSc skin. Furthermore, this study suggests a portion of PBMCs in SSc patients abnormally differentiates into CD14brightCD163+CD204+ subset. The subset specific to SSc may play an important role in the pathogenesis of this disease, as the source of CD163+ or CD204+ macrophages in the skin.

Antagonistic Effects of TNF-α on TGF-β Signaling Through Down-Regulation of TGF-β Receptor Type II in Human Dermal Fibroblasts

Transforming growth factor-β stimulates the production of the extracellular matrix, whereas TNF-α has antifibrotic activity. Understanding the molecular mechanism underlying the antagonistic activities of TNF-α against TGF-β is critical in the context of tissue repair and maintenance of tissue homeostasis. In the present study, we demonstrated a novel mechanism by which TNF-α blocks TGF-β-induced gene and signaling pathways in human dermal fibroblasts. We showed that TNF-α prevents TGF-β-induced gene trans activation, such as α2(I) collagen or tissue inhibitor of metalloproteinases 1, and TGF-β signaling pathways, such as Smad3, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases, without inducing levels of inhibitory Smad7 in human dermal fibroblasts. TNF-α down-regulates the expression of type II TGF-β receptor (TβRII) proteins, but not type I TGF-β receptor (TβRI), in human dermal fibroblasts. However, neither TβRII mRNA nor TβRII promoter activity was decreased by TNF-α. TNF-α-mediated decrease of TβRII protein expression was not inhibited by the treatment of fibroblasts with either a selective inhibitor of I-κB-α phosphorylation, BAY 11-7082, or a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, PD98059. Calpain inhibitor I (ALLN), a protease inhibitor, inhibits TNF-α-mediated down-regulation of TβRII. We found that TNF-α triggered down-regulation of TβRII, leading to desensitization of human dermal fibroblasts toward TGF-β. Furthermore, these events seemed to cause a dramatic down-regulation of α2(I) collagen and tissue inhibitor of metalloproteinases 1 in systemic sclerosis fibroblasts. These results indicated that TNF-α impaired the response of the cells to TGF-β by regulating the turnover of TβRII.