Search

Background: Toxic epidermal necrolysis (TEN) is a severe drug-induced cutaneous reaction. Although one of the primary histologic features of TEN is keratinocyte apoptosis, its exact mechanism remains unknown. Objectives: We investigated the role of microRNAs (miRNAs) in the pathogenesis of severe drug eruptions and evaluated the possibility that miRNA can be a disease marker. Methods: miRNAs were extracted from tissues and sera of patients. PCR array analyses were performed to identify pathogenic miRNAs. The results were confirmed with quantitative real-time PCR, in situ hybridization, transient transfection of small interfering RNAs or miRNA mimics into cultured keratinocytes, flow cytometry, immunoblotting, luciferase assay, and immunohistochemistry. Results: PCR array analysis and real-time PCR using tissue miRNAs demonstrated that the miR-18a-5p level was increased in the skin of patients with TEN in vivo. Transfection of the miR-18a-5p mimic into keratinocytes in vitro resulted in increased apoptotic cell numbers and caspase-9 activity, which were also increased in the skin of patients with TEN. The miR-18a-5p mimic also downregulated the expression of B-cell lymphoma/leukemia-2-like protein 10 (BCL2L10), an anti-intrinsic apoptotic molecule. A luciferase assay with the BCL2L10 3′ untranslated region showed BCL2L10 is directly targeted by miR-18a-5p. The protein and mRNA expressions of BCL2L10 were decreased in the skin of patients with TEN. Transfection with BCL2L10 small interfering RNA induced keratinocyte apoptosis and caspase activity. Furthermore, serum miR-18a-5p levels tended to be increased in patients with TEN and were correlated with areas of skin erythema or erosion in patients with drug eruptions. Conclusions: Our results indicated that downregulated BCL2L10 caused by miR-18a-5p overexpression mediates intrinsic keratinocyte apoptosis in patients with TEN. Serum miR-18a-5p levels can be a useful disease marker for drug eruptions. [Copyright &y& Elsevier]

Background: Acral lentiginous melanoma (ALM) is the most common type of melanoma in Japan. The association between ALM and acral nevus has not been elucidated. Objective: To investigate the prevalence and dermatoscopic patterns of plantar melanocytic nevi on the soles in the Japanese and to evaluate the relationship between acral nevi and ALM. Methods: All outpatients (N = 1697) and melanoma patients (N = 104) were included. We examined the number, size, and dermatoscopic images of nevi. Results: In the control group, the prevalence of plantar nevi was 10.9%, and the mean size was 3.8 ± 2.4 mm. The prevalence of nevi in patients with ALM and melanoma in situ on the soles was 8.6% and that of patients with melanoma on other sites was 14.5%. The main dermatoscopic pattern was “parallel furrow” in both groups. Limitations: This was a clinical observational study only. Conclusion: The number, size, and dermatoscopic patterns of nevi on the soles of patients with ALM and melanoma in situ on the soles did not differ from those of the control group. [Copyright &y& Elsevier]

Summary: Excessive extracellular matrix deposition in the skin, lung, and other organs is a hallmark of systemic sclerosis (SSc). Fibroblasts isolated from sclerotic lesions in patients with SSc and cultured in vitro are characterized by increased synthesis of collagen and other extracellular matrix components, consistent with the disease phenotype. Thus, cultured scleroderma fibroblasts serve as a principal experimental model for studying the mechanisms involved in extracellular matrix overproduction in SSc. The pathogenesis of SSc is still poorly understood, but increasing evidence suggests that transforming growth factor-β (TGF-β) is a key mediator of tissue fibrosis as a consequence of extracellular matrix accumulation in the pathology of SSc. TGF-β regulates diverse biological activities including cell growth, cell death or apoptosis, cell differentiation, and extracellular matrix synthesis. TGF-β is known to induce the expression of extracellular matrix proteins in mesenchymal cells and to stimulate the production of protease inhibitors that prevent enzymatic breakdown of the extracellular matrix. This review focuses on the possible role of autocrine TGF-β signaling in the pathogenesis of SSc. [Copyright &y& Elsevier]

Localized scleroderma (LSc) is a connective tissue disorder limited to skin and subcutaneous tissue, which may share pathogenic processes with systemic sclerosis (SSc). We previously demonstrated that upregulated expression of integrin αvβ5 might contribute to autocrine TGF-β signaling in SSc fibroblasts. Based on these data, we presently focused on αvβ5 and assessed its involvement in pathogenesis of LSc. We initially demonstrated that LSc fibroblasts might be activated by the stimulation of autocrine TGF-β. Consistent with SSc fibroblasts, expression levels of αvβ5 were elevated in LSc fibroblasts in vitro and in vivo. Anti-αvβ5 antibody partially reversed expression levels of type I procollagen and MMP-1 and constitutive DNA–Smad3 binding in LSc fibroblasts. In LSc fibroblasts pretreated with antisense TGF-β1, exogenous latent TGF-β1 stimulation increased expression of type I procollagen in an αvβ5-dependent manner. The luciferase activities of TMLC cells, Mv1Lu cells stably expressing a portion of the plasminogen activator inhibitor 1 promoter, co-cultured with LSc fibroblasts were significantly elevated compared with those co-cultured with normal fibroblasts and were significantly reduced in the presence of anti-αvβ5 antibody. Anti-αvβ5 antibody reversed the myofibroblastic features of LSc fibroblasts. These results indicate that upregulated expression of αvβ5 contributes to autocrine TGF-β signaling in LSc fibroblasts.Journal of Investigative Dermatology (2006) 126, 1761–1769. doi:10.1038/sj.jid.5700331; published online 4 May 2006 [ABSTRACT FROM AUTHOR]

In this study, we examined the genes targeted by IL-13 in human dermal fibroblasts using a cDNA microarray. We focused on the tenascin-C (TN-C) gene, which was identified as one of the genes induced by IL-13. IL-13 induced transcriptional activity of TN-C. IL-13-mediated TN-C expression was inhibited by treatment with wortmannin or LY294002, or Calphostin C. IL-13 induced the phosphorylation of the phosphoinositide 3-kinase (PI3K) regulatory subunit p85, induced tyrosine phosphorylation of Akt, upregulated Akt kinase activity, and activated protein kinase C (PKC)-δ and -ɛ. The IL-13-induced increase in TN-C protein expression was abrogated by the transfection of a dominant-negative mutant of Akt, PKC-δ, or PKC-ɛ. In conclusion, we showed that the PI3K/Akt and/or PKC signaling pathways are essential for the IL-13-mediated increase in TN-C. Both serum levels of IL-13 and the expression levels of TN-C in the dermis are increased in patients with systemic sclerosis. Our findings suggest that the expression of TN-C is upregulated in this disease due to IL-13 signaling, and that a blockade of the PI3K or PKC signaling pathway may also have therapeutic value by reducing the amount of TN-C produced during fibrosis. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β (TGF-β) stimulates the transcription of the α2(I) collagen gene. The dermal fibroblast activation in systemic sclerosis (SSc) may be a result of stimulation by autocrine TGF-β. In this study, we investigated whether p38 mitogen-activated protein kinase (MAPK) is involved in TGF-β-induced transcriptional activation of the human α2(I) collagen gene in normal dermal fibroblasts and in upregulated extracellular matrix (ECM) expression in SSc fibroblasts. Type I collagen expression induced by TGF-β was suppressed by the specific p38 MAPK inhibitors SB203580 or SB202190 in normal fibroblasts. TGF-β induced phosphorylation and activation of p38 MAPK in normal dermal fibroblasts. Transient transfection of dominant-negative mutant p38 MAPK into normal fibroblasts abolished TGF-β-induced promoter activity of the human α2(I) collagen gene in normal fibroblasts. Moreover, constitutive phosphorylation and activation of p38 MAPK was demonstrated in SSc fibroblasts, and the inhibition of p38 MAPK using specific p38 MAPK inhibitors or dominant-negative mutant p38 MAPK abolished the upregulated expression of type I collagen or fibronectin in SSc fibroblasts. These results strongly suggest the contribution of p38 MAPK signaling to the TGF-β-mediated regulation of the human α2(I) collagen gene in normal dermal fibroblasts and constitutive upregulated expression of type I collagen and fibronectin in SSc fibroblasts. [ABSTRACT FROM AUTHOR]

Most of the cultured scleroderma fibroblasts have been reported to be myofibroblasts that have the ability to expressα smooth muscle actin (αSMA). It is reported that, in human lung fibroblasts,αSMA is induced by transforming growth factor-β (TGF-β), which requires focal adhesion kinase (FAK) phosphorylation on its Tyr-397 site. In this study, we investigated howαSMA expression is upregulated in cultured scleroderma fibroblasts. 4-amino-5-(4-chlorophenyl)-7-(butyl)pyrazolo[3,4-d]pyrimidine, which is a pharmacologic inhibitor of FAK/Src, markedly diminished upregulatedαSMA expression in scleroderma fibroblasts as well as in normal fibroblasts stimulated with TGF-β. Likewise,αSMA expression was significantly reduced in sclerderma fibroblasts transfected with kinase-deficient FAK mutant. FAK phosphorylation levels on Tyr-397 in scleroderma fibroblasts were significantly higher than those in normal fibroblasts. BothαSMA expression and FAK phosphorylation levels in scleroderma fibroblasts were markedly diminished by the treatment with TGF-β antisense oligonucleotide. These results indicate that the constitutive phosphorylation of FAK, which is possibly because of the autocrine TGF-β signaling, may play an important role inαSMA expression in scleroderma fibroblasts. [ABSTRACT FROM AUTHOR]

We investigated the direct effect of hepatocyte growth factor (HGF) on the expression of type I collagen in normal and scleroderma dermal fibroblasts, and analyzed the mechanisms underlying the effectin vitro. HGF did not change the protein expression of type I procollagen in the medium of normal human fibroblasts, whereas it reduced the expression in scleroderma fibroblasts. But mRNA levels and the promoter activity ofα2(I) collagen gene were not significantly affected by HGF in either of the cells. On the other hand, matrix metalloproteinase-1 expression or activity was increased by HGF in both cells, but HGF had stronger effects in scleroderma fibroblasts than normal fibroblasts. Scleroderma fibroblasts overexpressed c-met protein, the receptor for HGF. The overexpression in scleroderma fibroblasts was abolished by the addition of antisense transforming growth factor (TGF)-β1 oligonucleotide. Our study indicated that HGF may reduce type I collagen accumulation only in scleroderma fibroblasts by enhancing collagenolysis activity, probably because of the overexpression of c-met because of autocrine TGF-β signaling. Thus, further investigation of the effects of HGF on collagen metabolism may contribute to the treatment of fibrosis in scleroderma. [ABSTRACT FROM AUTHOR]

Interleukin (IL)-13 is a novel lymphokine produced by activated Type 2 helper cells. In this study, we examined the target genes of IL-13 by the cDNA microarray analysis in human dermal fibroblasts. We focused on the human α2(I) collagen gene, which was one of the IL-13-induced genes by the microarray analysis. IL-13 induced type I collagen protein as well as mRNA in a dose-dependent manner. Actinomycin D, an RNA synthesis inhibitor, significantly blocked the IL-13-mediated up-regulation of α2(I) collagen mRNA expression, whereas cycloheximide, a protein synthesis inhibitor, did not block this up-regulation. In addition, IL-13 treatment induced the promoter activity of α2(I) collagen by nuclear run-on transcription assay and chloramphenicol acetyltransferase assay. IL-13-mediated transcriptional activation of α2(I) collagen gene or type I collagen protein up-regulation was inhibited by the treatment of fibroblasts with a selective phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, or STAT6 antisense oligonucleotide, but not by PD98059, a specific inhibitor of MEK/ERK, or SB202190 or SB203580, specific inhibitors of p38 MAPK; IL-13 induced the phosphorylation of PI3K p85 regulatory subunit and STAT6. These results suggest that IL-13 may play a role in the regulation of extracellular matrix and indicate the possible therapeutic value of the blockade of IL-13 signaling pathways via PI3K and STAT6 in fibrosis. [ABSTRACT FROM AUTHOR]

Epidermal growth factor (EGF) and fibronectin are known to play an important role in wound healing. In this study, we demonstrated that EGF upregulates the expression of fibronectin mRNA and protein in human dermal fibroblasts. Actinomycin D, an RNA synthesis inhibitor, significantly blocked basal mRNA expression, but the addition of EGF compensated the blockage. Cycloheximide, a protein synthesis inhibitor, did not block this upregulation by EGF. In addition, the treatment with EGF significantly reduced the degradation rate of fibronectin mRNA. But EGF did not increase fibronectin promoter activity. EGF-mediated induction of fibronectin expression was inhibited by the treatment of fibroblasts with protein kinase C (PKC) inhibitor, Calphostin C and Rottlerin. The transfection of a dominant-negative mutant of PKCδ into fibroblasts significantly reduced the induction of fibronectin protein expression by EGF. EGF enhanced PKCδ protein expression and also translocated PKCδ to the membrane. Rottlerin blocked the EGF-mediated reduction of mRNA degradation rate. These results indicate that EGF-mediated induction of fibronectin expression occurs at the post-transcriptional level and involves PKCδ signaling pathway. [ABSTRACT FROM AUTHOR]

We studied the prevalence and clinical significance of anti-U1 RNA antibodies in patients with systemic sclerosis. The presence of anti-U1 RNA antibodies was determined using immunoprecipitation in systemic sclerosis patients with anti-U1 RNP antibodies (n =36), antitopoisomerase I antibodies (n =20), or anticentromere antibodies (n =20), mixed connective tissue disease patients (n =23), systemic lupus erythematosus patients with anti-U1 RNP antibodies (n =26), and normal controls (n =20). Moreover, antigen specificities for anti-U1 RNP antibodies were examined in patients with systemic sclerosis by immunoblotting and enzyme-linked immunosorbent assay. Anti-U1 RNA antibodies was detected in 22 of 36 systemic sclerosis patients (61%) with anti-U1 RNP antibodies, 14 of 23 patients (61%) with mixed connective tissue disease, and eight of 26 systemic lupus erythematosus patients (31%) with anti-U1 RNP antibodies. Anti-U1 RNA antibodies were not detected in other groups. As for systemic sclerosis patients, the frequencies of pulmonary fibrosis and reduced percentage diffusion capacity for carbon monoxide were significantly greater in patients with anti-U1 RNA antibodies than in those without (76%vs 18%, p<0.005; 82%vs 27%, p<0.005, respectively). Moreover, patients with anti-U1 RNA antibodies had significantly lower percentage diffusion capacity for carbon monoxide and percentage vital capacity values than those without (51.9±16.8 vs 79.4±16.4, p<0.01; 83.8±21.4 vs 101.4±12.9, p<0.05, respectively). Regarding the antigen specificities of anti-U1 RNP antibodies in systemic sclerosis patients, the frequency of anti-70 kDa antibodies determined by immunoblotting was significantly higher in patients with anti-U1 RNA antibodies than in those without (77%vs 43%, p<0.05). This finding was also confirmed by enzyme-linked immunosorbent assay for anti-70 kDa antibodies (86%vs 43%, p<0.05). These results indicate that anti-U1 RNA antibodies may be a... [ABSTRACT FROM AUTHOR]

Dermatofibrosarcoma protuberans is a malignant mesenchymal tumor originating in the dermis. Although it is locally aggressive and recurs unless completely excised, it only rarely metastasizes. In this study, we investigated the mechanisms of increased proliferation of dermatofibrosarcoma protuberans cells. The cells showed increased DNA synthesis in serum-free medium, which was demonstrated by the incorporation of [3 H]-thymidine. Increased DNA synthesis of dermatofibrosarcoma protuberans cells was abolished by genistein, a tyrosine kinase inhibitor, or by PD98059, a specific extracellular signal related kinase pathway inhibitor, but not by calphostin C, a protein kinase C inhibitor. Immunoblotting analysis of dermatofibrosarcoma protuberans cells using a specific antibody against phosphorylated extracellular signal related kinase (Thr202 /Tyr204 ) showed that extracellular signal related kinase was expressed as constitutively phosphorylated molecules in dermatofibrosarcoma protuberans cells. Immunofluorescence analysis showed that the kinase was constitutively located in the nucleus of the cells. Furthermore, transfection of the dominant negative mutant extracellular signal related kinase into dermatofibrosarcoma protuberans cells abolished the increased mitogenic activity of the cells. These results suggest that an extracellular signal related kinase dependent pathway is implicated in the increased mitogenic activity of dermatofibrosarcoma protuberans cells. [ABSTRACT FROM AUTHOR]

Serum has been known to stimulate collagen production by dermal fibroblasts. As part of an ongoing study of the molecular mechanisms of collagen production, we have investigated transcriptional regulation of the human α2(I) collagen gene by serum in human dermal fibroblasts. Serum responsive elements were mapped by deletion analysis between bp -353 and -264, and between -148 and -108 in the α2(I) collagen promoter. Further functional analysis of the α2(I) collagen promoter containing various substitution mutations revealed that serum stimulation of this promoter is mediated equally by a GC-rich region located between bp -303 and -271 and by the TCCTCC motif located between bp -123 and -128, both of which constitute binding sites for transcription factor Spl and Sp3. No differences were observed in electrophoretic mobility shift assays between unstimulated and serum stimulated fibroblasts. The Spl inhibitor mithramycin blocked stimulation of the α2(I) collagen promoter activity by serum. Furthermore, immunoprecipitation analysis showed that serum stimulation increased Spl phosphorylation. In conclusion, this study characterized response elements that mediate serum stimulation of the human α2(I) collagen promoter and suggests that serum stimulation was mediated via Sp1/Sp3 binding sites in this promoter. [ABSTRACT FROM AUTHOR]

Summary Previous studies have identified four cis-response elements which mediate the basal transcriptional activity of the human α2(I) collagen gene. One of these elements, a pyrimidine-rich region (TCCCCC motif), was shown to be a repressor site, and the other three elements were shown to be activator sites. Furthermore, the repressor site and two of the activator sites were found to constitute binding sites for the transcription factors Sp1 and Sp3. In this study, we further determined the affinity and specificity of the binding of Sp1 and Sp3 to the human α2(I) collagen promoter and investigated the function of the pyrimidine-rich region which contains the TCCCCC motif. Functional analyses of Sp1 and Sp3 in Drosophila cells confirmed that Sp1 and Sp3 activate the human α2(I) collagen promoter via the GC boxes and the TCCTCC motif, but that binding of Sp1 or Sp3 to the repressor site does not activate or repress the collagen promoter activity. Com- petitive analyses using DNA mobility shift assays showed that the TCCCCC motif which constitutes the repressor site abolished the binding of Sp1 or Sp3 to the GC boxes or the TCCTCC motif, but not the binding of CCAAT-binding factor to the fourth cis-response element (CCAAT-binding factor site). Furthermore, the affinity of Sp1 or Sp3 for the TCCTCC motif was shown to be greater than that of the Sp1 consensus oligonucleotide. In vitro transcription analysis revealed that the addition of each activator site oligonucleotide or repressor site oligonucleotide had an inhibitory effect on the transcription of the collagen gene. These results suggest that the repressor site regulates the transcription of the collagen gene by taking away Sp1 or Sp3 from the activator sites. [ABSTRACT FROM AUTHOR]