The downregulation of microRNA let-7a contributes to the excessive expression of type I collagen in systemic and localized scleroderma.

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.

Upregulation of miR-18a-5p contributes to epidermal necrolysis in severe drug eruptions.

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.

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-ß1 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-ß1 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-ß and IL-17A may lead to a new therapeutic approach for this disease.

Increased accumulation of extracellular thrombospondin-2 due to low degradation activity stimulates type I collagen expression in scleroderma fibroblasts.

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.

Synthesized basement membranes direct the differentiation of mouse embryonic stem cells into pancreatic lineages.

We previously reported that embryonic stem (ES) cells cultured on M15 cells, a mesoderm-derived supportive cell line, were efficiently differentiated towards an endodermal fate, finally adopting the specific lineages of various digestive organs such as the pancreas and liver. We show here that the endoderm-inducing activity of M15 cells is in part mediated through the extracellular matrices, and that laminin alpha5 is one of the crucial components. In an attempt to establish a feeder-free ES-cell procedure for pancreatic differentiation, we used a synthesized basement membrane (sBM) substratum using an HEK293 cell line stably expressing laminin-511. On the sBM, mouse ES or induced pluripotent stem (iPS) cells sequentially differentiated into the definitive endoderm, pancreatic progenitor cells, and then insulin-expressing pancreatic beta-cells in vitro. Knockdown of ES cells with integrin beta1 (Itgb1) reduces differentiation towards pancreatic cells. Heparan sulfate proteoglycan 2 (HSPG2) knockdown and heparitinase treatment synergistically decreased the number of Pdx1-expressing cells. These findings indicate that components of the basement membrane have an important role in the differentiation of definitive endoderm lineages. This novel procedure will be useful for the study of pancreatic differentiation of ES or iPS cells and the generation of potential sources of surrogate cells for regenerative medicine.

microRNA-92a expression in the sera and dermal fibroblasts increases in patients with scleroderma.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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-ß. Furthermore, the forced overexpression of miR-92a in normal dermal fibroblasts using miR-92a mimic resulted in the down-regulation of MMP-1 expression. CONCLUSION: The increase of miR-92a in SSc may be due to the stimulation of intrinsic TGF-ß 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.

Increased serum levels of miR-1266 in patients with psoriasis vulgaris.

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.

In vivo partial reprogramming by bacteria promotes adult liver organ growth without fibrosis and tumorigenesis.

Ideal therapies for regenerative medicine or healthy aging require healthy organ growth and rejuvenation, but no organ-level approach is currently available. Using Mycobacterium leprae (ML) with natural partial cellular reprogramming capacity and its animal host nine-banded armadillos, we present an evolutionarily refined model of adult liver growth and regeneration. In infected armadillos, ML reprogram the entire liver and significantly increase total liver/body weight ratio by increasing healthy liver lobules, including hepatocyte proliferation and proportionate expansion of vasculature, and biliary systems. ML-infected livers are microarchitecturally and functionally normal without damage, fibrosis, or tumorigenesis. Bacteria-induced reprogramming reactivates liver progenitor/developmental/fetal genes and upregulates growth-, metabolism-, and anti-aging-associated markers with minimal change in senescence and tumorigenic genes, suggesting bacterial hijacking of homeostatic, regeneration pathways to promote de novo organogenesis. This may facilitate the unraveling of endogenous pathways that effectively and safely re-engage liver organ growth, with broad therapeutic implications including organ regeneration and rejuvenation.

CD437 induces apoptosis in ovarian adenocarcinoma cells via ER stress signaling.

A synthetic retinoid, CD437, has been shown to exert potent anti-tumor activity against various types of cancer cell lines, regardless of their sensitivities to natural retinoids. We herein demonstrate that CD437 induces endoplasmic reticulum (ER) stress, including the up-regulation of CHOP, BIP and GADD34 mRNA through ER stress transducer (PERK and IRE1alpha) activation in an ovarian adenocarcinoma cell line, SKOV3. It was also shown that CD437 induced the CHOP and GADD34 expressions in another four ovarian adenocarcinoma cell lines, indicating that CD437 functions as an ER stress inducer in these cell lines. Moreover, the siRNA-mediated knockdown of inducible CHOP expression prevented the cytotoxic effect of CD437. These results suggest that ER stress plays an important role in the mechanism by which CD437 induces apoptosis in ovarian adenocarcinoma cells.

Regulatory mechanisms of collagen expression by interleukin-22 signaling in scleroderma fibroblasts.

BACKGROUND: Various cytokines have been indicated to be involved in the pathogenesis of systemic sclerosis (SSc). IL-22 is one of the member of IL-10 cytokine family, and several studies have implicated IL-22 signaling in the pathogenesis of autoimmune diseases. OBJECTIVES: To clarify the role of IL-22 in the regulatory mechanism of ECM expression and to determine the contribution of IL-22 to the phenotype of SSc. METHODS: The effect of IL-22 on ECM expression in normal fibroblasts was determined by using PCR array, real-time PCR and immunoblotting. microRNA expression was evaluated by real-time PCR. The expression levels of IL-22 in the skin and sera were determined by using immunohistochemical staining and ELISA. RESULTS: IL-22 significantly increased the expression of type I collagen protein without changing its mRNA levels in cultured normal human dermal fibroblast. The expression of let-7a, one of the microRNAs which have negative effect on type I collagen expression, was significantly decreased by the treatment with IL-22 in dermal fibroblasts. There was no significant difference in the serum levels of IL-22 between SSc patients and control subjects. However, the expression of IL-22 was detected in the infiltrated lymphocytes in the SSc dermis, but not in normal dermis. IL-22 receptors were expressed in both normal and SSc dermal fibroblasts to the similar extent. CONCLUSION: IL-22 expressed in infiltrated lymphocytes may stimulate the up-regulation of type I collagen protein in dermal fibroblasts via let-7a down-regulation in SSc skin.

Regulation of SLD5 gene expression by miR-370 during acute growth of cancer cells.

SLD5 is a member of the GINS complex, essential for DNA replication in eukaryotes. It has been reported that SLD5 is involved in early embryogenesis in the mouse, and cell cycle progression and genome integrity in Drosophila. SLD5 may be involved in malignant tumor progression, but its relevance in human cancer has not been determined. Here, we found strong SLD5 expression in both human bladder cancer tissues from patients and cell lines. Knockdown of SLD5 using small interfering RNA resulted in reduction of cell growth both in vitro and an in vivo xenograft model. Moreover, we found that high levels of SLD5 in bladder cancer cells result from downregulation of microRNA (miR)-370 that otherwise suppresses its expression. High level expression of DNA-methyltransferase (DNMT) 1 and IL-6 were also observed in bladder cancer cells. Knockdown of IL-6 led to downregulation of DNMT1 and SLD5 expression, suggesting that IL-6-induced overexpression of DNMT1 suppresses miR-370, resulting in high SLD5 expression. Our findings could contribute to understanding tumorigenic processes and progression of human bladder cancer, whereby inhibition of SLD5 could represent a novel strategy to prevent tumor growth.

Endothelial Side Population Cells Contribute to Tumor Angiogenesis and Antiangiogenic Drug Resistance.

Angiogenesis plays a crucial role in tumor growth, with an undisputed contribution of resident endothelial cells (EC) to new blood vessels in the tumor. Here, we report the definition of a small population of vascular-resident stem/progenitor-like EC that contributes predominantly to new blood vessel formation in the tumor. Although the surface markers of this population are similar to other ECs, those from the lung vasculature possess colony-forming ability in vitro and contribute to angiogenesis in vivo These specific ECs actively proliferate in lung tumors, and the percentage of this population significantly increases in the tumor vasculature relative to normal lung tissue. Using genetic recombination and bone marrow transplant models, we show that these cells are phenotypically true ECs and do not originate from hematopoietic cells. After treatment of tumors with antiangiogenic drugs, these specific ECs selectively survived and remained in the tumor. Together, our results established that ECs in the peripheral vasculature are heterogeneous and that stem/progenitor-like ECs play an indispensable role in tumor angiogenesis as EC-supplying cells. The lack of susceptibility of these ECs to antiangiogenic drugs may account for resistance of the tumor to this drug type. Thus, inhibiting these ECs might provide a promising strategy to overcome antiangiogenic drug resistance. Cancer Res; 76(11); 3200-10. ©2016 AACR.

Down-regulation of microRNA-196a in the sera and involved skin of localized scleroderma patients.

BACKGROUND: Localized scleroderma (LSc) exhibits fibrosis of the skin and subcutaneous tissue. LSc shows an excessive deposition of type 1 collagen. OBJECTIVES: To elucidate the mechanism of type 1 collagen overexpression in LSc, we investigated the epigenetics, focusing on microRNA (miRNA). MATERIALS & METHODS: miRNA expression profile was determined by PCR array analysis. The expression of microRNA-196a (miR-196a) in the skin tissue was examined by in situ hybridization or real-time PCR. The serum levels of miR-196a were measured by real-time PCR. RESULTS: PCR array analysis demonstrated that the miR-196a level was markedly decreased in LSc skin tissue in vivo. The transfection of specific inhibitor for miR-196a into normal cultured human dermal fibroblasts led to the up-regulation of type 1 collagen protein in vitro. Furthermore, the serum levels of miR-196a were significantly decreased in LSc patients. CONCLUSION: Down-regulation of miR-196a and subsequent overexpression of type 1 collagen in dermal fibroblasts may play a key role in the pathogenesis of LSc. The serum levels of miR-196a may be useful as a diagnostic marker of LSc.

Down-regulation of miR-223 contributes to the formation of Gottron's papules in dermatomyositis via the induction of PKCɛ.

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.

microRNA-7 down-regulation mediates excessive collagen expression in localized scleroderma.

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.

Down-regulation of miR-124/-214 in cutaneous squamous cell carcinoma mediates abnormal cell proliferation via the induction of ERK.

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.

Expression of matrix metalloproteinase-13 is controlled by IL-13 via PI3K/Akt3 and PKC-δ in normal human dermal fibroblasts.

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.