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Sample records for fibroblasts expressing active

  1. Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity

    PubMed Central

    Hinz, Boris; Celetta, Giuseppe; Tomasek, James J.; Gabbiani, Giulio; Chaponnier, Christine

    2001-01-01

    To evaluate whether α-smooth muscle actin (α-SMA) plays a role in fibroblast contractility, we first compared the contractile activity of rat subcutaneous fibroblasts (SCFs), expressing low levels of α-SMA, with that of lung fibroblasts (LFs), expressing high levels of α-SMA, with the use of silicone substrates of different stiffness degrees. On medium stiffness substrates the percentage of cells producing wrinkles was similar to that of α-SMA–positive cells in each fibroblast population. On high stiffness substrates, wrinkle production was limited to a subpopulation of LFs very positive for α-SMA. In a second approach, we measured the isotonic contraction of SCF- and LF-populated attached collagen lattices. SCFs exhibited 41% diameter reduction compared with 63% by LFs. TGFβ1 increased α-SMA expression and lattice contraction by SCFs to the levels of LFs; TGFβ-antagonizing agents reduced α-SMA expression and lattice contraction by LFs to the level of SCFs. Finally, 3T3 fibroblasts transiently or permanently transfected with α-SMA cDNA exhibited a significantly higher lattice contraction compared with wild-type 3T3 fibroblasts or to fibroblasts transfected with α-cardiac and β- or γ-cytoplasmic actin. This took place in the absence of any change in smooth muscle or nonmuscle myosin heavy-chain expression. Our results indicate that an increased α-SMA expression is sufficient to enhance fibroblast contractile activity. PMID:11553712

  2. Tumor-produced, active Interleukin-1 {beta} regulates gene expression in carcinoma-associated fibroblasts

    SciTech Connect

    Dudas, Jozsef; Fullar, Alexandra; Bitsche, Mario; Schartinger, Volker; Kovalszky, Ilona; Sprinzl, Georg Mathias; Riechelmann, Herbert

    2011-09-10

    Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial-mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1{beta} (IL1-{beta}) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-{beta} expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-{beta} processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-{beta}. IL1-{beta} signaling was investigated by western blot and immunocytochemistry. IL1-{beta}-regulated genes were analyzed by real-time qPCR. SCC-25 cells produced 16 kD active IL1-{beta}, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NF{kappa}B{alpha}. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-{beta} reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-{beta}-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-{beta} in the tumor cells leads to IL1-{beta}-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression. -- Graphical abstract: SCC-25 cells produce active, processed IL1-{beta}. PDL fibroblasts possess receptor for IL1-{beta}, and its expression is increased 4.56-times in the

  3. Rationale Behind Targeting Fibroblast Activation Protein–Expressing Carcinoma-Associated Fibroblasts as a Novel Chemotherapeutic Strategy

    PubMed Central

    Brennen, W. Nathaniel; Isaacs, John T.; Denmeade, Samuel R.

    2013-01-01

    The tumor microenvironment has emerged as a novel chemotherapeutic strategy in the treatment of cancer. This is most clearly exemplified by the antiangiogenesis class of compounds. Therapeutic strategies that target fibroblasts within the tumor stroma offer another treatment option. However, despite promising data obtained in preclinical models, such strategies have not been widely used in the clinical setting, largely due to a lack of effective treatments that specifically target this population of cells. The identification of fibroblast activation protein α (FAP) as a target selectively expressed on fibroblasts within the tumor stroma or on carcinoma-associated fibroblasts led to intensive efforts to exploit this novel cellular target for clinical benefit. FAP is a membrane-bound serine protease of the prolyl oligopeptidase family with unique post-prolyl endopeptidase activity. Until recently, the majority of FAP-based therapeutic approaches focused on the development of small-molecule inhibitors of enzymatic activity. Evidence suggests, however, that FAP’s pathophysiological role in carcinogenesis may be highly contextual, depending on both the exact nature of the tumor microenvironment present and the cancer type in question to determine its tumor-promoting or tumor-suppressing phenotype. As an alternative strategy, we are taking advantage of FAP’s restricted expression and unique substrate preferences to develop a FAP-activated prodrug to target the activation of a cytotoxic compound within the tumor stroma. Of note, this strategy would be effective independently of FAP’s role in tumor progression because its therapeutic benefit would rely on FAP’s localization and activity within the tumor microenvironment rather than strictly on inhibition of its function. PMID:22323494

  4. Fibroblast activation protein is expressed by rheumatoid myofibroblast-like synoviocytes

    PubMed Central

    Bauer, Stefan; Jendro, Michael C; Wadle, Andreas; Kleber, Sascha; Stenner, Frank; Dinser, Robert; Reich, Anja; Faccin, Erica; Gödde, Stefan; Dinges, Harald; Müller-Ladner, Ulf; Renner, Christoph

    2006-01-01

    Fibroblast activation protein (FAP), as described so far, is a type II cell surface serine protease expressed by fibroblastic cells in areas of active tissue remodelling such as tumour stroma or healing wounds. We investigated the expression of FAP by fibroblast-like synoviocytes (FLSs) and compared the synovial expression pattern in rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Synovial tissue from diseased joints of 20 patients, 10 patients with refractory RA and 10 patients with end-stage OA, was collected during routine surgery. As a result, FLSs from intensively inflamed synovial tissues of refractory RA expressed FAP at high density. Moreover, FAP expression was co-localised with matrix metalloproteinases (MMP-1 and MMP-13) and CD44 splice variants v3 and v7/8 known to play a major role in the concert of extracellular matrix degradation. The pattern of signals appeared to constitute a characteristic feature of FLSs involved in rheumatoid arthritic joint-destructive processes. These FAP-expressing FLSs with a phenotype of smooth muscle actin-positive myofibroblasts were located in the lining layer of the synovium and differ distinctly from Thy-1-expressing and non-proliferating fibroblasts of the articular matrix. The intensity of FAP-specific staining in synovial tissue from patients with RA was found to be different when compared with end-stage OA. Because expression of FAP by RA FLSs has not been described before, the findings of this study highlight a novel element in cartilage and bone destruction of arthritic joints. Moreover, the specific expression pattern qualifies FAP as a therapeutic target for inhibiting the destructive potential of fibroblast-like synovial cells. PMID:17105646

  5. p53/PUMA expression in human pulmonary fibroblasts mediates cell activation and migration in silicosis

    PubMed Central

    Wang, Wei; Liu, Haijun; Dai, Xiaoniu; Fang, Shencun; Wang, Xingang; Zhang, Yingming; Yao, Honghong; Zhang, Xilong; Chao, Jie

    2015-01-01

    Phagocytosis of SiO2 into the lung causes an inflammatory cascade that results in fibroblast proliferation and migration, followed by fibrosis. Clinical evidence has indicated that the activation of alveolar macrophages by SiO2 produces rapid and sustained inflammation characterized by the generation of monocyte chemotactic protein 1, which, in turn, induces fibrosis. However, the details of events downstream of monocyte chemotactic protein 1 activity in pulmonary fibroblasts remain unclear. Here, to elucidate the role of p53 in fibrosis induced by silica, both the upstream molecular mechanisms and the functional effects on cell proliferation and migration were investigated. Experiments using primary cultured adult human pulmonary fibroblasts led to the following results: 1) SiO2 treatment resulted in a rapid and sustained increase in p53 and PUMA protein levels; 2) the MAPK and PI3K pathways were involved in the SiO2-induced alteration of p53 and PUMA expression; and 3) RNA interference targeting p53 and PUMA prevented the SiO2-induced increases in fibroblast activation and migration. Our study elucidated a link between SiO2-induced p53/PUMA expression in fibroblasts and cell migration, thereby providing novel insight into the potential use of p53/PUMA in the development of novel therapeutic strategies for silicosis treatment. PMID:26576741

  6. Gene expression and activity of cartilage degrading glycosidases in human rheumatoid arthritis and osteoarthritis synovial fibroblasts

    PubMed Central

    Pásztói, Mária; Nagy, György; Géher, Pál; Lakatos, Tamás; Tóth, Kálmán; Wellinger, Károly; Pócza, Péter; György, Bence; Holub, Marianna C; Kittel, Ágnes; Pálóczy, Krisztina; Mazán, Mercédesz; Nyirkos, Péter; Falus, András; Buzas, Edit I

    2009-01-01

    Introduction Similar to matrix metalloproteinases, glycosidases also play a major role in cartilage degradation. Carbohydrate cleavage products, generated by these latter enzymes, are released from degrading cartilage during arthritis. Some of the cleavage products (such as hyaluronate oligosaccharides) have been shown to bind to Toll-like receptors and provide endogenous danger signals, while others (like N-acetyl glucosamine) are reported to have chondroprotective functions. In the current study for the first time we systematically investigated the expression of glycosidases within the joints. Methods Expressions of β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, sperm adhesion molecule 1 and klotho genes were measured in synovial fibroblasts and synovial membrane samples of patients with rheumatoid arthritis and osteoarthritis by real-time PCR. β-D-Glucuronidase, β-D-glucosaminidase and β-D-galactosaminidase activities were characterized using chromogenic or fluorogenic substrates. Synovial fibroblast-derived microvesicles were also tested for glycosidase activity. Results According to our data, β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, and klotho are expressed in the synovial membrane. Hexosaminidase is the major glycosidase expressed within the joints, and it is primarily produced by synovial fibroblasts. HexA subunit gene, one of the two genes encoding for the alpha or the beta chains of hexosaminidase, was characterized by the strongest gene expression. It was followed by the expression of HexB subunit gene and the β-D-glucuronidase gene, while the expression of hyaluronidase-1 gene and the klotho gene was rather low in both synovial fibroblasts and synovial membrane samples. Tumor growth factor-β1 profoundly downregulated glycosidase expression in both rheumatoid arthritis and osteoarthritis derived synovial fibroblasts. In addition, expression of cartilage-degrading glycosidases was moderately downregulated by proinflammatory

  7. Integrated gene and miRNA expression analysis of prostate cancer associated fibroblasts supports a prominent role for interleukin-6 in fibroblast activation

    PubMed Central

    Giannoni, Elisa; D'Aiuto, Francesca; Maffezzini, Massimo; Valdagni, Riccardo; Chiarugi, Paola; Gandellini, Paolo; Zaffaroni, Nadia

    2015-01-01

    Tumor microenvironment coevolves with and simultaneously sustains cancer progression. In prostate carcinoma (PCa), cancer associated fibroblasts (CAF) have been shown to fuel tumor development and metastasis by mutually interacting with tumor cells. Molecular mechanisms leading to activation of CAFs from tissue-resident fibroblasts, circulating bone marrow-derived fibroblast progenitors or mesenchymal stem cells are largely unknown. Through integrated gene and microRNA expression profiling, we showed that PCa-derived CAF transcriptome strictly resembles that of normal fibroblasts stimulated in vitro with interleukin-6 (IL6), thus proving evidence, for the first time, that the cytokine is able per se to induce most of the transcriptional changes characteristic of patient-derived CAFs. Comparison with publicly available datasets, however, suggested that prostate CAFs may be alternatively characterized by IL6 and TGFβ-related signatures, indicating that either signal, depending on the context, may concur to fibroblast activation. Our analyses also highlighted novel pathways potentially relevant for induction of a reactive stroma. In addition, we revealed a role for muscle-specific miR-133b as a soluble factor secreted by activated fibroblasts to support paracrine activation of non-activated fibroblasts or promote tumor progression. Overall, we provided insights into the molecular mechanisms driving fibroblast activation in PCa, thus contributing to identify novel hits for the development of therapeutic strategies targeting the crucial interplay between tumor cells and their microenvironment. PMID:26375444

  8. Reactive oxygen species induce Cox-2 expression via TAK1 activation in synovial fibroblast cells

    PubMed Central

    Onodera, Yuta; Teramura, Takeshi; Takehara, Toshiyuki; Shigi, Kanae; Fukuda, Kanji

    2015-01-01

    Oxidative stress within the arthritis joint has been indicated to be involved in generating mediators for tissue degeneration and inflammation. COX-2 is a mediator in inflammatory action, pain and some catabolic reactions in inflamed tissues. Here, we demonstrated a direct relationship between oxidative stress and Cox-2 expression in the bovine synovial fibroblasts. Furthermore, we elucidated a novel mechanism, in which oxidative stress induced phosphorylation of MAPKs and NF-κB through TAK1 activation and resulted in increased Cox-2 and prostaglandin E2 expression. Finally, we demonstrated that ROS-induced Cox-2 expression was inhibited by supplementation of an antioxidant such as N-acetyl cysteamine and hyaluronic acid in vitro and in vivo. From these results, we conclude that oxidative stress is an important factor for generation of Cox-2 in synovial fibroblasts and thus its neutralization may be an effective strategy in palliative therapy for chronic joint diseases. PMID:26110105

  9. Methylation-mediated BMPER expression in fibroblast activation in vitro and lung fibrosis in mice in vivo

    PubMed Central

    Huan, Caijuan; Yang, Ting; Liang, Jiurong; Xie, Ting; Cheng, Luis; Liu, Ningshan; Kurkciyan, Adrianne; Monterrosa Mena, Jessica; Wang, Chen; Dai, Huaping; Noble, Paul W.; Jiang, Dianhua

    2015-01-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease. Although the pathogenesis is poorly understood, evidence suggests that genetic and epigenetic alterations, such as DNA methylation, may play a key role. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily and are important regulators in IPF. Here we identified BMP endothelial cell precursor-derived regulator (BMPER) as a key regulator of fibroblast activation. BMPER is a secreted glycoprotein that binds directly to BMPs and may regulate TGF-β/BMP signaling, but its role in lung fibrosis is not clear. BMPER is highly expressed in human IPF lung fibroblasts compared to normal lung fibroblasts. Demethylation agent 5′-azacytidine decreased BMPER expression in fibroblasts, and attenuated the invasion and migration of IPF lung fibroblasts. Furthermore, siRNA-mediated reduction of BMPER in the human lung fibroblasts impaired cell migration and invasion. 5′-azacytidine treatment additionally regulated BMPER expression and reduced lung fibrosis in mice in vivo. These findings demonstrate that methylation of specific genes in fibroblasts may offer a new therapeutic strategy for IPF by modulating fibroblast activation. PMID:26442443

  10. AMP-activated protein kinase suppresses matrix metalloproteinase-9 expression in mouse embryonic fibroblasts.

    PubMed

    Morizane, Yuki; Thanos, Aristomenis; Takeuchi, Kimio; Murakami, Yusuke; Kayama, Maki; Trichonas, George; Miller, Joan; Foretz, Marc; Viollet, Benoit; Vavvas, Demetrios G

    2011-05-01

    Matrix metalloproteinase-9 (MMP-9) plays a critical role in tissue remodeling under both physiological and pathological conditions. Although MMP-9 expression is low in most cells and is tightly controlled, the mechanism of its regulation is poorly understood. We utilized mouse embryonic fibroblasts (MEFs) that were nullizygous for the catalytic α subunit of AMP-activated protein kinase (AMPK), which is a key regulator of energy homeostasis, to identify AMPK as a suppressor of MMP-9 expression. Total AMPKα deletion significantly elevated MMP-9 expression compared with wild-type (WT) MEFs, whereas single knock-out of the isoforms AMPKα1 and AMPKα2 caused minimal change in the level of MMP-9 expression. The suppressive role of AMPK on MMP-9 expression was mediated through both its activity and presence. The AMPK activators 5-amino-4-imidazole carboxamide riboside and A769662 suppressed MMP-9 expression in WT MEFs, and AMPK inhibition by the overexpression of dominant negative (DN) AMPKα elevated MMP-9 expression. However, in AMPKα(-/-) MEFs transduced with DN AMPKα, MMP-9 expression was suppressed. AMPKα(-/-) MEFs showed increased phosphorylation of IκBα, expression of IκBα mRNA, nuclear localization of nuclear factor-κB (NF-κB), and DNA-binding activity of NF-κB compared with WT. Consistently, selective NF-κB inhibitors BMS345541 and SM7368 decreased MMP-9 expression in AMPKα(-/-) MEFs. Overall, our results suggest that both AMPKα isoforms suppress MMP-9 expression and that both the activity and presence of AMPKα contribute to its function as a regulator of MMP-9 expression by inhibiting the NF-κB pathway. PMID:21402702

  11. Fibroblast Activation Protein Expression by Stromal Cells and Tumor-Associated Macrophages in Human Breast Cancer

    PubMed Central

    Julia, Tchou; Zhang Paul, J; Yingtao, Bi; Celine, Satija; Rajrupa, Marjumdar; Stephen, TL; Lo, A; Haiying, Chen; Carolyn, Mies; June, Carl H; Jose, Conejo-Garcia; Ellen, Puré

    2013-01-01

    Summary Fibroblast activation protein (FAP) has long been known to be expressed in the stroma of breast cancer. However, very little is known if the magnitude of FAP expression within the stroma may have prognostic value and reflect the heterogeneous biology of the tumor cell. An earlier study had suggested that stromal FAP expression in breast cancer was inversely proportional to prognosis. We, therefore, hypothesized that stromal FAP expression may correlate with clinicopathologic variables and may serve as an adjunct prognostic factor in breast cancer. We evaluated the expression of FAP in a panel of breast cancer tissues (n=52) using a combination of immunostain analyses at the tissue and single cell level using freshly frozen or freshly digested human breast tumor samples respectively. Our results showed that FAP expression was abundantly expressed in the stroma across all breast cancer subtypes without significant correlation with clinicopathologic factors. We further identified a subset of FAP positive or FAP+ stromal cells that also expressed CD45, a pan-leukocyte marker. Using freshly dissociated human breast tumor specimens (n=5), we demonstrated that some of these FAP+ CD45+ cells were CD11b+CD14+MHC-II+ indicating that they were likely tumor associated macrophages (TAMs). Although FAP+CD45+ cells have been demonstrated in the mouse tumor stroma, our results demonstrating that human breast TAMs expressed FAP was novel and suggested that existing and future FAP directed therapy may have dual therapeutic benefits targeting both stromal mesenchymal cells and immune cells such as TAMs. More work is needed to explore the role of FAP as a potential targetable molecule in breast cancer treatment. PMID:24074532

  12. Myofibroblasts are distinguished from activated skin fibroblasts by the expression of AOC3 and other associated markers.

    PubMed

    Hsia, Lin-Ting; Ashley, Neil; Ouaret, Djamila; Wang, Lai Mun; Wilding, Jennifer; Bodmer, Walter F

    2016-04-12

    Pericryptal myofibroblasts in the colon and rectum play an important role in regulating the normal colorectal stem cell niche and facilitating tumor progression. Myofibroblasts previously have been distinguished from normal fibroblasts mostly by the expression of α smooth muscle actin (αSMA). We now have identified AOC3 (amine oxidase, copper containing 3), a surface monoamine oxidase, as a new marker of myofibroblasts by showing that it is the target protein of the myofibroblast-reacting mAb PR2D3. The normal and tumor tissue distribution and the cell line reactivity of AOC3 match that expected for myofibroblasts. We have shown that the surface expression of AOC3 is sensitive to digestion by trypsin and collagenase and that anti-AOC3 antibodies can be used for FACS sorting of myofibroblasts obtained by nonenzymatic procedures. Whole-genome microarray mRNA-expression profiles of myofibroblasts and skin fibroblasts revealed four additional genes that are significantly differentially expressed in these two cell types: NKX2-3 and LRRC17 in myofibroblasts and SHOX2 and TBX5 in skin fibroblasts. TGFβ substantially down-regulated AOC3 expression in myofibroblasts but in skin fibroblasts it dramatically increased the expression of αSMA. A knockdown of NKX2-3 in myofibroblasts caused a decrease of myofibroblast-related gene expression and increased expression of the fibroblast-associated gene SHOX2, suggesting that NKX2-3 is a key mediator for maintaining myofibroblast characteristics. Our results show that colorectal myofibroblasts, as defined by the expression of AOC3, NKX2-3, and other markers, are a distinctly different cell type from TGFβ-activated fibroblasts. PMID:27036009

  13. Tumoral Immune Suppression by Macrophages Expressing Fibroblast Activation Protein-Alpha and Heme Oxygenase-1

    PubMed Central

    Arnold, James N.; Magiera, Lukasz; Kraman, Matthew; Fearon, Douglas T.

    2013-01-01

    The depletion of tumor stromal cells that are marked by their expression of the membrane protein fibroblast activation protein-α (FAP) overcomes immune suppression and allows an anti-cancer cell immune response to control tumor growth. In subcutaneous tumors established with immunogenic Lewis lung carcinoma cells expressing ovalbumin (LL2/OVA), the FAP+ population comprises CD45+ and CD45− cells. In the present study, we further characterize the tumoral FAP+/CD45+ population as a minor sub-population of F4/80hi/CCR2+/CD206+ M2 macrophages. Using bone marrow chimeric mice in which the primate diphtheria toxin receptor (DTR) is restricted either to the FAP+/CD45+ or to the FAP+/CD45− subset, we demonstrate by conditionally depleting each subset that both independently contribute to the immune suppressive tumor microenvironment. A basis for the function of the FAP+/CD45+ subset is shown to be the immune inhibitory enzyme, heme oxygenase-1 (HO-1). The FAP+/CD45+ cells are the major tumoral source of HO-1, and an inhibitor of HO-1, Sn mesoporphyrin, causes the same extent of immune-dependent arrest of LL2/OVA tumor growth as does the depletion of these cells. Since this observation of immune suppression by HO-1 expressed by the FAP+/CD45+ stromal cell is replicated in a transplanted model of pancreatic ductal adenocarcinoma, we conclude that pharmacologically targeting this enzyme may improve cancer immunotherapy. PMID:24778275

  14. Abrogation of fibroblast activation protein enzymatic activity attenuates tumor growth.

    PubMed

    Cheng, Jonathan D; Valianou, Matthildi; Canutescu, Adrian A; Jaffe, Eileen K; Lee, Hyung-Ok; Wang, Hao; Lai, Jack H; Bachovchin, William W; Weiner, Louis M

    2005-03-01

    Tumor-associated fibroblasts are functionally and phenotypically distinct from normal fibroblasts that are not in the tumor microenvironment. Fibroblast activation protein is a 95 kDa cell surface glycoprotein expressed by tumor stromal fibroblasts, and has been shown to have dipeptidyl peptidase and collagenase activity. Site-directed mutagenesis at the catalytic site of fibroblast activation protein, Ser624 --> Ala624, resulted in an approximately 100,000-fold loss of fibroblast activation protein dipeptidyl peptidase (DPP) activity. HEK293 cells transfected with wild-type fibroblast activation protein, enzymatic mutant (S624A) fibroblast activation protein, or vector alone, were inoculated subcutaneously into immunodeficient mouse to assess the contribution of fibroblast activation protein enzymatic activity to tumor growth. Overexpression of wild-type fibroblast activation protein showed growth potentiation and enhanced tumorigenicity compared with both fibroblast activation protein S624A and vector-transfected HEK293 xenografts. HEK293 cells transfected with fibroblast activation protein S624A showed tumor growth rates and tumorigenicity potential similar only to vector-transfected HEK293. In vivo assessment of fibroblast activation protein DPP activity of these tumors showed enhanced enzymatic activity of wild-type fibroblast activation protein, with only baseline levels of fibroblast activation protein DPP activity in either fibroblast activation protein S624A or vector-only xenografts. These results indicate that the enzymatic activity of fibroblast activation protein is necessary for fibroblast activation protein-driven tumor growth in the HEK293 xenograft model system. This establishes the proof-of-principle that the enzymatic activity of fibroblast activation protein plays an important role in the promotion of tumor growth, and provides an attractive target for therapeutics designed to alter fibroblast activation protein-induced tumor growth by targeting

  15. Solar simulated irradiation modulates gene expression and activity of antioxidant enzymes in cultured human dermal fibroblasts.

    PubMed

    Leccia, M T; Yaar, M; Allen, N; Gleason, M; Gilchrest, B A

    2001-08-01

    Exposure of skin to solar irradiation generates reactive oxygen species that damage DNA, membranes, mitochondria and proteins. To protect against such damage, skin cells have evolved antioxidant enzymes including glutathione peroxidase (GSH-Px), copper and zinc-dependent superoxide dismutase (SOD1), the mitochondrial manganese-dependent superoxide dismutase (SOD2), and catalase. This report examines the effect of a single low or moderate dose exposure to solar-simulating combined UVB and UVA irradiation on the gene expression and activities of these antioxidant enzymes in cultured normal human fibroblasts. We find that both doses initially decrease GSH-Px, SOD2 and catalase activities, but within 5 days after irradiation the activities of the enzymes return to pre-irradiation level (catalase) or are induced slightly (SOD1, GSH-Px) or substantially (SOD2) above the basal level. For SOD1, SOD2 and catalase, the higher dose also detectably modulates the mRNA level of these enzymes. Our results indicate that the effects of a single physiologic solar simulated irradiation dose persist for at least several days and suggest that skin cells prepare for subsequent exposure to damaging irradiation by upregulating this antioxidant defense system, in particular the mitochondrial SOD2. Our findings are consistent with the existence of a broad-based SOS-like response in irradiated human skin. PMID:11493316

  16. B lymphocytes and B-cell activating factor promote collagen and profibrotic markers expression by dermal fibroblasts in systemic sclerosis

    PubMed Central

    2013-01-01

    Introduction B lymphocytes might play a pathogenic role in dermal fibrosis in systemic sclerosis (SSc). B-cell activating factor (BAFF), a key cytokine for B-cell activation, is increased in the serum and the skin of patients with SSc. However, the ability of B cells directly to stimulate dermal fibroblasts and the role of BAFF are not fully understood. We therefore investigated the involvement of B cells and BAFF in the expression of collagen and profibrotic markers by dermal fibroblasts. Methods Cocultures of blood B cells from healthy blood donors and normal or SSc dermal fibroblasts stimulated with anti-IgM and BAFF were performed. Alpha-SMA, TIMP1, MMP9, COL1A1, COL1A2, and COL3A1 mRNA expression were determined by quantitative RT-PCR. Soluble collagen, BAFF, IL-6, IL-1β, TGF-β1, and CCL2 protein secretion were assessed. Results Coculture of blood B cells and dermal fibroblasts isolated from SSc patients induced IL-6, TGF-β1, CCL2, and collagen secretion, as well as Alpha-SMA, TIMP1, and MMP9 expression in dermal fibroblasts. Transwell assays demonstrated that this induction was dependent on cell-cell contact. Addition of anti-IgM and BAFF to the coculture increased IL-6, CCL2, TGF-β1, and collagen secretion. B cell- and BAFF-induced collagen secretion was highly reduced by anti-TGF-β1 antibodies. Conclusions Our results showed for the first time a direct role of B cells on the production of collagen by dermal fibroblasts, which is further enhanced by BAFF. Thus, these results demonstrate a new pathogenic role of B cells and BAFF in fibrosis and systemic sclerosis. PMID:24289101

  17. Gene expression profile in the activation of subperitoneal fibroblasts reflects prognosis of patients with colon cancer.

    PubMed

    Yokota, Mitsuru; Kojima, Motohiro; Higuchi, Youichi; Nishizawa, Yuji; Kobayashi, Akihiro; Ito, Masaaki; Saito, Norio; Ochiai, Atsushi

    2016-03-15

    Tumors can create a heterogenetic tumor microenvironment. We recently identified the pathologically unique cancer microenvironment formed by peritoneal invasion (CMPI), and revealed that subperitoneal fibroblasts (SPFs) within peritoneal tissue play a crucial role in tumor progression through their interaction with cancer cells. Therefore, the genes in SPFs altered by cancer stimulation may include some biologically important factors associated with patient prognosis. In this study, we aimed to identify new biomarkers using genes specifically upregulated in SPFs by cancer-cell-conditioned medium (CCCM) stimulation (SPFs CCCM response genes; SCR genes) in colon cancer (CC). We constructed two frameworks using SCR gene data: a publicly released microarray dataset, and validation cases with freshly frozen CC samples to identify genes related to short recurrence-free survival (RFS). In the first framework, we selected differentially expressed genes between the high and low SCR gene expression groups. In the second framework, genes significantly related to short RFS were selected by univariate analysis using all SCR genes, and multivariate analysis was performed to select robust genes associated with short RFS. We identified CTGF, CALD1, INHBA and TAGLN in the first framework, and PDLIM5, MAGI1, SPTBN1 and TAGLN in the second framework. Among these seven genes, high expression of three genes (CALD1, TAGLN and SPTBN1) showed a poor prognosis in our validation cases. In a public microarray dataset, SCR gene expression was associated with the expression of ECM component, EMT, and M2-macrophage associated genes, which was concordant with the pathological features of CMPI. Thus, we successfully identified new prognostic factors. PMID:26370611

  18. Abietic acid inhibits UVB-induced MMP-1 expression in human dermal fibroblast cells through PPARα/γ dual activation.

    PubMed

    Jeon, Youngsic; Jung, Yujung; Youm, Jong-Kyung; Kang, Ki Sung; Kim, Yong Kee; Kim, Su-Nam

    2015-02-01

    Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors and consist of three isotypes: PPARα, PPARβ/δ and PPARγ. PPARs are expressed in various cell types in the skin, including keratinocytes, fibroblasts and infiltrating immune cells. Thus, these receptors are highly studied in dermato-endocrine research, and their ligands are targets for the treatment of various skin disorders, such as photoageing and chronological ageing of skin. Intensive studies have revealed that PPARα/γ functions in photoageing and age-related inflammation by regulating matrix metalloproteinases (MMPs) via nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1). However, the detailed mechanism of PPARα/γ's role in photoageing has not yet been elucidated. In this study, we confirmed that abietic acid (AA) is a PPARα/γ dual ligand and significantly decreased UVB-induced MMP-1 expression by downregulating UVB-induced MAPK signalling and downstream transcription factors, subsequently reducing IκBα degradation and blocking NF-κB p65 nuclear translocation in Hs68 human dermal fibroblast cells. Treatment of cells with AA and GW6471 or bisphenol A diglycidyl ether (BADGE), PPARα or PPARγ antagonists, respectively, reversed the effect on UVB-induced MMP-1 expression and inflammatory signalling pathway activation. Taken together, our data suggest that AA acts as a PPARα/γ dual activator to inhibit UVB-induced MMP-1 expression and age-related inflammation by suppressing NF-κB and the MAPK/AP-1 pathway and can be a useful agent for improving skin photoageing. PMID:25496486

  19. Activation of protease-activated receptors (PARs)-1 and -2 promotes alpha-smooth muscle actin expression and release of cytokines from human lung fibroblasts

    PubMed Central

    Asokananthan, Nithiananthan; Lan, Rommel S; Graham, Peter T; Bakker, Anthony J; Tokanović, Ana; Stewart, Geoffrey A

    2015-01-01

    Previous studies have shown that protease-activated receptors (PARs) play an important role in various physiological processes. In the present investigation, we determined the expression of PARs on human lung fibroblasts (HLF-1) and whether they were involved in cellular differentiation and pro-inflammatory cytokine and prostaglandin (PGE2) secretion. PAR-1, PAR-2, PAR-3, and PAR-4 were detected in fibroblasts using RT-PCR, immunocytochemistry, and flow cytometry. Increased expression of PAR-4, but not other PARs, was observed in fibroblasts stimulated with phorbol myristate acetate. The archetypical activators of PARs, namely, thrombin and trypsin, as well as PAR-1 and PAR-2 agonist peptides, stimulated transient increases in intracellular Ca2+, and promoted increased α-smooth muscle actin expression. The proteolytic and peptidic PAR activators also stimulated the release of IL-6 and IL-8, as well as PGE2, with a rank order of potency of PAR-1 > PAR-2. The combined stimulation of PAR-1 and PAR-2 resulted in an additive release of both IL-6 and IL-8. In contrast, PAR-3 and PAR-4 agonist peptides, as well as all the PAR control peptides examined, were inactive. These results suggest an important role for PARs associated with fibroblasts in the modulation of inflammation and remodeling in the airway. PMID:25663523

  20. Transforming growth factor-β inhibits IQ motif containing guanosine triphosphatase activating protein 1 expression in lung fibroblasts via the nuclear factor-κB signaling pathway.

    PubMed

    Zong, Chuanyue; Zhang, Xianlong; Xie, Ying; Cheng, Jiawen

    2015-07-01

    IQ motif containing guanosine triphosphatase activating protein 1 (IQGAP1) is associated with idiopathic pulmonary fibrogenesis (IPF); however, characterization of the expression of IQGAP1 in lung fibroblasts has remained elusive. The present study therefore evaluated IQGAP1 expression in mouse and human lung fibroblasts under fibrotic conditions via western blot analysis. It was revealed that IQGAP1 expression levels were significantly decreased in lung fibroblasts isolated from bleomycin-challenged mice than in those of control mice. Transforming growth factor-β (TGF-β) induced differentiation, as well as decreased expression of IQGAP1 in WI-38 cells human lung fibroblasts. Furthermore, inhibition of nuclear factor (NF)-κB activation restored the TGF-β-induced inhibition of IQGAP1 expression in WI-38 cells. In lysophosphatidic acid (LPA)-challenged WI-38 cells, the expression of IQGAP1 was also decreased, while neutralized anti-TGF-β antibody treatment restored the LPA-induced inhibition of IQGAP1 expression. These data indicated that TGF-β inhibited IQGAP1 expression in lung fibroblasts via the NF-κB signaling pathway, presenting a potential novel therapeutic target for the treatment of IPF. PMID:25684348

  1. Acute cholesterol depletion impairs functional expression of tissue factor in fibroblasts: modulation of tissue factor activity by membrane cholesterol

    PubMed Central

    Mandal, Samir K.; Iakhiaev, Alexei; Pendurthi, Usha R.; Mohan Rao, L. Vijaya

    2010-01-01

    Cholesterol, in addition to providing rigidity to the fluid membrane, plays a critical role in receptor function, endocytosis, recycling, and signal transduction. In the present study, we examined the effect of membrane cholesterol on functional expression of tissue factor (TF), a cellular receptor for clotting factor VIIa. Depletion of cholesterol in human fibroblasts (WI-38) with methyl-β-cyclodextrin–reduced TF activity at the cell surface. Binding studies with radiolabeled VIIa and TF monoclonal antibody (mAB) revealed that reduced TF activity in cholesterol-depleted cells stems from the impairment of VIIa interaction with TF rather than the loss of TF receptors at the cell surface. Repletion of cholesterol-depleted cells with cholesterol restored TF function. Loss of caveolar structure on cholesterol removal is not responsible for reduced TF activity. Solubilization of cellular TF in different detergents indicated that a substantial portion of TF in fibroblasts is associated with noncaveolar lipid rafts. Cholesterol depletion studies showed that the TF association with these rafts is cholesterol dependent. Overall, the data presented herein suggest that membrane cholesterol functions as a positive regulator of TF function by maintaining TF receptors, probably in noncaveolar lipid rafts, in a high-affinity state for VIIa binding. PMID:15328160

  2. Resveratrol suppresses myofibroblast activity of human buccal mucosal fibroblasts through the epigenetic inhibition of ZEB1 expression

    PubMed Central

    Yu, Cheng-Chia; Wang, Bing-Yen; Huang, Yu-Hao; Hsieh, Yang-Chih; Kuo, Yu-Liang; Chang, Wen-Wei

    2016-01-01

    Oral submucous fibrosis (OSF) is a precancerous condition of the oral mucosa without specific therapeutic drugs. We previously demonstrated that the zinc finger E-box binding homeobox 1 (ZEB1) plays a pathogenic role in the induction of the myofibroblast activity of buccal mucosal fibroblasts (BMFs) and contributes to the pathogenesis of OSF. Resveratrol is a natural polyphenolic flavonoid with anti-fibrosis activity in various tissues and has the capability to inhibit ZEB1 in oral cancer cells. We examined the effect of resveratrol on the myofibroblast activity of human primary fibrotic BMFs (fBMFs) derived from OSF tissues. With the collagen contraction assay, resveratrol displayed anti-myofibroblast activity in three fBMF lines. Resveratrol also inhibited the expression of fibrogenic genes at the mRNA and protein levels in a dose- and time-dependent manner. The downregulation of ZEB1 in fBMFs by resveratrol was mediated by epigenetic mechanisms, such as the upregulated expression of miR-200c and the enhancer of zeste homolog 2 (EZH2), as well as the trimethylated lysine 27 of histone H3 (H3K27me3). Resveratrol also increased the binding of H3K27me3 to the ZEB1 promoter. The knockdown of EZH2 in fBMFs caused the upregulation of ZEB1 and suppressed the inhibitory effect of resveratrol. Furthermore, the reversed expression pattern between EZH2 and ZEB1 was observed in 6/8 OSF tissues with twofold upregulation of ZEB1 expression compared with the adjacent normal mucosa. In conclusion, our data suggest that resveratrol epigenetically inhibits ZEB1 expression to suppress the myofibroblast activity of fBMFs and may serve as a dietary supplement for OSF patients. PMID:26934322

  3. NF-κB Activation Limits Airway Branching through Inhibition of Sp1-Mediated Fibroblast Growth Factor-10 Expression

    PubMed Central

    Benjamin, John T.; Carver, Billy J.; Plosa, Erin J.; Yamamoto, Yasutoshi; Miller, J. Davin; Liu, Jin-Hua; van der Meer, Riet; Blackwell, Timothy S.; Prince, Lawrence S.

    2015-01-01

    Bronchopulmonary dysplasia (BPD) is a frequent complication of preterm birth. This chronic lung disease results from arrested saccular airway development and is most common in infants exposed to inflammatory stimuli. In experimental models, inflammation inhibits expression of fibroblast growth factor-10 (FGF-10) and impairs epithelial–mesenchymal interactions during lung development; however, the mechanisms connecting inflammatory signaling with reduced growth factor expression are not yet understood. In this study we found that soluble inflammatory mediators present in tracheal fluid from preterm infants can prevent saccular airway branching. In addition, LPS treatment led to local production of mediators that inhibited airway branching and FGF-10 expression in LPS-resistant C.C3-Tlr4Lpsd/J fetal mouse lung explants. Both direct NF-κB activation and inflammatory cytokines (IL-1β and TNF-α) that activate NF-κB reduced FGF-10 expression, whereas chemokines that signal via other inflammatory pathways had no effect. Mutational analysis of the FGF-10 promoter failed to identify genetic elements required for direct NF-κB–mediated FGF-10 inhibition. Instead, NF-κB activation appeared to interfere with the normal stimulation of FGF-10 expression by Sp1. Chromatin immunoprecipitation and nuclear coimmunoprecipitation studies demonstrated that the RelA subunit of NF-κB and Sp1 physically interact at the FGF-10 promoter. These findings indicate that inflammatory signaling through NF-κB disrupts the normal expression of FGF-10 in fetal lung mesenchyme by interfering with the transcriptional machinery critical for lung morphogenesis. PMID:20861353

  4. Inflammatory Gene Expression Upon TGF-β1-Induced p38 Activation in Primary Dupuytren's Disease Fibroblasts

    PubMed Central

    Bujak, Maro; Ratkaj, Ivana; Markova-Car, Elitza; Jurišić, Davor; Horvatić, Anita; Vučinić, Srđan; Lerga, Jonatan; Baus-Lončar, Mirela; Pavelić, Krešimir; Kraljević Pavelić, Sandra

    2015-01-01

    Objectives: Inflammation is an underlying mechanism behind fibrotic processes and differentiation of cells into myofibroblasts. Presented study therefore provides new data on activation of autoimmune and inflammatory immune response genes that accompany activation of p38 and cell differentiation in primary cells derived from Dupuytren's disease (DD) patients. Methods: Primary non-Dupuytren's disease cells (ND) were isolated from macroscopically unaffected palmar fascia adjacent to diseased tissue obtained from patients diagnosed with the last stage of DD and cultured in vitro. Gene expression, collagen gel contraction assay and analysis of secreted proteins were performed in ND cells treated with TGF-β1 and/or inhibitor of p38 phosphorylation. Results: During differentiation of ND fibroblasts, increased expression of immune response genes PAI-1, TIMP-1, CCL11, and IL-6 was found. These changes were accompanied by increased cell contractility and activation of p38 and its target kinase MK2. Inhibition of p38 phosphorylation reversed these processes in vitro. Conclusions: TGF-β1 induced p38 phosphorylation in ND cells grown from macroscopically unaffected palmar fascia adjacent to diseased tissue from DD patients. This was accompanied by activation of the cytokine genes CCL-11 and IL-6 and secretion of extracellular matrix regulatory proteins PAI-1 and TIMP-1. A combined approach directed toward inflammation and p38 MAPK-mediated processes in DD might be considered for improving management of DD patients and prevention of recurrence. PMID:26697433

  5. Kynurenine Modulates MMP-1 and Type-I Collagen Expression Via Aryl Hydrocarbon Receptor Activation in Dermal Fibroblasts.

    PubMed

    Poormasjedi-Meibod, Malihe-Sadat; Salimi Elizei, Sanam; Leung, Victor; Baradar Jalili, Reza; Ko, Frank; Ghahary, Aziz

    2016-12-01

    Dermal fibrosis is characterized by a high deposition of extracellular matrix (ECM) and tissue cellularity. Unfortunately all means of treating this condition are unsatisfactory. We have previously reported the anti-fibrotic effects of Kynurenine (Kyn), a tryptophan metabolite, in fibrotic rabbit ear model. Here, we report the mechanism by which Kyn modulates the expression of key ECM components in dermal fibroblasts. The results showed that Kyn activates aryl hydrocarbon receptor (AHR) nuclear translocation and up-regulates cytochrome-P450 (CYP1A-1) expression, the AHR target gene. A specific AHR antagonist, 6,2',4'-trimethoxyflavone, inhibited the Kyn-dependent modulation of CYP1A-1, MMP-1, and type-I collagen expression. Establishing the anti-fibrogenic effect of Kyn and its mechanism of action, we then developed nano-fibrous Kyn slow-releasing dressings and examined their anti-fibrotic efficacy in vitro and in a rat model. Our results showed the feasibility of incorporating Kyn into PVA/PLGA nanofibers, prolonging the Kyn release up to 4 days tested. Application of medicated-dressings significantly improved the dermal fibrosis indicated by MMP-1 induction, alpha-smooth muscle actin and type-I collagen suppression, and reduced tissue cellularity, T-cells and myofibroblasts. This study clarifies the mechanism by which Kyn modulates ECM expression and reports the development of a new slow-releasing anti-fibrogenic dressing. J. Cell. Physiol. 231: 2749-2760, 2016. © 2016 Wiley Periodicals, Inc. PMID:26992058

  6. Activating the Expression of Human K-rasG12D Stimulates Oncogenic Transformation in Transgenic Goat Fetal Fibroblast Cells

    PubMed Central

    Gong, Jianhua; Wang, Zhongde; Polejaeva, Irina; Salgia, Ravi; Kao, Chien-Min; Chen, Chin-Tu; Chen, Guangchun; Chen, Liaohai

    2014-01-01

    Humane use of preclinical large animal cancer models plays a critical role in understanding cancer biology and developing therapeutic treatments. Among the large animal candidates, goats have great potentials as sustainable sources for large animal cancer model development. Goats are easier to handle and cheaper to raise. The genome of the goats has been sequenced recently. It has been known that goats develop skin, adrenal cortex, breast and other types of cancers. Technically, goats are subject to somatic cell nuclear transfer more efficiently and exhibit better viability through the cloning process. Towards the development of a goat cancer model, we created a transgenic goat fetal fibroblast (GFF) cell as the donor cell for SCNT. Human mutated K-ras (hK-rasG12D) was chosen as the transgene, as it is present in 20% of cancers. Both hK-rasG12D and a herpes simplex viral thymidine kinase (HSV1-tk) reporter genes, flanked by a pair of LoxP sites, were knocked in the GFF endogenous K-ras locus through homologous recombination. Following Cre-mediated activation (with a 95% activation efficiency), hK-rasG12D and HSV1-tk were expressed in the transgenic GFF cells, evidently through the presence of corresponding mRNAs, and confirmed by HSV1-tk protein function assay. The hK-rasG12D expressing GFF cells exhibited enhanced proliferation rates and an anchorage-independent growth behavior. They were able to initiate tumor growth in athymic nude mice. In conclusion, after activating hK-rasG12D gene expression, hK-rasG12D transgenic GFF cells were transformed into tumorgenesis cells. Transgenic goats via SCNT using the above-motioned cells as the donor cells have been established. PMID:24594684

  7. Chondrocytes, synoviocytes and dermal fibroblasts all express PH-20, a hyaluronidase active at neutral pH

    PubMed Central

    El Hajjaji, Hafida; Cole, Ada Asbury; Manicourt, Daniel-Henri

    2005-01-01

    Hyaluronan (HA), an important component of connective tissues, is highly metabolically active, but the mechanisms involved in its catabolism are still largely unknown. We hypothesized that a protein similar to sperm PH-20, the only mammalian hyaluronidase known to be active at neutral pH, could be expressed in connective tissue cells. An mRNA transcript similar to that of PH-20 was found in chondrocytes, synoviocytes, and dermal fibroblasts, and its levels were enhanced upon stimulation with IL-1. In cell layers extracted with Triton X-100 – but not with octylglucoside – and in culture media, a polyclonal antipeptide anti-PH-20 antibody identified protein bands with a molecular weight similar to that of sperm PH-20 (60 to 65 kDa) and exhibiting a hyaluronidase activity at neutral pH. Further, upon stimulation with IL-1, the amounts of the neutral-active hyaluronidase increased in both cell layers and culture media. These findings contribute potential important new insights into the biology of connective tissues. It is likely that PH-20 facilitates cell-receptor-mediated uptake of HA, while overexpression or uncontrolled expression of the enzyme can cause great havoc to connective tissues: not only does HA fragmentation compromise the structural integrity of tissues, but also the HA fragments generated are highly angiogenic and are potent inducers of proinflammatory cytokines. On the other hand, the enzyme activity may account for the progressive depletion of HA seen in osteoarthritis cartilage, a depletion that is believed to play an important role in the apparent irreversibility of this disease process. PMID:15987477

  8. Expression and function of microRNA-188-5p in activated rheumatoid arthritis synovial fibroblasts

    PubMed Central

    Ruedel, Anke; Dietrich, Peter; Schubert, Thomas; Hofmeister, Simone; Hellerbrand, Claus; Bosserhoff, Anja-Katrin

    2015-01-01

    Activated synovial fibroblasts in rheumatoid arthritis (RASF) play a critical role in the pathology of rheumatoid arthritis (RA). Recent studies suggested that deregulation of microRNAs (miRs) affects the development and progression of RA. Therefore, we aimed to identify de-regulated miRs in RASF and to identify target genes that may contribute to the aggressive phenotype of RASF. Quantitative real-time PCR revealed a marked downregulation of miR-188-5p in synovial tissue samples of RA patients as well as in RASF. Exposure to the cytokine interleukine-1β lead to a further downregulation of miR-188-5p expression levels compared to control cells. Re-expression of miR-188-5p in RASF by transient transfection significantly inhibited cell migration. However, miR-188-5p re-expression had no effects on glycosaminoglycan degradation or expression of repellent factors, which have been previously shown to affect the invasive behavior of RASF. In search for target genes of miR-188-5p in RASF we performed gene expression profiling in RASF and found a strong regulatory effect of miR-188-5p on the hyaluronan binding protein KIAA1199 as well as collagens COL1A1 and COL12A1, which was confirmed by qRT-PCR. In silico analysis revealed that KIAA1199 carries a 3’UTR binding site for miR-188-5p. COL1A1and COL12A1 showed no binding site in the mRNA region, suggesting an indirect regulation of these two genes by miR-188-5p. In summary, our study showed that miR-188-5p is down-regulated in RA in vitro and in vivo, most likely triggered by an inflammatory environment. MiR-188-5p expression is correlated to the activation state of RASF and inhibits migration of these cells. Furthermore, miR-188-5p is directly and indirectly regulating the expression of genes, which may play a role in extracellular matrix formation and destruction in RA. Herewith, this study identified potential novel therapeutic targets to inhibit the development and progression of RA. PMID:26191188

  9. Expression and function of microRNA-188-5p in activated rheumatoid arthritis synovial fibroblasts

    PubMed Central

    Ruedel, Anke; Dietrich, Peter; Schubert, Thomas; Hofmeister, Simone; Hellerbrand, Claus; Bosserhoff, Anja Katrin

    2015-01-01

    Activated synovial fibroblasts in rheumatoid arthritis (RASF) play a critical role in the pathology of rheumatoid arthritis (RA). Recent studies suggested that deregulation of microRNAs (miRs) affects the development and progression of RA. Therefore, we aimed to identify de-regulated miRs in RASF and to identify target genes that may contribute to the aggressive phenotype of RASF. Quantitative real-time PCR revealed a marked downregulation of miR-188-5p in synovial tissue samples of RA patients as well as in RASF. Exposure to the cytokine interleukine-1β lead to a further downregulation of miR-188-5p expression levels compared to control cells. Re-expression of miR-188-5p in RASF by transient transfection significantly inhibited cell migration. However, miR-188-5p re-expression had no effects on glycosaminoglycan degradation or expression of repellent factors, which have been previously shown to affect the invasive behavior of RASF. In search for target genes of miR-188-5p in RASF we performed gene expression profiling in RASF and found a strong regulatory effect of miR-188-5p on the hyaluronan binding protein KIAA1199 as well as collagens COL1A1 and COL12A1, which was confirmed by qRT-PCR. In silico analysis revealed that KIAA1199 carries a 3’UTR binding site for miR-188-5p. COL1A1 and COL12A1 showed no binding site in the mRNA region, suggesting an indirect regulation of these two genes by miR-188-5p. In summary, our study showed that miR-188-5p is down-regulated in RA in vitro and in vivo, most likely triggered by an inflammatory environment. MiR-188-5p expression is correlated to the activation state of RASF and inhibits migration of these cells. Furthermore, miR-188-5p is directly and indirectly regulating the expression of genes, which may play a role in extracellular matrix formation and destruction in RA. Herewith, this study identified potential novel therapeutic targets to inhibit the development and progression of RA. PMID:26261542

  10. Expression and function of microRNA-188-5p in activated rheumatoid arthritis synovial fibroblasts.

    PubMed

    Ruedel, Anke; Dietrich, Peter; Schubert, Thomas; Hofmeister, Simone; Hellerbrand, Claus; Bosserhoff, Anja-Katrin

    2015-01-01

    Activated synovial fibroblasts in rheumatoid arthritis (RASF) play a critical role in the pathology of rheumatoid arthritis (RA). Recent studies suggested that deregulation of microRNAs (miRs) affects the development and progression of RA. Therefore, we aimed to identify de-regulated miRs in RASF and to identify target genes that may contribute to the aggressive phenotype of RASF. Quantitative real-time PCR revealed a marked downregulation of miR-188-5p in synovial tissue samples of RA patients as well as in RASF. Exposure to the cytokine interleukine-1β lead to a further downregulation of miR-188-5p expression levels compared to control cells. Re-expression of miR-188-5p in RASF by transient transfection significantly inhibited cell migration. However, miR-188-5p re-expression had no effects on glycosaminoglycan degradation or expression of repellent factors, which have been previously shown to affect the invasive behavior of RASF. In search for target genes of miR-188-5p in RASF we performed gene expression profiling in RASF and found a strong regulatory effect of miR-188-5p on the hyaluronan binding protein KIAA1199 as well as collagens COL1A1 and COL12A1, which was confirmed by qRT-PCR. In silico analysis revealed that KIAA1199 carries a 3'UTR binding site for miR-188-5p. COL1A1and COL12A1 showed no binding site in the mRNA region, suggesting an indirect regulation of these two genes by miR-188-5p. In summary, our study showed that miR-188-5p is down-regulated in RA in vitro and in vivo, most likely triggered by an inflammatory environment. MiR-188-5p expression is correlated to the activation state of RASF and inhibits migration of these cells. Furthermore, miR-188-5p is directly and indirectly regulating the expression of genes, which may play a role in extracellular matrix formation and destruction in RA. Herewith, this study identified potential novel therapeutic targets to inhibit the development and progression of RA. PMID:26191188

  11. Expression and function of microRNA-188-5p in activated rheumatoid arthritis synovial fibroblasts.

    PubMed

    Ruedel, Anke; Dietrich, Peter; Schubert, Thomas; Hofmeister, Simone; Hellerbrand, Claus; Bosserhoff, Anja Katrin

    2015-01-01

    Activated synovial fibroblasts in rheumatoid arthritis (RASF) play a critical role in the pathology of rheumatoid arthritis (RA). Recent studies suggested that deregulation of microRNAs (miRs) affects the development and progression of RA. Therefore, we aimed to identify de-regulated miRs in RASF and to identify target genes that may contribute to the aggressive phenotype of RASF. Quantitative real-time PCR revealed a marked downregulation of miR-188-5p in synovial tissue samples of RA patients as well as in RASF. Exposure to the cytokine interleukine-1β lead to a further downregulation of miR-188-5p expression levels compared to control cells. Re-expression of miR-188-5p in RASF by transient transfection significantly inhibited cell migration. However, miR-188-5p re-expression had no effects on glycosaminoglycan degradation or expression of repellent factors, which have been previously shown to affect the invasive behavior of RASF. In search for target genes of miR-188-5p in RASF we performed gene expression profiling in RASF and found a strong regulatory effect of miR-188-5p on the hyaluronan binding protein KIAA1199 as well as collagens COL1A1 and COL12A1, which was confirmed by qRT-PCR. In silico analysis revealed that KIAA1199 carries a 3'UTR binding site for miR-188-5p. COL1A1 and COL12A1 showed no binding site in the mRNA region, suggesting an indirect regulation of these two genes by miR-188-5p. In summary, our study showed that miR-188-5p is down-regulated in RA in vitro and in vivo, most likely triggered by an inflammatory environment. MiR-188-5p expression is correlated to the activation state of RASF and inhibits migration of these cells. Furthermore, miR-188-5p is directly and indirectly regulating the expression of genes, which may play a role in extracellular matrix formation and destruction in RA. Herewith, this study identified potential novel therapeutic targets to inhibit the development and progression of RA. PMID:26261542

  12. Proteasome activity or expression is not altered by activation of the heat shock transcription factor Hsf1 in cultured fibroblasts or myoblasts.

    PubMed

    Taylor, David M; Kabashi, Edor; Agar, Jeffrey N; Minotti, Sandra; Durham, Heather D

    2005-01-01

    Heat shock proteins (Hsps) with chaperoning function work together with the ubiquitin-proteasome pathway to prevent the accumulation of misfolded, potentially toxic proteins, as well as to control catabolism of the bulk of cytoplasmic, cellular protein. There is evidence for the involvement of both systems in neurodegenerative disease, and a therapeutic target is the heat shock transcription factor, Hsf1, which mediates upregulation of Hsps in response to cellular stress. The mechanisms regulating expression of proteasomal proteins in mammalian cells are less well defined. To assess any direct effect of Hsf1 on expression of proteasomal subunits and activity in mammalian cells, a plasmid encoding a constitutively active form of Hsf1 (Hsf1act) was expressed in mouse embryonic fibroblasts lacking Hsf1 and in cultured human myoblasts. Plasmid encoding an inactivatible form of Hsf1 (Hsf1inact) served as control. In cultures transfected with plasmid hsf1act, robust expression of the major stress-inducible Hsp, Hsp70, occurred but not in cultures transfected with hsf1inact. No significant changes in the level of expression of representative proteasomal proteins (structural [20Salpha], a nonpeptidase beta subunit [20Sbeta3], or 2 regulatory subunits [19S subunit 6b, 11 Salpha]) or in chymotrypsin-, trypsin-, and caspaselike activities of the proteasome were measured. Thus, stress-induced or pharmacological activation of Hsf1 in mammalian cells would upregulate Hsps but not directly affect expression or activity of proteasomes. PMID:16184768

  13. Asiatic Acid Isolated From Centella Asiatica Inhibits TGF-β1-induced Collagen Expression in Human Keloid Fibroblasts via PPAR-γ Activation

    PubMed Central

    Bian, Difei; Zhang, Jizhou; Wu, Xin; Dou, Yannong; Yang, Yan; Tan, Qian; Xia, Yufeng; Gong, Zhunan; Dai, Yue

    2013-01-01

    Keloids are fibroproliferative disorders characterized by exuberant extracellular matrix deposition and transforming growth factor (TGF)-β/Smad pathway plays a pivotal role in keloid pathogenesis. Centella asiatica extract has been applied in scar management for ages. As one of its major components, asiatic acid (AA) has been recently reported to inhibit liver fibrosis by blocking TGF-β/Smad pathway. However, its effect on keloid remains unknown. In order to investigate the effects of AA on cell proliferation, invasion and collagen synthesis, normal and keloid fibroblasts were exposed to TGF-β1 with or without AA. Relevant experiments including 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2-deoxyuridine (EdU) incorporation assay, Transwell invasion assay, enzyme-linked immunosorbent assay, Western blot, quantitative polymerase chain reaction and RNA interference assay were conducted. As a result, keloid fibroblasts showed higher responsiveness to TGF-β1 stimulation than normal fibroblasts in terms of invasion and collagen synthesis. AA could suppress TGF-β1-induced expression of collagen type I, inhibit Smad 2/3 phosphorylation and plasminogen activator inhibitor-1 (PAI-1) expression, while elevate Smad 7 protein level. Noteworthy, the effects of AA on keloid fibroblasts could be abrogated by PPAR-γ antagonist GW9662 and by silencing of PPAR-γ. The present study demonstrated that AA inhibited TGF-β1-induced collagen and PAI-1 expression in keloid fibroblasts through PPAR-γ activation, which suggested that AA was one of the active constituents of C. asiatica responsible for keloid management, and could be included in the arsenal for combating against keloid. PMID:24250248

  14. Modulatory Effects of Connexin-43 Expression on Gap Junction Intercellular Communications with Mast Cells and Fibroblasts

    PubMed Central

    Pistorio, Ashey L.; Ehrlich, H. Paul

    2011-01-01

    The influence of mast cells upon aberrant wound repair and excessive fibrosis has supportive evidence, but the mechanism for these mast cell activities is unclear. It is proposed that heterocellular gap junctional intercellular communication (GJIC) between fibroblasts and mast cells directs some fibroblast activities. An in vitro model was used employing a rodent derived peritoneal mast cell line (RMC-1) and human dermal derived fibroblasts. The influence of the expression of the gap junction channel structural protein, connexin 43 (Cx-43) on heterocellular GJIC, the expression of microtubule β-tubulin and microfilament α smooth muscle actin (SMA) were investigated. The knockdown of Cx-43 by siRNA in RMC-1 cells completely blocked GJIC between RMC-1 cells. SiRNA knockdown of Cx-43 within fibroblasts only dampened GJIC between fibroblasts. It appears Cx-43 is the only expressed connexin in RMC-1 cells. Fibroblasts express other connexins that participate in GJIC between fibroblasts in the absence of Cx-43 expression. Heterocellular GJIC between RMC-1 cells and fibroblasts transformed fibroblasts into myofibroblasts, expressing α SMA within cytoplasmic stress fibers. The knockdown of Cx-43 in RMC-1 cells increased β-tubulin expression, but its knockdown in fibroblasts reduced β-tubulin expression. Knocking down the expression of Cx-43 in fibroblasts limited α SMA expression. Cx-43 participation is critical for heterocellular GJIC between mast cells and fibroblasts, which may herald a novel direction for controlling fibrosis. PMID:21328609

  15. Palmitate induces COX-2 expression via the sphingolipid pathway-mediated activation of NF-κB, p38, and ERK in human dermal fibroblasts.

    PubMed

    Oh, Eunhye; Yun, Mihee; Kim, Seong Keun; Seo, Gimoon; Bae, Joon Sung; Joo, Kwon; Chae, Gue Tae; Lee, Seong-Beom

    2014-05-01

    It has been suggested that free fatty acids (FFA) such as palmitate, which are secreted from enlarged adipocytes in the subcutaneous fat of obese subjects, serve as a link between obesity and altered skin functions. Cyclooxygenease-2 (COX-2) and prostanoids participate in the induction of impaired dermal function. In the current study, we investigated the issue of whether palmitate induces COX-2 expression via the sphingolipid pathway-mediated activation of NF-κB or mitogen-activated protein kinase (MAPK) pathways in human dermal fibroblasts. Palmitate treatment significantly induced COX-2 expression and prostaglandin E2 (PGE2) release in human dermal fibroblasts. In addition, pre-treatment with triacsin C, an inhibitor of acyl-CoA synthetase in de novo ceramide synthesis, was found to reduce palmitate-induced COX-2 expression and PGE2 release in human dermal fibroblast. The findings also show that palmitate-induced COX-2 expression and PGE2 release are mediated by the NF-κB, p38, and extracellular signal-regulated kinase (ERK) MAPK pathways. These findings point to a new mechanism for explaining the link between increased FFAs in obesity and impaired dermal function. PMID:24337700

  16. Pulp Fibroblasts Control Nerve Regeneration through Complement Activation.

    PubMed

    Chmilewsky, F; About, I; Chung, S-H

    2016-07-01

    Dentin-pulp regeneration is closely linked to the presence of nerve fibers in the pulp and to the healing mechanism by sprouting of the nerve fiber's terminal branches beneath the carious injury site. However, little is known about the initial mechanisms regulating this process in carious teeth. It has been recently demonstrated that the complement system activation, which is one of the first immune responses, contributes to tissue regeneration through the local production of anaphylatoxins such as C5a. While few pulp fibroblasts in intact teeth and in untreated fibroblast cultures express the C5a receptor (C5aR), here we show that all dental pulp fibroblasts, localized beneath the carious injury site, do express this receptor. This observation is consistent with our in vitro results, which showed expression of C5aR in lipoteichoic acid-stimulated pulp fibroblasts. The interaction of C5a, produced after complement synthesis and activation from pulp fibroblasts, with the C5aR of these cells mediated the local brain-derived neurotropic factor (BDNF) secretion. Overall, this activation guided the neuronal growth toward the lipoteichoic acid-stimulated fibroblasts. Thus, our findings highlight a new mechanism in one of the initial steps of the dentin-pulp regeneration process, linking pulp fibroblasts to the nerve sprouting through the complement system activation. This may provide a useful future therapeutic tool in targeting the fibroblasts in the dentin-pulp regeneration process. PMID:27053117

  17. Increased CCT-eta expression is a marker of latent and active disease and a modulator of fibroblast contractility in Dupuytren's contracture.

    PubMed

    Satish, Latha; O'Gorman, David B; Johnson, Sandra; Raykha, Christina; Gan, Bing Siang; Wang, James H-C; Kathju, Sandeep

    2013-07-01

    Dupuytren's contracture (DC) is a fibroproliferative disorder of unknown etiology characterized by a scar-like contracture that develops in the palm and/or digits. We have previously reported that the eta subunit of the chaperonin containing T-complex polypeptide (CCT-eta) is increased in fibrotic wound healing, and is essential for the accumulation of α-smooth muscle actin (α-SMA) in fibroblasts. The purpose of this study was to determine if CCT-eta is similarly implicated in the aberrant fibrosis seen in DC and to investigate the role of CCT-eta in the behavior of myo/fibroblasts in DC. Fibroblasts were obtained from DC-affected palmar fascia, from adjacent phenotypically normal palmar fascia in the same DC patients (PF), and from non-DC palmar fascial tissues in patients undergoing carpal tunnel (CT) release. Inherent contractility in these three populations was examined using fibroblast-populated collagen lattices (FPCLs) and by cell traction force microscopy. Expression of CCT-eta and α-SMA protein was determined by Western blot. The effect of CCT-eta inhibition on the contractility of DC cells was determined by deploying an siRNA versus CCT-eta. DC cells were significantly more contractile than both matching palmar fascial (PF) cells and CT cells in both assays, with PF cells demonstrating an intermediate contractility in the FPCL assay. Whereas α-SMA protein was significantly increased only in DC cells compared to PF and CT cells, CCT-eta protein was significantly increased in both PF and DC cells compared to CT cells. siRNA-mediated depletion of CCT-eta inhibited the accumulation of both CCT-eta and α-SMA protein in DC cells, and also significantly decreased the contractility of treated DC cells. These observations suggest that increased expression of CCT-eta appears to be a marker for latent and active disease in these patients and to be essential for the increased contractility exhibited by these fibroblasts. PMID:23292503

  18. Expression and targeting of human fibroblast activation protein in a human skin/severe combined immunodeficient mouse breast cancer xenograft model.

    PubMed

    Tahtis, Kiki; Lee, Fook-Thean; Wheatley, Jennifer M; Garin-Chesa, Pilar; Park, John E; Smyth, Fiona E; Obata, Yuichi; Stockert, Elisabeth; Hall, Cathrine M; Old, Lloyd J; Rettig, Wolfgang J; Scott, Andrew M

    2003-08-01

    Antigens and receptors that are highly expressed on tumor stromal cells, such as fibroblast activation protein (FAP), are attractive targets for antibody-based therapies because the supporting stroma and vessel network is essential for a solid neoplasm to grow beyond a size of 1-2 mm. The in vivo characterization of antibodies targeting human stromal or vessel antigens is hindered by the lack of an appropriate mouse model system because xenografts in standard mouse models express stromal and vessels elements of murine origin. This limitation may be overcome by the development of a human skin/mouse chimeric model, which is established by transplanting human foreskin on to the lateral flank of severe combined immunodeficient mice. The subsequent inoculation of breast carcinoma MCF-7 cells within the dermis of the transplanted human skin resulted in the production of xenografts expressing stromal and vessel elements of human origin. Widespread expression of human FAP-positive reactive stromal fibroblasts within xenografts was seen up to 2 months posttransplantation and postinjection of cells. Human blood vessel antigen expression also persisted at 2 months posttransplantation and postinjection of cells with murine vessels coexisting with the human vascular supply. The model was subsequently used to evaluate the biodistribution properties of an iodine-131-labeled humanized anti-FAP monoclonal antibody (BIBH-7). The results showed high specific targeting of the stromal compartment of the xenograft, indicating that the model provides a useful and novel approach for the in vivo assessment of the immunotherapeutic potential of molecules targeting human stroma and angiogenic systems. PMID:12939462

  19. Compound K inhibits MMP-1 expression through suppression of c-Src-dependent ERK activation in TNF-α-stimulated dermal fibroblast.

    PubMed

    Lee, Chang Seok; Bae, Il-Hong; Han, Jiwon; Choi, Gye-young; Hwang, Kyung-Hwan; Kim, Dong-Hyun; Yeom, Myeong-Hun; Park, Young-Ho; Park, Miyoung

    2014-11-01

    Compound K (CK) is one of the major metabolites of ginsenosides exhibiting a variety of pharmacological properties such as anti-ageing, anti-oxidation and anti-inflammatory activities. However, the protective efficacy of CK in abnormal skin conditions with inflammatory responses was not examined. Here, we investigated the effects of CK on matrix metalloproteinase-1 (MMP-1) and type I procollagen production in tumor necrosis factor-α (TNF-α)-stimulated human skin fibroblasts HS68 cells and human skin equivalents. We found that CK suppressed MMP-1 secretion and increased the level of reduced type I procollagen secretion, caused by the inhibition of extracellular signal-regulated kinase (ERK) activation, but not p38 and c-Jun N-terminal kinase (JNK) activation in TNF-α-stimulated HS68 cells. Then, we focused on the involvement of the c-Src and epidermal growth factor receptor (EGFR) as upstream signalling molecules for ERK activation by TNF-α in HS68 cells. CK suppressed the phosphorylation of c-Src/EGFR by TNF-α, which led to the inactivation of downstream signalling molecules including AKT and MEK. In addition, CK suppressed AP-1 (c-jun and c-fos) phosphorylation as downstream transcription factors of active ERK for MMP-1 expression in TNFα-stimulated HS68 cells. These results showed novel mechanisms by which CK inhibits TNF-α-induced MMP-1 expression through the inactivation of c-Src/EGFR-dependent ERK/AP-1 signalling pathway, resulting in the inhibition of collagen degradation in human fibroblast cells. Therefore, CK may be a promising protective agent for the treatment of inflammatory skin conditions such as skin ageing and atopic dermatitis. PMID:25181017

  20. Fibroblast activation in cancer: when seed fertilizes soil.

    PubMed

    Kuzet, Sanya-Eduarda; Gaggioli, Cedric

    2016-09-01

    In solid cancers, activated fibroblasts acquire the capacity to provide fertile soil for tumor progression. Specifically, cancer-associated fibroblasts (CAFs) establish a strong relationship with cancer cells. This provides advantages to both cell types: whereas cancer cells initiate and sustain CAF activation, CAFs support cancer cell growth, motility and invasion. This results in tumor progression, metastasis and chemoresistance. Numerous studies have detailed the mechanisms involved in fibroblast activation and cancer progression, some of which are reviewed in this article. Cancer cells and CAFs are "partners in crime", and their interaction is supported by inflammation. An understanding of the enemy, the cancer cell population and its "allies" should provide novel opportunities for targeted-drug development. Graphical Abstract Molecular mechanism of fibroblast activation. a Normal fibroblasts are the most common cell type in the extracellular matrix and are responsible for the synthesis of collagens and fibrilar proteins. Under normal conditions, fibroblasts maintain tissue homeostasis and contribute to proper cell communication and function. Fibroblasts can be activated by a diverse set of factors secreted from cancer or immune cells. Not only growth factors such as TGF-β, PDGF, HGF and FGF but also interleukins, metalloproteinases and reactive oxygen species can promote activation. Likewise, transcriptional factors such as NF-κB and HSF-1 play an important role, as do the gene family of metalloproteinase inhibitors, Timp and the NF-κB subunit, p62. Interestingly, fibroblasts themselves can stimulate cancer cells to support activation further. b Once activated, fibroblasts undergo a phenotype switch and become cancer-associated fibroblasts (CAFs) expressing various markers such as α-SMA, FSP1, vimentin and periostatin. c Recently, the LIF/GP130/IL6-R pathway has been identified as a signaling cascade involved in fibroblast activation. Upon LIF stimulation

  1. Peroxisome proliferator-activated receptor-γ agonist inhibits collagen synthesis in human keloid fibroblasts by suppression of early growth response-1 expression through upregulation of miR-543 expression

    PubMed Central

    Zhu, Hua-Yu; Bai, Wen-Dong; Wang, Hong-Tao; Xie, Song-Tao; Tao, Ke; Su, Lin-Lin; Liu, Jia-Qi; Yang, Xue-Kang; Li, Jun; Wang, Yun-Chuan; He, Ting; Han, Jun-Tao; Hu, Da-Hai

    2016-01-01

    A keloid is a benign skin tumor formed by an overgrowth of granulation tissue in affected patients. Peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists were reported to be able to regulate extracellular matrix production in human dermal fibroblasts. This study explored the underlying molecular mechanism of PPAR-γ agonist troglitazone treatment for fibroblasts obtained from keloid patients. The data revealed that troglitazone treatment of keloid fibroblasts (KFs) downregulated the expression of early growth response-1 (Egr1) and collagen-1 (Col1). Level of Egr1 were closely associated with KF-induced fibrosis. The miRNA profiling data revealed that miR-543 was transcriptionally activated after troglitazone treatment. Bioinformatic analysis and experimental data showed that miR-543 was able to target Egr1. ELISA data confirmed that Col1 protein in the supernatant were modulated by the feedback regulatory axis of PPAR-γ agonist-induced miR-543 to inhibit Egr1 expression, whereas PPAR-γ antagonist treatment abolished such effect on Col1 suppression in KFs. This study demonstrated that the PPAR-γ agonist-mediated miR-543 and Egr1 signaling plays an important role in the suppression of collagen synthesis in KFs. Future in vivo studies are needed to confirm these in vitro data. PMID:27429849

  2. Peroxisome proliferator-activated receptor-γ agonist inhibits collagen synthesis in human keloid fibroblasts by suppression of early growth response-1 expression through upregulation of miR-543 expression.

    PubMed

    Zhu, Hua-Yu; Bai, Wen-Dong; Wang, Hong-Tao; Xie, Song-Tao; Tao, Ke; Su, Lin-Lin; Liu, Jia-Qi; Yang, Xue-Kang; Li, Jun; Wang, Yun-Chuan; He, Ting; Han, Jun-Tao; Hu, Da-Hai

    2016-01-01

    A keloid is a benign skin tumor formed by an overgrowth of granulation tissue in affected patients. Peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists were reported to be able to regulate extracellular matrix production in human dermal fibroblasts. This study explored the underlying molecular mechanism of PPAR-γ agonist troglitazone treatment for fibroblasts obtained from keloid patients. The data revealed that troglitazone treatment of keloid fibroblasts (KFs) downregulated the expression of early growth response-1 (Egr1) and collagen-1 (Col1). Level of Egr1 were closely associated with KF-induced fibrosis. The miRNA profiling data revealed that miR-543 was transcriptionally activated after troglitazone treatment. Bioinformatic analysis and experimental data showed that miR-543 was able to target Egr1. ELISA data confirmed that Col1 protein in the supernatant were modulated by the feedback regulatory axis of PPAR-γ agonist-induced miR-543 to inhibit Egr1 expression, whereas PPAR-γ antagonist treatment abolished such effect on Col1 suppression in KFs. This study demonstrated that the PPAR-γ agonist-mediated miR-543 and Egr1 signaling plays an important role in the suppression of collagen synthesis in KFs. Future in vivo studies are needed to confirm these in vitro data. PMID:27429849

  3. Dipeptides Increase Functional Activity of Human Skin Fibroblasts.

    PubMed

    Malinin, V V; Durnova, A O; Polyakova, V O; Kvetnoi, I M

    2015-05-01

    We analyzed the effect of dipeptide Glu-Trp and isovaleroyl-Glu-Trp in concentrations of 0.2, 2 and 20 μg/ml and Actovegin preparation on functional activity of human skin fibroblasts. Dipeptides, especially Glu-Trp, produce a stimulating effect on human skin fibroblasts and their effect is equivalent to that of Actovegin. Dipeptides stimulate cell renewal processes by activating synthesis of Ki-67 and reducing expression of caspase-9 and enhance antioxidant function of the cells by stimulating the expression of Hsp-90 and inducible NO-synthase. These findings suggest that dipeptides are promising candidates for preparations stimulating reparative processes. PMID:26033609

  4. Enhancement of fibroblast activation protein α-based vaccines and adenovirus boost immunity by cyclophosphamide through inhibiting IL-10 expression in 4T1 tumor bearing mice.

    PubMed

    Xia, Qiu; Geng, Fei; Zhang, Fang-Fang; Liu, Chen-Lu; Xu, Ping; Lu, Zhen-Zhen; Zhang, Hai-Hong; Kong, Wei; Yu, Xiang-Hui

    2016-08-31

    Fibroblast activation protein α (FAPα) is expressed in cancer-associated fibroblasts (CAFs) of more than 90% of malignant epithelia carcinomas. CAFs are the main type of cells in the tumor microenvironment which offer nutrition and protection to the tumor and regulate immunosuppression. To eliminate CAFs, a vaccine targeting FAPα may be used with a heterologous prime-boost strategy to enhance the FAPα-specific cellular immunity. Here, a FAP vaccine using a recombinant adenovirus (rAd) vector was constructed as well as a DNA vaccine reported in our previous work. Although the DNA prime-rAd boost strategy enhanced FAPα-specific immune responses, improvement of anti-tumor immunity effects was not observed. Examination of immunosuppressive factors revealed that high expression of the IL-10 cytokine was considered the main cause of the failure of the prime-boost strategy. However, heterologous vaccination in combination with a low-dose of cyclophosphamide (CY), which was reported to reduce IL-10 production and promote a shift from immunosuppression to immunopotentiation, resulted in enhanced effects in terms of numbers of effector T cells and tumor growth inhibition rates, compared to the CY alone or DNA alone group. Tumor growth was inhibited markedly when the prime-boost strategy was combined with CY in both the prophylactic and therapeutic settings and the survival time of 4T1 tumor bearing mice was also prolonged significantly. With the reduction of IL-10, enhancement of the anti-tumor effect by the prime-boost strategy was observed. These results suggest that FAPα-targeted rAd boosting in combination with CY is an attractive approach to overcoming immunosuppression in cancer vaccines. PMID:27498213

  5. EPAC expression and function in cardiac fibroblasts and myofibroblasts

    SciTech Connect

    Olmedo, Ivonne; Muñoz, Claudia; Guzmán, Nancy; Catalán, Mabel; Vivar, Raúl; Ayala, Pedro; Humeres, Claudio; Aránguiz, Pablo; García, Lorena; Velarde, Victoria; Díaz-Araya, Guillermo

    2013-10-15

    In the heart, cardiac fibroblasts (CF) and cardiac myofibroblasts (CMF) are the main cells responsible for wound healing after cardiac insult. Exchange protein activated by cAMP (EPAC) is a downstream effector of cAMP, and it has been not completely studied on CF. Moreover, in CMF, which are the main cells responsible for cardiac healing, EPAC expression and function are unknown. We evaluated in both CF and CMF the effect of transforming growth factor β1 (TGF-β1) on EPAC-1 expression. We also studied the EPAC involvement on collagen synthesis, adhesion, migration and collagen gel contraction. Method: Rat neonatal CF and CMF were treated with TGF-β1 at different times and concentrations. EPAC-1 protein levels and Rap1 activation were measured by western blot and pull down assay respectively. EPAC cellular functions were determined by adhesion, migration and collagen gel contraction assay; and collagen expression was determined by western blot. Results: TGF-β1 through Smad and JNK significantly reduced EPAC-1 expression in CF, while in CMF this cytokine increased EPAC-1 expression through ERK1/2, JNK, p38, AKT and Smad3. EPAC activation was able to induce higher Rap1-GTP levels in CMF than in CF. EPAC and PKA, both cAMP effectors, promoted CF and CMF adhesion on fibronectin, as well as CF migration; however, this effect was not observed in CMF. EPAC but not PKA activation mediated collagen gel contraction in CF, while in CMF both PKA and EPAC mediated collagen gel contraction. Finally, the EPAC and PKA activation reduced collagen synthesis in CF and CMF. Conclusion: TGF-β1 differentially regulates the expression of EPAC in CF and CMF; and EPAC regulates differentially CF and CMF functions associated with cardiac remodeling. - Highlights: • TGF-β1 regulates EPAC-1 expression in cardiac fibroblast and myofibroblast. • Rap-1GTP levels are higher in cardiac myofibroblast than fibroblast. • EPAC-1 controls adhesion, migration and collagen synthesis in cardiac

  6. Constitutive Smad signaling and Smad-dependent collagen gene expression in mouse embryonic fibroblasts lacking peroxisome proliferator-activated receptor-{gamma}

    SciTech Connect

    Ghosh, Asish K Wei, Jun; Wu, Minghua; Varga, John

    2008-09-19

    Transforming growth factor-{beta} (TGF-{beta}), a potent inducer of collagen synthesis, is implicated in pathological fibrosis. Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is a nuclear hormone receptor that regulates adipogenesis and numerous other biological processes. Here, we demonstrate that collagen gene expression was markedly elevated in mouse embryonic fibroblasts (MEFs) lacking PPAR-{gamma} compared to heterozygous control MEFs. Treatment with the PPAR-{gamma} ligand 15d-PGJ{sub 2} failed to down-regulate collagen gene expression in PPAR-{gamma} null MEFs, whereas reconstitution of these cells with ectopic PPAR-{gamma} resulted in their normalization. Compared to control MEFs, PPAR-{gamma} null MEFs displayed elevated levels of the Type I TGF-{beta} receptor (T{beta}RI), and secreted more TGF-{beta}1 into the media. Furthermore, PPAR-{gamma} null MEFs showed constitutive phosphorylation of cellular Smad2 and Smad3, even in the absence of exogenous TGF-{beta}, which was abrogated by the ALK5 inhibitor SB431542. Constitutive Smad2/3 phosphorylation in PPAR-{gamma} null MEFs was associated with Smad3 binding to its cognate DNA recognition sequences, and interaction with coactivator p300 previously implicated in TGF-{beta} responses. Taken together, these results indicate that loss of PPAR-{gamma} in MEFs is associated with upregulation of collagen synthesis, and activation of intracellular Smad signal transduction, due, at least in part, to autocrine TGF-{beta} stimulation.

  7. Retrograde fibroblast growth factor 22 (FGF22) signaling regulates insulin-like growth factor 2 (IGF2) expression for activity-dependent synapse stabilization in the mammalian brain.

    PubMed

    Terauchi, Akiko; Johnson-Venkatesh, Erin M; Bullock, Brenna; Lehtinen, Maria K; Umemori, Hisashi

    2016-01-01

    Communication between pre- and postsynaptic cells promotes the initial organization of synaptic specializations, but subsequent synaptic stabilization requires transcriptional regulation. Here we show that fibroblast growth factor 22 (FGF22), a target-derived presynaptic organizer in the mouse hippocampus, induces the expression of insulin-like growth factor 2 (IGF2) for the stabilization of presynaptic terminals. FGF22 is released from CA3 pyramidal neurons and organizes the differentiation of excitatory nerve terminals formed onto them. Local application of FGF22 on the axons of dentate granule cells (DGCs), which are presynaptic to CA3 pyramidal neurons, induces IGF2 in the DGCs. IGF2, in turn, localizes to DGC presynaptic terminals and stabilizes them in an activity-dependent manner. IGF2 application rescues presynaptic defects of Fgf22(-/-) cultures. IGF2 is dispensable for the initial presynaptic differentiation, but is required for the following presynaptic stabilization both in vitro and in vivo. These results reveal a novel feedback signal that is critical for the activity-dependent stabilization of presynaptic terminals in the mammalian hippocampus. PMID:27083047

  8. Retrograde fibroblast growth factor 22 (FGF22) signaling regulates insulin-like growth factor 2 (IGF2) expression for activity-dependent synapse stabilization in the mammalian brain

    PubMed Central

    Terauchi, Akiko; Johnson-Venkatesh, Erin M; Bullock, Brenna; Lehtinen, Maria K; Umemori, Hisashi

    2016-01-01

    Communication between pre- and postsynaptic cells promotes the initial organization of synaptic specializations, but subsequent synaptic stabilization requires transcriptional regulation. Here we show that fibroblast growth factor 22 (FGF22), a target-derived presynaptic organizer in the mouse hippocampus, induces the expression of insulin-like growth factor 2 (IGF2) for the stabilization of presynaptic terminals. FGF22 is released from CA3 pyramidal neurons and organizes the differentiation of excitatory nerve terminals formed onto them. Local application of FGF22 on the axons of dentate granule cells (DGCs), which are presynaptic to CA3 pyramidal neurons, induces IGF2 in the DGCs. IGF2, in turn, localizes to DGC presynaptic terminals and stabilizes them in an activity-dependent manner. IGF2 application rescues presynaptic defects of Fgf22-/- cultures. IGF2 is dispensable for the initial presynaptic differentiation, but is required for the following presynaptic stabilization both in vitro and in vivo. These results reveal a novel feedback signal that is critical for the activity-dependent stabilization of presynaptic terminals in the mammalian hippocampus. DOI: http://dx.doi.org/10.7554/eLife.12151.001 PMID:27083047

  9. Role of IGF-1 pathway in lung fibroblast activation

    PubMed Central

    2013-01-01

    Background IGF-1 is elevated in pulmonary fibrosis and acute lung injury, where fibroblast activation is a prominent feature. We previously demonstrated that blockade of IGF pathway in murine model of lung fibrosis improved outcome and decreased fibrosis. We now expand that study to examine effects of IGF pathway on lung fibroblast behaviors that could contribute to fibrosis. Methods We first examined mice that express αSMA promoter upstream of GFP reporter treated with A12, a blocking antibody to IGF-1 receptor, after bleomycin induced lung injury. We then examined the effect of IGF-1 alone, or in combination with the pro-fibrotic cytokine TGFβ on expression of markers of myofibroblast activation in vitro, including αSMA, collagen α1, type 1, collagen α1, type III, and TGFβ expression. Results After bleomycin injury, we found decreased number of αSMA-GFP + cells in A12 treated mice, validated by αSMA immunofluorescent staining. We found that IGF-1, alone or in combination with TGF-β, did not affect αSMA RNA expression, promoter activity, or protein levels when fibroblasts were cultured on stiff substrate. IGF-1 stimulated Col1a1 and Col3a1 expression on stiff substrate. In contrast, IGF-1 treatment on soft substrate resulted in upregulation of αSMA gene and protein expression, as well as Col1a1 and Col3a1 transcripts. In conclusion, IGF-1 stimulates differentiation of fibroblasts into a myofibroblast phenotype in a soft matrix environment and has a modest effect on αSMA stress fiber organization in mouse lung fibroblasts. PMID:24103846

  10. IMPACT OF AGE AND AUTOANTIBODY STATUS ON THE GENE EXPRESSION OF SCLERODERMA FIBROBLASTS IN RESPONSE TO SILICA STIMULATION.

    PubMed

    Yang, Y; Wei, P; Guo, X J; Zhou, D; Zhang, W Z; Assassi, S; Zhou, X D

    2013-09-01

    Environmental factors are believed to play an important role in the pathogenesis of systemic sclerosis (SSc). Silica exposure has been implicated as potentially hazardous in epidemiological studies of SSc. It can activate fibroblasts to express profibrotic genes at certain conditions. The aim of this study is to examine whether the fibroblasts of SSc patients respond to silica particles with specific gene expressions differentially from normal control fibroblasts. The fibroblasts obtained from skin biopsies of 96 SSc patients and 104 controls were examined. Silica particles were used to perturb the cultures of the fibroblasts in time-course and dose-response assays. The transcript levels of COL1A2, COL3A1, MIVIP1, MMP3, TIMP3 and CTGF genes of the fibroblasts were measured with quantitative RT-PCR. The results showed that the expressions of all six genes in SSc fibroblasts under silica perturbation appeared significantly different from normal control fibroblasts. In age stratified analysis, compared to control fibroblasts, SSc fibroblasts from patients at age 30-40 years and 50-60 years displayed significantly decreased expressions of MMP1 gene in all dosage assays and increased expression of COL3A1 genes started at low dosages perturbation of silica particles, respectively. In autoantibody stratified analysis, specific gene expression patterns were significantly associated with autoantibody-subgroups of fibroblasts. A common feature of SSc fibroblasts was unstable and a wide range of gene expression changes in response to silica perturbation. Our studies may suggest an altered intrinsic dynamic control in SSc fibroblasts. In addition, sensitivity and specificity of SSc fibroblasts to potentially hazardous environmental trigger is age and autoantibody-subgroup-dependent. The fibroblasts of SSc patients at age 30-60 years may be more sensitive to silica perturbation toward a profibrotic gene expression. PMID:25435887

  11. Quiescent Fibroblasts Exhibit High Metabolic Activity

    PubMed Central

    Lemons, Johanna M. S.; Feng, Xiao-Jiang; Bennett, Bryson D.; Legesse-Miller, Aster; Johnson, Elizabeth L.; Raitman, Irene; Pollina, Elizabeth A.; Rabitz, Herschel A.; Rabinowitz, Joshua D.; Coller, Hilary A.

    2010-01-01

    Many cells in mammals exist in the state of quiescence, which is characterized by reversible exit from the cell cycle. Quiescent cells are widely reported to exhibit reduced size, nucleotide synthesis, and metabolic activity. Much lower glycolytic rates have been reported in quiescent compared with proliferating lymphocytes. In contrast, we show here that primary human fibroblasts continue to exhibit high metabolic rates when induced into quiescence via contact inhibition. By monitoring isotope labeling through metabolic pathways and quantitatively identifying fluxes from the data, we show that contact-inhibited fibroblasts utilize glucose in all branches of central carbon metabolism at rates similar to those of proliferating cells, with greater overflow flux from the pentose phosphate pathway back to glycolysis. Inhibition of the pentose phosphate pathway resulted in apoptosis preferentially in quiescent fibroblasts. By feeding the cells labeled glutamine, we also detected a “backwards” flux in the tricarboxylic acid cycle from α-ketoglutarate to citrate that was enhanced in contact-inhibited fibroblasts; this flux likely contributes to shuttling of NADPH from the mitochondrion to cytosol for redox defense or fatty acid synthesis. The high metabolic activity of the fibroblasts was directed in part toward breakdown and resynthesis of protein and lipid, and in part toward excretion of extracellular matrix proteins. Thus, reduced metabolic activity is not a hallmark of the quiescent state. Quiescent fibroblasts, relieved of the biosynthetic requirements associated with generating progeny, direct their metabolic activity to preservation of self integrity and alternative functions beneficial to the organism as a whole. PMID:21049082

  12. Functional TRPV1 expression in human corneal fibroblasts.

    PubMed

    Yang, Yuanquan; Yang, Hua; Wang, Zheng; Mergler, Stefan; Wolosin, J Mario; Reinach, Peter S

    2013-02-01

    Corneal wound healing in mice subsequent to an alkali burn results in dysregulated inflammation and opacification. Transient receptor potential vanilloid subtype 1 (TRPV1) channel activation in all tissue layers by endogenous ligands contributes to this sight compromising outcome since in TRPV1 knockout mice wound healing results instead in tissue transparency restoration. However, it is not known if primary human stromal fibroblasts exhibit such expression even though functional TRPV1 expression is evident in an immortalized human corneal epithelial cell line. In primary human corneal fibroblasts (HCF), TRPV1 gene expression and localization were identified based on the results of quantitative RT-PCR and immunocytochemistry, respectively. Western blot analysis identified a 100 kD protein corresponding to TRPV1 protein expression in a positive control. Single-cell fluorescence imaging detected in fura2-AM loaded cells Ca(2+) transients that rose 1.8-fold above the baseline induced by a selective TRPV1 agonist, capsaicin (CAP), which were blocked by a TRPV1 antagonist, capsazepine (CPZ) or exposure to a Ca(2+) free medium. The whole-cell mode of the planar patch-clamp technique identified TRPV1-induced currents that rose 1.76-fold between -60 and +130 mV. CAP-induced time dependent changes in the phosphorylation status of mitogen activated protein kinase (MAPK) signaling mediators that led to a 2.5-fold increase in IL-6 release after 24 h. This rise did not occur either in TRPV1 siRNA gene silenced cells or during exposure to SB203580 (10 μM), a selective p38 MAPK inhibitor. Taken together, identification of functional TRPV1 expression in HCF suggests that in vivo its activation by injury contributes to corneal opacification and inflammation during wound healing. These undesirable effects may result in part from increases in IL-6 expression mediated by p-p38 MAPK signaling. PMID:23232207

  13. S100A4 expression is increased in stricture fibroblasts from patients with fibrostenosing Crohn's disease and promotes intestinal fibroblast migration.

    PubMed

    Cunningham, Michael F; Docherty, Neil G; Burke, John P; O'Connell, P Ronan

    2010-08-01

    Fibroblasts represent the key cell type in fibrostenosing Crohn's disease (FCD) pathogenesis. S100A4 is an EF-hand calcium-binding protein family member, implicated in epithelial-mesenchymal transition and as a marker of activated T lymphocytes and fibroblasts in chronic tissue remodeling. The aim of this study was to examine the expression profile of S100A4 in the resected ileum of patients with FCD. Mucosa, seromuscular explants, and transmural biopsies were harvested from diseased and proximal, macroscopically normal margins of ileocecal resections from patients with FCD. Samples were processed for histochemistry, immunohistochemistry, real-time RT-PCR, Western blotting, and transmission electron microscopy. Primary explant cultures of seromuscular fibroblasts were exposed to transforming growth factor (TGF)-beta1 (1 ng/ml), and S100A4 expression and scratch wound-healing activity were assessed at 24 h. CCD-18Co fibroblasts were transfected with S100A4 small interfering RNA, treated with TGF-beta1 (1 ng/ml) for 30 min or 24 h, and then assessed for S100A4 and Smad3 expression and scratch wound-healing activity. S100A4 expression was increased in stricture mucosa, in the lamina propria, and in CD3-positive intraepithelial CD3-positive T lymphocytes. Fibroblastic S100A4 staining was observed in seromuscular scar tissue. Stricture fibroblast explant culture showed significant upregulation of S100A4 expression. TGF-beta1 increased S100A4 expression in cultured ileal fibroblasts. In CCD-18Co fibroblasts, S100A4 small interfering RNA inhibited scratch wound healing and modestly inhibited Smad3 activation. S100A4 expression is increased in fibroblasts, as well as immune cells, in Crohn's disease stricture and induced by TGF-beta1. Results from knockdown experiments indicate a potential role for S100A4 in mediating intestinal fibroblast migration. PMID:20489045

  14. Plasminogen Activator Inhibitor-1 Suppresses Profibrotic Responses in Fibroblasts from Fibrotic Lungs*

    PubMed Central

    Marudamuthu, Amarnath S.; Shetty, Shwetha K.; Bhandary, Yashodhar P.; Karandashova, Sophia; Thompson, Michael; Sathish, Venkatachalem; Florova, Galina; Hogan, Taryn B.; Pabelick, Christina M.; Prakash, Y. S.; Tsukasaki, Yoshikazu; Fu, Jian; Ikebe, Mitsuo; Idell, Steven; Shetty, Sreerama

    2015-01-01

    Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive interstitial scarification. A hallmark morphological lesion is the accumulation of myofibroblasts or fibrotic lung fibroblasts (FL-fibroblasts) in areas called fibroblastic foci. We previously demonstrated that the expression of both urokinase-type plasminogen activator (uPA) and the uPA receptor are elevated in FL-fibroblasts from the lungs of patients with IPF. FL-fibroblasts isolated from human IPF lungs and from mice with bleomycin-induced pulmonary fibrosis showed an increased rate of proliferation compared with normal lung fibroblasts (NL-fibroblasts) derived from histologically “normal” lung. Basal expression of plasminogen activator inhibitor-1 (PAI-1) in human and murine FL-fibroblasts was reduced, whereas collagen-I and α-smooth muscle actin were markedly elevated. Conversely, alveolar type II epithelial cells surrounding the fibrotic foci in situ, as well as those isolated from IPF lungs, showed increased activation of caspase-3 and PAI-1 with a parallel reduction in uPA expression. Transduction of an adenovirus PAI-1 cDNA construct (Ad-PAI-1) suppressed expression of uPA and collagen-I and attenuated proliferation in FL-fibroblasts. On the contrary, inhibition of basal PAI-1 in NL-fibroblasts increased collagen-I and α-smooth muscle actin. Fibroblasts isolated from PAI-1-deficient mice without lung injury also showed increased collagen-I and uPA. These changes were associated with increased Akt/phosphatase and tensin homolog proliferation/survival signals in FL-fibroblasts, which were reversed by transduction with Ad-PAI-1. This study defines a new role of PAI-1 in the control of fibroblast activation and expansion and its role in the pathogenesis of fibrosing lung disease and, in particular, IPF. PMID:25648892

  15. Activation of GPR30 inhibits cardiac fibroblast proliferation.

    PubMed

    Wang, Hao; Zhao, Zhuo; Lin, Marina; Groban, Leanne

    2015-07-01

    The incidence of left ventricular diastolic dysfunction significantly increases in postmenopausal women suggesting the association between estrogen loss and diastolic dysfunction. The in vivo activation of G protein-coupled estrogen receptor (GPR30) attenuates the adverse effects of estrogen loss on cardiac fibrosis and diastolic dysfunction in mRen2.Lewis rats. This study was designed to address the effects of GPR30 on cardiac fibroblast proliferation in rats. The expression of GPR30 in cardiac fibroblasts isolated from adult Sprague-Dawley rats was confirmed by RT-PCR, Western blot analysis, and immunofluorescence staining. Results from BrdU incorporation assays, cell counting, carboxyfluorescein diacetate succinimidyl ester labeling in conjunction with flow cytometry, and Ki-67 staining showed that treatment with G1, a specific agonist of GPR30, inhibited cardiac fibroblast proliferation in a dose-dependent manner, which was associated with decreases in CDK1 and cyclin B1 protein expressions. In the GPR30-KO cells, BrdU incorporation, and CDK1 and cyclin B1 expressions significantly increased when compared to GPR30-intact cells. G1 had no effect on BrdU incorporation, CDK1 and cyclin B1 mRNA levels in GPR30-KO cells. In vivo studies showed increases in CDK1 and cyclin B1 mRNA levels, Ki-67-positive cells, and the immunohistochemistry staining of vimentin, a fibroblast marker, in the left ventricles from ovariectomized mRen2.Lewis rats versus hearts from ovary-intact littermates; 2 weeks of G1 treatment attenuated these adverse effects of estrogen loss. This study demonstrates that GPR30 is expressed in rat cardiac fibroblasts, and activation of GPR30 limits proliferation of these cells likely via suppression of the cell cycle proteins, cyclin B1, and CDK1. PMID:25893735

  16. Gene Expression Profiling of IL-17A-Treated Synovial Fibroblasts from the Human Temporomandibular Joint

    PubMed Central

    Hattori, Toshio; Ogura, Naomi; Akutsu, Miwa; Kawashima, Mutsumi; Watanabe, Suguru; Ito, Ko; Kondoh, Toshirou

    2015-01-01

    Synovial fibroblasts contribute to the inflammatory temporomandibular joint under pathogenic stimuli. Synovial fibroblasts and T cells participate in the perpetuation of joint inflammation in a mutual activation feedback, via secretion of cytokines and chemokines that stimulate each other. IL-17 is an inflammatory cytokine produced primarily by Th17 cells which plays critical role in the pathogenesis of numerous autoimmune and inflammatory diseases. Here, we investigated the roles of IL-17A in temporomandibular joint disorders (TMD) using genome-wide analysis of synovial fibroblasts isolated from patients with TMD. IL-17 receptors were expressed in synovial fibroblasts as assessed using real-time PCR. Microarray analysis indicated that IL-17A treatment of synovial fibroblasts upregulated the expression of IL-6 and chemokines. Real-time PCR analysis showed that the gene expression of IL-6, CXCL1, IL-8, and CCL20 was significantly higher in IL-17A-treated synovial fibroblasts compared to nontreated controls. IL-6 protein production was increased by IL-17A in a time- and a dose-dependent manner. Additionally, IL-17A simulated IL-6 protein production in synovial fibroblasts samples isolated from three patients. Furthermore, signal inhibitor experiments indicated that IL-17-mediated induction of IL-6 was transduced via activation of NFκB and phosphatidylinositol 3-kinase/Akt. These results suggest that IL-17A is associated with the inflammatory progression of TMD. PMID:26839464

  17. Keratinocyte Microvesicles Regulate the Expression of Multiple Genes in Dermal Fibroblasts.

    PubMed

    Huang, Ping; Bi, Jiarui; Owen, Gethin R; Chen, Weimin; Rokka, Anne; Koivisto, Leeni; Heino, Jyrki; Häkkinen, Lari; Larjava, Hannu

    2015-12-01

    Extracellular vesicles released from cells regulate many normal and pathological conditions. Little is known about the role of epidermal keratinocyte microvesicles (KC-MVs) in epithelial-stromal interaction that is essential for wound healing. We investigated, therefore, whether MV-like structures are present in human wounds and whether they affect wound healing-associated gene expression in dermal fibroblasts. In human wounds, MV-like vesicles were observed during active epithelial migration and early granulation tissue formation. When KC-MVs derived from keratinocyte-like cells (HaCaT) were added to fibroblast cultures, expression of 21 genes was significantly regulated (P<0.05) out of 80 genes investigated, including matrix metalloproteinase-1 and -3, interleukin-6 and -8, and genes associated with transforming growth factor-β signaling. Similar changes were observed at the protein level. MVs from normal epidermal keratinocytes showed similar response to HaCaT cells. KC-MVs activated ERK1/2, JNK, Smad, and p38 signaling pathways in fibroblasts with ERK1/2 signaling having the most prominent role in the MV-induced gene expression changes. KC-MVs stimulated fibroblast migration and induced fibroblast-mediated endothelial tube formation but did not affect collagen gel contraction by fibroblasts. The results demonstrate that keratinocyte microvesicles have a strong and a specific regulatory effect on fibroblasts that may modulate several aspects of wound healing. PMID:26288358

  18. Cytokine-mediated PGE2 expression in human colonic fibroblasts.

    PubMed

    Kim, E C; Zhu, Y; Andersen, V; Sciaky, D; Cao, H J; Meekins, H; Smith, T J; Lance, P

    1998-10-01

    We investigated prostanoid biogenesis in human colonic fibroblasts (CCD-18Co and 5 primary fibroblast cultures) and epithelial cell lines (NCM460, T84, HT-29, and LS 174T) and the effect of PGE2 on fibroblast morphology. Cytokine-stimulated PGE2 production was measured. PGH synthase-1 and -2 (PGHS-1 and -2) protein and mRNA expression were evaluated. Basal PGE2 levels were low in all cell types (0.15-6.47 ng/mg protein). Treatment for 24 h with interleukin-1beta (IL-1beta; 10 ng/ml) or tumor necrosis factor-alpha (50 ng/ml), respectively, elicited maximal 25- and 6-fold inductions of PGE2 synthesis in CCD-18Co cultures and similar results in primary fibroblast cultures; maximal inductions with IL-1beta in colonic epithelial cell lines were from zero to fivefold. Treatment of CCD-18Co fibroblasts with IL-1beta caused maximal 21- and 53-fold increases, respectively, in PGHS-2 protein and mRNA levels without altering PGHS-1 expression. PGE2 (0.1 micromol/l) elicited a dramatic shape change in selected fibroblasts. Colonic fibroblasts are potentially important as cytokine targets and a source of and target for colonic prostanoids in vivo. PMID:9755052

  19. Hypoxia induces expression of connective tissue growth factor in scleroderma skin fibroblasts

    PubMed Central

    Hong, K-H; Yoo, S-A; Kang, S-S; Choi, J-J; Kim, W-U; Cho, C-S

    2006-01-01

    Connective tissue growth factor (CTGF) plays a role in the fibrotic process of systemic sclerosis (SSc). Because hypoxia is associated with fibrosis in several profibrogenic conditions, we investigated whether CTGF expression in SSc fibroblasts is regulated by hypoxia. Dermal fibroblasts from patients with SSc and healthy controls were cultured in the presence of hypoxia or cobalt chloride (CoCl2), a chemical inducer of hypoxia-inducible factor (HIF)-1α. Expression of CTGF was evaluated by Northern and Western blot analyses. Dermal fibroblasts exposed to hypoxia (1% O2) or CoCl2 (1–100 µM) enhanced expression of CTGF mRNA. Skin fibroblasts transfected with HIF-1α showed the increased levels of CTGF protein and mRNA, as well as nuclear staining of HIF-1α, which was enhanced further by treatment of CoCl2. Simultaneous treatment of CoCl2 and transforming growth factor (TGF)-β additively increased CTGF mRNA in dermal fibroblasts. Interferon-γ inhibited the TGF-β-induced CTGF mRNA expression dose-dependently in dermal fibroblasts, but they failed to hamper the CoCl2-induced CTGF mRNA expression. In addition, CoCl2 treatment increased nuclear factor (NF)-κB binding activity for CTGF mRNA, while decreasing IκBα expression in dermal fibroblasts. Our data suggest that hypoxia, caused possibly by microvascular alterations, up-regulates CTGF expression through the activation of HIF-1α in dermal fibroblasts of SSc patients, and thereby contributes to the progression of skin fibrosis. PMID:17034590

  20. Increased fibroblast telomerase expression precedes myofibroblast α-smooth muscle actin expression in idiopathic pulmonary fibrosis

    PubMed Central

    Waisberg, Daniel Reis; Parra, Edwin Roger; Barbas-Filho, João Valente; Fernezlian, Sandra; Capelozzi, Vera Luiza

    2012-01-01

    OBJECTIVE: This study sought to identify the relationship between fibroblast telomerase expression, myofibroblasts, and telomerase-mediated regulatory signals in idiopathic pulmonary fibrosis. METHODS: Thirty-four surgical lung biopsies, which had been obtained from patients with idiopathic pulmonary fibrosis and histologically classified as usual interstitial pneumonia, were examined. Immunohistochemistry was used to evaluate fibroblast telomerase expression, myofibroblast α-smooth muscle actin expression and the tissue expression of interleukin-4, transforming growth factor-β, and basic fibroblast growth factor. The point-counting technique was used to quantify the expression of these markers in unaffected, collapsed, mural fibrosis, and honeycombing areas. The results were correlated to patient survival. RESULTS: Fibroblast telomerase expression and basic fibroblast growth factor tissue expression were higher in collapsed areas, whereas myofibroblast expression and interleukine-4 tissue expression were higher in areas of mural fibrosis. Transforming growth factor-β expression was higher in collapsed, mural fibrosis and honeycombing areas in comparison to unaffected areas. Positive correlations were found between basic fibroblast growth factor tissue expression and fibroblast telomerase expression and between interleukin-4 tissue expression and myofibroblast α-smooth muscle actin expression. Negative correlations were observed between interleukin-4 expression and basic fibroblast growth factor tissue expression in areas of mural fibrosis. Myofibroblast α-smooth muscle actin expression and interleukin-4 tissue expression in areas of mural fibrosis were negatively associated with patient survival. CONCLUSION: Fibroblast telomerase expression is higher in areas of early remodeling in lung tissues demonstrating typical interstitial pneumonia, whereas myofibroblast α-smooth muscle actin expression predominates in areas of late remodeling. These events seem to be

  1. Hepatic Aryl Hydrocarbon Receptor Attenuates Fibroblast Growth Factor 21 Expression.

    PubMed

    Girer, Nathaniel G; Murray, Iain A; Omiecinski, Curtis J; Perdew, Gary H

    2016-07-15

    The Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in many physiological processes. Several studies indicate that AHR is also involved in energy homeostasis. Fibroblast growth factor 21 (FGF21) is an important regulator of the fasting and feeding responses. When administered to various genetic and diet-induced mouse models of obesity, FGF21 can attenuate obesity-associated morbidities. Here, we explore the role of AHR in hepatic Fgf21 expression through the use of a conditional, hepatocyte-targeted AHR knock-out mouse model (Cre(Alb)Ahr(Fx/Fx)). Compared with the congenic parental strain (Ahr(Fx/Fx)), non-fasted Cre(Alb)Ahr(Fx/Fx) mice exhibit a 4-fold increase in hepatic Fgf21 expression, as well as elevated expression of the FGF21-target gene Igfbp1 Furthermore, in vivo agonist activation of AHR reduces hepatic Fgf21 expression during a fast. The Fgf21 promoter contains several putative dioxin response elements (DREs). Using EMSA, we demonstrate that the AHR-ARNT heterodimer binds to a specific DRE that overlaps binding sequences for peroxisome proliferator-activated receptor α (PPARα), carbohydrate response element-binding protein (ChREBP), and cAMP response element-binding protein, hepatocyte specific (CREBH). In addition, we reveal that agonist-activated AHR impairs PPARα-, ChREBP-, and CREBH-mediated promoter activity in Hepa-1 cells. Accordingly, agonist treatment in Hepa-1 cells ablates potent ER stress-driven Fgf21 expression, and pre-treatment with AHR antagonist blocks this effect. Finally, we show that pre-treatment of primary human hepatocytes with AHR agonist diminishes PPARα-, glucose-, and ER stress-driven induction of FGF21 expression, indicating the effect is not mouse-specific. Together, our data show that AHR contributes to hepatic energy homeostasis, partly through the regulation of FGF21 expression and signaling. PMID:27226639

  2. Over Expression of Nucleophosmin and Nucleolin Contributes to the Suboptimal Activation of a G2/M Checkpoint in Ataxia Telangiectasia Fibroblasts.

    PubMed

    Nalabothula, Narasimharao; Chakravarty, Devulapalli; Pierce, Adam; Carrier, France

    2010-01-01

    Ataxia Telangiectasia (AT) cells exhibit suboptimal activation of radiation-induced cell cycle checkpoints despite having a wild type p53 genotype. Reducing or eliminating this delay could restore p53 function and reinstate normal cellular response to genotoxic stress. Here we show that the levels of Nuclephosmin (NPM), NPM phosphorylated at Serine 125, p53, p53 phosphorylated at Serine 15 and Serine 392 and the levels of Nucleolin (NCL) are high in AT fibroblasts compared to normal cells. Transfection of a functional ATM into AT fibroblasts reduced p53, phospo-p53, phospho-NPM and NCL levels to wild type fibroblasts levels. Our data indicate that ATM regulates phospho-NPM and NCL indirectly through the Protein Phosphatase 1 (PP1). Both, NPM and NCL interact with p53 and hinder its phosphorylation at Serine 15 in response to bleomycin. Moreover, NPM and NCL are phosphorylated by several of the same kinases targeting p53 and could potentially compete with p53 for phosphorylation in AT cells. In addition, our data indicate that down regulation of NCL and to a lesser extent NPM increase the number of AT cells arrested in G2/M in response to bleomycin. Together this data indicate that the lack of PP1 activation in AT cells result in increased NPM and NCL protein levels which prevents p53 phosphorylation in response to bleomycin and contributes to a defective G2/M checkpoint. PMID:21499441

  3. Expression of the E-cadherin repressors Snail, Slug and Zeb1 in urothelial carcinoma of the urinary bladder: relation to stromal fibroblast activation and invasive behaviour of carcinoma cells.

    PubMed

    Schulte, Julia; Weidig, Michaela; Balzer, Philipp; Richter, Petra; Franz, Marcus; Junker, Kerstin; Gajda, Mieczyslaw; Friedrich, Karlheinz; Wunderlich, Heiko; Östman, Arne; Petersen, Iver; Berndt, Alexander

    2012-12-01

    Epithelial-mesenchymal transition (EMT) is regulated by interaction of carcinoma and stromal cells and crucial for progression of urinary bladder carcinoma (UBC). Therefore, the influence of activated fibroblasts on the expression of E-cadherin repressors as well as EMT and invasion in UBC was investigated. A correlative analysis of the immunohistochemical expression of fibroblast (ASMA, S100A4, FAP, SDF1, PDGFRβ) and EMT (Snail, Slug, Zeb1, E-cadherin) markers was performed on 49 UBC cases of different stages. The impact of distinguishable growth factor stimulated fibroblasts on invasion, EMT, and E-cadherin repressor expression was investigated in an invasion model. In situ, invasiveness was significantly correlated to the loss of membranous E-cadherin (E-cad_m) and increased Snail, Slug, Zeb1 in tumour cells, as well as to increased ASMA, S100A4, and PDGFRβ in stromal cells. A significant correlation to nodal metastasis could be evidenced for the loss of E-Cad_m, and for an increase in S100A4 and PDGFRβ. Comparison of stromal and EMT markers revealed significant correlations of ASMA to Snail and Slug; of S100A4 to the loss of E-cad_m and Zeb1; and of PDGFRβ to the loss of E-Cad_m, Slug and Zeb1. In vitro, TGFβ1 induced myofibroblasts were the strongest attractants, while aFGF or TGFβ1/aFGF stimulated fibroblasts were the most potent EMT inductors. As shown here for the first time, distinct sub-populations of fibroblasts are to various extents associated with EMT and tumour progression in UBC. These relevant findings might be the basis for the identification of new diagnostic markers and therapeutic targets selectively affecting tumour supporting CAF effects. PMID:22820858

  4. Neurotensin Decreases the Proinflammatory Status of Human Skin Fibroblasts and Increases Epidermal Growth Factor Expression

    PubMed Central

    Miguel Neves, Bruno; Cruz, Maria Teresa; Carvalho, Eugénia

    2014-01-01

    Fibroblasts colonization into injured areas during wound healing (WH) is responsible for skin remodelling and is also involved in the modulation of inflammation, as fibroblasts are immunologically active. Herein, we aimed to determine neurotensin effect on the immunomodulatory profile of fibroblasts, both in homeostatic and inflammatory conditions. Neurotensin mediated responses occurred through NTR1 or NTR3 receptors, while under inflammatory conditions NTR1 expression increase seemed to modulate neurotensin responses. Among different immunomodulatory genes, CCL11, IL-8, and IL-6 were the most expressed genes, while CCL4 and EGF were the less expressed genes. After neurotensin exposure, IL-8 mRNA expression was increased while CCL11 was decreased, suggesting a proinflammatory upregulation and chemoattractant ability downregulation of fibroblasts. Under inflammatory conditions, gene expression was significantly increased. After neurotensin exposure, CCL4 and IL-6 mRNA expression were decreased while CCL11 was increased, suggesting again a decrease in the chemoattractant capacity of fibroblasts and in their proinflammatory status. Furthermore, the expression of EGF, a crucial growth factor for skin cells proliferation and WH, was increased in all conditions. Overall, neurotensin, released by nerve fibers or skin cells, may be involved in the decrease of the chemotaxis and the proinflammatory status in the proliferation and remodelling phases of WH. PMID:25180119

  5. EGFR-mediated expression of aquaporin-3 is involved in human skin fibroblast migration

    PubMed Central

    Cao, Cong; Sun, Yun; Healey, Sarah; Bi, Zhigang; Hu, Gang; Wan, Shu; Kouttab, Nicola; Chu, Wenming; Wan, Yinsheng

    2006-01-01

    AQP3 (aquaporin-3), known as an integral membrane channel in epidermal keratinocytes, facilitates water and glycerol movement into and out of the skin. Here, we demonstrate that AQP3 is also expressed in cultured human skin fibroblasts, which under normal wound healing processes migrate from surrounding tissues to close the wound. EGF (epidermal growth factor), which induced fibroblast migration, also induced AQP3 expression in a time- and dose-dependent manner. CuSO4 and NiCl2, previously known as AQP3 water transport inhibitors, as well as two other bivalent heavy metals Mn2+ and Co2+, inhibited EGF-induced cell migration in human skin fibroblasts. AQP3 knockdown by small interfering RNA inhibited EGF-induced AQP3 expression and cell migration. Furthermore, an EGFR (EGF receptor) kinase inhibitor, PD153035, blocked EGF-induced AQP3 expression and cell migration. MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK inhibitor U0126 and PI3K (phosphoinositide 3-kinase) inhibitor LY294002 also inhibited EGF-induced AQP3 expression and cell migration. Collectively, our findings show for the first time that AQP3 is expressed in human skin fibroblasts and that EGF induces AQP3 expression via EGFR, PI3K and ERK signal transduction pathways. We have provided evidence for a novel role of AQP3 in human skin fibroblast cell migration, which occurs during normal wound healing. PMID:16848764

  6. Lipopolysaccharide activated TLR4/NF-κB signaling pathway of fibroblasts from uterine fibroids.

    PubMed

    Guo, Jing; Zheng, Lihua; Chen, Li; Luo, Ning; Yang, Weihong; Qu, Xiaoyan; Liu, Mingmin; Cheng, Zhongping

    2015-01-01

    Uterine fibroids (UF) are the most common benign tumor of the female reproductive tract. The aim of this study was to explore the role of lipopolysaccharide (LPS)-induced activation of TLR4/NF-κB signaling pathway on stromal fibroblasts in the pathogenesis of UF. Here, TLR4/NF-κB signaling pathway was more activated in UF, and UF cells (UFC) and UF derived fibroblasts (TAF) than in smooth muscle tissues, smooth muscle cell (SMC) and myometrial fibroblasts (fib) respectively. After lipopolysaccharide (LPS) stimulation, the activity of fib was enhanced, characterized by the increased expression of fibroblast activation protein (FAP), and increased secretion of collagen I and transforming growth factor-β (TGF-β). Moreover, TLR4 inhibitor (VIPER) and siTLR4 can represses LPS-activated fibroblasts and TLR4/NF-κB signaling transduction pathways in fib and UFC cells. Co-cultured with LPS-activated fibroblast enhanced fibroblast activation and TLR4/NF-κB signaling. In conclusion, LPS treatment activated TLR4/NF-κB signaling pathway on fibroblasts, which may involve in the development of UF. Our study indicated reproductive tract infection may be associated with fibroid pathogenesis through TLR4/NF-κB signaling. Targeting NF-κB with inhibitors may hold promises of treating uterine fibroid. PMID:26617709

  7. Lipopolysaccharide activated TLR4/NF-κB signaling pathway of fibroblasts from uterine fibroids

    PubMed Central

    Guo, Jing; Zheng, Lihua; Chen, Li; Luo, Ning; Yang, Weihong; Qu, Xiaoyan; Liu, Mingmin; Cheng, Zhongping

    2015-01-01

    Uterine fibroids (UF) are the most common benign tumor of the female reproductive tract. The aim of this study was to explore the role of lipopolysaccharide (LPS)-induced activation of TLR4/NF-κB signaling pathway on stromal fibroblasts in the pathogenesis of UF. Here, TLR4/NF-κB signaling pathway was more activated in UF, and UF cells (UFC) and UF derived fibroblasts (TAF) than in smooth muscle tissues, smooth muscle cell (SMC) and myometrial fibroblasts (fib) respectively. After lipopolysaccharide (LPS) stimulation, the activity of fib was enhanced, characterized by the increased expression of fibroblast activation protein (FAP), and increased secretion of collagen I and transforming growth factor-β (TGF-β). Moreover, TLR4 inhibitor (VIPER) and siTLR4 can represses LPS-activated fibroblasts and TLR4/NF-κB signaling transduction pathways in fib and UFC cells. Co-cultured with LPS-activated fibroblast enhanced fibroblast activation and TLR4/NF-κB signaling. In conclusion, LPS treatment activated TLR4/NF-κB signaling pathway on fibroblasts, which may involve in the development of UF. Our study indicated reproductive tract infection may be associated with fibroid pathogenesis through TLR4/NF-κB signaling. Targeting NF-κB with inhibitors may hold promises of treating uterine fibroid. PMID:26617709

  8. Tropoelastin regulates chemokine expression in fibroblasts in Costello syndrome

    SciTech Connect

    Tatano, Yutaka; Fujinawa, Reiko; Kozutsumi, Yasunori; Takahashi, Tsutomu; Tsuji, Daisuke; Takeuchi, Naohiro; Tsuta, Kohji; Takada, Goro; Sakuraba, Hitoshi; Itoh, Kohji

    2008-08-08

    Costello syndrome is a multiple congenital anomaly associated with growth and mental retardation, cardiac and skeletal anomalies, and a predisposition to develop neoplasia. Comprehensive expression analysis revealed remarkable up-regulation of several cytokines and chemokines including Gro family proteins, interleukin-1{beta} (IL-1{beta}), IL-8 and MCP-1 but down-regulation of extracellular matrix components including collagens and proteoglycans of skin fibroblasts derived from a Japanese Costello syndrome patient characterized by significantly reduced tropoelastin mRNA, impaired elastogenesis and enhanced cell proliferation. In contrast, decreases in these chemokines and IL-1{beta} expression were observed in Costello fibroblastic cell lines stably expressing the bovine tropoelastin (btEln) gene and in restored elastic fibers. These results strongly suggest that the human TE gene (ELN) transfer could be applicable for the gene therapy of a group of Costello syndrome patients with reduced ELN gene expression.

  9. Staphylococcus aureus induces TGF-β1 and bFGF expression through the activation of AP-1 and NF-κB transcription factors in bovine mammary gland fibroblasts.

    PubMed

    Wu, Jianmei; Ding, Yulin; Bi, Yannan; Wang, Yi; Zhi, Yu; Wang, Jinling; Wang, Fenglong

    2016-06-01

    Staphylococcus aureus is a common Gram-positive pathogen that causes bovine mastitis, a persistent infection of the bovine mammary gland. To better understand the importance of bovine mammary fibroblasts (BMFBs) and the roles of the TLR-NF-κB and TLR-AP-1 signaling pathways in the regulation of S. aureus-associated mastitis and mammary fibosis, BMFBs cultured in vitro were stimulated with different concentrations of heat-inactivated S. aureus to analyze the gene and protein expression of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), transforming growth factor beta 1 (TGF-β1), basic fibroblast growth factor (bFGF) as well as the protein expression of nuclear factor-kappa B (NF-κB) and activation protein-1 (AP-1) by means of quantitative polymerase chain reaction (qPCR) and western blotting, respectively. Specific NF-κB and AP-1 inhibitors were also used to investigate their effects on the regulation of TGF-β1 and bFGF expression. The results indicated that, in addition to increasing mRNA and protein expression of TLR2 and TLR4, S. aureus could also upregulate TGF-β1 and bFGF mRNA expression and secretion through the activation of NF-κB and AP-1. The increase in TGF-β1 and bFGF expression was shown to be inhibited by AP-1- and NF-κB-specific inhibitors. Taken together, S. aureus induces TGF-β1 and bFGF expression through the activation of AP-1 and NF-κB in BMFBs. This information offers new potential targets for the treatment of bovine mammary fibrosis. PMID:26948281

  10. Calcium pantothenate modulates gene expression in proliferating human dermal fibroblasts.

    PubMed

    Wiederholt, Tonio; Heise, Ruth; Skazik, Claudia; Marquardt, Yvonne; Joussen, Sylvia; Erdmann, Kati; Schröder, Henning; Merk, Hans F; Baron, Jens Malte

    2009-11-01

    Topical application of pantothenate is widely used in clinical practice for wound healing. Previous studies identified a positive effect of pantothenate on migration and proliferation of cultured fibroblasts. However, these studies were mainly descriptive with no molecular data supporting a possible model of its action. In this study, we first established conditions for an in vitro model of pantothenate wound healing and then analysed the molecular effects of pantothenate. To test the functional effect of pantothenate on dermal fibroblasts, cells were cultured and in vitro proliferation tests were performed using a standardized scratch test procedure. For all three donors analysed, a strong stimulatory effect of pantothenate at a concentration of 20 microg/ml on the proliferation of cultivated dermal fibroblasts was observed. To study the molecular mechanisms resulting in the proliferative effect of pantothenate, gene expression was analysed in dermal fibroblasts cultivated with 20 microg/ml of pantothenate compared with untreated cells using the GeneChip Human Exon 1.0 ST Array. A number of significantly regulated genes were identified including genes coding for interleukin (IL)-6, IL-8, Id1, HMOX-1, HspB7, CYP1B1 and MARCH-II. Regulation of these genes was subsequently verified by quantitative real-time polymerase chain reaction analysis. Induction of HMOX-1 expression by pantothenol and pantothenic acid in dermal cells was confirmed on the protein level using immunoblots. Functional studies revealed the enhanced suppression of free radical formation in skin fibroblasts cultured with panthenol. In conclusion, these studies provided new insight in the molecular mechanisms linked to the stimulatory effect of pantothenate and panthenol on the proliferation of dermal fibroblasts. PMID:19397697

  11. Pentadecapeptide BPC 157 enhances the growth hormone receptor expression in tendon fibroblasts.

    PubMed

    Chang, Chung-Hsun; Tsai, Wen-Chung; Hsu, Ya-Hui; Pang, Jong-Hwei Su

    2014-01-01

    BPC 157, a pentadecapeptide derived from human gastric juice, has been demonstrated to promote the healing of different tissues, including skin, muscle, bone, ligament and tendon in many animal studies. However, the underlying mechanism has not been fully clarified. The present study aimed to explore the effect of BPC 157 on tendon fibroblasts isolated from Achilles tendon of male Sprague-Dawley rat. From the result of cDNA microarray analysis, growth hormone receptor was revealed as one of the most abundantly up-regulated genes in tendon fibroblasts by BPC 157. BPC 157 dose- and time-dependently increased the expression of growth hormone receptor in tendon fibroblasts at both the mRNA and protein levels as measured by RT/real-time PCR and Western blot, respectively. The addition of growth hormone to BPC 157-treated tendon fibroblasts dose- and time-dependently increased the cell proliferation as determined by MTT assay and PCNA expression by RT/real-time PCR. Janus kinase 2, the downstream signal pathway of growth hormone receptor, was activated time-dependently by stimulating the BPC 157-treated tendon fibroblasts with growth hormone. In conclusion, the BPC 157-induced increase of growth hormone receptor in tendon fibroblasts may potentiate the proliferation-promoting effect of growth hormone and contribute to the healing of tendon. PMID:25415472

  12. Fibroblast activation protein predicts prognosis in clear cell renal cell carcinoma.

    PubMed

    López, José I; Errarte, Peio; Erramuzpe, Asier; Guarch, Rosa; Cortés, Jesús M; Angulo, Javier C; Pulido, Rafael; Irazusta, Jon; Llarena, Roberto; Larrinaga, Gorka

    2016-08-01

    Clear cell renal cell carcinoma is a complex disease with only partial response to therapy and scarce reliable clinical parameters indicative of progression and survival. Fibroblast activation protein expression has been correlated with prognosis in several malignancies but never in renal cancer. We aim to analyze the immunohistochemical expression of fibroblast activation protein in 208 clear cell renal cell carcinomas and to evaluate its impact on the prognosis and survival. A positive cytoplasmic immunostaining of this protein in the stromal fibroblasts associated to cancer cells is associated with large tumor diameter (≥4cm), high-grade (G3/4) tumors, and high-stage (≥pT3) tumors. Fibroblast activation protein-positive cases had significantly shorter survivals after 5 (P=.00015), 10 (P=.0000042), and 15 (P=.000043) years of follow-up, with a hazard ratio of 0.31. Multivariate analysis showed that fibroblast activation protein (P=.00117) was stronger than grade and stage in predicting clinical aggressiveness in clear cell renal cell carcinoma. This study confirms the usefulness of fibroblast activation protein detection in the stromal fibroblast associated to cancer in clear cell renal cell carcinoma and adds a new immunohistochemical marker to predict clinical behavior in these patients. PMID:27063470

  13. CX3CL1 expression induced by Candida albicans in oral fibroblasts.

    PubMed

    Ohta, Kouji; Nishi, Hiromi; Fukui, Akiko; Shigeishi, Hideo; Takechi, Masaaki; Kamata, Nobuyuki

    2010-11-01

    Oral fibroblasts as well as keratinocytes are thought to influence host inflammatory responses against Candida albicans. However, little is known about chemokine expressions in oral fibroblasts against C. albicans infection. We therefore examined whether C. albicans induced several chemokines including fractalkine/CX3CL1 (CX3CL1), a unique chemokine that has properties of both chemoattractants and adhesion molecules, in fibroblasts and keratinocytes. The addition of C. albicans live cells to human immortalized oral keratinocytes (RT7) resulted in increases in the mRNA levels of multiple chemokines, but not of CX3CL1. In contrast, live and heat-killed C. albicans caused an increase in CX3CL1 mRNA and protein expression in human immortalized oral fibroblasts (GT1). CX3CL1 mRNA expression in GT1 cells was also enhanced by stimulation with a nonalbicans species of Candida. Further, the CX3CL1 chemokine domain showed antifungal activity against C. albicans. CX3CL1 secreted by oral fibroblasts appears to play an important role in the oral immune response to C. albicans infection. PMID:20880200

  14. Analysis of K-Ras Nuclear Expression in Fibroblasts and Mesangial Cells

    PubMed Central

    Fuentes-Calvo, Isabel; Blázquez-Medela, Ana M.; Santos, Eugenio; López-Novoa, José M.; Martínez-Salgado, Carlos

    2010-01-01

    Background Ras GTPases are considered cytoplasmic proteins that must be localized to cell membranes for activation, and there are few evidences of the presence of any Ras isoform in nuclei of eukaryotic cells. Methodology/Principal Findings Using conventional antibodies and inmunocytochemistry, differential centrifugation and western blot, we have observed the putative presence of K-Ras isoform in the nuclei of fibroblasts and mesangial cells. In order to avoid cross-reactions with other Ras isoforms, and using antibodies against K-Ras (R-3400, H3845-M01, sc-30) or pan-Ras (05-516, OP40) in cells that only expressed the K-Ras isoform (fibroblasts obtained from H-ras−/−,N-ras−/− mice) we also detected some nuclear positive expression. To further probe the identity of nuclear K-Ras, we have generated K-Ras knockout (K-ras−/−) embrionary fibroblasts by mating of K-ras+/− heterozygote mice. Using specific antibodies, only H- and N-Ras isoforms were observed in the cytoplasm of K-ras−/− fibroblasts. However, both K-Ras4A and K-Ras4B positive signals were detected by immunocytochemistry and Western blot with two commercial antibodies (sc-522 and sc-521 against each isoforms, respectively) in both cytoplasm and nuclei from K-ras−/− fibroblasts. Conclusions/Significance We show that the presence of K-Ras4B in fibroblast nuclei, already described by other authors, is probably due to a cross-reaction of the antibody with an undetermined nucleolar protein. Although this study also shows the possible nuclear expression of K-Ras isoform in fibroblasts or in mesangial cells, it also reveals the importance of being cautious in these studies about distribution of protein isoforms due to some important limitations imposed by the unspecificity of the antibodies or contaminations in cellular preparations. PMID:20090846

  15. Expression pattern of matrix metalloproteinase and TIMP genes in fibroblasts derived from Ets-1 knock-out mice compared to wild-type mouse fibroblasts.

    PubMed

    Hahne, Jens Claus; Fuchs, Tanja; El Mustapha, Haddouti; Okuducu, Ali Fuat; Bories, Jean Christophe; Wernert, Nicolas

    2006-07-01

    Matrix-degrading proteases play a key role in normal development, wound healing, many diseases such as rheumatoid arthritis and, in particular, tumour invasion. In invasive tumours, these enzymes are expressed by fibroblasts of the tumour stroma. Their expression and activity are tightly regulated at several levels, an important one being transcription. Previous in vitro and in vivo findings pointed to a major role of the Ets-1 transcription factor for this level of regulation. In the present study, we tried to prove this role in fibroblasts. We stimulated wild-type mouse fibroblasts with physiological doses of basic fibroblast growth factor (bFGF, known to induce different proteases and expressed by tumour cells) and compared the results to those obtained in Ets-1 -/- fibroblasts derived from Ets-1 knock-out mice. We found that basal Ets-1 levels are necessary not only for a fast induction of MMPs 2, 3 and 13 by bFGF but also for maintenance of the bFGF-induced expression of tissue inhibitors of metalloproteinases (TIMPs) 1, 2 and 3, which are known not only to inhibit but also participate as activators of certain pro-MMPs. PMID:16786167

  16. Fn14, a Downstream Target of the TGF-β Signaling Pathway, Regulates Fibroblast Activation

    PubMed Central

    Yang, Min; Lai, Wen; Ye, Litong; Chen, Jing; Hou, Xinghua; Ding, Hong; Zhang, Wenwei; Wu, Yueheng; Liu, Xiaoying; Huang, Shufang; Yu, Xiyong; Xiao, Dingzhang

    2015-01-01

    Fibrosis, the hallmark of human injuries and diseases such as serious burns, is characterized by excessive collagen synthesis and myofibroblast accumulation. Transforming growth factor-β (TGF-β), a potent inducer of collagen synthesis, has been implicated in fibrosis in animals. In addition to TGF-β, fibroblast growth factor-inducible molecule 14 (Fn14) has been reported to play an important role in fibrotic diseases, such as cardiac fibrosis. However, the function and detailed regulatory mechanism of Fn14 in fibrosis are unclear. Here, we investigated the effect of Fn14 on the activation of human dermal fibroblasts. In normal dermal fibroblasts, TGF-β signaling increased collagen production and Fn14 expression. Furthermore, Fn14 siRNA blocked extracellular matrix gene expression; even when TGF-β signaling was activated by TGF-β1, fibroblast activation remained blocked in the presence of Fn14 siRNA. Overexpressing Fn14 increased extracellular matrix gene expression. In determining the molecular regulatory mechanism, we discovered that SMAD4, an important TGF-β signaling co-mediator, bound to the Fn14 promoter and activated Fn14 transcription. Taken together, these results indicate that the TGF-β signaling pathway activates Fn14 expression through the transcription factor SMAD4 and that activated Fn14 expression increases extracellular matrix synthesis and fibroblast activation. Therefore, Fn14 may represent a promising approach to preventing the excessive accumulation of collagen or ECM in skin fibrosis. PMID:26625141

  17. Targeting Carcinoma-Associated Fibroblasts Within the Tumor Stroma With a Fibroblast Activation Protein-Activated Prodrug

    PubMed Central

    2012-01-01

    Background Fibroblasts undergo a morphological transformation to a reactive phenotype in the tumor microenvironment characterized by the expression of proteins such as fibroblast activation protein (FAP), a post-prolyl endopeptidase with expression largely restricted to carcinoma-associated fibroblasts. Thapsigargin (TG) is a highly toxic natural plant product that triggers a rise in intracellular calcium levels and apoptosis. FAP is therefore a provocative target for the activation of prodrugs consisting of a FAP-specific peptide coupled to a potent cytotoxic analog of TG. Methods The efficacy of FAP-activated peptidyl-TG prodrugs was tested in vitro in cell proliferation assays and effects on intracellular calcium in human cancer cell lines. The effects of FAP-activated prodrugs on tumor growth and host toxicity were tested in Balb-C nude MCF-7 and LNCaP xenograft mice (n = 9–11 per group). P values were calculated using permutation tests based on 50 000 permutations. Mixed effects models were used to account for correlations among replicate measures. All statistical tests were two-sided. Results FAP-activated prodrugs killed human cancer cells at low nanomolar concentrations (MCF-7 cells: IC50 = 3.5nM). Amino acid-12ADT analogs from FAP-cleaved prodrugs, but not uncleaved prodrugs, produced a rapid rise in intracellular calcium within minutes of exposure. Immunohistochemical analysis of xenografts exposed to FAP-prodrugs documented stromal-selective cell death of fibroblasts, pericytes, and endothelial cells of sufficient magnitude to inhibit growth of MCF-7 and LNCaP xenografts with minimal systemic toxicity, whereas non-FAP cleavable prodrugs were inactive. MCF-7 and LNCaP xenografts treated with a FAP-activated prodrug had maximal treated-to-control tumor volume ratios of 0.36 (treated: mean = 0.206mm3, 95% CI = 0.068 to 0.344mm3; control: mean = 0.580mm3, 95% CI = 0.267 to 0.893mm3) and 0.24 (treated: mean = 0.131mm3, 95% CI = 0.09 to 0.180mm3; control

  18. Adventitial fibroblasts are activated in the early stages of atherosclerosis in the apolipoprotein E knockout mouse

    SciTech Connect

    Xu Fang; Ji Jian; Li Li; Chen Rong; Hu Weicheng . E-mail: huweicheng@sdu.edu.cn

    2007-01-19

    The role of the adventitia in vascular function and vascular lesion formation has been largely ignored. This study observed the activation of the adventitia and specifically the fibroblasts in the development of atherosclerosis in the apoE(-/-) mouse. The results showed a gradual increase in expression of collagen types I and III after 2, 4, and 8 weeks of hyperlipidic diet. The earliest expression of monocyte chemoattractant protein-1 (MCP-1) protein and mRNA was detected in the adventitial fibroblast before the formation of intimal lesions. Proliferation, too, was first found in the adventitial fibroblasts. We hypothesize that the adventitial fibroblast is activated in the early stage of atherosclerosis. Adventitial inflammation may be an early event in the development of atherosclerotic lesions.

  19. Conjunctival Interleukin-13 Expression in Mucous Membrane Pemphigoid and Functional Effects of Interleukin-13 on Conjunctival Fibroblasts in Vitro

    PubMed Central

    Saw, Valerie P.J.; Offiah, Ifeoma; Dart, Robin J.; Galatowicz, Grazyna; Dart, John K.G.; Daniels, Julie T.; Calder, Virginia L.

    2009-01-01

    Interleukin-13 (IL-13) is the dominant effector cytokine of fibrosis in pulmonary and liver disease. Excessive conjunctival fibrosis in the immunobullous disease ocular mucous membrane pemphigoid (MMP) causes blindness; the pathogenesis of scarring in this disease is incompletely understood. To determine whether IL-13 is involved in conjunctival fibrosis in MMP, we studied the expression of IL-13 in ocular MMP patients before and after systemic immunosuppression and examined the effects of IL-13 on normal human conjunctival fibroblasts. We found high stromal cell expression of IL-13 in active ocular MMP by immunohistochemistry; 80% of these cells were CD3-positive T cells. Following immunosuppression, in clinically uninflamed, treated, ocular MMP patients, the number of IL-13 positive cells was significantly reduced, but this was still fourfold greater than in normal conjunctiva. IL-13 stimulated collagen lattice contraction and migration, and decreased production of mmp-3 and mmp-10 by human conjunctival fibroblasts. The addition of T cell culture supernatant to IL-13 synergistically augmented fibroblast migration. IL-13 also up-regulated surface expression of HLA-DR, CD80, CD40, and CD154 by conjunctival fibroblasts, suggesting a potential mechanism for fibroblast-T cell cross talk, via which fibroblasts may actively engage in perpetuating chronic inflammation and continued fibrosis. Together, these findings suggest that IL-13 is involved in conjunctival fibrosis in MMP, and that IL-13 has both profibrotic and pro-inflammatory effects on human conjunctival fibroblasts. PMID:19910508

  20. Manipulating Cx43 expression triggers gene reprogramming events in dermal fibroblasts from oculodentodigital dysplasia patients.

    PubMed

    Esseltine, Jessica L; Shao, Qing; Huang, Tao; Kelly, John J; Sampson, Jacinda; Laird, Dale W

    2015-11-15

    Oculodentodigital dysplasia (ODDD) is primarily an autosomal dominant disorder linked to over 70 GJA1 gene [connexin43 (Cx43)] mutations. For nearly a decade, our laboratory has been investigating the relationship between Cx43 and ODDD by expressing disease-linked mutants in reference cells, tissue-relevant cell lines, 3D organ cultures and by using genetically modified mouse models of human disease. Although salient features of Cx43 mutants have been revealed, these models do not necessarily reflect the complexity of the human context. To further overcome these limitations, we have acquired dermal fibroblasts from two ODDD-affected individuals harbouring D3N and V216L mutations in Cx43, along with familial controls. Using these ODDD patient dermal fibroblasts, which naturally produce less GJA1 gene product, along with RNAi and RNA activation (RNAa) approaches, we show that manipulating Cx43 expression triggers cellular gene reprogramming. Quantitative RT-PCR, Western blot and immunofluorescent analysis of ODDD patient fibroblasts show unusually high levels of extracellular matrix (ECM)-interacting proteins, including integrin α5β1, matrix metalloproteinases as well as secreted ECM proteins collagen-I and laminin. Cx43 knockdown in familial control cells produces similar effects on ECM expression, whereas Cx43 transcriptional up-regulation using RNAa decreases production of collagen-I. Interestingly, the enhanced levels of ECM-associated proteins in ODDD V216L fibroblasts is not only a consequence of increased ECM gene expression, but also due to an apparent deficit in collagen-I secretion which may further contribute to impaired collagen gel contraction in ODDD fibroblasts. These findings further illuminate the altered function of Cx43 in ODDD-affected individuals and highlight the impact of manipulating Cx43 expression in human cells. PMID:26349540

  1. Cellular retinol-binding protein-1 is transiently expressed in granulation tissue fibroblasts and differentially expressed in fibroblasts cultured from different organs.

    PubMed Central

    Xu, G.; Redard, M.; Gabbiani, G.; Neuville, P.

    1997-01-01

    We have reported that cellular retinol-binding protein-1 (CRBP-1) is transiently expressed by arterial smooth muscle cells during experimental intimal repair (P. Neuville, A. Geinoz, G. Benzonana, M. Redard, F. Gabbiani, P. Ropraz, G. Gabbiani: Am J Pathol 1997, 150:509-521). We have examined here the expression of CRBP-1 during wound healing after a full-thickness rat skin wound. CRBP-1 was transiently expressed by a significant proportion of fibroblastic cells including myofibroblasts. Expression started 4 days after wounding, reached a maximum at 12 days, and persisted up to 30 days when a scar was formed. After wound closure, most CRBP-1-containing fibroblastic cells underwent apoptosis. We have further investigated CRBP-1 expression in rat fibroblasts cultured from different organs. CRBP-1 was abundant in lung and heart fibroblasts and was detected in decreasing amounts in muscle, tendon, subcutaneous tissue, and granulation tissue fibroblasts. Dermis fibroblasts contained no detectable levels of CRBP-1. All-trans retinoic acid and transforming growth factor-beta1 inhibited cell proliferation and increased CRBP-1 expression in fibroblastic populations except dermis fibroblasts. We demonstrate that during granulation tissue formation a subpopulation of fibroblastic cells express CRBP-1 de novo. We also demonstrate that CRBP-1 expression by fibroblasts is regulated in vitro by retinoic acid and transforming growth factor-beta1. Our results suggest that CRBP-1 and possibly retinoic acid play a role in the evolution of granulation tissue. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 7 PMID:9403724

  2. Combined CSL and p53 downregulation promotes cancer-associated fibroblast activation

    PubMed Central

    Procopio, Maria-Giuseppina; Laszlo, Csaba; Labban, Dania Al; Kim, Dong Eun; Bordignon, Pino; Jo, Seunghee; Goruppi, Sandro; Menietti, Elena; Ostano, Paola; Ala, Ugo; Provero, Paolo; Hoetzenecker, Wolfram; Neel, Victor; Kilarski, Witek; Swartz, Melody A.; Brisken, Cathrin; Lefort, Karine; Dotto, G. Paolo

    2015-01-01

    Stromal fibroblast senescence has been linked to aging-associated cancer risk. However, density and proliferation of cancer-associated fibroblasts (CAF) are frequently increased. Loss or down-modulation of the Notch effector CSL/RBP-Jκ in dermal fibroblasts is sufficient for CAF activation and ensuing keratinocyte-derived tumors. We report that CSL silencing induces senescence of primary fibroblasts from dermis, oral mucosa, breast and lung. CSL functions in these cells as direct repressor of multiple senescence- and CAF-effector genes. It also physically interacts with p53, repressing its activity. CSL is down-modulated in stromal fibroblasts of premalignant skin actinic keratosis lesions and squamous cell carcinomas (SCC), while p53 expression and function is down-modulated only in the latter, with paracrine FGF signaling as likely culprit. Concomitant loss of CSL and p53 overcomes fibroblast senescence, enhances expression of CAF effectors and promotes stromal and cancer cell expansion. The findings support a CAF activation/stromal co-evolution model under convergent CSL/p53 control. PMID:26302407

  3. Can Pulp Fibroblasts Kill Cariogenic Bacteria? Role of Complement Activation.

    PubMed

    Jeanneau, C; Rufas, P; Rombouts, C; Giraud, T; Dejou, J; About, I

    2015-12-01

    Complement system activation has been shown to be involved in inflammation and regeneration processes that can be observed within the dental pulp after moderate carious decay. Studies simulating carious injuries in vitro have shown that when human pulp fibroblasts are stimulated by lipoteichoic acid (LTA), they synthetize all complement components. Complement activation leads to the formation of the membrane attack complex (MAC), which is known for its bacterial lytic effect. This work was designed to find out whether human pulp fibroblasts can kill Streptococcus mutans and Streptococcus sanguinis via complement activation. First, histological staining of carious tooth sections showed that the presence of S. mutans correlated with an intense MAC staining. Next, to simulate bacterial infection in vitro, human pulp fibroblasts were incubated in serum-free medium with LTA. Quantification by an enzymatic assay showed a significant increase of MAC formation on bacteria grown in this LTA-conditioned medium. To determine whether the MAC produced by pulp fibroblasts was functional, bacteria sensitivity to LTA-conditioned medium was evaluated using agar well diffusion assay and succinyl dehydrogenase (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide [MTT]) assay. Both assays showed that S. mutans and S. sanguinis were sensitive to LTA-conditioned medium. Finally, to evaluate whether MAC formation on cariogenic bacteria, by pulp fibroblasts, can be directly induced by the presence of these bacteria, a specific coculture model of human pulp fibroblasts and bacteria was developed. Immunofluorescence revealed an intense MAC labeling on bacteria after direct contact with pulp fibroblasts. The observed MAC formation and its lethal effects were significantly reduced when CD59, an inhibitor of MAC formation, was added. Our findings demonstrate that the MAC produced by LTA-stimulated pulp fibroblasts is functional and can kill S. mutans and S. sanguinis. Taken together

  4. Palladin Mediates Stiffness-Induced Fibroblast Activation in the Tumor Microenvironment

    PubMed Central

    McLane, Joshua S.; Ligon, Lee A.

    2015-01-01

    Mechanical properties of the tumor microenvironment have emerged as key factors in tumor progression. It has been proposed that increased tissue stiffness can transform stromal fibroblasts into carcinoma-associated fibroblasts. However, it is unclear whether the three to five times increase in stiffness seen in tumor-adjacent stroma is sufficient for fibroblast activation. In this study we developed a three-dimensional (3D) hydrogel model with precisely tunable stiffness and show that a physiologically relevant increase in stiffness is sufficient to lead to fibroblast activation. We found that soluble factors including CC-motif chemokine ligand (CCL) chemokines and fibronectin are necessary for this activation, and the combination of C-C chemokine receptor type 4 (CCR4) chemokine receptors and β1 and β3 integrins are necessary to transduce these chemomechanical signals. We then show that these chemomechanical signals lead to the gene expression changes associated with fibroblast activation via a network of intracellular signaling pathways that include focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K). Finally, we identify the actin-associated protein palladin as a key node in these signaling pathways that result in fibroblast activation. PMID:26200861

  5. Follistatin-like 1 promotes cardiac fibroblast activation and protects the heart from rupture.

    PubMed

    Maruyama, Sonomi; Nakamura, Kazuto; Papanicolaou, Kyriakos N; Sano, Soichi; Shimizu, Ippei; Asaumi, Yasuhide; van den Hoff, Maurice J; Ouchi, Noriyuki; Recchia, Fabio A; Walsh, Kenneth

    2016-01-01

    Follistatin-like 1 (Fstl1) is a secreted protein that is acutely induced in heart following myocardial infarction (MI). In this study, we investigated cell type-specific regulation of Fstl1 and its function in a murine model of MI Fstl1 was robustly expressed in fibroblasts and myofibroblasts in the infarcted area compared to cardiac myocytes. The conditional ablation of Fstl1 in S100a4-expressing fibroblast lineage cells (Fstl1-cfKO mice) led to a reduction in injury-induced Fstl1 expression and increased mortality due to cardiac rupture during the acute phase. Cardiac rupture was associated with a diminished number of myofibroblasts and decreased expression of extracellular matrix proteins. The infarcts of Fstl1-cfKO mice displayed weaker birefringence, indicative of thin and loosely packed collagen. Mechanistically, the migratory and proliferative capabilities of cardiac fibroblasts were attenuated by endogenous Fstl1 ablation. The activation of cardiac fibroblasts by Fstl1 was mediated by ERK1/2 but not Smad2/3 signaling. This study reveals that Fstl1 is essential for the acute repair of the infarcted myocardium and that stimulation of early fibroblast activation is a novel function of Fstl1. PMID:27234440

  6. Dynamic Regulation of Platelet-Derived Growth Factor Receptor α Expression in Alveolar Fibroblasts during Realveolarization

    PubMed Central

    Chen, Leiling; Acciani, Thomas; Le Cras, Tim; Lutzko, Carolyn

    2012-01-01

    Although the importance of platelet-derived growth factor receptor (PDGFR)-α signaling during normal alveogenesis is known, it is unclear whether this signaling pathway can regulate realveolarization in the adult lung. During alveolar development, PDGFR-α–expressing cells induce α smooth muscle actin (α-SMA) and differentiate to interstitial myofibroblasts. Fibroblast growth factor (FGF) signaling regulates myofibroblast differentiation during alveolarization, whereas peroxisome proliferator-activated receptor (PPAR)-γ activation antagonizes myofibroblast differentiation in lung fibrosis. Using left lung pneumonectomy, the roles of FGF and PPAR-γ signaling in differentiation of myofibroblasts from PDGFR-α–positive precursors during compensatory lung growth were assessed. FGF receptor (FGFR) signaling was inhibited by conditionally activating a soluble dominant-negative FGFR2 transgene. PPAR-γ signaling was activated by administration of rosiglitazone. Changes in α-SMA and PDGFR-α protein expression were assessed in PDGFR-α–green fluorescent protein (GFP) reporter mice using immunohistochemistry, flow cytometry, and real-time PCR. Immunohistochemistry and flow cytometry demonstrated that the cell ratio and expression levels of PDGFR-α–GFP changed dynamically during alveolar regeneration and that α-SMA expression was induced in a subset of PDGFR-α–GFP cells. Expression of a dominant-negative FGFR2 and administration of rosiglitazone inhibited induction of α-SMA in PDGFR-α–positive fibroblasts and formation of new septae. Changes in gene expression of epithelial and mesenchymal signaling molecules were assessed after left lobe pneumonectomy, and results demonstrated that inhibition of FGFR2 signaling and increase in PPAR-γ signaling altered the expression of Shh, FGF, Wnt, and Bmp4, genes that are also important for epithelial–mesenchymal crosstalk during early lung development. Our data demonstrate for the first time that a comparable

  7. Biased Allelic Expression in Human Primary Fibroblast Single Cells

    PubMed Central

    Borel, Christelle; Ferreira, Pedro G.; Santoni, Federico; Delaneau, Olivier; Fort, Alexandre; Popadin, Konstantin Y.; Garieri, Marco; Falconnet, Emilie; Ribaux, Pascale; Guipponi, Michel; Padioleau, Ismael; Carninci, Piero; Dermitzakis, Emmanouil T.; Antonarakis, Stylianos E.

    2015-01-01

    The study of gene expression in mammalian single cells via genomic technologies now provides the possibility to investigate the patterns of allelic gene expression. We used single-cell RNA sequencing to detect the allele-specific mRNA level in 203 single human primary fibroblasts over 133,633 unique heterozygous single-nucleotide variants (hetSNVs). We observed that at the snapshot of analyses, each cell contained mostly transcripts from one allele from the majority of genes; indeed, 76.4% of the hetSNVs displayed stochastic monoallelic expression in single cells. Remarkably, adjacent hetSNVs exhibited a haplotype-consistent allelic ratio; in contrast, distant sites located in two different genes were independent of the haplotype structure. Moreover, the allele-specific expression in single cells correlated with the abundance of the cellular transcript. We observed that genes expressing both alleles in the majority of the single cells at a given time point were rare and enriched with highly expressed genes. The relative abundance of each allele in a cell was controlled by some regulatory mechanisms given that we observed related single-cell allelic profiles according to genes. Overall, these results have direct implications in cellular phenotypic variability. PMID:25557783

  8. IDO-Expressing Fibroblasts Protect Islet Beta Cells From Immunological Attack and Reverse Hyperglycemia in Non-Obese Diabetic Mice.

    PubMed

    Zhang, Yun; Jalili, Reza B; Kilani, Ruhangiz T; Elizei, Sanam Salimi; Farrokhi, Ali; Khosravi-Maharlooei, Mohsen; Warnock, Garth L; Ao, Ziliang; Marzban, Lucy; Ghahary, Aziz

    2016-09-01

    Indoleamine 2,3-dioxygenase (IDO) induces immunological tolerance in physiological and pathological conditions. Therefore, we used dermal fibroblasts with stable IDO expression as a cell therapy to: (i) Investigate the factors determining the efficacy of this cell therapy for autoimmune diabetes in non-obese diabetic (NOD) mice; (ii) Scrutinize the potential immunological mechanisms. Newly diabetic NOD mice were randomly injected with either 10 × 10(6) (10M) or 15 × 10(6) (15M) IDO-expressing dermal fibroblasts. Blood glucose levels (BGLs), body weight, plasma kynurenine levels, insulitis severity, islet beta cell function, autoreactive CD8(+) T cells, Th17 cells and regulatory T cells (Tregs) were then investigated in these mice. IL-1β and cleaved caspase-3 levels were assessed in islets co-cultured with IDO-expressing fibroblasts. BGLs in 83% mice treated with 15M IDO-expressing fibroblasts recovered to normal up to 120 days. However, only 17% mice treated with 10M IDO-expressing cells were reversed to normoglycemia. A 15M IDO-expressing fibroblasts significantly reduced infiltrated immune cells in islets and recovered the functionality of remaining islet beta cells in NOD mice. Additionally, they successfully inhibited autoreactive CD8(+) T cells and Th17 cells as well as increased Tregs in different organs of NOD mice. Islet beta cells co-cultured with IDO-expressing fibroblasts had reduced IL-1β levels and cell apoptosis. Both cell number and IDO enzymatic activity contributes to the efficiency of IDO cell therapy. Optimized IDO-expressing fibroblasts successfully reverse the progression of diabetes in NOD mice through induction of Tregs as well as inhibition of beta cell specific autoreactive CD8(+) T cells and Th17 cells. J. Cell. Physiol. 231: 1964-1973, 2016. © 2016 Wiley Periodicals, Inc. PMID:26743772

  9. PDGFRβ expression and function in fibroblasts derived from pluripotent cells is linked to DNA demethylation

    PubMed Central

    Hewitt, Kyle J.; Shamis, Yulia; Knight, Elana; Smith, Avi; Maione, Anna; Alt-Holland, Addy; Sheridan, Steven D.; Haggarty, Stephen J.; Garlick, Jonathan A.

    2012-01-01

    Platelet-derived growth factor receptor-beta (PDGFRβ) is required for the development of mesenchymal cell types, and plays a diverse role in the function of fibroblasts in tissue homeostasis and regeneration. In this study, we characterized the expression of PDGFRβ in fibroblasts derived from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), and showed that this expression is important for cellular functions such as migration, extracellular matrix production and assembly in 3D self-assembled tissues. To determine potential regulatory regions predictive of expression of PDGFRβ following differentiation from ESCs and iPSCs, we analyzed the DNA methylation status of a region of the PDGFRB promoter that contains multiple CpG sites, before and after differentiation. We demonstrated that this promoter region is extensively demethylated following differentiation, and represents a developmentally regulated, differentially methylated region linked to PDGFRβ expression. Understanding the epigenetic regulation of genes such as PDGFRB, and identifying sites of active DNA demethylation, is essential for future applications of iPSC-derived fibroblasts for regenerative medicine. PMID:22344267

  10. Cardiomyocyte Marker Expression in Mouse Embryonic Fibroblasts by Cell-Free Cardiomyocyte Extract and Epigenetic Manipulation

    PubMed Central

    Talaei-Khozani, Tahereh; Heidari, Fatemeh; Esmaeilpour, Tahereh; Vojdani, Zahra; Mostafavi-Pour, Zohrah; Rohani, Leili

    2014-01-01

    Background: The regenerative capacity of the mammalian heart is quite limited. Recent reports have focused on reprogramming mesenchymal stem cells into cardiomyocytes. We investigated whether fibroblasts could transdifferentiate into myocardium. Methods: Mouse embryonic fibroblasts were treated with Trichostatin A (TSA) and 5-Aza-2-Deoxycytidine (5-aza-dC). The treated cells were permeabilized with streptolysin O and exposed to the mouse cardiomyocyte extract and cultured for 1, 10, and 21 days. Cardiomyocyte markers were detected by immunohistochemistry. Alkaline phosphatase activity and OCT4 were also detected in cells treated by chromatin-modifying agents. Results: The cells exposed to a combination of 5-aza-dC and TSA and permeabilized in the presence of the cardiomyocyte extract showed morphological changes. The cells were unable to express cardiomyocyte markers after 24 h. Immunocytochemical assays showed a notable degree of myosin heavy chain and α-actinin expressions after 10 days. The expression of the natriuretic factor and troponin T occurred after 21 days in these cells. The cells exposed to chromatin-modifying agents also expressed cardiomyocyte markers; however, the proportion of reprogrammed cells was clearly smaller than that in the cultures exposed to 5-aza-dC , TSA, and extract. Conclusion: It seems that the fibroblasts were able to eliminate the previous epigenetic markers and form new ones according to the factors existing in the extract. Since no beating was observed, at least up to 21 days, the cells may need an appropriate extracellular matrix for their function. PMID:24753644

  11. Expression of human. alpha. sub 2 -macroglobulin cDNA in baby hamster kidney fibroblasts: Secretion of high levels of active. alpha. sub 2 -macroglobulin

    SciTech Connect

    Boel, E.; Mortensen, S.B. ); Kristensen, T.; Sottrup-Jensen, L. ); Petersen, C.M. )

    1990-05-01

    Human {alpha}{sub 2}-macroglobulin ({alpha}{sub 2}M) is a unique 720-kDa proteinase inhibitor with a broad specificity. Unlike most other proteinase inhibitors, it does not inhibit proteolytic activity by blocking the active site of the proteinase. During complex formation with a proteinase {alpha}{sub 2}M entraps the proteinase molecule in a reaction that involves large conformational changes in {alpha}{sub 2}M. The authors describe the molecular cloning of {alpha}{sub 2}M cDNA from the human hepatoblastoma cell line HepG2. The cDNA was subcloned under control of the adenovirus major late promoter in a mammalian expression vector and introduced into the baby hamster kidney (BHK) cell line. Transformed clones were isolated and tested for production of human {alpha}{sub 2}M with a specific enzyme-linked immunosorbent assay. Human recombinant {alpha}{sub 2}M (r{alpha}{sub 2}M), secreted and purified form isolated transfected BHK cell lines, was structurally and functionally compared to {alpha}{sub 2}M purified from human serum. The results show that r{alpha}{sub 2}M was secreted from the BHK cells as an active proteinase-binding tetramer with functional thiol esters. Cleavage reactions of r{alpha}{sub 2}M with methylamine and trypsin showed that the recombinant product, which was correctly processed at the N-terminus, exhibited molecular characteristics similar to those of the human serum derived reference.

  12. Irradiated fibroblasts promote epithelial–mesenchymal transition and HDGF expression of esophageal squamous cell carcinoma

    SciTech Connect

    Bao, Ci-Hang; Wang, Xin-Tong; Ma, Wei; Wang, Na-Na; Nesa, Effat un; Wang, Jian-Bo; Wang, Cong; Jia, Yi-Bin; Wang, Kai; Tian, Hui; Cheng, Yu-Feng

    2015-03-06

    Recent evidence suggested that nonirradiated cancer-associated fibroblasts (CAFs) promoted aggressive phenotypes of cancer cells through epithelial–mesenchymal transition (EMT). Hepatoma-derived growth factor (HDGF) is a radiosensitive gene of esophageal squamous cell carcinoma (ESCC). This study aimed to investigate the effect of irradiated fibroblasts on EMT and HDGF expression of ESCC. Our study demonstrated that coculture with nonirradiated fibroblasts significantly increased the invasive ability of ESCC cells and the increased invasiveness was further accelerated when they were cocultured with irradiated fibroblasts. Scattering of ESCC cells was also accelerated by the supernatant from irradiated fibroblasts. Exposure of ESCC cells to supernatant from irradiated fibroblasts resulted in decreased E-cadherin, increased vimentin in vitro and β-catenin was demonstrated to localize to the nucleus in tumor cells with irradiated fibroblasts in vivo models. The expression of HDGF and β-catenin were increased in both fibroblasts and ESCC cells of irradiated group in vitro and in vivo models. Interestingly, the tumor cells adjoining the stromal fibroblasts displayed strong nuclear HDGF immunoreactivity, which suggested the occurrence of a paracrine effect of fibroblasts on HDGF expression. These data suggested that irradiated fibroblasts promoted invasion, growth, EMT and HDGF expression of ESCC. - Highlights: • Irradiated CAFs accelerated invasiveness and scattering of ESCC cell lines. • Irradiated CAFs promoted EMT of ESCC cells. • Irradiated fibroblasts induced nuclear β-catenin relocalization in ESCC cells. • Irradiated fibroblasts increased HDGF expression in vitro and in vivo.

  13. Cell proliferation in vitro modulates fibroblast collagenase activity

    SciTech Connect

    Lindblad, W.J.; Flood, L.

    1986-05-01

    Collagenase enzyme activity is regulated by numerous control mechanisms which prevent excessive release and activation of this protease. A primary mechanism for regulating enzyme extracellular activity may be linked to cell division, therefore they have examined the release of collagenase by fibroblasts in vitro in response to cellular proliferation. Studies were performed using fibroblasts derived from adult rat dermis maintained in DMEM containing 10% newborn calf serum, 25 mM tricine buffer, and antibiotics. Cells between subculture 10 and 19 were used with enzyme activity determined with a /sup 14/C-labelled soluble Type I collagen substrate with and without trypsin activation. Fibroblasts, trypsinized and plated at low density secreted 8.5 fold more enzyme than those cells at confluence (975 vs. 115 dpm/..mu..g DNA). This diminution occurred gradually as the cells went from logrithmic growth towards confluence. Confluent fibroblast monolayers were scraped in a grid arrangement, stimulating the remaining cells to divide, without exposure to trypsin. Within 24-48 hr postscraping enzyme levels had increased 260-400%, accompanied by enhanced incorporation of /sup 3/H-thymidine and /sup 3/H-uridine into cell macromolecules. The burst of enzyme release began to subside 12 hr later. These results support a close relationship between fibroblast proliferation and collagenase secretion.

  14. Differential gene expression in tumorigenic and nontumorigenic HeLa x normal human fibroblast hybrid cells.

    PubMed

    Tsujimoto, H; Nishizuka, S; Redpath, J L; Stanbridge, E J

    1999-12-01

    Fusion of tumorigenic HeLa cells with human skin fibroblasts results in chromosomally stable hybrids that are nontumorigenic and no longer express the HeLa tumor-associated marker intestinal alkaline phosphatase (IAP). Previous studies of spontaneous tumorigenic segregants from the nontumorigenic hybrids implicated the loss of one copy of human fibroblast chromosome 11 in the concomitant reexpression of tumorigenicity. In an attempt to identify genes involved in the control of tumorigenic expression, we performed differential display screening of nontumorigenic hybrid cells and tumorigenic segregants. Subsequent northern blot analyses reproducibly showed 17 differentially expressed genes, eight of which were expressed differentially in the nontumorigenic hybrids and nine of which were expressed differentially in the tumorigenic hybrids. The former were genes for 80K-L protein (a substrate of protein kinase C), AXL/UFO (a receptor tyrosine kinase), insulin-like growth factor binding protein 3, apolipoprotein AI regulatory protein, collagen type I alpha-2 chain, transforming growth factor-beta-induced gene product 3 (BIGH3), pregnancy-specific beta-1-glycoprotein, and fibroblast activation protein alpha. The latter nine genes were genes for serum/glucocorticoid-regulated kinase (SGK; a serine/threonine protein kinase), PTPCAAX1 (a tyrosine phosphatase), CXCR-4 (a G-protein-coupled membrane receptor), L-kynurenine hydrolase, beta-1, 4-galactosyltransferase, keratin 8, keratin 17, and H19 and a novel gene. The differential expression of these genes provided several interesting candidates for regulation of tumorigenic expression, including those involved in signal transduction and the extracellular matrix, cytoskeletal proteins, cell-surface enzyme, and the H19 gene. PMID:10569806

  15. Fibroblast activation protein α in tumor microenvironment: Recent progression and implications (Review)

    PubMed Central

    ZI, FUMING; HE, JINGSONG; HE, DONGHUA; LI, YI; YANG, LI; CAI, ZHEN

    2015-01-01

    Accumulated evidence has demonstrated that the microenvironment of a given tumor is important in determining its drug resistance, tumorigenesis, progression and metastasis. These microenvironments, like tumor cells, are vital targets for cancer therapy. The cross-talk between tumor cells and cancer-associated fibroblasts (CAFs, alternatively termed activated fibroblasts) is crucial in regulating the drug resistance, tumorigenesis, neoplastic progression, angiogenesis, invasion and metastasis of a tumor. Fibroblast activation protein α (FAPα) is a transmembrane serine protease and is highly expressed on CAFs present in >90% of human epithelial neoplasms. FAPα activity, alongside that of gelatinase and type I collagenase, has become increasingly important in cancer therapy due to its effectiveness in modulating tumor behavior. In this review, recent progression in the knowledge of the role of FAPα in tumor microenvironments is discussed. PMID:25593080

  16. Sirtuin 1 suppresses nuclear factor κB induced transactivation and pro-inflammatory cytokine expression in cat fibroblast cells

    PubMed Central

    ISHIKAWA, Shingo; TAKEMITSU, Hiroshi; HABARA, Makoto; MORI, Nobuko; YAMAMOTO, Ichiro; ARAI, Toshiro

    2015-01-01

    Nuclear factor κB (NF-κB) is a key factor in the development of chronic inflammation and is deeply involved in age-related and metabolic diseases development. These diseases have become a serious problem in cats. Sirtuin 1 (SIRT1) is associated with aging and metabolism through maintaining inflammation via NF-κB. In addition, fibroblasts are considered an important factor in the development of chronic inflammation. Therefore, we aimed to examine the effect of cat SIRT1 (cSIRT1) on NF-κB in cat fibroblast cells. The up-regulation of NF-κB transcriptional activity and pro-inflammatory cytokine mRNA expression by p65 subunit of NF-κB and lipopolysaccharide was suppressed by cSIRT1 in cat fibroblast cells. Our findings show that cSIRT1 is involved in the suppression of inflammation in cat fibroblast cells. PMID:26165138

  17. Neuropeptide Y is a physiological substrate of fibroblast activation protein: Enzyme kinetics in blood plasma and expression of Y2R and Y5R in human liver cirrhosis and hepatocellular carcinoma.

    PubMed

    Wong, Pok Fai; Gall, Margaret G; Bachovchin, William W; McCaughan, Geoffrey W; Keane, Fiona M; Gorrell, Mark D

    2016-01-01

    Fibroblast activation protein (FAP) is a dipeptidyl peptidase (DPP) and endopeptidase that is weakly expressed in normal adult human tissues but is greatly up-regulated in activated mesenchymal cells of tumors and chronically injured tissue. The identities and locations of target substrates of FAP are poorly defined, in contrast to the related protease DPP4. This study is the first to characterize the physiological substrate repertoire of the DPP activity of endogenous FAP present in plasma. Four substrates, neuropeptide Y (NPY), peptide YY, B-type natriuretic peptide and substance P, were analyzed by mass spectrometry following proteolysis in human or mouse plasma, and by in vivo localization in human liver tissues with cirrhosis and hepatocellular carcinoma (HCC). NPY was the most efficiently cleaved substrate of both human and mouse FAP, whereas all four peptides were efficiently cleaved by endogenous DPP4, indicating that the in vivo degradomes of FAP and DPP4 differ. All detectable DPP-specific proteolysis and C-terminal processing of these neuropeptides was attributable to FAP and DPP4, and plasma kallikrein, respectively, highlighting their combined physiological significance in the regulation of these neuropeptides. In cirrhotic liver and HCC, NPY and its receptor Y2R, but not Y5R, were increased in hepatocytes near the parenchymal-stromal interface where there is an opportunity to interact with FAP expressed on nearby activated mesenchymal cells in the stroma. These novel findings provide insights into the substrate specificity of FAP, which differs greatly from DPP4, and reveal a potential function for FAP in neuropeptide regulation within liver and cancer biology. PMID:26621486

  18. Regulation of matrix metalloproteinase-9 expression between gingival fibroblast cells from old and young rats

    SciTech Connect

    Kim, Su-Jung; Chung, Yong-Koo; Chung, Tae-Wook; Kim, Jeong-Ran; Moon, Sung-Kwon; Kim, Cheorl-Ho Park, Young-Guk

    2009-01-09

    Gingival fibroblast cells (rGF) from aged rats have an age-related decline in proliferative capacity compared with young rats. We investigated G1 phase cell cycle regulation and MMP-9 expression in both young and aged rGF. G1 cell cycle protein levels and activity were significantly reduced in response to interleukin-1{beta} (IL-1{beta}) stimulation with increasing in vitro age. Tumor necrosis factor-{alpha} (TNF-{alpha})-induced matrix metalloproteinase-9 (MMP-9) expression was also decreased in aged rGF in comparison with young rGF. Mutational analysis and gel shift assays demonstrated that the lower MMP-9 expression in aged rGF is associated with lower activities of transcription factors NF-{kappa}B and AP-1. These results suggest that cell cycle dysregulation and down-regulation of MMP-9 expression in rGF may play a role in gingival remodeling during in vitro aging.

  19. Atrial natriuretic peptide suppresses endothelin gene expression and proliferation in cardiac fibroblasts through a GATA4-dependent mechanism

    PubMed Central

    Glenn, Denis J.; Rahmutula, Dolkun; Nishimoto, Minobu; Liang, Faquan; Gardner, David G.

    2009-01-01

    Aims Atrial natriuretic peptide (ANP) is a hormone that has both antihypertrophic and antifibrotic properties in the heart. We hypothesized that myocyte-derived ANP inhibits endothelin (ET) gene expression in fibroblasts. Methods and results We have investigated the mechanism(s) involved in the antiproliferative effect of ANP on cardiac fibroblasts in a cell culture model. We found that cardiac myocytes inhibited DNA synthesis in co-cultured cardiac fibroblasts as did treatment with the ET-1 antagonist BQ610. The effect of co-culture was reversed by antibody directed against ANP or the ANP receptor antagonist HS-142-1. ANP inhibited the expression of the ET-1 gene and ET-1 gene promoter activity in cultured fibroblasts. The site of the inhibition was localized to a GATA-binding site positioned between −132 and −135 upstream from the transcription start site. GATA4 expression was demonstrated in cardiac fibroblasts, GATA4 bound the ET-1 promoter both in vitro and in vivo, and siRNA-mediated knockdown of GATA4 inhibited ET-1 expression. ET-1 treatment resulted in increased levels of phospho-serine105 GATA4 in cardiac fibroblasts and this induction was partially suppressed by co-treatment with ANP. Conclusion Collectively, these findings suggest that locally produced ET-1 serves as an autocrine stimulator of fibroblast proliferation, that ANP produced in neighbouring myocytes serves as a paracrine inhibitor of this proliferation, and that the latter effect operates through a reduction in GATA4 phosphorylation and coincident reduction in GATA4-dependent transcriptional activity. PMID:19546173

  20. Rho A and the Rho kinase pathway regulate fibroblast contraction: Enhanced contraction in constitutively active Rho A fibroblast cells

    SciTech Connect

    Nobe, Koji; Nobe, Hiromi; Yoshida, Hiroko; Kolodney, Michael S.; Paul, Richard J.; Honda, Kazuo

    2010-08-20

    Research highlights: {yields} Mechanisms of fibroblast cell contraction in collagen matrix. {yields} Assessed an isometric force development using 3D-reconstituted-fibroblast fiber. {yields} Constitutively active Rho A induced the over-contraction of fibroblast cells. {yields} Rho A and Rho kinase pathway has a central role in fibroblast cell contraction. -- Abstract: Fibroblast cells play a central role in the proliferation phase of wound healing processes, contributing to force development. The intracellular signaling pathways regulating this non-muscle contraction are only partially understood. To study the relations between Rho A and contractile responses, constitutively active Rho A (CA-Rho A) fibroblast cells were reconstituted into fibers and the effects of calf serum (CS) on isometric force were studied. CS-induced force in CA-Rho A fibroblast fibers was twice as large as that in wild type (NIH 3T3) fibroblast fibers. During this response, the translocation of Rho A from the cytosol to the membrane was detected by Rho A activity assays and Western blot analysis. Pre-treatment with a Rho specific inhibitor (C3-exoenzyme) suppressed translocation as well as contraction. These results indicate that Rho A activation is essential for fibroblast contraction. The Rho kinase inhibitor ( (Y27632)) inhibited both NIH 3T3 and CA-Rho A fibroblast fiber contractions. Activation of Rho A is thus directly coupled with Rho kinase activity. We conclude that the translocation of Rho A from the cytosol to the membrane and the Rho kinase pathway can regulate wound healing processes mediated by fibroblast contraction.

  1. Oleic, Linoleic and Linolenic Acids Increase ROS Production by Fibroblasts via NADPH Oxidase Activation

    PubMed Central

    Hatanaka, Elaine; Dermargos, Alexandre; Hirata, Aparecida Emiko; Vinolo, Marco Aurélio Ramirez; Carpinelli, Angelo Rafael; Newsholme, Philip; Armelin, Hugo Aguirre; Curi, Rui

    2013-01-01

    The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47phox phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47phox mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts. PMID:23579616

  2. Oleic, linoleic and linolenic acids increase ros production by fibroblasts via NADPH oxidase activation.

    PubMed

    Hatanaka, Elaine; Dermargos, Alexandre; Hirata, Aparecida Emiko; Vinolo, Marco Aurélio Ramirez; Carpinelli, Angelo Rafael; Newsholme, Philip; Armelin, Hugo Aguirre; Curi, Rui

    2013-01-01

    The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47 (phox) phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47 (phox) mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts. PMID:23579616

  3. Epidermal β-catenin activation remodels the dermis via paracrine signalling to distinct fibroblast lineages

    PubMed Central

    Lichtenberger, Beate M.; Mastrogiannaki, Maria; Watt, Fiona M.

    2016-01-01

    Sustained epidermal Wnt/β-catenin signalling expands the stem cell compartment and induces ectopic hair follicles (EFs). This is accompanied by extensive fibroblast proliferation and extracellular matrix (ECM) remodelling in the underlying dermis. Here we show that epidermal Hedgehog (Hh) and Transforming growth factor-beta (TGF-β) signalling mediate the dermal changes. Pharmacological inhibition or genetic deletion of these pathways prevents β-catenin-induced dermal reprogramming and EF formation. Epidermal Shh stimulates proliferation of the papillary fibroblast lineage, whereas TGF-β2 controls proliferation, differentiation and ECM production by reticular fibroblasts. Hh inhibitors do not affect TGF-β target gene expression in reticular fibroblasts, and TGF-β inhibition does not prevent Hh target gene induction in papillary fibroblasts. However, when Hh signalling is inhibited the reticular dermis does not respond to epidermal β-catenin activation. We conclude that the dermal response to epidermal Wnt/β-catenin signalling depends on distinct fibroblast lineages responding to different paracrine signals. PMID:26837596

  4. Epidermal β-catenin activation remodels the dermis via paracrine signalling to distinct fibroblast lineages.

    PubMed

    Lichtenberger, Beate M; Mastrogiannaki, Maria; Watt, Fiona M

    2016-01-01

    Sustained epidermal Wnt/β-catenin signalling expands the stem cell compartment and induces ectopic hair follicles (EFs). This is accompanied by extensive fibroblast proliferation and extracellular matrix (ECM) remodelling in the underlying dermis. Here we show that epidermal Hedgehog (Hh) and Transforming growth factor-beta (TGF-β) signalling mediate the dermal changes. Pharmacological inhibition or genetic deletion of these pathways prevents β-catenin-induced dermal reprogramming and EF formation. Epidermal Shh stimulates proliferation of the papillary fibroblast lineage, whereas TGF-β2 controls proliferation, differentiation and ECM production by reticular fibroblasts. Hh inhibitors do not affect TGF-β target gene expression in reticular fibroblasts, and TGF-β inhibition does not prevent Hh target gene induction in papillary fibroblasts. However, when Hh signalling is inhibited the reticular dermis does not respond to epidermal β-catenin activation. We conclude that the dermal response to epidermal Wnt/β-catenin signalling depends on distinct fibroblast lineages responding to different paracrine signals. PMID:26837596

  5. Induction of fibroblast apolipoprotein E expression during apoptosis, starvation-induced growth arrest and mitosis.

    PubMed Central

    Quinn, Carmel M; Kågedal, Katarina; Terman, Alexei; Stroikin, Uri; Brunk, Ulf T; Jessup, Wendy; Garner, Brett

    2004-01-01

    Apolipoprotein E (apoE) mediates the hepatic clearance of plasma lipoproteins, facilitates cholesterol efflux from macrophages and aids neuronal lipid transport. ApoE is expressed at high levels in hepatocytes, macrophages and astrocytes. In the present study, we identify nuclear and cytosolic pools of apoE in human fibroblasts. Fibroblast apoE mRNA and protein levels were up-regulated during staurosporine-induced apoptosis and this was correlated with increased caspase-3 activity and apoptotic morphological alterations. Because the transcription of apoE and specific pro-apoptotic genes is regulated by the nuclear receptor LXR (liver X receptor) alpha, we analysed LXRalpha mRNA expression by quantitative real-time PCR and found it to be increased before apoE mRNA induction. The expression of ABCA1 (ATP-binding cassette transporter A1) mRNA, which is also regulated by LXRalpha, was increased in parallel with apoE mRNA, indicating that LXRalpha probably promotes apoE and ABCA1 transcription during apoptosis. Fibroblast apoE levels were increased under conditions of serum-starvation-induced growth arrest and hyperoxia-induced senescence. In both cases, an increased nuclear apoE level was observed, particularly in cells that accumulated lipofuscin. Nuclear apoE was translocated to the cytosol when mitotic nuclear disassembly occurred and this was associated with an increase in total cellular apoE levels. ApoE amino acid sequence analysis indicated several potential sites for phosphorylation. In vivo studies, using 32P-labelling and immunoprecipitation, revealed that fibroblast apoE can be phosphorylated. These studies reveal novel associations and potential roles for apoE in fundamental cellular processes. PMID:14656220

  6. Expression of microRNAs in fibroblast of pterygium

    PubMed Central

    Lee, Joon H.; Jung, Sun-Ah; Kwon, Young-A; Chung, Jae-Lim; Kim, Ungsoo Samuel

    2016-01-01

    AIM To screen microRNAs (miRNAs) and set up target miRNAs in pterygium. METHODS Primary fibroblasts were isolated from pterygium and Tenon's capsule and cultured. Immunocytochemical analysis and Western blotting were performed to confirm the culture of fibroblasts. In all, 1733 miRNAs were screened in the first step by using GeneChip® miRNA3.0 Array. Specific miRNAs involved in the pathogenesis of pterygium were subsequently determined using the following criteria: 1) high reproducibility in a repetitive test; 2) base log value of >7.0 for both control and pterygial fibroblasts; and 3) log ratio of >1.0 between pterygial fibroblasts and control fibroblasts. RESULTS Primary screening showed that 887/1733 miRNAs were up-regulated and 846/1733 miRNAs were down-regulated in pterygial fibroblasts compared with those in control fibroblasts. Of the 1733 miRNAs screened, 4 miRNAs, namely, miRNA-143a-3p, miRNA-181a-2-3p, miRNA-377-5p and miRNA-411a-5p, met the above-mentioned criteria. Primary screening showed that these 4 miRNAs were up-regulated in pterygial fibroblasts compared with control fibroblasts and that miRNA-143a-3p had the highest mean ratio compared with the miRNAs in control fibroblasts. CONCLUSION miRNA-143a-3p, miRNA-181a-2-3p, miRNA-377-5p and miRNA-411a-5p are up-regulated in pterygial fibroblasts compared with control fibroblasts, suggesting their involvement in the pathogenesis of pterygium. PMID:27500101

  7. Thrombin stimulates fibroblast procollagen production via proteolytic activation of protease-activated receptor 1.

    PubMed Central

    Chambers, R C; Dabbagh, K; McAnulty, R J; Gray, A J; Blanc-Brude, O P; Laurent, G J

    1998-01-01

    Thrombin is a multifunctional serine protease that has a crucial role in blood coagulation. It is also a potent mesenchymal cell mitogen and chemoattractant and might therefore have an important role in the recruitment and local proliferation of mesenchymal cells at sites of tissue injury. We hypothesized that thrombin might also affect the deposition of connective tissue proteins at these sites by directly stimulating fibroblast procollagen production. To address this hypothesis, the effect of thrombin on procollagen production and gene expression by human foetal lung fibroblasts was assessed over 48 h. Thrombin stimulated procollagen production at concentrations of 1 nM and above, with maximal increases of between 60% and 117% at 10 nM thrombin. These effects of thrombin were, at least in part, due to increased steady-state levels of alpha1(I) procollagen mRNA. They could furthermore be reproduced with thrombin receptor-activating peptides for the protease-activated receptor 1 (PAR-1) and were completely abolished when thrombin was rendered proteolytically inactive with the specific inhibitors d-Phe-Pro-ArgCH2Cl and hirudin, indicating that thrombin is mediating these effects via the proteolytic activation of PAR-1. These results suggest that thrombin might influence the deposition of connective tissue proteins during normal wound healing and the development of tissue fibrosis by stimulating fibroblast procollagen production. PMID:9639571

  8. Collagen expression in fibroblasts with a novel LMNA mutation

    SciTech Connect

    Nguyen, Desiree; Leistritz, Dru F.; Turner, Lesley; MacGregor, David; Ohson, Kamal; Dancey, Paul; Martin, George M.; Oshima, Junko . E-mail: picard@u.washington.edu

    2007-01-19

    Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies, and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy.

  9. Collagen Expression in Fibroblasts with a Novel LMNA Mutation

    PubMed Central

    Nguyen, Desiree; Leistritz, Dru F.; Turner, Lesley; MacGregor, David; Ohson, Kamal; Dancey, Paul; Martin, George M.; Oshima, Junko

    2007-01-01

    Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy. PMID:17150192

  10. Expression of fibroblast growth factor 23 by canine soft tissue sarcomas.

    PubMed

    Hardcastle, M R; Dittmer, K E

    2016-09-01

    Tumour-induced osteomalacia (TIO) is a rare paraneoplastic syndrome of humans. Some mesenchymal tumours (often resembling haemangiopericytomas) express molecules that normally regulate phosphorus metabolism; most frequently, fibroblast growth factor 23. Patients develop renal phosphate wasting and inappropriately low serum concentrations of 1, 25 (OH)2 vitamin D3 , leading to osteomalacia. Surgical removal of the tumour is curative. The authors examined expression of canine fibroblast growth factor 23 in 49 soft tissue sarcomas, and control tissues from normal adult dogs. RNA extracted from bone or formalin-fixed, paraffin-embedded tissues was analysed by end point and quantitative reverse transcriptase-polymerase chain reaction. Fibroblast growth factor 23 expression was detected in bone, lung, kidney, lymph node and thymus. Fifteen of 49 sarcomas (31%) expressed fibroblast growth factor 23, three of these had high relative expression and some features resembling phosphatonin-expressing mesenchymal tumours of humans. Further work is required to determine whether TIO may occur in dogs. PMID:24923416

  11. Rapid fibroblast activation in mammalian cells induced by silicon nanowire arrays

    NASA Astrophysics Data System (ADS)

    Ha, Qing; Yang, Gao; Ao, Zhuo; Han, Dong; Niu, Fenglan; Wang, Shutao

    2014-06-01

    Activated tumor-associated fibroblasts (TAFs) with abundant fibroblast activation protein (FAP) expression attract tremendous attention in tumor progression studies. In this work, we report a rapid 24 h FAP activation method for fibroblasts using silicon nanowires (SiNWs) as culture substrates instead of growth factors or chemokines. In contrast with cells cultured on flat silicon which rarely express FAP, SiNW cultivated cells exhibit FAP levels similar to those found in cancerous tissue. We demonstrated that activated cells grown on SiNWs maintain their viability and proliferation in a time-dependent manner. Moreover, environmental scanning electron microscopy (ESEM) and focused ion beam and scanning electron microscopy (FIB-SEM) analysis clearly revealed that activated cells on SiNWs adapt to the structure of their substrates by filling inter-wire cavities via filopodia in contrast to cells cultured on flat silicon which spread freely. We further illustrated that the expression of FAP was rarely detected in activated cells after being re-cultured in Petri dishes, suggesting that the unique structure of SiNWs may have a certain influence on FAP activation.Activated tumor-associated fibroblasts (TAFs) with abundant fibroblast activation protein (FAP) expression attract tremendous attention in tumor progression studies. In this work, we report a rapid 24 h FAP activation method for fibroblasts using silicon nanowires (SiNWs) as culture substrates instead of growth factors or chemokines. In contrast with cells cultured on flat silicon which rarely express FAP, SiNW cultivated cells exhibit FAP levels similar to those found in cancerous tissue. We demonstrated that activated cells grown on SiNWs maintain their viability and proliferation in a time-dependent manner. Moreover, environmental scanning electron microscopy (ESEM) and focused ion beam and scanning electron microscopy (FIB-SEM) analysis clearly revealed that activated cells on SiNWs adapt to the structure of

  12. Hyaluronan Inhibits Tlr-4-Dependent RANKL Expression in Human Rheumatoid Arthritis Synovial Fibroblasts

    PubMed Central

    Hirabara, Shinya; Ishiguro, Naoki; Kojima, Toshihisa

    2016-01-01

    The Toll-like receptor (TLR) signaling pathway is activated in synovial fibroblast cells in patients with rheumatoid arthritis (RA). The receptor activator of nuclear factor-κB (RANK) and its ligand, RANKL, are key molecules involved in the differentiation of osteoclasts and joint destruction in RA. Hyaluronan (HA) is a major extracellular component and an important immune regulator. In this study, we show that lipopolysaccharide (LPS) stimulation significantly increases RANKL expression via a TLR-4 signaling pathway. We also demonstrate that HA suppresses LPS-induced RANKL expression, which is dependent on CD44, but not intercellular adhesion molecule-1 (ICAM-1). Our study provides evidence for HA-mediated suppression of TLR-4-dependent RANKL expression. This could present an alternative target for the treatment of destructed joint bones and cartilages in RA. PMID:27054952

  13. Transient Gene and miRNA Expression Profile Changes of Confluent Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Lu, Tao; Wong, Michael; Feiveson, Alan; Stodieck, Louis; Karouia, Fathi; Wang, Xiaoyu; Wu, Honglu

    2015-01-01

    Microgravity or an altered gravity environment from the static 1 gravitational constant has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies conducted in space or using simulated microgravity on the ground have focused on the growth or differentiation of the cells. Whether non-dividing cultured cells will sense the presence of microgravity in space has not been specifically addressed. In an experiment conducted on the International Space Station, confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 days for investigations of gene and miRNA (microRNA) expression profile changes in these cells. A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework for tissues, and plays a critical role in wound healing and other functions. Results of the experiment showed that on Day 3, both the flown and ground cells were still proliferating slowly even though they were confluent, as measured by the expression of the protein Ki-67 positive cells, and the cells in space grew slightly faster. Gene and miRNA expression data indicated activation of NF(sub kappa)B (nuclear factor kappa-light-chain-enhancer of activated B cells) and other growth related pathways involving HGF and VEGF in the flown cells. On Day 14 when the cells were mostly non-dividing, the gene and miRNA expression profiles between the flight and ground samples were indistinguishable. Comparison of gene and miRNA expressions in the Day 3 samples in respect to Day 14 revealed that most of the changes observed on Day 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeleton changes by immunohistochemistry staining of the cells with antibodies for alpha-tubulin showed no difference between the flight and ground samples. Results of our study suggest that in true non-dividing human fibroblast cells, microgravity in

  14. Novel Biphasic Role of LipoxinA4 on Expression of Cyclooxygenase-2 in Lipopolysaccharide-Stimulated Lung Fibroblasts

    PubMed Central

    Zheng, Shengxing; Wang, Qian; He, Qian; Song, Xiaorong; Ye, Duyun; Gao, Fang; Jin, Shengwei; Lian, QingQuan

    2011-01-01

    Fibroblasts are important to host defence and immunity, can also as initiators of inflammation as well. As the endogenous “braking signal”, Lipoxins can regulate anti-inflammation and the resolution of inflammation. We investigated the effect of lipoxinA4 on the expression of cyclooxygenase-2 in lipopolysaccharide-stimulated lung fibroblasts. We demonstrated that the expression of cyclooxygenase-2 protein was significantly increased and peaked initially at 6 hours, with a second increase, with maximal levels occurring 24 hours after lipopolysaccharide challenge. ProstaglandinE2 levels also peaked at 6 hours, and prostaglandinD2 levels were increased at both 6 and 24 hours. Exogenous lipoxinA4 inhibited the first peak of cyclooxygenase-2 expression as well as the production of prostaglandinE2 induced by lipopolysaccharide in a dose-dependent manner. In contrast, exogenous lipoxinA4 increased the second peak of cyclooxygenase-2 expression as well as the production of prostaglandinD2 induced by lipopolysaccharide in a dose-dependent manner. LipoxinA4 receptor mRNA expression was markedly stimulated by lipopolysaccharide but inhibited by lipoxinA4. We present evidence for a novel biphasic role of lipoxinA4 on the expression of cyclooxygenase-2 in lipopolysaccharide-stimulated lung fibroblasts, whereby LXA4 has an anti-inflammatory and proresolving activity in lung fibroblasts following LPS stimulation. PMID:21765620

  15. Adiponectin Attenuates Lung Fibroblasts Activation and Pulmonary Fibrosis Induced by Paraquat

    PubMed Central

    He, Ya-rong; Lau, Wayne Bond; Zeng, Zhi; Liang, Zong-an

    2015-01-01

    -dependent manner, via suppression of lung fibroblast activation. Functional AdipoR1 are expressed by human WI-38 lung fibroblasts, suggesting potential future clinical applicability of APN against pulmonary fibrosis. PMID:25945502

  16. Adiponectin attenuates lung fibroblasts activation and pulmonary fibrosis induced by paraquat.

    PubMed

    Yao, Rong; Cao, Yu; He, Ya-rong; Lau, Wayne Bond; Zeng, Zhi; Liang, Zong-an

    2015-01-01

    -dependent manner, via suppression of lung fibroblast activation. Functional AdipoR1 are expressed by human WI-38 lung fibroblasts, suggesting potential future clinical applicability of APN against pulmonary fibrosis. PMID:25945502

  17. Force generation and protease gene expression in organotypic co-cultures of fibroblasts and keratinocytes.

    PubMed

    Wall, Ivan B; Bhadal, Navneet; Broad, Simon; Whawell, Simon A; Mudera, Vivek; Lewis, Mark P

    2009-12-01

    Fibroblast-epithelium interactions are crucial for successful tissue engineering of skin and oral mucosal equivalents. In this study, we assessed early force generation in organotypic fibroblast-epithelium co-cultures, using normal human keratinocytes (NHK) and HPV16-transformed (UP) cells. During the initial 2 h period, organotypic co-cultures containing both epithelial cell types produced significantly more force than fibroblasts alone (p < 0.05). After 2 h, the epithelial contribution became diminished and did not significantly contribute to intrinsic force generation by fibroblasts, and no differences were observed when using UP vs. NHK. We then measured protease gene expression at the end of the experimental period. Distinct differences were evident in protease expression both between NHK-human skin fibroblast (HSF) vs. UP-HSF co-cultures and compared to fibroblasts alone. We conclude that whilst the very early contractile response of fibroblasts is enhanced by the overlying epithelium, this becomes diminished as the fibroblast response becomes predominant and it does contribute to tissue remodelling via regulation of protease expression. PMID:19701934

  18. MicroRNA Expression Profiles in Cultured Human Fibroblasts in Space

    NASA Technical Reports Server (NTRS)

    Wu, Honglu; Lu, Tao; Jeevarajan, John; Rohde, Larry; Zhang, Ye

    2014-01-01

    Microgravity, or an altered gravity environment from the static 1g, has been shown to influence global gene expression patterns and protein levels in living organisms. However, it is unclear how these changes in gene and protein expressions are related to each other or are related to other factors regulating such changes. A different class of RNA, the small non-coding microRNA (miRNA), can have a broad effect on gene expression networks by mainly inhibiting the translation process. Previously, we investigated changes in the expression of miRNA and related genes under simulated microgravity conditions on the ground using the NASA invented bioreactor. In comparison to static 1 g, simulated microgravity altered a number of miRNAs in human lymphoblastoid cells. Pathway analysis with the altered miRNAs and RNA expressions revealed differential involvement of cell communication and catalytic activity, as well as immune response signaling and NGF activation of NF-kB pathways under simulated microgravity condition. The network analysis also identified several projected networks with c- Rel, ETS1 and Ubiquitin C as key factors. In a flight experiment on the International Space Station (ISS), we will investigate the effects of actual spaceflight on miRNA expressions in nondividing human fibroblast cells in mostly G1 phase of the cell cycle. A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework for tissues, and plays a critical role in wound healing and other functions. In addition to miRNA expressions, we will investigate the effects of spaceflight on the cellular response to DNA damages from bleomycin treatment.

  19. Diversity of Interstitial Lung Fibroblasts Is Regulated by Platelet-Derived Growth Factor Receptor α Kinase Activity.

    PubMed

    Green, Jenna; Endale, Mehari; Auer, Herbert; Perl, Anne-Karina T

    2016-04-01

    Epithelial-mesenchymal cell interactions and factors that control normal lung development are key players in lung injury, repair, and fibrosis. A number of studies have investigated the roles and sources of epithelial progenitors during lung regeneration; such information, however, is limited in lung fibroblasts. Thus, understanding the origin, phenotype, and roles of fibroblast progenitors in lung development, repair, and regeneration helps address these limitations. Using a combination of platelet-derived growth factor receptor α-green fluorescent protein (PDGFRα-GFP) reporter mice, microarray, real-time polymerase chain reaction, flow cytometry, and immunofluorescence, we characterized two distinct interstitial resident fibroblasts, myo- and matrix fibroblasts, and identified a role for PDGFRα kinase activity in regulating their activation during lung regeneration. Transcriptional profiling of the two populations revealed a myo- and matrix fibroblast gene signature. Differences in proliferation, smooth muscle actin induction, and lipid content in the two subpopulations of PDGFRα-expressing fibroblasts during alveolar regeneration were observed. Although CD140α(+)CD29(+) cells behaved as myofibroblasts, CD140α(+)CD34(+) appeared as matrix and/or lipofibroblasts. Gain or loss of PDGFRα kinase activity using the inhibitor nilotinib and a dominant-active PDGFRα-D842V mutation revealed that PDGFRα was important for matrix fibroblast differentiation. We demonstrated that PDGFRα signaling promotes alveolar septation by regulating fibroblast activation and matrix fibroblast differentiation, whereas myofibroblast differentiation was largely PDGFRα independent. These studies provide evidence for the phenotypic and functional diversity as well as the extent of specificity of interstitial resident fibroblasts differentiation during regeneration after partial pneumonectomy. PMID:26414960

  20. A voltage-activated proton current in human cardiac fibroblasts

    SciTech Connect

    El Chemaly, Antoun; Guinamard, Romain; Demion, Marie; Fares, Nassim; Jebara, Victor; Faivre, Jean-Francois; Bois, Patrick . E-mail: patrick.bois@univ-poitiers.fr

    2006-02-10

    A voltage-activated proton current in human cardiac fibroblasts, measured using the whole-cell recording configuration of the patch-clamp technique, is reported. Increasing the pH of the bathing solution shifted the current activation threshold to more negative potentials and increased both the current amplitude and its rate of activation. Changing the pH gradient by one unit caused a 51 mV shift in the reversal potential of the current, demonstrating a high selectivity for protons of the channel carrying the current. Extracellularly applied Zn{sup 2+} reversibly inhibited the current. Activation of the current contributes to the resting membrane conductance under conditions of intracellular acidosis. It is proposed that this current in cardiac fibroblasts is involved in the regulation of the intracellular pH and the membrane potential under physiological conditions as well as in response to pathological conditions such as ischemia.

  1. Inhibition of cyclooxygenase-2 prevents intra-abdominal adhesions by decreasing activity of peritoneal fibroblasts

    PubMed Central

    Wei, Guangbing; Chen, Xin; Wang, Guanghui; Jia, Pengbo; Xu, Qinhong; Ping, Gaofeng; Wang, Kang; Li, Xuqi

    2015-01-01

    Background Postoperative intra-abdominal adhesions are common complications after abdominal surgery. The exact molecular mechanisms that are responsible for these complications remain unclear, and there are no effective methods for preventing adhesion formation or reformation. The aim of the study reported here was to investigate the preventive effects and underlying potential molecular mechanisms of selective cyclooxygenase-2 (COX-2) inhibitors in a rodent model of postoperative intra-abdominal adhesions. Materials and methods The expression of COX-2 in postoperative intra-abdominal adhe-sions and normal peritoneal tissue was examined by immunohistochemistry and Western blot analysis. Assays were performed to elucidate the effect of COX-2 inhibition on hypoxia-induced fibroblast activity in vitro and on intra-abdominal adhesion formation in vivo. Results Hypoxia-induced COX-2 expression in peritoneal fibroblasts was increased in postoperative intra-abdominal adhesions. Inhibition of COX-2 attenuated the activating effect of hypoxia on normal peritoneal fibroblasts in vitro. Data indicate that selective COX-2 inhibitor prevents in vivo intra-abdominal adhesion by inhibition of basic fibroblast growth factor and transforming growth factor-beta expression, but not through an antiangiogenic mechanism. Furthermore, using selective COX-2 inhibitors to prevent intra-abdominal adhesions did not adversely affect the weight, bowel motility, or healing of intestinal anastomoses in a rat model. Conclusion These results show that hypoxia-induced COX-2 expression in peritoneal fibroblasts is involved in the formation of intra-abdominal adhesions. Inhibition of COX-2 prevents postoperative intra-abdominal adhesions through suppression of inflammatory cytokines. PMID:26109851

  2. Toll-Like Receptors Expressed by Synovial Fibroblasts Perpetuate Th1 and Th17 Cell Responses in Rheumatoid Arthritis

    PubMed Central

    Zheng, Li; Shi, Lianjie; Liu, Hongjiang; Zhang, Xuewu; Zhu, Huaqun; Tang, Sumei; Zhu, Lei; Xu, Liling; Yang, Yuqin; Li, Zhanguo

    2014-01-01

    Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial fibroblast hyperplasia and bone and cartilage erosion. Synovial fibroblast- and T cell-mediated inflammation plays crucial roles in the pathogenesis of RA. However how this inflammation is initiated, propagated, and maintained remains controversial. Here, we systemically examined the contribution of toll-like receptors (TLRs) to the inflammatory mediator production as well as Th1 and Th17 cell hyperactivity in RA. Our results show that rheumatoid arthritis synovial fibroblasts (RASF) express a series of TLRs, including TLR2, TLR3, TLR4, and TLR9, with the predominant expression of TLR3. Moreover, the expression levels of these TLRs were higher than those in osteoarthritis synovial fibroblasts (OASF). Ligation of TLR3, as well as TLR2 and TLR4, resulted in vigorous production of inflammatory cytokines, matrix metalloproteinases (MMPs), and vascular endothelial growth factor (VEGF) in RASF, with activation of the NF-κB, MAPK, and IRF3 pathways. More important, activation of these TLRs expressed by RASF exacerbated inflammatory Th1 and Th17 cell expansion both in cell-cell contact-dependent and inflammatory cytokine-dependent manners, which induced more IFN-γ and IL-17 accumulation. Targeting TLRs may modulate the inflammation in RA and provide new therapeutic strategies for overcoming this persistent disease. PMID:24936783

  3. Interferon Regulatory Factor 4 Contributes to Transformation of v-Rel-Expressing Fibroblasts

    PubMed Central

    Hrdličková, Radmila; Nehyba, Jiří; Bose, Henry R.

    2001-01-01

    The avian homologue of the interferon regulatory factor 4 (IRF-4) and a novel splice variant lacking exon 6, IRF-4ΔE6, were isolated and characterized. Chicken IRF-4 is expressed in lymphoid organs, less in small intestine, and lungs. IRF-4ΔE6 mRNA, though less abundant than full-length IRF-4, was detected in lymphoid tissues, with the highest levels observed in thymic cells. IRF-4 is highly expressed in v-Rel-transformed lymphocytes, and the expression of IRF-4 is increased in v-Rel- and c-Rel-transformed fibroblasts relative to control cells. The expression of IRF-4 from retrovirus vectors morphologically transformed primary fibroblasts, increased their saturation density, proliferation, and life span, and promoted their growth in soft agar. IRF-4 and v-Rel cooperated synergistically to transform fibroblasts. The expression of IRF-4 antisense RNA eliminated formation of soft agar colonies by v-Rel and reduced the proliferation of v-Rel-transformed cells. v-Rel-transformed fibroblasts produced interferon 1 (IFN1), which inhibits fibroblast proliferation. Infection of fibroblasts with retroviruses expressing v-Rel resulted in an increase in the mRNA levels of IFN1, the IFN receptor, STAT1, JAK1, and 2′,5′-oligo(A) synthetase. The exogenous expression of IRF-4 in v-Rel-transformed fibroblasts decreased the production of IFN1 and suppressed the expression of several genes in the IFN transduction pathway. These results suggest that induction of IRF-4 expression by v-Rel likely facilitates transformation of fibroblasts by decreasing the induction of this antiproliferative pathway. PMID:11533227

  4. Electrical Stimulation Modulates the Expression of Multiple Wound Healing Genes in Primary Human Dermal Fibroblasts.

    PubMed

    Park, Hyun Jin; Rouabhia, Mahmoud; Lavertu, Denis; Zhang, Ze

    2015-07-01

    This study profiled multiple human dermal fibroblast wound-healing genes in response to electrical stimulation (ES) by using an RT(2) profiler PCR-Array system. Primary human skin fibroblasts were seeded on heparin (HE)-bioactivated polypyrrole (PPy)/poly(l-lactic acid) (PLLA) conductive membranes, cultured, and subsequently exposed to ES of 50 or 200 mV/mm for 6 h. Following ES, the cells were used to extract RNA for gene profiling, and culture supernatants were used to measure the level of the different wound healing mediators. A total of 57 genes were affected (activated/repressed) by ES; among these, 49 were upregulated and 8 were downregulated. ES intensities at 50 and 200 mV/mm activated/repressed different genes. The ES-modulated genes are involved in cell adhesion, remodeling and spreading, cytoskeletal activity, extracellular matrix metabolism, production of inflammatory cytokines/chemokines and growth factors, as well as signal transduction. The expression of several genes was supported by protein production. Protein analyses showed that ES increased CCL7, KGF, and TIMP2, but reduced MMP2. This study demonstrated that ES modulates the expression of a variety of genes involved in the wound healing process, confirming that ES is a useful tool in regenerative medicine. PMID:25873313

  5. METHYL METHANESULFONATE-INDUCED GENE EXPRESSION CHANGES IN HUMAN SKIN FIBROBLASTS

    EPA Science Inventory

    METHYL METHANESULFONATE-INDUCED GENE EXPRESSION CHANGES IN HUMAN SKIN FIBROBLASTS. Geremy W. Knapp, Alan Tennant, and Russell D. Owen. Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, U. S. Environmental Protection Agency, Re...

  6. AGE-RELATED GENE EXPRESSION CHANGES IN HUMAN SKIN FIBROBLASTS INDUCED BY MMS

    EPA Science Inventory

    Age-Related Gene Expression Changes In Human Skin Fibroblasts Induced By methyl methanesulfonate. Geremy W. Knapp, Alan H. Tennant, and Russell D. Owen. Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, U. S. Environmental Prote...

  7. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts

    SciTech Connect

    Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath; Zeichner-David, Margarita; Reyes-Gasga, Jose; Molina-Guarneros, Juan; Garcia-Hernandez, Ana Lilia; Suarez-Franco, Jose Luis; Chavarria, Ivet Gil; Villarreal-Ramirez, Eduardo; Arzate, Higinio . E-mail: harzate@servidor.unam.mx

    2007-07-06

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

  8. Activation of Sirtuin-1 Promotes Renal Fibroblast Activation and Aggravates Renal Fibrogenesis.

    PubMed

    Ponnusamy, Murugavel; Zhuang, Michelle A; Zhou, Xiaoxu; Tolbert, Evelyn; Bayliss, George; Zhao, Ting C; Zhuang, Shougang

    2015-08-01

    Although activation of sirtuin-1 (SIRT1) has been shown to protect the kidney from acute injury, its role in renal fibrosis remains controversial since both inhibition and activation of SIRT1 have been reported to attenuate renal fibrosis. To resolve this conflict, we further examined the effect of SIRT1 activators on the activation of renal interstitial fibroblasts and development of renal fibrosis in vivo and in vitro. In a murine model of renal fibrosis induced by unilateral ureteral obstruction, administration of SRT1720 (N-[2-[3-(piperazin-1-ylmethyl)imidazo[2,1-b][1,3]thiazol-6-yl]phenyl]quinoxaline-2-carboxamide), a potent activator of SIRT1, accelerated deposition of collagen fibrils and increased expression of fibroblast activation markers (α-smooth muscle actin [α-SMA], collagen I, and fibronectin) in the obstructive kidney of mice. In cultured rat renal interstitial fibroblasts (NRK-49F), exposure of cells to SRT1720 or YK-3-237 (B-[2-methoxy-5-[(1E)-3-oxo-3-(3,4,5-trimethoxyphenyl)-1-propen-1-yl]phenyl]-boronic acid), another SIRT1 activator, also resulted in enhanced expression of α-SMA and fibronectin. Mechanistic studies showed that augmentation of renal fibrogenesis by SRT1720 is associated with elevated phosphorylation of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor β (PDGFRβ). SRT1720 treatment also increased the phosphorylation of signal transducer and activator of transcription 3 and protein kinase B in the fibrotic kidney and NRK-49F cells. However, SRT1720 treatment did not affect expression of proliferating cell nuclear protein, a proliferation marker and activation of extracellular signal regulated kinase 1/2 in vitro and in vivo. These results indicate that SIRT1-activating compounds can provoke renal fibrogenesis through a mechanism involved in the activation of EGFR and PDGFR signaling pathways and suggest that long-term use of SIRT1 activators risks the development and progression of chronic

  9. Expression of TGF-β3 in isolated fibroblasts from foreskin

    PubMed Central

    Mahmoudi Rad, Mahnaz; Mahmoudi Rad, Niki; Mirdamadi, Yasaman

    2015-01-01

    Background: The multifunctional transforming growth factor beta (TGF-β) is a glycoprotein that exists in three isoforms. TGF-β3 expression increases in fetal wound healing and reduces fibronectin and collagen I and III deposition, and also improves the architecture of the neodermis which is a combination of blood vessels and connective tissue during wound healing. Fibroblasts are key cells in the wound healing process. TGF-β3 plays a critical role in scar-free wound healing and fibroblast actions in the wound healing process. The aim of this study was to express the TGF-β3 gene (tgf-b3) in human foreskin fibroblasts (HFF’s). Methods: We obtained HFF’s from a newborn and a primary fibroblast culture was prepared. The cells were transfected with TGF-β3-pCMV6-XL5 plasmid DNA by both lipofection and electroporation. Expression of TGF-β3 was measured by enzyme-linked immunosorbent assay (ELISA). Results: The highest TGF-β3 expression (8.3-fold greater than control) was obtained by lipofection after 72 hours using 3 µl of transfection reagent. Expression was 1.4-fold greater than control by electroporation. Conclusions: In this study, we successfully increased TGF-β3 expression in primary fibroblast cells. In the future, grafting these transfected fibroblasts onto wounds can help the healing process without scarring. PMID:26989741

  10. IL-27 enhances innate immunity of human pulmonary fibroblasts and epithelial cells through upregulation of TLR4 expression.

    PubMed

    Su, Yufeng; Yao, Hua; Wang, Hong; Xu, Fang; Li, Dagen; Li, Dairong; Zhang, Xuemei; Yin, Yibing; Cao, Ju

    2016-01-15

    Lung tissue cells play an active role in the pathogenesis of pulmonary inflammatory diseases by releasing a variety of cytokines and chemokines. However, how lung tissue cells respond to microbial stimuli during pulmonary infections remains unclear. In this study, we found that patients with community-acquired pneumonia displayed increased IL-27 levels in bronchoalveolar lavage fluid and serum. We subsequently examined the immunopathological mechanisms for the activation of primary human lung fibroblasts and bronchial epithelial cells by IL-27. We demonstrated that IL-27 priming enhanced LPS-induced production of IL-6 and IL-8 from lung fibroblasts and bronchial epithelia cells via upregulating Toll-like receptor-4 (TLR4) expression. IL-27 upregulated TLR4 expression in lung fibroblasts through activation of Janus-activated kinase (JAK) and Jun NH2-terminal kinase (JNK) signaling pathways, and inhibition of the JAK pathway could partially decrease IL-27-induced TLR4 expression, while inhibition of JNK pathway could completely suppress IL-27-induced TLR4 expression. Our data suggest that IL-27 modulates innate immunity of lung tissue cells through upregulating TLR4 expression during pulmonary infections. PMID:26608531

  11. Thyroid Hormone Regulates Hepatic Expression of Fibroblast Growth Factor 21 in a PPARα-dependent Manner*

    PubMed Central

    Adams, Andrew C.; Astapova, Inna; Fisher, ffolliott M.; Badman, Michael K.; Kurgansky, Katherine E.; Flier, Jeffrey S.; Hollenberg, Anthony N.; Maratos-Flier, Eleftheria

    2010-01-01

    Thyroid hormone has profound and diverse effects on liver metabolism. Here we show that tri-iodothyronine (T3) treatment in mice acutely and specifically induces hepatic expression of the metabolic regulator fibroblast growth factor 21 (FGF21). Mice treated with T3 showed a dose-dependent increase in hepatic FGF21 expression with significant induction at doses as low as 100 μg/kg. Time course studies determined that induction is seen as early as 4 h after treatment with a further increase in expression at 6 h after injection. As FGF21 expression is downstream of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα), we treated PPARα knock-out mice with T3 and found no increase in expression, indicating that hepatic regulation of FGF21 by T3 in liver is via a PPARα-dependent mechanism. In contrast, in white adipose tissue, FGF21 expression was suppressed by T3 treatment, with other T3 targets unaffected. In cell culture studies with an FGF21 reporter construct, we determined that three transcription factors are required for induction of FGF21 expression: thyroid hormone receptor β (TRβ), retinoid X receptor (RXR), and PPARα. These findings indicate a novel regulatory pathway whereby T3 positively regulates hepatic FGF21 expression, presenting a novel therapeutic target for diseases such as non-alcoholic fatty liver disease. PMID:20236931

  12. Eicosapentaenoic acid inhibits UV-induced MMP-1 expression in human dermal fibroblasts.

    PubMed

    Kim, Hyeon Ho; Shin, Chung Min; Park, Chi-Hyun; Kim, Kyu Han; Cho, Kwang Hyun; Eun, Hee Chul; Chung, Jin Ho

    2005-08-01

    Ultraviolet (UV) irradiation regulates UV-responsive genes, including matrix metalloproteinases (MMPs). Moreover, UV-induced MMPs cause connective tissue damage and the skin to become wrinkled and aged. Here, we investigated the effect of eicosapentaenoic acid (EPA), a dietary omega-3 fatty acid, on UV-induced MMP-1 expression in human dermal fibroblasts (HDFs). We found that UV radiation increases MMP-1 expression and that this is mediated by p44 and p42 MAP kinase (ERK) and Jun-N-terminal kinase (JNK) activation but not by p38 activation. Pretreatment of HDFs with EPA inhibited UV-induced MMP-1 expression in a dose-dependent manner and also inhibited the UV-induced activation of ERK and JNK by inhibiting ERK kinase (MEK1) and SAPK/ERK kinase 1 (SEK1) activation, respectively. Moreover, inhibition of ERK and JNK by EPA resulted in the decrease of c-Fos expression and c-Jun phosphorylation/expression induced by UV, respectively, which led to the inhibition of UV-induced activator protein-1 DNA binding activity. This inhibitory effect of EPA on MMP-1 was not mediated by an antioxidant effect. We also found that EPA inhibited 12-O-tetradecanoylphorbol-13-acetate- or tumor necrosis factor-alpha-induced MMP-1 expression in HDFs and UV-induced MMP-1 expression in HaCaT cells. In conclusion, our results demonstrate that EPA can inhibit UV-induced MMP-1 expression by inhibiting the MEK1/ERK/c-Fos and SEK1/JNK/c-Jun pathways. Therefore, EPA is a potential agent for the prevention and treatment of skin aging. PMID:15930517

  13. Transient Gene and MicroRNA Expression Profile Changes of Confluent Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Lu, Tao; Wong, Michael; Wang, Xiaoyu; Stodieck, Louis; Karouia, Fathi; Story, Michael; Wu, Honglu

    2016-01-01

    Microgravity, or an altered gravity environment from the Earth1g, has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies conducted in space or using simulated microgravity on the ground have focused on the growth or differentiation of these cells. Whether non-proliferating cultured cells will sense the presence of microgravity in space has not been specifically addressed. In an experiment conducted onboard the International Space Station (ISS), confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 days, respectively, for investigations of gene and miRNA expression profile changes in these cells. Results of the experiment showed that on Day 3, both the flown and ground cells were still proliferating slowly, as measured by the percentage of Ki-67 positive cells. Gene and miRNA expression data indicated activation of NF(kappa)B and other growth related pathways involving HGF and Vegf along with down regulation of the Let-7 miRNA family. On Day 14 when the cells were mostly non-proliferating, the gene and miRNA expression profiles between the flight and ground samples were indistinguishable. Comparison of gene and miRNA expressions in the Day 3 samples with respect to Day 14 revealed that most of the changes observed on Day 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeletal changes via immunohistochemistry staining of the cells with antibodies for alpha-tubulin and fibronectin showed no difference between flown and ground samples. Taken together, our study suggests that in true non-dividing human fibroblast cells in culture, microgravity experienced in space has little effect on the gene and miRNA expression profiles.

  14. Transient gene and microRNA expression profile changes of confluent human fibroblast cells in space

    NASA Astrophysics Data System (ADS)

    Wu, Honglu; Story, Michael; Karouia, Fathi; Stodieck, Louis; Zhang, Ye; Lu, Tao

    2016-07-01

    Microgravity, or an altered gravity environment from the Earth1g, has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies conducted in space or using simulated microgravity on the ground have focused on the growth or differentiation of these cells. Whether non-proliferating cultured cells will sense the presence of microgravity in space has not been specifically addressed. In an experiment conducted onboard the International Space Station (ISS), confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 days, respectively, for investigations of gene and miRNA expression profile changes in these cells. Results of the experiment showed that on Day 3, both the flown and ground cells were still proliferating slowly, as measured by the percentage of Ki-67 positive cells. Gene and miRNA expression data indicated activation of NFkB and other growth related pathways involving HGF and Vegf along with down regulation of the Let-7 miRNA family. On Day 14 when the cells were mostly non-proliferating, the gene and miRNA expression profiles between the flight and ground samples were indistinguishable. Comparison of gene and miRNA expressions in the Day 3 samples with respect to Day 14 revealed that most of the changes observed on Day 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeletal changes via immunohistochemistry staining of the cells with antibodies for αa-tubulin and fibronectin showed no difference between flown and ground samples. Taken together, our study suggests that in true non-dividing human fibroblast cells in culture, microgravity experienced in space has little effect on the gene and miRNA expression profiles.

  15. Alteration of Connective Tissue Growth Factor (CTGF) Expression in Orbital Fibroblasts from Patients with Graves’ Ophthalmopathy

    PubMed Central

    Chang, Pei-Chen; Wei, Yau-Huei

    2015-01-01

    Graves’ ophthalmopathy (GO) is a disfiguring and sometimes blinding disease, which is characterized by inflammation and swelling of orbital tissues, with fibrosis and adipogenesis being predominant features. The aim of this study is to investigate whether the expression levels of fibrosis-related genes, especially that of connective tissue growth factor (CTGF), are altered in orbital fibroblasts of patients with GO. The role of oxidative stress in the regulation of CTGF expression in GO orbital fibroblasts is also examined. By a SYBR Green-based real time quantitative PCR (RT-QPCR), we demonstrated that the mRNA expression levels of fibronectin, apolipoprotein J, and CTGF in cultured orbital fibroblasts from patients with GO were significantly higher than those of age-matched normal controls (p = 0.007, 0.037, and 0.002, respectively). In addition, the protein expression levels of fibronectin, apolipoprotein J, and CTGF analyzed by Western blot were also significantly higher in GO orbital fibroblasts (p = 0.046, 0.032, and 0.008, respectively) as compared with the control. Furthermore, after treatment of orbital fibroblasts with a sub-lethal dose of hydrogen peroxide (200 μM H2O2), we found that the H2O2-induced increase of CTGF expression was more pronounced in the GO orbital fibroblasts as compared with those in normal controls (20% vs. 7%, p = 0.007). Importantly, pre-incubation with antioxidants including N-acetylcysteine (NAC) and vitamin C, respectively, resulted in significant attenuation of the induction of CTGF in GO orbital fibroblasts in response to H2O2 (p = 0.004 and 0.015, respectively). Taken together, we suggest that oxidative stress plays a role in the alteration of the expression of CTGF in GO orbital fibroblasts that may contribute to the pathogenesis and progression of GO. Antioxidants may be used in combination with the therapeutic agents for effective treatment of GO. PMID:26599235

  16. Transient gene and microRNA expression profile changes of confluent human fibroblast cells in spaceflight.

    PubMed

    Zhang, Ye; Lu, Tao; Wong, Michael; Wang, Xiaoyu; Stodieck, Louis; Karouia, Fathi; Story, Michael; Wu, Honglu

    2016-06-01

    Microgravity, or an altered gravity environment different from the 1 g of the Earth, has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies that have been conducted in space or by using simulated microgravity on the ground have focused on the growth or differentiation of these cells. It has not been specifically addressed whether nonproliferating cultured cells will sense the presence of microgravity in space. In an experiment conducted onboard the International Space Station, confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 d, respectively, to investigate changes in gene and microRNA (miRNA) expression profiles in these cells. Results of the experiment showed that on d 3, both the flown and ground cells were still proliferating slowly, as measured by the percentage of Ki-67(+) cells. Gene and miRNA expression data indicated activation of NF-κB and other growth-related pathways that involve hepatocyte growth factor and VEGF as well as the down-regulation of the Let-7 miRNA family. On d 14, when the cells were mostly nonproliferating, the gene and miRNA expression profile of the flight sample was indistinguishable from that of the ground sample. Comparison of gene and miRNA expressions in the d 3 samples, with respect to d 14, revealed that most of the changes observed on d 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeletal changes via immunohistochemistry staining of the cells with antibodies for α-tubulin and fibronectin showed no difference between the flown and ground samples. Taken together, our study suggests that in true nondividing human fibroblast cells in culture, microgravity experienced in space has little effect on gene and miRNA expression profiles.-Zhang, Y., Lu, T., Wong, M., Wang, X., Stodieck, L., Karouia, F., Story, M., Wu, H. Transient gene and microRNA expression profile changes of

  17. Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts

    SciTech Connect

    Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith , E-Mail: Judith.hall@ncl.ac.uk

    2005-09-16

    In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of the TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian.

  18. Smad ubiquitination regulatory factor 2 expression is enhanced in hypertrophic scar fibroblasts from burned children

    PubMed Central

    Finnerty, Celeste C; He, Jing; Herndon, David N

    2013-01-01

    Transforming growth factor-β1 (TGF-β1) plays a key role in hypertrophic scar formation. A lot of studies have shown that TGF-β1 stimulates fibroblast proliferation, collagen production, and α-smooth muscle actin (α-SMA) expression, inhibits matrix degradation and eventually leads to scar formation. Smad proteins are important intracellular mediators of TGF-β1 signaling, and Smad ubiquitination regulatory factor 2 (Smurf2), an ubiquitin ligase for Smads, plays critical roles in the regulation of TGF-β1/Smad signaling. It was reported that Smurf2 was abnormally expressed during the process of liver fibrosis and lung fibrosis. Hypertrophic scarring is a fibroproliferative disorder of the dermis that occurs following wounding. However, little is known about the expression of Smurf2 in hypertrophic scarring. We hypothesized that TGF-β1 signaling cannot be disrupted after wound epithelialization probably due to abnormal expression of Smurf2 in hypertrophic scar fibroblasts. In the present study, we found that hypertrophic scar fibroblasts exhibited increased Smurf2 protein and mRNA levels compared with normal fibroblasts, and the expression of Smurf2 gradually increased in hypertrophic scar fibroblasts after TGF-β1 stimulation. Furthermore, we transfected Smurf2 siRNA into hypertrophic scar fibroblasts, and we found that silencing the expression of Smurf2 in hypertrophic scar fibroblasts dramatically reduced TGF-β1 production, inhibited TGF-β1-induced α-SMA expression and inhibited TGF-β1-induced collagen I synthesis. Our results suggest that the enhanced expression of Smurf2 is involved in the progression of hypertrophic scarring. PMID:21920670

  19. Hydroxycamptothecin-induced apoptotic gene expression profiling by PCR array in human Tenon's capsule fibroblasts.

    PubMed

    Tang, Wei; Zhang, Yinong; Zhang, Qing; Wang, Qinghua; Wu, Zhifeng

    2015-01-01

    Studies have indicated that hydroxycamptothecin (HCPT) induces apoptosis of fibroblasts. In this study, we investigated the apoptotic gene expression profiling in HCPT-treated -human Tenon's capsule fibroblasts (HTCFs) and identify the most implicated gene in apoptotic signaling of HTCFs by HCPT. Method HTCFs were incubated with HCPT at 0, 0.25 and 4 mg/L for 24, 48 and 72 h, respectively. Anti-proliferative effects were measured by MTT assay. Apoptosis was determined using the Annexin V-FITC/PI apoptosis detection kit and apoptotic cells were identified by flow cytometry. A PCR array was employed to analyze the most implicated apoptotic genes during HCPT-induced apoptosis in HTCFs. Results from our studies showed that HCPT induced apoptosis in HTCFs in a concentration-and time-dependent manner. The apoptotic HTCFs was increased by 9.38% with a multiplicity at 4 mg/L HCPT. PCR array demonstrated remarkable changes in 88 apoptotic genes, including 9 up-regulated genes and 36 down-regulated genes. HCPT treatment induced the upregulation of CHOP and downregulation of XIAP in HTCFs. To conclude, our results support HCPT induced the apoptosis of HTCFs, involving the activation of mitochondrial and endoplasmic reticulum stresses as well as the downregulation expression of XIAP. PMID:26045770

  20. Cleavage of Type I Collagen by Fibroblast Activation Protein-α Enhances Class A Scavenger Receptor Mediated Macrophage Adhesion

    PubMed Central

    Mazur, Anna; Holthoff, Emily; Vadali, Shanthi; Kelly, Thomas; Post, Steven R.

    2016-01-01

    Pathophysiological conditions such as fibrosis, inflammation, and tumor progression are associated with modification of the extracellular matrix (ECM). These modifications create ligands that differentially interact with cells to promote responses that drive pathological processes. Within the tumor stroma, fibroblasts are activated and increase the expression of type I collagen. In addition, activated fibroblasts specifically express fibroblast activation protein-α (FAP), a post-prolyl peptidase. Although FAP reportedly cleaves type I collagen and contributes to tumor progression, the specific pathophysiologic role of FAP is not clear. In this study, the possibility that FAP-mediated cleavage of type I collagen modulates macrophage interaction with collagen was examined using macrophage adhesion assays. Our results demonstrate that FAP selectively cleaves type I collagen resulting in increased macrophage adhesion. Increased macrophage adhesion to FAP-cleaved collagen was not affected by inhibiting integrin-mediated interactions, but was abolished in macrophages lacking the class A scavenger receptor (SR-A/CD204). Further, SR-A expressing macrophages localize with activated fibroblasts in breast tumors of MMTV-PyMT mice. Together, these results demonstrate that FAP-cleaved collagen is a substrate for SR-A-dependent macrophage adhesion, and suggest that by modifying the ECM, FAP plays a novel role in mediating communication between activated fibroblasts and macrophages. PMID:26934296

  1. Tumour cell-derived Wnt7a recruits and activates fibroblasts to promote tumour aggressiveness

    PubMed Central

    Avgustinova, Alexandra; Iravani, Marjan; Robertson, David; Fearns, Antony; Gao, Qiong; Klingbeil, Pamela; Hanby, Andrew M.; Speirs, Valerie; Sahai, Erik; Calvo, Fernando; Isacke, Clare M.

    2016-01-01

    Stromal fibroblast recruitment to tumours and activation to a cancer-associated fibroblast (CAF) phenotype has been implicated in promoting primary tumour growth and progression to metastatic disease. However, the mechanisms underlying the tumour:fibroblast crosstalk that drive the intertumoural stromal heterogeneity remain poorly understood. Using in vivo models we identify Wnt7a as a key factor secreted exclusively by aggressive breast tumour cells, which induces CAF conversion. Functionally, this results in extracellular matrix remodelling to create a permissive environment for tumour cell invasion and promotion of distant metastasis. Mechanistically, Wnt7a-mediated fibroblast activation is not dependent on classical Wnt signalling. Instead, we demonstrate that Wnt7a potentiates TGFβ receptor signalling both in 3D in vitro and in vivo models, thus highlighting the interaction between two of the key signalling pathways in development and disease. Importantly, in clinical breast cancer cohorts, tumour cell Wnt7a expression correlates with a desmoplastic, poor-prognosis stroma and poor patient outcome. PMID:26777421

  2. Low α2β1 Integrin Function Enhances the Proliferation of Fibroblasts from Patients with Idiopathic Pulmonary Fibrosis by Activation of the β-Catenin Pathway

    PubMed Central

    Xia, Hong; Seeman, Jeremy; Hong, Jian; Hergert, Polla; Bodem, Vidya; Jessurun, Jose; Smith, Karen; Nho, Richard; Kahm, Judy; Gaillard, Philippe; Henke, Craig

    2013-01-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive and incurable fibroproliferative disorder characterized by unrelenting proliferation of fibroblasts and their deposition of collagen within alveoli, resulting in permanently scarred, nonfunctional airspaces. Normally, polymerized collagen suppresses fibroblast proliferation and serves as a physiological restraint to limit fibroproliferation after tissue injury. The IPF fibroblast, however, is a pathologically altered cell that has acquired the capacity to elude the proliferation-suppressive effects of polymerized collagen. The mechanism for this phenomenon remains incompletely understood. Here, we demonstrate that expression of α2β1 integrin, a major collagen receptor, is pathologically low in IPF fibroblasts interacting with polymerized collagen. Low integrin expression in IPF fibroblasts is associated with a failure to induce PP2A phosphatase activity, resulting in abnormally high levels of phosphorylated (inactive) GSK-3β and high levels of active β-catenin in the nucleus. Knockdown of β-catenin in IPF fibroblasts inhibits their ability to proliferate on collagen. Interdiction of α2β1 integrin in control fibroblasts reproduces the IPF phenotype and leads to the inability of these cells to activate PP2A, resulting in high levels of phosphorylated GSK-3β and active β-catenin and in enhanced proliferation on collagen. Our findings indicate that the IPF fibroblast phenotype is characterized by low α2β1 integrin expression, resulting in a failure of integrin to activate PP2A phosphatase, which permits inappropriate activation of the β-catenin pathway. PMID:22642910

  3. TGF-β1–Containing Exosomes from Injured Epithelial Cells Activate Fibroblasts to Initiate Tissue Regenerative Responses and Fibrosis

    PubMed Central

    Borges, Fernanda T.; Melo, Sonia A.; Özdemir, Berna C.; Kato, Noritoshi; Revuelta, Ignacio; Miller, Caroline A.; Gattone, Vincent H.; LeBleu, Valerie S.

    2013-01-01

    Hypoxia is associated with tissue injury and fibrosis but its functional role in fibroblast activation and tissue repair/regeneration is unknown. Using kidney injury as a model system, we demonstrate that injured epithelial cells produce an increased number of exosomes with defined genetic information to activate fibroblasts. Exosomes released by injured epithelial cells promote proliferation, α-smooth muscle actin expression, F-actin expression, and type I collagen production in fibroblasts. Fibroblast activation is dependent on exosomes delivering TGF-β1 mRNA among other yet to be identified moieties. This study suggests that TGF-β1 mRNA transported by exosomes constitutes a rapid response to initiate tissue repair/regenerative responses and activation of fibroblasts when resident parenchyma is injured. The results also inform potential utility of exosome-targeted therapies to control tissue fibrosis. PMID:23274427

  4. Type 1 neurofibromatosis: selective expression of extracellular matrix genes by Schwann cells, perineurial cells, and fibroblasts in mixed cultures.

    PubMed Central

    Jaakkola, S; Peltonen, J; Riccardi, V; Chu, M L; Uitto, J

    1989-01-01

    Cutaneous neurofibromas, characteristic lesions of neurofibromatosis 1, are composed of an abundant extracellular matrix and nerve connective tissue-derived cell types: Schwann cells, perineurial cells, and fibroblasts. In this study, the extracellular matrix gene expression by these cells was examined under culture conditions that allowed them to be metabolically active and readily identifiable by morphologic and immunocytochemical criteria. Northern hybridizations demonstrated expression of genes for type I, III, IV, and VI collagens, as well as for fibronectin, laminin, and elastin. In situ hybridizations revealed that all three cell types expressed pro alpha 1 (I), pro alpha 2 (VI), and laminin B1 chain genes. However, fibroblasts did not contain [35S]cDNA-mRNA hybrids specific for type IV collagen, whereas both Schwann cells and perineurial cells expressed these genes. Perineurial cells and fibroblasts readily expressed the fibronectin gene whereas Schwann cells were essentially devoid of the corresponding mRNA. Perineurial cells also expressed the gene for laminin A chain. The results indicate that the extracellular matrix gene expression profiles of Schwann cells, perineurial cells, and fibroblasts are distinct: all three cell types are capable of expressing some of the genes for extracellular matrix components, such as type I and VI collagens, whereas Schwann cells and perineurial cells may have the primary role in synthesizing basement membrane zone components, type IV collagen and laminin. These observations potentially relate to the mechanisms of growth and development of human neurofibromas. The results attest to the applicability of the methodology utilized here to study other human tumors with mixed cell populations. Images PMID:2500456

  5. Disrupted TSH Receptor Expression in Female Mouse Lung Fibroblasts Alters Subcellular IGF-1 Receptor Distribution.

    PubMed

    Atkins, Stephen J; Lentz, Stephen I; Fernando, Roshini; Smith, Terry J

    2015-12-01

    A relationship between the actions of TSH and IGF-1 was first recognized several decades ago. The close physical and functional associations between their respective receptors (TSHR and IGF-1R) has been described more recently in thyroid epithelium and human orbital fibroblasts as has the noncanonical behavior of IGF-1R. Here we report studies conducted in lung fibroblasts from female wild-type C57/B6 (TSHR(+/+)) mice and their littermates in which TSHR has been knocked out (TSHR(-/-)). Flow cytometric analysis revealed that cell surface IGF-1R levels are substantially lower in TSHR(-/-) fibroblasts compared with TSHR(+/+) fibroblasts. Confocal immunofluorescence microscopy revealed similar divergence with regard to both cytoplasmic and nuclear IGF-1R. Western blot analysis demonstrated both intact IGF-1R and receptor fragments in both cellular compartments. In contrast, IGF-1R mRNA levels were similar in fibroblasts from mice without and with intact TSHR expression. IGF-1 treatment of TSHR(+/+) fibroblasts resulted in reduced nuclear and cytoplasmic staining for IGF-1Rα, whereas it enhanced the nuclear signal in TSHR(-/-) cells. In contrast, IGF-1 enhanced cytoplasmic IGF-1Rβ in TSHR(-/-) fibroblasts while increasing the nuclear signal in TSHR(+/+) cells. These findings indicate the intimate relationship between TSHR and IGF-1R found earlier in human orbital fibroblasts also exists in mouse lung fibroblasts. Furthermore, the presence of TSHR in these fibroblasts influenced not only the levels of IGF-1R protein but also its subcellular distribution and response to IGF-1. They suggest that the mouse might serve as a suitable model for delineating the molecular mechanisms overarching these two receptors. PMID:26389690

  6. PGE2 Reduces MMP-14 and Increases Plasminogen Activator Inhibitor-1 in Cardiac Fibroblasts

    PubMed Central

    Kassem, Kamal M.; Clevenger, Margarette H.; Szandzik, David L.; Peterson, Edward; Harding, Pamela

    2014-01-01

    Prostaglandin E2 (PGE2) is elevated during cardiac injury and we have previously shown that mice lacking the PGE EP4 receptor display dilated cardiomyopathy (DCM) with increased expression of the membrane type matrix metalloproteinase, MMP-14. We thus hypothesized that PGE2 regulates expression of MMP-14 and also affects fibroblast migration. Primary cultures of neonatal rat ventricular fibroblasts (NVFs) were used to test the effects of PGE2. Gene and protein expression was assessed by real time RT-PCR and Western blot, MMP activity was determined by zymography and migration of NVF was assessed by motility in a transwell system. PGE2 reduced expression of MMP-14 and these effects were antagonized by an EP4 antagonist. An EP4 agonist mimicked the effect of PGE2. PGE2 also increased mRNA and protein levels of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of MMP activation. However, PGE2-stimulation of PAI-1 was mediated by the EP1/EP3 receptor and not EP4. Migration of NVF was assessed by motility in a transwell system. Treatment of NVFs with PGE2 reduced the number of cells migrating towards 10% FCS. Treatment with the EP2 agonist also reduced migration but did not affect MMP-14 expression or PAI-1. Our results suggest that PGE2 utilizes different receptors and mechanisms to ultimately decrease MMP expression and NVF migration. PMID:25263346

  7. PGE2 reduces MMP-14 and increases plasminogen activator inhibitor-1 in cardiac fibroblasts.

    PubMed

    Kassem, Kamal M; Clevenger, Margarette H; Szandzik, David L; Peterson, Edward; Harding, Pamela

    2014-10-01

    Prostaglandin E2 (PGE2) is elevated during cardiac injury and we have previously shown that mice lacking the PGE2 EP4 receptor display dilated cardiomyopathy (DCM) with increased expression of the membrane type matrix metalloproteinase, MMP-14. We thus hypothesized that PGE2 regulates expression of MMP-14 and also affects fibroblast migration. Primary cultures of neonatal rat ventricular fibroblasts (NVFs) were used to test the effects of PGE2. Gene and protein expression was assessed by real time RT-PCR and Western blot, MMP activity was determined by zymography and migration of NVF was assessed by motility in a transwell system. PGE2 reduced expression of MMP-14 and these effects were antagonized by an EP4 antagonist. An EP4 agonist mimicked the effect of PGE2. PGE2 also increased mRNA and protein levels of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of MMP activation. However, PGE2-stimulation of PAI-1 was mediated by the EP1/EP3 receptor and not EP4. Migration of NVF was assessed by motility in a transwell system. Treatment of NVFs with PGE2 reduced the number of cells migrating toward 10% FCS. Treatment with the EP2 agonist also reduced migration but did not affect MMP-14 expression or PAI-1. Our results suggest that PGE2 utilizes different receptors and mechanisms to ultimately decrease MMP expression and NVF migration. PMID:25263346

  8. FGF inhibits neurite outgrowth over monolayers of astrocytes and fibroblasts expressing transfected cell adhesion molecules.

    PubMed

    Williams, E J; Mittal, B; Walsh, F S; Doherty, P

    1995-11-01

    We have cultured cerebellar neurons on monolayers of cortical astrocytes in control medium or medium containing recombinant basic fibroblast growth factor (FGF). FGF was found to inhibit neurite outgrowth, with a significant effect seen at 0.5 ng/ml and a maximal effect at 10 ng/ml. FGF increased the production of arachidonic acid (AA) in cerebellar neurons, and when added directly to cultures or generated endogenously via activation of phospholipase A2 using melittin, this second messenger could mimic the inhibitory effect of FGF. FGF and AA could also specifically inhibit neurite outgrowth stimulated by three cell adhesion molecules (NCAM, N-cadherin and L1) expressed in transfected fibroblasts, or in the case of L1 bound to a tissue culture substratum. These data demonstrate that, in certain cellular contexts, FGF can act as an inhibitory cue for axonal growth and that arachidonic acid is the second messenger responsible for this activity. We discuss the possibility that arachidonic acid inhibits neurite outgrowth by desensitising the second messenger pathway underlying neuronal responsiveness to cell adhesion molecules. PMID:8586663

  9. Therapeutic transdifferentiation of human fibroblasts into endothelial cells using forced expression of lineage-specific transcription factors

    PubMed Central

    Wong, Wing Tak; Cooke, John P

    2016-01-01

    Transdifferentiation is the direct conversion from one somatic cell type into another desired somatic cell type. This reprogramming method offers an attractive approach for regenerative medicine. Here, we demonstrate that neonatal fibroblasts can be transdifferentiated into endothelial cells using only four endothelial transcription factors, namely, ETV2, FLI1, GATA2, and KLF4. We observed a significant up-regulation of endothelial genes including KDR, CD31, CD144, and vWF in human neonatal foreskin (BJ) fibroblasts infected with the lentiviral construct encoding the open reading frame of the four transcription factors. We observed morphological changes in BJ fibroblasts from the fibroblastic spindle shape into a more endothelial-like cobblestone structures. Fluorescence-activated cell sorting analysis revealed that ~16% of the infected cells with the lentiviral constructs encoding 4F expressed CD31. The sorted cells were allowed to expand for 2 weeks and these cells were immunostained and found to express endothelial markers CD31. The induced endothelial cells also incorporated fluorescence-labeled acetylated low-density lipoprotein and efficiently formed capillary-like networks when seeded on Matrigel. These results suggested that the induced endothelial cells were functional in vitro. Taken together, we successfully demonstrated the direct conversion of human neonatal fibroblasts into endothelial cells by transduction of lentiviral constructs encoding endothelial lineage-specific transcription factors ETV2, FLI1, GATA2, and KLF4. The directed differentiation of fibroblasts into endothelial cells may have significant utility in diseases characterized by fibrosis and loss of microvasculature. PMID:27081470

  10. Human fibroblasts in idiopathic retroperitoneal fibrosis express HLA-DR antigens.

    PubMed

    Lee, I

    1991-09-01

    Idiopathic retroperitoneal fibrosis (IRF) is a rare human disease characterized by non-neoplastic fibroblastic proliferation associated with chronic inflammatory cells; its pathogenesis is obscure. We undertook an immunohistochemical study for the expression of HLA-DR antigens and other immune-related markers by retroperitoneal proliferating fibroblasts and inflammatory cells from 2 IRF patients. Patterns of immunoreactivity were compared with those expressed by human nodular fasciitis (NF) and granulation tissue. In IRF, most fibroblasts immunostained strongly for HLA-DR antigens, whereas fibroblasts in NF and granulation tissue did, not immunostain at all. The fibroblasts did not immunostain for interleukin 2 receptor, C3b receptor, CD-4, CD-8, or Leu-M1 in any of the tissue studied. Most macrophages and lymphocytes in IRF and NF immunostained Strangly for HLA-DR antigens. In IRF, the CD-4 and CD-8 immunostained T-lymphocytes appeared equally distributed. The expression of HLA-DR antigens by fibroblasts in IRF indicates that this rare disease may indeed be an immune-associated hypersensitivity disorder. PMID:1777134

  11. Pellino1-mediated TGF-β1 synthesis contributes to mechanical stress induced cardiac fibroblast activation.

    PubMed

    Song, Juan; Zhu, Yun; Li, Jiantao; Liu, Jiahao; Gao, Yun; Ha, Tuanzhu; Que, Linli; Liu, Li; Zhu, Guoqing; Chen, Qi; Xu, Yong; Li, Chuanfu; Li, Yuehua

    2015-02-01

    Activation of cardiac fibroblasts is a key event in the progression of cardiac fibrosis that leads to heart failure. However, the molecular mechanisms underlying mechanical stress-induced cardiac fibroblast activation are complex and poorly understood. This study demonstrates that Pellino1, an E3 ubiquitin ligase, was activated in vivo in pressure overloaded rat hearts and in cultured neonatal rat cardiac fibroblasts (NRCFs) exposed to mechanical stretch in vitro. Suppression of the expression and activity of Pellino1 by adenovirus-mediated delivery of shPellino1 (adv-shpeli1) attenuated pressure overload-induced cardiac dysfunction and cardiac hypertrophy and decreased cardiac fibrosis in rat hearts. Transfection of adv-shpeli1 also significantly attenuated mechanical stress-induced proliferation, differentiation and collagen synthesis in NRCFs. Pellino1 silencing also abrogated mechanical stretch-induced polyubiquitination of tumor necrosis factor-alpha receptor association factor-6 (TRAF6) and receptor-interacting protein 1 (RIP1) and consequently decreased the DNA binding activity of nuclear factor-kappa B (NF-κB) in NRCFs. In addition, Pellino1 silencing prevented stretch-induced activation of p38 and activator protein 1 (AP-1) binding activity in NRCFs. Chromatin Immunoprecipitation (ChIP) and luciferase reporter assays showed that Pellino1 silencing prevented the binding of NF-κB and AP-1 to the promoter region of transforming growth factor-β1 (TGF-β1) thus dampening TGF-β1 transactivation. Our data reveal a previously unrecognized role of Pellino1 in extracellular matrix deposition and cardiac fibroblast activation in response to mechanical stress and provides a novel target for treatment of cardiac fibrosis and heart failure. PMID:25446187

  12. Fibroblast Growth Factor Receptors (FGFRs) in Human Sperm: Expression, Functionality and Involvement in Motility Regulation.

    PubMed

    Saucedo, Lucía; Buffa, Gabriela N; Rosso, Marina; Guillardoy, Tomás; Góngora, Adrian; Munuce, María J; Vazquez-Levin, Mónica H; Marín-Briggiler, Clara

    2015-01-01

    Fibroblast growth factors receptors (FGFRs) have been widely characterized in somatic cells, but there is scarce evidence of their expression and function in mammalian gametes. The objective of the present study was to evaluate the expression of FGFRs in human male germ cells, to determine sperm FGFR activation by the FGF2 ligand and their participation in the regulation of sperm motility. The expression of FGFR1, 2, 3 and 4 mRNAs and proteins in human testis and localization of these receptors in germ cells of the seminiferous epithelium was demonstrated. In ejaculated sperm, FGFRs were localized to the acrosomal region and flagellum. Sperm exposure to FGF2 caused an increase in flagellar FGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB or Akt) signaling pathways. Incubation with FGF2 led to a significant increase in the percentage of total and progressive sperm motility, as well as in sperm kinematics. All responses were prevented by sperm preincubation with BGJ398, a specific inhibitor of FGFR tyrosine kinase activity. In addition to confirming the expression of FGFRs in germ cells of the human testis, our study describes for the first time the presence, localization and functionality of human sperm FGFRs, and provides evidence of the beneficial effect of FGF2 upon sperm motility. PMID:25970615

  13. Fibroblast Growth Factor Receptors (FGFRs) in Human Sperm: Expression, Functionality and Involvement in Motility Regulation

    PubMed Central

    Saucedo, Lucía; Buffa, Gabriela N.; Rosso, Marina; Guillardoy, Tomás; Góngora, Adrian; Munuce, María J.

    2015-01-01

    Fibroblast growth factors receptors (FGFRs) have been widely characterized in somatic cells, but there is scarce evidence of their expression and function in mammalian gametes. The objective of the present study was to evaluate the expression of FGFRs in human male germ cells, to determine sperm FGFR activation by the FGF2 ligand and their participation in the regulation of sperm motility. The expression of FGFR1, 2, 3 and 4 mRNAs and proteins in human testis and localization of these receptors in germ cells of the seminiferous epithelium was demonstrated. In ejaculated sperm, FGFRs were localized to the acrosomal region and flagellum. Sperm exposure to FGF2 caused an increase in flagellar FGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB or Akt) signaling pathways. Incubation with FGF2 led to a significant increase in the percentage of total and progressive sperm motility, as well as in sperm kinematics. All responses were prevented by sperm preincubation with BGJ398, a specific inhibitor of FGFR tyrosine kinase activity. In addition to confirming the expression of FGFRs in germ cells of the human testis, our study describes for the first time the presence, localization and functionality of human sperm FGFRs, and provides evidence of the beneficial effect of FGF2 upon sperm motility. PMID:25970615

  14. Fibroblasts from phenotypically normal palmar fascia exhibit molecular profiles highly similar to fibroblasts from active disease in Dupuytren's Contracture

    PubMed Central

    2012-01-01

    Background Dupuytren's contracture (DC) is a fibroproliferative disorder characterized by the progressive development of a scar-like collagen-rich cord that affects the palmar fascia of the hand and leads to digital flexion contractures. DC is most commonly treated by surgical resection of the diseased tissue, but has a high reported recurrence rate ranging from 27% to 80%. We sought to determine if the transcriptomic profiles of fibroblasts derived from DC-affected palmar fascia, adjacent phenotypically normal palmar fascia, and non-DC palmar fascial tissues might provide mechanistic clues to understanding the puzzle of disease predisposition and recurrence in DC. Methods To achieve this, total RNA was obtained from fibroblasts derived from primary DC-affected palmar fascia, patient-matched unaffected palmar fascia, and palmar fascia from non-DC patients undergoing carpal tunnel release (6 patients in each group). These cells were grown on a type-1 collagen substrate (to better mimic their in vivo environments). Microarray analyses were subsequently performed using Illumina BeadChip arrays to compare the transcriptomic profiles of these three cell populations. Data were analyzed using Significance Analysis of Microarrays (SAM v3.02), hierarchical clustering, concordance mapping and Venn diagram. Results We found that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected fascia of DC patients exhibited a much greater overlap than fibroblasts derived from the palmar fascia of patients undergoing carpal tunnel release. Quantitative real time RT-PCR confirmed the differential expression of select genes validating the microarray data analyses. These data are consistent with the hypothesis that predisposition and recurrence in DC may stem, at least in part, from intrinsic similarities in the basal gene expression of diseased and phenotypically unaffected palmar fascia fibroblasts. These data also demonstrate that a collagen

  15. Fibroblast growth factor 7 inhibits cholesterol 7{alpha}-hydroxylase gene expression in hepatocytes

    SciTech Connect

    Sun, Zhichao; Yu, Xuemei; Wu, Weibin; Jia, Dongwei; Chen, Yinle; Ji, Lingling; Liu, Xijun; Peng, Xiaomin; Li, Yintao; Yang, Lili; Ruan, Yuanyuan; Gu, Jianxin; Ren, Shifang; Zhang, Songwen

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer FGF7 strongly and rapidly down-regulates the expression of CYP7A1 in hepatocytes. Black-Right-Pointing-Pointer FGF7 suppresses the expression of CYP7A1 via FGFR2 and downstream JNK activation. Black-Right-Pointing-Pointer Blocking FGF7 abrogates HSC-induced inhibition of CYP7A1 expression in hepatocytes. -- Abstract: Cholesterol 7{alpha}-hydroxylase (CYP7A1) is the initial and rate-limiting enzyme for bile acid synthesis. Transcription of the CYP7A1 gene is regulated by bile acids, nuclear receptors and cytokines. Fibroblast growth factor 7 (FGF7) secreted from activated hepatic stellate cells (HSC) during chronic liver fibrosis regulates hepatocyte survival and liver regeneration. In the carbon tetrachloride (CCl{sub 4})-induced fibrotic mouse liver, we demonstrated that the expression of CYP7A1 was largely decreased while the expression of FGF7 was significantly increased. We further demonstrated that FGF7 inhibited CYP7A1 gene expression in hepatocytes. Knockdown study by short interfering RNA, kinase inhibition and phosphorylation assays revealed that the suppression of CYP7A1 expression by FGF7 was mediated by FGFR2 and its downstream JNK signaling cascade. The FGF7 neutralizing antibody restored CYP7A1 expression in Hep3B cells treated with conditioned medium from HSC. In summary, the data suggest that FGF7 is a novel regulator of CYP7A1 expression in hepatocytes and may prevent hepatocytes from accumulating toxic bile acids during liver injury and fibrosis.

  16. CTGF increases vascular endothelial growth factor-dependent angiogenesis in human synovial fibroblasts by increasing miR-210 expression

    PubMed Central

    Liu, S-C; Chuang, S-M; Hsu, C-J; Tsai, C-H; Wang, S-W; Tang, C-H

    2014-01-01

    Connective tissue growth factor (CTGF, a.k.a. CCN2) is inflammatory mediator and abundantly expressed in osteoarthritis (OA). Angiogenesis is essential for OA progression. Here, we investigated the role of CTGF in vascular endothelial growth factor (VEGF) production and angiogenesis in OA synovial fibroblasts (OASFs). We showed that expression of CTGF and VEGF in synovial fluid were higher in OA patients than in controls. Directly applying CTGF to OASFs increased VEGF production then promoted endothelial progenitor cells tube formation and migration. CTGF induced VEGF by raising miR-210 expression via PI3K, AKT, ERK, and nuclear factor-κB (NF-κB)/ELK1 pathways. CTGF-mediating miR-210 upregulation repressed glycerol-3-phosphate dehydrogenase 1-like (GPD1L) expression and PHD activity and subsequently promoted hypoxia-inducible factor (HIF)-1α-dependent VEGF expression. Knockdown of CTGF decreased VEGF expression and abolished OASF-conditional medium-mediated angiogenesis in vitro as well as angiogenesis in chick chorioallantoic membrane and Matrigel-plug nude mice model in vivo. Taken together, our results suggest CTGF activates PI3K, AKT, ERK, and NF-κB/ELK1 pathway, leading to the upregulation of miR-210, contributing to inhibit GPD1L expression and prolyl hydroxylases 2 activity, promoting HIF-1α-dependent VEGF expression and angiogenesis in human synovial fibroblasts. PMID:25341039

  17. Individual Differences in the Expression of Conditioned Fear Are Associated with Endogenous Fibroblast Growth Factor 2

    ERIC Educational Resources Information Center

    Graham, Bronwyn M.; Richardson, Rick

    2016-01-01

    These experiments examined the relationship between the neurotrophic factor fibroblast growth factor 2 (FGF2) and individual differences in the expression of conditioned fear. Experiments 1 and 2 demonstrated that rats naturally expressing low levels of contextual or cued fear have higher levels of hippocampal FGF2 relative to rats that express…

  18. The effects of ripasudil (K-115), a Rho kinase inhibitor, on activation of human conjunctival fibroblasts.

    PubMed

    Futakuchi, Akiko; Inoue, Toshihiro; Fujimoto, Tomokazu; Inoue-Mochita, Miyuki; Kawai, Motofumi; Tanihara, Hidenobu

    2016-08-01

    The most common cause of glaucoma surgery failure is scar formation induced by activation of wound-healing responses and resultant fibrosis at the surgical site. We investigated the effects of ripasudil, a Rho kinase inhibitor, on activation of human conjunctival fibroblasts (HConF). HConF were pretreated with different concentrations of ripasudil for 1 h before addition of transforming growth factor (TGF)-β2, followed by incubation for 48 h. TGF-β2-treated fibroblasts exhibited a significant increase in expression of α-smooth muscle actin (α-SMA), a marker of fibroblast-to-myofibroblast differentiation, and this increase was significantly suppressed, in a dose-dependent manner, by pretreatment with ripasudil. Ripasudil pretreatment also significantly attenuated TGF-β2-induced fibronectin production and collagen gel contraction. TGF-β2 increased both the number of viable cells and the number of cells in the G2/M phase of the cell cycle; these effects were attenuated by pretreatment with ripasudil. In addition, we explored the effects of ripasudil on stimulation of HConF by activated macrophages. Human monocytic cell line THP-1 cells were differentiated into M1 or M2 macrophage-like cells, and HConF were treated with conditioned media derived from these macrophages in the presence or absence of ripasudil. Conditioned medium from M2 macrophage-like cells induced a significant increase in α-SMA expression, viable cell numbers, and gel contraction, all of which were significantly suppressed by ripasudil. Thus, overall, ripasudil attenuated activation of human conjunctival fibroblasts. Ripasudil may be of therapeutic utility, preventing excessive scarring after glaucoma filtration surgery. PMID:27394186

  19. Lentiviral Engineered Fibroblasts Expressing Codon-Optimized COL7A1 Restore Anchoring Fibrils in RDEB

    PubMed Central

    Georgiadis, Christos; Syed, Farhatullah; Petrova, Anastasia; Abdul-Wahab, Alya; Lwin, Su M.; Farzaneh, Farzin; Chan, Lucas; Ghani, Sumera; Fleck, Roland A.; Glover, Leanne; McMillan, James R.; Chen, Mei; Thrasher, Adrian J.; McGrath, John A.; Di, Wei-Li; Qasim, Waseem

    2016-01-01

    Cells therapies, engineered to secrete replacement proteins, are being developed to ameliorate otherwise debilitating diseases. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects of type VII collagen, a protein essential for anchoring fibril formation at the dermal-epidermal junction. Whereas allogeneic fibroblasts injected directly into the dermis can mediate transient disease modulation, autologous gene-modified fibroblasts should evade immunological rejection and support sustained delivery of type VII collagen at the dermal-epidermal junction. We demonstrate the feasibility of such an approach using a therapeutic grade, self-inactivating-lentiviral vector, encoding codon-optimized COL7A1, to transduce RDEB fibroblasts under conditions suitable for clinical application. Expression and secretion of type VII collagen was confirmed with transduced cells exhibiting supranormal levels of protein expression, and ex vivo migration of fibroblasts was restored in functional assays. Gene-modified RDEB fibroblasts also deposited type VII collagen at the dermal-epidermal junction of human RDEB skin xenografts placed on NOD-scid IL2Rgammanull recipients, with reconstruction of human epidermal structure and regeneration of anchoring fibrils at the dermal-epidermal junction. Fibroblast-mediated restoration of protein and structural defects in this RDEB model strongly supports proposed therapeutic applications in man. PMID:26763448

  20. Lentiviral Engineered Fibroblasts Expressing Codon-Optimized COL7A1 Restore Anchoring Fibrils in RDEB.

    PubMed

    Georgiadis, Christos; Syed, Farhatullah; Petrova, Anastasia; Abdul-Wahab, Alya; Lwin, Su M; Farzaneh, Farzin; Chan, Lucas; Ghani, Sumera; Fleck, Roland A; Glover, Leanne; McMillan, James R; Chen, Mei; Thrasher, Adrian J; McGrath, John A; Di, Wei-Li; Qasim, Waseem

    2016-01-01

    Cells therapies, engineered to secrete replacement proteins, are being developed to ameliorate otherwise debilitating diseases. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects of type VII collagen, a protein essential for anchoring fibril formation at the dermal-epidermal junction. Whereas allogeneic fibroblasts injected directly into the dermis can mediate transient disease modulation, autologous gene-modified fibroblasts should evade immunological rejection and support sustained delivery of type VII collagen at the dermal-epidermal junction. We demonstrate the feasibility of such an approach using a therapeutic grade, self-inactivating-lentiviral vector, encoding codon-optimized COL7A1, to transduce RDEB fibroblasts under conditions suitable for clinical application. Expression and secretion of type VII collagen was confirmed with transduced cells exhibiting supranormal levels of protein expression, and ex vivo migration of fibroblasts was restored in functional assays. Gene-modified RDEB fibroblasts also deposited type VII collagen at the dermal-epidermal junction of human RDEB skin xenografts placed on NOD-scid IL2Rgamma(null) recipients, with reconstruction of human epidermal structure and regeneration of anchoring fibrils at the dermal-epidermal junction. Fibroblast-mediated restoration of protein and structural defects in this RDEB model strongly supports proposed therapeutic applications in man. PMID:26763448

  1. Leptospiral lipopolysaccharide stimulates the expression of toll-like receptor 2 and cytokines in pig fibroblasts.

    PubMed

    Guo, Yijie; Fukuda, Tomokazu; Donai, Kenichiro; Kuroda, Kengo; Masuda, Mizuki; Nakamura, Shuichi; Yoneyama, Hiroshi; Isogai, Emiko

    2015-02-01

    Pigs throughout the world are afflicted with leptospirosis, causing serious economic losses and potential hazards to human health. Although it has been known that leptospiral lipopolysaccharide (L-LPS) is involved in an immunological reaction between an antigen and a host cell, little is known about how the immune system of pigs can respond to L-LPS. Here, we stimulated pig fibroblasts by L-LPS and then quantitatively measured gene and protein expression levels of two toll-like receptors (TLRs), TLR2 and TLR4, by real-time PCR and Western blotting. As a result, expression of TLR2 was found to be significantly up-regulated within 24 h after L-LPS stimulation whereas induction of TLR4 expression was relatively weak. We also revealed that of myeloid differentiation primary response gene 88 (MyD88), interleukin 6 (IL-6) and IL-8 gene expressions were markedly up-regulated by L-LPS stimulation. These results may suggest that the pig cell can activate TLR2 rather than TLR4 by L-LPS stimulation, thereby inducing expression of cytokines. PMID:25039909

  2. Fibroblasts behavior after N-acetylcysteine and amino acids exposure: extracellular matrix gene expression.

    PubMed

    Avantaggiato, Anna; Palmieri, Annalisa; Bertuzzi, Gianluigi; Carinci, Francesco

    2014-06-01

    Reactive oxygen species (ROS) are chemically reactive molecules with impaired electrons that make them unstable and able to react easily with a great variety of molecules. The main targets of ROS are DNA, proteins, and membrane phospholipids. In the skin, ROS are able to affect the production of collagen and elastin, the main components of the extracellular matrix (ECM). This action contributes to the skin's aging. N-Acetylcysteine (NAC) is an acetylated cysteine residue with excellent anti-oxidant activity that boosts glutathione (GSH) levels. This study evaluates the effect of a solution of NAC and amino acids, which is used in aesthetic medicine as an intra-dermal injective treatment, on fibroblast behavior. To this aim, the expression levels of some ECM-related genes (HAS1, HYAL1 ELN, ELANE, MMP2, MMP3, MMP13, COL1A1, COL3A1) were analyzed on cultured dermal fibroblasts using real-time reverse transcription polymerase chain reaction (RT-PCR). All but two collagen genes were up-regulated after 24 hr of treatment. PMID:24438160

  3. The correction of biochemical abnormalities in fibroblasts of a Zellweger patient by gene expression

    SciTech Connect

    Shimozawa, N.; Suzuki, Y.; Oril, T.

    1994-09-01

    Zellweger syndrome is a prototype of peroxisome-deficient disorders and a fatal autosomal recessive disease with no effective therapy. We identified nine genetic complementation groups of these disorders among several laboratories, and mutations in peroxisome assembly factor-1 (PAF-1) and the 70-kDa peroxisomal membrane protein (PMP70) genes have been described in Zellweger patients from our group F and Roscher`s group 1, respectively. We now succeed the permanent recovery of generalized peroxisomal abnormalities in fibroblasts of a Zellweger patient from the group F by the stable transfection of human cDNA encoding PAF-1. In the transfectants, a number of peroxisomal dysfunctions such as lignocelic acid oxidation, dihydroxyacetone phosphate acyltransferase activity and biogenesis of peroxisomal {beta}-oxidation enzymes were restored, as well as morphological absence of peroxisomes. These findings are useful for basic studies on gene therapy of peroxisomal disorders in the cultured cellular system. Further study on expression of human PMP70 cDNA in fibroblasts from Roscher`s group 1 will be also necessary to confirm whether the PMP70 is responsible for Zellweger syndrome.

  4. The expression of pregnancy-specific {beta}1-glycoprotein genes in Meckel-Gruber syndrome fibroblasts

    SciTech Connect

    Wu, Shao-Ming; Cham, Wai-Yee

    1994-09-01

    Meckel-Gruber syndrome (MS) is an autosomal recessive disorder with multiple congenital malformations. The only available prenatal diagnostic marker for this disorder is the amniotic fluid level of pregnancy-specific {beta}1-glycoprotein (PSG). PSG is a family of proteins which are expressed at high levels during pregnancy. Increasing maternal serum PSG levels correlate with the progression of pregnancy and can be used as indicators for pregnancy outcome and fetal well-being. The amniotic fluid PSG level is about one-tenth of that of the maternal serum level in normal pregnancy, but are elevated in all cases of MS examined so far. On the other hand, the maternal serum PSG level and third trimester placental PSG content are normal in most cases of MS. This study aims at comparing the expression of PSG in fibroblasts derived from a fetus afflicted with MS. Total cellular RNA was extracted from two MS cultured fibroblast lines (M3206 and GM7817) and four age- and sex-matched control fibroblast lines obtained from the Human Genetic Mutant Cell Repository, Camden, NJ. The expression of eight PSG genes namely, PSG1, PSG2, PSG3, PSG4, PSG5, PSG6, PSG9 and PSG11, were examined with reverse transcription-polymerase chain reaction (RT-PCR). All PSG transcripts present in the cell were first amplified using universal primers in a 28-cycle PCR. Specific PSG gene products were then amplified with PSG gene-specific primers. Results showed that there is no significant difference in PSG expression between control and disease fibroblasts. In both cases, the most abundant transcript was the type II transcript of PSG5 followed by the type I transcripts of PSG1 and PG4. PSG9, PSG11 and PSG 3 were expressed at very low levels or not expressed at all in MS as well as in normal control fibroblasts. These results showed that PSG gene expression was not altered in MS fibroblasts.

  5. Recruitment of Matrix Metalloproteinase-9 (MMP-9) to the Fibroblast Cell Surface by Lysyl Hydroxylase 3 (LH3) Triggers Transforming Growth Factor-β (TGF-β) Activation and Fibroblast Differentiation*

    PubMed Central

    Dayer, Cynthia; Stamenkovic, Ivan

    2015-01-01

    Solid tumor growth triggers a wound healing response. Similar to wound healing, fibroblasts in the tumor stroma differentiate into myofibroblasts (also referred to as cancer-associated fibroblasts) primarily, but not exclusively, in response to transforming growth factor-β (TGF-β). Myofibroblasts in turn enhance tumor progression by remodeling the stroma. Among proteases implicated in stroma remodeling, matrix metalloproteinases (MMPs), including MMP-9, play a prominent role. Recent evidence indicates that MMP-9 recruitment to the tumor cell surface enhances tumor growth and invasion. In the present work, we addressed the potential relevance of MMP-9 recruitment to and activity at the surface of fibroblasts. We show that recruitment of MMP-9 to the fibroblast cell surface occurs through its fibronectin-like (FN) domain and that the molecule responsible for the recruitment is lysyl hydroxylase 3 (LH3). Functional assays suggest that both pro- and active MMP-9 trigger α-smooth muscle actin expression in cultured fibroblasts, reflecting myofibroblast differentiation, possibly as a result of TGF-β activation. Moreover, the recombinant FN domain inhibited both MMP-9-induced TGF-β activation and α-smooth muscle actin expression by displacing MMP-9 from the fibroblast cell surface. Together our results uncover LH3 as a new docking receptor of MMP-9 on the fibroblast cell surface and demonstrate that the MMP-9 FN domain is essential for the interaction. They also show that the recombinant FN domain inhibits MMP-9-induced TGF-β activation and fibroblast differentiation, providing a potentially attractive therapeutic reagent toward attenuating tumor progression where MMP-9 activity is strongly implicated. PMID:25825495

  6. Modulation of phospholipase A2 activity in human fibroblasts.

    PubMed Central

    Solito, E.; Parente, L.

    1989-01-01

    1. Human embryonic skin fibroblasts (HSF) incubated overnight with either human recombinant interleukin-1 alpha (rIL-1 alpha) or interleukin-1 beta (rIL-1 beta) released large amounts of prostaglandin E2 (PGE2). 2. rIL-1 beta, bradykinin (Bk) and arachidonic acid (AA) significantly stimulated PGE2 release from HSF incubated overnight in the presence of either interleukin. 3. Hydrocortisone inhibited the PGE2 release induced by rIL-1 beta and Bk, but not by AA. 4. The steroid inhibitory effect was reversed by actinomycin D as well as by an anti-lipocortin monoclonal antibody. 5. The results suggest that in HSF, rIL-1 beta is able to stimulate both cyclo-oxygenase and phospholipase A2 (PLA2) activity. 6. The stimulation of PLA2 activity by rIL-1 beta is inhibited by hydrocortisone, probably via induction of lipocortin-like proteins. PMID:2785834

  7. Wnt1/βcatenin injury response activates the epicardium and cardiac fibroblasts to promote cardiac repair

    PubMed Central

    Duan, Jinzhu; Gherghe, Costin; Liu, Dianxin; Hamlett, Eric; Srikantha, Luxman; Rodgers, Laurel; Regan, Jenna N; Rojas, Mauricio; Willis, Monte; Leask, Andrew; Majesky, Mark; Deb, Arjun

    2012-01-01

    Wnts are required for cardiogenesis but the role of specific Wnts in cardiac repair remains unknown. In this report, we show that a dynamic Wnt1/βcatenin injury response activates the epicardium and cardiac fibroblasts to promote cardiac repair. Acute ischaemic cardiac injury upregulates Wnt1 that is initially expressed in the epicardium and subsequently by cardiac fibroblasts in the region of injury. Following cardiac injury, the epicardium is activated organ-wide in a Wnt-dependent manner, expands, undergoes epithelial–mesenchymal transition (EMT) to generate cardiac fibroblasts, which localize in the subepicardial space. The injured regions in the heart are Wnt responsive as well and Wnt1 induces cardiac fibroblasts to proliferate and express pro-fibrotic genes. Disruption of downstream Wnt signalling in epicardial cells decreases epicardial expansion, EMT and leads to impaired cardiac function and ventricular dilatation after cardiac injury. Furthermore, disruption of Wnt/βcatenin signalling in cardiac fibroblasts impairs wound healing and decreases cardiac performance as well. These findings reveal that a pro-fibrotic Wnt1/βcatenin injury response is critically required for preserving cardiac function after acute ischaemic cardiac injury. PMID:22085926

  8. Largazole, a class I histone deacetylase inhibitor, enhances TNF-α-induced ICAM-1 and VCAM-1 expression in rheumatoid arthritis synovial fibroblasts

    SciTech Connect

    Ahmed, Salahuddin; Riegsecker, Sharayah; Beamer, Maria; Rahman, Ayesha; Bellini, Joseph V.; Bhansali, Pravin; Tillekeratne, L.M. Viranga

    2013-07-15

    In the present study, we evaluated the effect of largazole (LAR), a marine-derived class I HDAC inhibitor, on tumor necrosis factor-α (TNF-α)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and matrix metalloproteinase-2 (MMP-2) activity. LAR (1–5 μM) had no adverse effect on the viability of RA synovial fibroblasts. Among the different class I HDACs screened, LAR (0.5–5 μM) inhibited the constitutive expression of HDAC1 (0–30%). Surprisingly, LAR increased class II HDAC [HDAC6] by ∼ 220% with a concomitant decrease in HDAC5 [30–58%] expression in RA synovial fibroblasts. SAHA (5 μM), a pan-HDAC inhibitor, also induced HDAC6 expression in RA synovial fibroblasts. Pretreatment of RA synovial fibroblasts with LAR further enhanced TNF-α-induced ICAM-1 and VCAM-1 expression. However, LAR inhibited TNF-α-induced MMP-2 activity in RA synovial fibroblasts by 35% when compared to the TNF-α-treated group. Further, the addition of HDAC6 specific inhibitor Tubastatin A with LAR suppressed TNF-α + LAR-induced ICAM-1 and VCAM-1 expression and completely blocked MMP-2 activity, suggesting a role of HDAC6 in LAR-induced ICAM-1 and VCAM-1 expression. LAR also enhanced TNF-α-induced phospho-p38 and phospho-AKT expression, but inhibited the expression of phospho-JNK and nuclear translocation of NF-κBp65 in RA synovial fibroblasts. These results suggest that LAR activates p38 and Akt pathways and influences class II HDACs, in particular HDAC6, to enhance some of the detrimental effects of TNF-α in RA synovial fibroblasts. Understanding the exact role of different HDAC isoenzymes in RA pathogenesis is extremely important in order to develop highly effective HDAC inhibitors for the treatment of RA. - Highlights: • Largazole enhances TNF-α-induced ICAM-1 and VCAM-1. • Largazole upregulates class II HDAC (HDAC6) in RA synovial fibroblasts. • Largazole also induces the expression of phospho-p38

  9. A specific, transmembrane interface regulates fibroblast activation protein (FAP) homodimerization, trafficking and exopeptidase activity.

    PubMed

    Wonganu, Benjamaporn; Berger, Bryan W

    2016-08-01

    Fibroblast activation protein (FAP) is a cell-surface serine protease which promotes invasiveness of certain epithelial cancers and is therefore a potential target for cancer drug development and delivery. Unlike dipeptidyl peptidase IV (DPPIV), FAP exhibits prolyl endopeptidase activity and is active as a homodimer with specificity for type I collagen. The mechanism that regulates FAP homodimerization and its relation to prolyl endopeptidase activity is not completely understood. Here, we investigate key residues in the FAP TM domain that may be significant for FAP homodimerization. Mutations to predicted TM interfacial residues (G10L, S14L, and A18L) comprising a small-X3-small motif reduced FAP TM-CYTO dimerization relative to wild type as measured using the AraTM assay, whereas predicted off-interface residues showed no significant change from wild type. The results implied that the predicted small-X3-small dimer interface affect stabilization of FAP TM-CYTO homodimerization. Compared with FAPwild-type, the interfacial TM residue G10L significantly decreased FAP endopeptidase activity more than 25%, and also reduced cell-surface versus intracellular expression relative to other interfacial residues S14L and A18L. Thus, our results suggest FAP dimerization is important for both trafficking and protease activity, and is dependent on a specific TM interface. PMID:27155568

  10. Lysyl oxidase expression in cardiac fibroblasts is regulated by α2β1 integrin interactions with the cellular microenvironment.

    PubMed

    Gao, Albert E; Sullivan, Kelly E; Black, Lauren D

    2016-06-17

    Lysyl oxidase (LOX) catalyzes crosslink formation between fibrillar collagens and elastins and an increase in LOX activity has been associated with cardiac fibrosis following myocardial infarction (MI). It has been previously reported that LOX expression is regulated by growth factors and cytokines including transforming growth factor (TGF-β1); however, it is unclear how the biophysical and biochemical properties of the cellular microenvironment affect LOX expression. In this study, we isolated rat cardiac fibroblasts (CF) and infarct cardiac fibroblasts (ICF), from healthy and 1-week post-MI left ventricular tissue respectively, and cultured them under varied substrate conditions in vitro to assess their influence on LOX expression. Culture of ICF on collagen I-coated plates increased LOX expression versus uncoated plates with an additional increase observed with the presence of TGF-β1. To further investigate the effect of integrin interactions with collagen I on LOX expression, we inhibited the α2β1 integrin from binding to collagen I and found gene and protein expression of LOX to be downregulated. Together, this demonstrates that the interaction of α2β1 integrin to collagen I in the cellular microenvironment can regulate expression of LOX. Further studies investigating additional integrin interactions may identify therapeutic targets for treating cardiac fibrosis. PMID:27169768

  11. Krüppel-Like Factor 4 Is a Regulator of Proinflammatory Signaling in Fibroblast-Like Synoviocytes through Increased IL-6 Expression

    PubMed Central

    Ruan, Jianwei; Xie, Jiangwen; Lv, Guoju

    2016-01-01

    Human fibroblast-like synoviocytes play a vital role in joint synovial inflammation in rheumatoid arthritis (RA). Proinflammatory cytokines induce fibroblast-like synoviocyte activation and dysfunction. The inflammatory mediator Krüppel-like factor 4 is upregulated during inflammation and plays an important role in endothelial and macrophage activation during inflammation. However, the role of Krüppel-like factor 4 in fibroblast-like synoviocyte activation and RA inflammation remains to be defined. In this study, we identify the notion that Krüppel-like factor 4 is higher expressed in synovial tissues and fibroblast-like synoviocytes from RA patients than those from osteoarthritis patients. In vitro, the expression of Krüppel-like factor 4 in RA fibroblast-like synoviocytes is induced by proinflammatory cytokine tumor necrosis factor-α. Overexpression of Krüppel-like factor 4 in RA fibroblast-like synoviocytes robustly induced interleukin-6 production in the presence or absence of tumor necrosis factor-α. Conversely, knockdown of Krüppel-like factor 4 markedly attenuated interleukin-6 production in the presence or absence of tumor necrosis factor-α. Krüppel-like factor 4 not only can bind to and activate the interleukin-6 promoter, but also may interact directly with nuclear factor-kappa B. These results suggest that Krüppel-like factor 4 may act as a transcription factor mediating the activation of fibroblast-like synoviocytes in RA by inducing interleukin-6 expression in response to tumor necrosis factor-α. PMID:27413250

  12. A heterocyclic molecule kartogenin induces collagen synthesis of human dermal fibroblasts by activating the smad4/smad5 pathway.

    PubMed

    Wang, Jing; Zhou, Jia; Zhang, Ning; Zhang, Xiaoling; Li, Qingfeng

    2014-07-18

    Declined production of collagen by fibroblasts is one of the major causes of aging appearance. However, only few of compounds found in cosmetic products are able to directly increase collagen synthesis. A novel small heterocyclic compound called kartogenin (KGN) was found to stimulate collagen synthesis of mesenchymal stem cells (MSCs). So, we hypothesized and tested that if KGN could be applied to stimulate the collagen synthesis of fibroblasts. Human dermal fibroblasts in vitro were treated with various concentrations of KGN, with dimethyl sulfoxide (DMSO) serving as the negative control. Real-time reverse-transcription polymerase chain reaction, Western blot, and immunofluorescence analyses were performed to examine the expression of collagen and transforming growth factor beta (TGF-β) signaling pathway. The production of collagen was also tested in vivo by Masson's trichrome stain and immunohistochemistry in the dermis of mice administrated with KGN. Results showed that without obvious influence on fibroblasts' apoptosis and viability, KGN stimulated type-I collagen synthesis of fibroblasts at the mRNA and protein levels in a time-dependent manner, but KGN did not induce expression of α-skeletal muscle actin (α-sma) or matrix metallopeptidase1 (MMP1), MMP9 in vitro. Smad4/smad5 of the TGF-β signaling pathway was activated by KGN while MAPK signaling pathway remained unchanged. KGN also increased type-I collagen synthesis in the dermis of BALB/C mice. Our results indicated that KGN promoted the type-I collagen synthesis of dermal fibroblasts in vitro and in the dermis of mice through activation of the smad4/smad5 pathway. This molecule could be used in wound healing, tissue engineering of fibroblasts, or aesthetic and reconstructive procedures. PMID:24928394

  13. Genetic polymorphism directs IL-6 expression in fibroblasts but not selected other cell types

    PubMed Central

    Noss, Erika H.; Nguyen, Hung N.; Chang, Sook Kyung; Watts, Gerald F. M.; Brenner, Michael B.

    2015-01-01

    Interleukin (IL)-6 blockade is an effective treatment for rheumatoid arthritis (RA), and synovial fibroblasts are a major IL-6 producer in the inflamed joint. We found that human RA and osteoarthritis (OA) synovial fibroblasts derived from independent donors reproducibly segregated into low, medium, and high IL-6 producers, independent of stimulus, cell passage, or disease state. IL-6 expression pattern correlated strongly with total mRNA expression, not mRNA stability, suggesting transcriptional rather than posttranscriptional regulation. High-fibroblast IL-6 expression was significantly associated with the IL-6 proximal promoter single nucleotide polymorphism (SNP) rs1800795 minor allele (CC) genotype. In contrast, no association between this SNP and IL-6 production was detected in CD14+ monocytes, another major producer of synovial IL-6. Luciferase expression assays confirmed that this SNP was associated with differential IL-6 expression in fibroblasts. To date, several association studies examining rs1800795 allele frequency and disease risk have reported seemingly conflicting results ranging from no association to association with either the major or minor allele across a spectrum of conditions, including cancer and autoimmune, cardiovascular, infectious, and metabolic diseases. This study points to a prominent contribution from promoter genetic variation in fibroblast IL-6 regulation, but not in other IL-6–producing cell types. We propose that some of the heterogeneity in these clinical studies likely reflects the cellular source of IL-6 in specific diseases, much of which may be produced by nonhematopoietic cells. These results highlight that functional analysis of disease-associated SNPs on gene expression and pathologic processes must consider variation in diverse cell types. PMID:26578807

  14. Problem-Based Test: The Effect of Fibroblast Growth Factor on Gene Expression

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2011-01-01

    This paper shows the results of an experiment in which the effects of fibroblast growth factor (FGF), actinomycin D (Act D; an inhibitor of transcription), and cycloheximide (CHX; an inhibitor of translation) were studied on the expression of two genes: a gene called "Fnk" and the gene coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).…

  15. Resveratrol inhibits collagen I synthesis by suppressing IGF-1R activation in intestinal fibroblasts

    PubMed Central

    Li, Ping; Liang, Mei-Lan; Zhu, Ying; Gong, Yao-Yao; Wang, Yun; Heng, Ding; Lin, Lin

    2014-01-01

    I induced by IGF-1. Moreover, silencing SIRT1 restored collagen I expression in fibroblasts challenged with resveratrol. However, disruption of SIRT1 did not influence the anti-fibrotic effects of resveratrol and IGF-1-induced collagen I expression. Further analysis revealed that resveratrol significantly decreased phosphorylation of IGF-1R and its downstream signaling molecules by inhibiting IGF-1 binding to its receptor. CONCLUSION: Our data suggest that resveratrol effectively inhibits collagen I synthesis in IGF-1-stimulated fibroblasts, partly by inhibiting IGF-1R activation, and SIRT1 is also responsible for the process. PMID:24782617

  16. Paracrine effect of GTP cyclohydrolase and angiopoietin-1 interaction in stromal fibroblasts on tumor Tie2 activation and breast cancer growth.

    PubMed

    Chen, Liye; Zeng, Xin; Kleibeuker, Esther; Buffa, Francesca; Barberis, Alessandro; Leek, Russell D; Roxanis, Ioannis; Zhang, Wei; Worth, Andrew; Beech, John S; Harris, Adrian L; Cai, Shijie

    2016-02-23

    Cancer-associated fibroblasts (CAFs) play a key role in promoting tumor growth, acting through complex paracrine regulation. GTP cyclohydrolase (GTPCH) expression for tetrahydrobiopterin synthesis in tumor stroma is implicated in angiogenesis and tumor development. However, the clinical significance of GTPCH expression in breast cancer is still elusive and how GTPCH regulates stromal fibroblast and tumor cell communication remains unknown. We found that GTPCH was upregulated in breast CAFs and epithelia, and high GTPCH RNA was significantly correlated with larger high grade tumors and worse prognosis. In cocultures, GTPCH expressing fibroblasts stimulated breast cancer cell proliferation and motility, cancer cell Tie2 phosphorylation and consequent downstream pathway activation. GTPCH interacted with Ang-1 in stromal fibroblasts and enhanced Ang-1 expression and function, which in turn phosphorylated tumor Tie2 and induced cell proliferation. In coimplantation xenografts, GTPCH in fibroblasts enhanced tumor growth, upregulating Ang-1 and alpha-smooth muscle actin mainly in fibroblast-like cells. GTPCH inhibition resulted in the attenuation of tumor growth and angiogenesis. GTPCH/Ang-1 interaction in stromal fibroblasts and activation of Tie2 on breast tumor cells could play an important role in supporting breast cancer growth. GTPCH may be an important mechanism of paracrine tumor growth and hence a target for therapy in breast cancer. PMID:26814432

  17. Paracrine effect of GTP cyclohydrolase and angiopoietin-1 interaction in stromal fibroblasts on tumor Tie2 activation and breast cancer growth

    PubMed Central

    Kleibeuker, Esther; Buffa, Francesca; Barberis, Alessandro; Leek, Russell D.; Roxanis, Ioannis; Zhang, Wei; Worth, Andrew; Beech, John S.; Harris, Adrian L.; Cai, Shijie

    2016-01-01

    Cancer-associated fibroblasts (CAFs) play a key role in promoting tumor growth, acting through complex paracrine regulation. GTP cyclohydrolase (GTPCH) expression for tetrahydrobiopterin synthesis in tumor stroma is implicated in angiogenesis and tumor development. However, the clinical significance of GTPCH expression in breast cancer is still elusive and how GTPCH regulates stromal fibroblast and tumor cell communication remains unknown. We found that GTPCH was upregulated in breast CAFs and epithelia, and high GTPCH RNA was significantly correlated with larger high grade tumors and worse prognosis. In cocultures, GTPCH expressing fibroblasts stimulated breast cancer cell proliferation and motility, cancer cell Tie2 phosphorylation and consequent downstream pathway activation. GTPCH interacted with Ang-1 in stromal fibroblasts and enhanced Ang-1 expression and function, which in turn phosphorylated tumor Tie2 and induced cell proliferation. In coimplantation xenografts, GTPCH in fibroblasts enhanced tumor growth, upregulating Ang-1 and alpha-smooth muscle actin mainly in fibroblast-like cells. GTPCH inhibition resulted in the attenuation of tumor growth and angiogenesis. GTPCH/Ang-1 interaction in stromal fibroblasts and activation of Tie2 on breast tumor cells could play an important role in supporting breast cancer growth. GTPCH may be an important mechanism of paracrine tumor growth and hence a target for therapy in breast cancer. PMID:26814432

  18. Bacillus Calmette Guerin Induces Fibroblast Activation Both Directly and through Macrophages in a Mouse Bladder Cancer Model

    PubMed Central

    Lodillinsky, Catalina; Langle, Yanina; Guionet, Ariel; Góngora, Adrián; Baldi, Alberto; Sandes, Eduardo O.; Casabé, Alberto; Eiján, Ana María

    2010-01-01

    Background Bacillus Calmette-Guerin (BCG) is the most effective treatment for non-muscle invasive bladder cancer. However, a failure in the initial response or relapse within the first five years of treatment has been observed in 20% of patients. We have previously observed that in vivo administration of an inhibitor of nitric oxide improved the response to BCG of bladder tumor bearing mice. It was described that this effect was due to a replacement of tumor tissue by collagen depots. The aim of the present work was to clarify the mechanism involved in this process. Methodology/Principal Findings We demonstrated that BCG induces NIH-3T3 fibroblast proliferation by activating the MAPK and PI3K signaling pathways and also differentiation determined by alpha-smooth muscle actin (alpha-SMA) expression. In vivo, intratumoral inoculation of BCG also increased alpha-SMA and collagen expression. Oral administration of L-NAME enhanced the pro-fibrotic effect of BCG. Peritoneal macrophages obtained from MB49 tumor-bearing mice treated in vivo with combined treatment of BCG with L-NAME also enhanced fibroblast proliferation. We observed that FGF-2 is one of the factors released by BCG-activated macrophages that is able to induce fibroblast proliferation. The involvement of FGF-2 was evidenced using an anti-FGF2 antibody. At the same time, this macrophage population improved wound healing rate in normal mice and FGF-2 expression was also increased in these wounds. Conclusions/Significance Our findings suggest that fibroblasts are targeted by BCG both directly and through activated macrophages in an immunotherapy context of a bladder murine model. We also described, for the first time, that FGF-2 is involved in a dialog between fibroblasts and macrophages induced after BCG treatment. The fact that L-NAME administration improves the BCG effect on fibroblasts, NO inhibition, might represent a new approach to add to the conventional BCG therapy. PMID:21042580

  19. Angiotensin II increases CTGF expression via MAPKs/TGF-{beta}1/TRAF6 pathway in atrial fibroblasts

    SciTech Connect

    Gu, Jun; Liu, Xu; Wang, Quan-xing; Tan, Hong-wei; Guo, Meng; Jiang, Wei-feng; Zhou, Li

    2012-10-01

    The activation of transforming growth factor-{beta}1(TGF-{beta}1)/Smad signaling pathway and increased expression of connective tissue growth factor (CTGF) induced by angiotensin II (AngII) have been proposed as a mechanism for atrial fibrosis. However, whether TGF{beta}1/non-Smad signaling pathways involved in AngII-induced fibrogenetic factor expression remained unknown. Recently tumor necrosis factor receptor associated factor 6 (TRAF6)/TGF{beta}-associated kinase 1 (TAK1) has been shown to be crucial for the activation of TGF-{beta}1/non-Smad signaling pathways. In the present study, we explored the role of TGF-{beta}1/TRAF6 pathway in AngII-induced CTGF expression in cultured adult atrial fibroblasts. AngII (1 {mu}M) provoked the activation of P38 mitogen activated protein kinase (P38 MAPK), extracellular signal-regulated kinase 1/2(ERK1/2) and c-Jun NH(2)-terminal kinase (JNK). AngII (1 {mu}M) also promoted TGF{beta}1, TRAF6, CTGF expression and TAK1 phosphorylation, which were suppressed by angiotensin type I receptor antagonist (Losartan) as well as p38 MAPK inhibitor (SB202190), ERK1/2 inhibitor (PD98059) and JNK inhibitor (SP600125). Meanwhile, both TGF{beta}1 antibody and TRAF6 siRNA decreased the stimulatory effect of AngII on TRAF6, CTGF expression and TAK1 phosphorylation, which also attenuated AngII-induced atrial fibroblasts proliferation. In summary, the MAPKs/TGF{beta}1/TRAF6 pathway is an important signaling pathway in AngII-induced CTGF expression, and inhibition of TRAF6 may therefore represent a new target for reversing Ang II-induced atrial fibrosis. -- Highlights: Black-Right-Pointing-Pointer MAPKs/TGF{beta}1/TRAF6 participates in AngII-induced CTGF expression in atrial fibroblasts. Black-Right-Pointing-Pointer TGF{beta}1/TRAF6 participates in AngII-induced atrial fibroblasts proliferation. Black-Right-Pointing-Pointer TRAF6 may represent a new target for reversing Ang II-induced atrial fibrosis.

  20. A three-dimensional human model of the fibroblast activation that accompanies bronchopulmonary dysplasia identifies Notch-mediated pathophysiology.

    PubMed

    Sucre, Jennifer M S; Wilkinson, Dan; Vijayaraj, Preethi; Paul, Manash; Dunn, Bruce; Alva-Ornelas, Jackelyn A; Gomperts, Brigitte N

    2016-05-15

    Bronchopulmonary dysplasia (BPD) is a leading complication of premature birth and occurs primarily in infants delivered during the saccular stage of lung development. Histopathology shows decreased alveolarization and a pattern of fibroblast proliferation and differentiation to the myofibroblast phenotype. Little is known about the molecular pathways and cellular mechanisms that define BPD pathophysiology and progression. We have developed a novel three-dimensional human model of the fibroblast activation associated with BPD, and using this model we have identified the Notch pathway as a key driver of fibroblast activation and proliferation in response to changes in oxygen. Fetal lung fibroblasts were cultured on sodium alginate beads to generate lung organoids. After exposure to alternating hypoxia and hyperoxia, the organoids developed a phenotypic response characterized by increased α-smooth muscle actin (α-SMA) expression and other genes known to be upregulated in BPD and also demonstrated increased expression of downstream effectors of the Notch pathway. Inhibition of Notch with a γ-secretase inhibitor prevented the development of the pattern of cellular proliferation and α-SMA expression in our model. Analysis of human autopsy tissue from the lungs of infants who expired with BPD demonstrated evidence of Notch activation within fibrotic areas of the alveolar septae, suggesting that Notch may be a key driver of BPD pathophysiology. PMID:26968771

  1. Prostacyclin Analogue Beraprost Inhibits Cardiac Fibroblast Proliferation Depending on Prostacyclin Receptor Activation through a TGF β-Smad Signal Pathway

    PubMed Central

    Yao, Wenjuan; Zhu, Hongyan; Xu, Xiaole; Meng, Guoliang; Zhang, Wei

    2014-01-01

    Previous studies showed that prostacyclin inhibited fibrosis. However, both receptors of prostacyclin, prostacyclin receptor (IP) and peroxisome proliferator-activated receptor (PPAR), are abundant in cardiac fibroblasts. Here we investigated which receptor was vital in the anti-fibrosis effect of prostacyclin. In addition, the possible mechanism involved in protective effects of prostacyclin against cardiac fibrosis was also studied. We found that beraprost, a prostacyclin analogue, inhibited angiotensin II (Ang II)-induced neonatal rat cardiac fibroblast proliferation in a concentration-dependent and time-dependent manner. Beraprost also suppressed Ang II-induced collagen I mRNA expression and protein synthesis in cardiac fibroblasts. After IP expression was knocked down by siRNA, Ang II-induced proliferation and collagen I synthesis could no longer be rescued by beraprost. However, treating cells with different specific inhibitors of PPAR subtypes prior to beraprost and Ang II stimulation, all of the above attenuating effects of beraprost were still available. Moreover, beraprost significantly blocked transforming growth factor β (TGF β) expression as well as Smad2 phosphorylation and reduced Smad-DNA binding activity. Beraprost also increased phosphorylation of cAMP response element binding protein (CREB) at Ser133 in the nucleus. Co-immunoprecipitation analysis revealed that beraprost increased CREB but decreased Smad2 binding to CREB-binding protein (CBP) in nucleus. In conclusion, beraprost inhibits cardiac fibroblast proliferation by activating IP and suppressing TGF β-Smad signal pathway. PMID:24852754

  2. A potent immunotoxin targeting fibroblast activation protein for treatment of breast cancer in mice.

    PubMed

    Fang, Jinxu; Xiao, Liang; Joo, Kye-Il; Liu, Yarong; Zhang, Chupei; Liu, Shuanglong; Conti, Peter S; Li, Zibo; Wang, Pin

    2016-02-15

    Fibroblast activation protein (FAP) is highly expressed in the tumor-associated fibroblasts (TAFs) of most human epithelial cancers. FAP plays a critical role in tumorigenesis and cancer progression, which makes it a promising target for novel anticancer therapy. However, mere abrogation of FAP enzymatic activity by small molecules is not very effective in inhibiting tumor growth. In this study, we have evaluated a novel immune-based approach to specifically deplete FAP-expressing TAFs in a mouse 4T1 metastatic breast cancer model. Depletion of FAP-positive stromal cells by FAP-targeting immunotoxin αFAP-PE38 altered levels of various growth factors, cytokines, chemokines and matrix metalloproteinases, decreased the recruitment of tumor-infiltrating immune cells in the tumor microenvironment and suppressed tumor growth. In addition, combined treatment with αFAP-PE38 and paclitaxel potently inhibited tumor growth in vivo. Our findings highlight the potential use of immunotoxin αFAP-PE38 to deplete FAP-expressing TAFs and thus provide a rationale for the use of this immunotoxin in cancer therapy. PMID:26334777

  3. Basic calcium phosphate crystal-induced Egr-1 expression stimulates mitogenesis in human fibroblasts

    SciTech Connect

    Zeng, Xiao R.; Sun Yubo; Wenger, Leonor; Cheung, Herman S. . E-mail: hcheung@med.miami.edu

    2005-05-13

    Previously, we have reported that basic calcium phosphate (BCP) crystals stimulate mitogenesis and synthesis of matrix metalloproteinases in cultured human foreskin and synovial fibroblasts. However, the detailed mechanisms involved are still unclear. In the present study, using RT-PCR and Egr-1 promoter analysis we showed that BCP crystals could stimulate early growth response gene Egr-1 transcription through a PKC{alpha}-dependent p44/p42 MAPK pathway. Using a retrovirus gene expression system (Clontech) to overexpress Egr-1 in human fibroblast BJ-1 cells resulted in promotion of mitogenesis measured either by MTT cell proliferation analysis or by direct cell counting. The results demonstrate that Egr-1 may play a key role in mediating BCP crystal-induced synovial fibroblast mitogenesis.

  4. Fibroblast adhesion to recombinant tropoelastin expressed as a protein A-fusion protein.

    PubMed Central

    Grosso, L E; Parks, W C; Wu, L J; Mecham, R P

    1991-01-01

    A bovine tropoelastin cDNA encoding exons 15-36 that includes the elastin-receptor binding site was expressed in Escherichia coli as a fusion protein with Protein A from Staphylococcus aureus. After isolation of the fusion protein by affinity chromatography on Ig-Sepharose, the tropoelastin domain was separated from plasmid-pR1T2T-encoded Protein A (Protein A') by CNBr cleavage. Cell-adhesion assays demonstrated specific adhesion to the recombinant tropoelastin. Furthermore, the data indicate that interactions involving the bovine elastin receptor mediate nuchalligament fibroblast adhesion to the recombinant protein. In agreement with earlier studies of fibroblast chemotaxis to bovine tropoelastin, nuchal-ligament fibroblast adhesion demonstrated developmental regulation of the elastin receptor. Images Fig. 2. Fig. 3. PMID:1996952

  5. Significantly reduced expression of the proteoglycan decorin in Alzheimer's disease fibroblasts

    PubMed Central

    Brandan, Enrique; Melo, Francisco; García, María; Contreras, Maribel

    1996-01-01

    Aims—To investigate whether proteoglycan synthesis is altered in skin fibroblasts in patients with Alzheimer's disease compared with normal subjects. Methods—Cell lines obtained from donors with Alzheimer's disease and healthy controls were incubated with radioactive sulphate. The proteoglycans synthesised were determined and analysed by chromatographic, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and glycosaminoglycans-lyase treatment. The amount of decorin synthesised by each cell line was quantified using western blot analysis. Transcripts for human decorin were determined using northern blot analysis. Results—No significant changes in total sulphate incorporation and glycos-aminoglycan (GAG) composition were detected in the incubation media of these cells. However, chromatographic and SDS-PAGE analysis of the proteoglycans secreted by the cell lines showed that a dermatan sulphate proteoglycan of 150-125 kilodaltons was substantially reduced in Alzheimer's disease fibroblasts. The molecular characteristics of this proteoglycan correspond to decorin. Western blot analysis indicated that decorin was reduced in Alzheimer's disease incubation medium compared with normal medium. Northern blotting indicated that in Alzheimer's disease fibroblasts decorin transcripts were significantly reduced compared with normal fibroblasts. Glypican concentrations, a cell surface heparan sulphate proteoglycan, remained the same. Conclusions—These results strongly suggest that the expression and synthesis of decorin is affected in Alzheimer's disease skin fibroblasts. Images PMID:16696102

  6. Celecoxib suppresses fibroblast growth factor-2 expression in pancreatic ductal adenocarcinoma PANC-1 cells.

    PubMed

    Li, Jing; Luo, Miaosha; Wang, Yan; Shang, Boxin; Dong, Lei

    2016-09-01

    The inhibition of cyclooxygenase (COX)-2 has been reported to suppress growth and induce apoptosis in human pancreatic cancer cells. Nevertheless, the precise biological mechanism of how celecoxib, a selective COX-2 inhibitor, regulates the growth and invasion of pancreatic tumors is not completely understood. It has been shown that fibroblast growth factor-2 (FGF-2) and its receptor levels correlate with the inhibition of cancer cell proliferation, migration and invasion in pancreatic ductal adenocarcinoma (PDAC). Therefore, the aim of the present study was to examine the hypothesis that the antitumor activity of celecoxib in PDAC may be exerted through modulation of FGF-2 function. In the present study, we evaluated the effects of celecoxib on the proliferation, migration, invasion and apoptosis of the PANC-1 cell line. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were used to examine the expression of FGF-2, FGFR-2, ERK1/2 and MMPs. In the present study, FGF-2 and FGFR-2 were expressed in PANC-1 cells and FGF-2 exerted a stimulatory effect on phosphorylated extracellular signal regulated kinase (p-ERK) expression. Celecoxib treatment suppressed FGF-2 and FGFR-2 expression and decreased MMP-2, MMP-9 and p-ERK expression in the PANC-1 cells. Furthermore, celecoxib treatment caused the resistance of PANC-1 cells to FGF-2 induced proliferation, migration and invasion ability, as well as the increase in their apoptotic rate. Our data provide evidence that targeting FGF-2 with celecoxib may be used as an effective treatment in PDAC. PMID:27430377

  7. Effect of advanced glycation endproducts on gene expression profiles of human dermal fibroblasts.

    PubMed

    Molinari, J; Ruszova, E; Velebny, V; Robert, L

    2008-06-01

    The Maillard reaction and its end products, AGE-s (Advanced Glycation End products) are rightly considered as one of the important mechanisms of post-translational tissue modifications with aging. We studied the effect of two AGE-products prepared by the glycation of lysozyme and of BSA, on the expression profile of a large number of genes potentially involved in the above mentioned effects of AGE-s. The two AGE-products were added to human skin fibroblasts and gene expression profiles investigated using microarrays. Among the large number of genes monitored the expression of 16 genes was modified by each AGE-preparations, half of them only by both of them. Out of these 16 genes, 12 were more strongly affected, again not all the same for both preparations. Both of them upregulated MMP and serpin-expression and downregulated some of the collagen-chain coding genes, as well as the cadherin- and fibronectin genes. The BSA-AGE preparation downregulated 10 of the 12 genes strongly affected, only the serpin-1 and MMP-9 genes were upregulated. The lysozyme-AGE preparation upregulated selectively the genes coding for acid phosphatase (ACP), integrin chain alpha5 (ITGA5) and thrombospondin (THBS) which were unaffected by the BSA-AGE preparation. It was shown previously that the lysozyme-AGE strongly increased the rate of proliferation and also cell death, much more than the BSA-AGE preparation. These differences between these two AGE-preparations tested suggest the possibility of different receptor-mediated transmission pathways activated by these two preparations. Most of the gene-expression modifications are in agreement with biological effects of Maillard products, especially interference with normal tissue structure and increased tissue destruction. PMID:18297408

  8. Cancer Associated Fibroblasts express pro-inflammatory factors in human breast and ovarian tumors

    SciTech Connect

    Erez, Neta; Glanz, Sarah; Raz, Yael; Avivi, Camilla; Barshack, Iris

    2013-08-02

    Highlights: •CAFs in human breast and ovarian tumors express pro-inflammatory factors. •Expression of pro-inflammatory factors correlates with tumor invasiveness. •Expression of pro-inflammatory factors is associated with NF-κb activation in CAFs. -- Abstract: Inflammation has been established in recent years as a hallmark of cancer. Cancer Associated Fibroblasts (CAFs) support tumorigenesis by stimulating angiogenesis, cancer cell proliferation and invasion. We previously demonstrated that CAFs also mediate tumor-enhancing inflammation in a mouse model of skin carcinoma. Breast and ovarian carcinomas are amongst the leading causes of cancer-related mortality in women and cancer-related inflammation is linked with both these tumor types. However, the role of CAFs in mediating inflammation in these malignancies remains obscure. Here we show that CAFs in human breast and ovarian tumors express high levels of the pro-inflammatory factors IL-6, COX-2 and CXCL1, previously identified to be part of a CAF pro-inflammatory gene signature. Moreover, we show that both pro-inflammatory signaling by CAFs and leukocyte infiltration of tumors are enhanced in invasive ductal carcinoma as compared with ductal carcinoma in situ. The pro-inflammatory genes expressed by CAFs are known NF-κB targets and we show that NF-κB is up-regulated in breast and ovarian CAFs. Our data imply that CAFs mediate tumor-promoting inflammation in human breast and ovarian tumors and thus may be an attractive target for stromal-directed therapeutics.

  9. Bezafibrate upregulates carnitine palmitoyltransferase II expression and promotes mitochondrial energy crisis dissipation in fibroblasts of patients with influenza-associated encephalopathy.

    PubMed

    Yao, Min; Yao, Dengbin; Yamaguchi, Miyoko; Chida, Junji; Yao, Dengfu; Kido, Hiroshi

    2011-11-01

    Influenza-associated encephalopathy (IAE) is characterized by persistently high fever, febrile convulsions, severe brain edema and high mortality. We reported previously that a large proportion of patients with disabling or fatal IAE exhibit a thermolabile phenotype of compound variants for [1055T>G/F352C] and [1102G>A/V368I] of carnitine palmitoyltransferase II (CPT II) and mitochondrial energy crisis during high fever. In the present study, we studied the effect of bezafibrate, a hypolipidemic pan-agonist of peroxisome proliferator-activated receptor (PPAR), on CPT II expression and mitochondrial energy metabolism in fibroblasts of IAE patients and wild type (WT) fibroblasts from a healthy volunteer at 37°C and 41°C. Although heat stress markedly upregulated CPT II, CPT IA and PPAR-δ mRNA expression levels, CPT II activity, β-oxidation and ATP levels in WT and IAE fibroblasts at 41°C were paradoxically downregulated probably due to the thermal instability of the corresponding enzymes. Bezafibrate significantly enhanced the expression levels of the above mRNAs and cellular functions of these enzymes in fibroblasts at 37°C. Bezafibrate-induced increase in CPT II activity also tended to restore the downregulated ATP levels, though moderately, and improved mitochondrial membrane potential even at 41°C to the levels at 37°C in fibroblasts of IAE patients. L-carnitine, a substrate of CPT II, boosted the effects of bezafibrate on cellular ATP levels in WT and IAE fibroblasts, even in severe IAE fibroblasts with thermolabile compound variations of F352C+V368I at 37°C and 41°C. The results suggest the potential usefulness of bezafibrate for the treatment of IAE. PMID:21816645

  10. Sustained activation of fibroblast transforming growth factor-beta/Smad signaling in a murine model of scleroderma.

    PubMed

    Takagawa, Shinsuke; Lakos, Gabriella; Mori, Yasuji; Yamamoto, Toshiyuki; Nishioka, Kiyoshi; Varga, John

    2003-07-01

    Transforming growth factor-beta is responsible for triggering a cascade of events leading to fibrosis in scleroderma. The Smads are intracellular signal transducers recently shown to mediate fibroblast activation and other profibrotic responses elicited by transforming growth factor-betain vitro. To understand better the involvement of Smads in the pathogenesis of fibrosis, we examined Smad expression and activation in situ in a murine model of scleroderma. Bleomycin injections induced striking dermal infiltration with macrophages by 3 d, and progressive fibrosis by 2 wk. Infiltrating macrophages and resident fibroblasts expressed Smad3, the positive mediator for transforming growth factor-beta responses. Importantly, in bleomycin-injected skin, fibroblasts showed predominantly nuclear localization of Smad3 and intense staining for phospho-Smad2/3. Furthermore, phosphorylated Smad2/3 in fibroblasts was detected even after the resolution of inflammation. Expression of Smad7, the endogenous inhibitor of transforming growth factor-beta/Smad signaling, was strongly induced in dermal cells by transforming growth factor-beta, but not by bleomycin injections. Collectively, these results indicate that bleomycin-induced murine scleroderma is associated with rapid and sustained induction of transforming growth factor-beta/Smad signaling in resident dermal fibroblasts. Despite apparent activation of the intracellular transforming growth factor-beta signaling pathway in the lesional dermis, the expression of transforming growth factor-beta-inducible Smad7 was not upregulated. In light of the critical function of Smad7 as an endogenous inhibitor of Smad signaling that restricts the duration and magnitude of transforming growth factor-beta responses, and as a mediator of apoptosis, relative Smad7 deficiency observed in the present studies may account for sustained activation of transforming growth factor-beta/Smad signaling in lesional tissues. These findings raise the

  11. Alkaline pH activates the transport activity of GLUT1in L929 fibroblast cells

    PubMed Central

    Gunnink, Stephen M.; Kerk, Samuel A.; Kuiper, Benjamin D.; Alabi, Ola D.; Kuipers, David P.; Praamsma, Riemer C.; Wrobel, Kathryn E.; Louters, Larry L.

    2016-01-01

    The widely expressed mammalian glucose transporter, GLUT1, can be acutely activated in L929 fibroblast cells by a variety of conditions, including glucose deprivation, or treatment with various respiration inhibitors. Known thiol reactive compounds including phenylarsine oxide and nitroxyl are the fastest acting stimulators of glucose uptake, implicating cysteine biochemistry as critical to the acute activation of GLUT1. In this study, we report that in L929 cells glucose uptake increases 6-fold as the pH of the uptake solution is increased from 6 to 9 with the half-maximal activation at pH 7.5; consistent with the pKa of cysteine residues. This pH effect is essentially blocked by the pretreatment of the cells with either iodoacetamide or cinnamaldehyde, compounds that form covalent adducts with reduced cysteine residues. In addition, the activation by alkaline pH is not additive at pH 8 with known thiol reactive activators such as phenylarsine oxide or hydroxylamine. Kinetic analysis in L929 cells at pH 7 and 8 indicate that alkaline conditions both increases the Vmax and decreases the Km of transport. This is consistent with the observation that pH activation is additive to methylene blue, which activates uptake by increasing the Vmax, as well as to berberine, which activates uptake by decreasing the Km. This suggests that cysteine biochemistry is utilized in both methylene blue and berberine activation of glucose uptake. In contrast a pH increase from 7 to 8 in HCLE cells does not further activate glucose uptake. HCLE cells have a 25-fold higher basal glucose uptake rate than L929 cells and the lack of a pH effect suggests that the cysteine biochemistry has already occurred in HCLE cells. The data are consistent with pH having a complex mechanism of action, but one likely mediated by cysteine biochemistry. PMID:24333987

  12. Alkaline pH activates the transport activity of GLUT1 in L929 fibroblast cells.

    PubMed

    Gunnink, Stephen M; Kerk, Samuel A; Kuiper, Benjamin D; Alabi, Ola D; Kuipers, David P; Praamsma, Riemer C; Wrobel, Kathryn E; Louters, Larry L

    2014-04-01

    The widely expressed mammalian glucose transporter, GLUT1, can be acutely activated in L929 fibroblast cells by a variety of conditions, including glucose deprivation, or treatment with various respiration inhibitors. Known thiol reactive compounds including phenylarsine oxide and nitroxyl are the fastest acting stimulators of glucose uptake, implicating cysteine biochemistry as critical to the acute activation of GLUT1. In this study, we report that in L929 cells glucose uptake increases 6-fold as the pH of the uptake solution is increased from 6 to 9 with the half-maximal activation at pH 7.5; consistent with the pKa of cysteine residues. This pH effect is essentially blocked by the pretreatment of the cells with either iodoacetamide or cinnamaldehyde, compounds that form covalent adducts with reduced cysteine residues. In addition, the activation by alkaline pH is not additive at pH 8 with known thiol reactive activators such as phenylarsine oxide or hydroxylamine. Kinetic analysis in L929 cells at pH 7 and 8 indicate that alkaline conditions both increases the Vmax and decreases the Km of transport. This is consistent with the observation that pH activation is additive to methylene blue, which activates uptake by increasing the Vmax, as well as to berberine, which activates uptake by decreasing the Km. This suggests that cysteine biochemistry is utilized in both methylene blue and berberine activation of glucose uptake. In contrast a pH increase from 7 to 8 in HCLE cells does not further activate glucose uptake. HCLE cells have a 25-fold higher basal glucose uptake rate than L929 cells and the lack of a pH effect suggests that the cysteine biochemistry has already occurred in HCLE cells. The data are consistent with pH having a complex mechanism of action, but one likely mediated by cysteine biochemistry. PMID:24333987

  13. Control of fibroblast fibronectin expression and alternative splicing via the PI3K/Akt/mTOR pathway

    SciTech Connect

    White, Eric S.; Sagana, Rommel L.; Booth, Adam J.; Yan, Mei; Cornett, Ashley M.; Bloomheart, Christopher A.; Tsui, Jessica L.; Wilke, Carol A.; Moore, Bethany B.; Ritzenthaler, Jeffrey D.; Roman, Jesse; Muro, Andres F.

    2010-10-01

    Fibronectin (FN), a ubiquitous glycoprotein that plays critical roles in physiologic and pathologic conditions, undergoes alternative splicing which distinguishes plasma FN (pFN) from cellular FN (cFN). Although both pFN and cFN can be incorporated into the extracellular matrix, a distinguishing feature of cFN is the inclusion of an alternatively spliced exon termed EDA (for extra type III domain A). The molecular steps involved in EDA splicing are well-characterized, but pathways influencing EDA splicing are less clear. We have previously found an obligate role for inhibition of the tumor suppressor phosphatase and tensin homologue on chromosome 10 (PTEN), the primary regulator of the PI3K/Akt pathway, in fibroblast activation. Here we show TGF-{beta}, a potent inducer of both EDA splicing and fibroblast activation, inhibits PTEN expression and activity in mesenchymal cells, corresponding with enhanced PI3K/Akt signaling. In pten{sup -/-} fibroblasts, which resemble activated fibroblasts, inhibition of Akt attenuated FN production and decreased EDA alternative splicing. Moreover, inhibition of mammalian target of rapamycin (mTOR) in pten{sup -/-} cells also blocked FN production and EDA splicing. This effect was due to inhibition of Akt-mediated phosphorylation of the primary EDA splicing regulatory protein SF2/ASF. Importantly, FN silencing in pten{sup -/-} cells resulted in attenuated proliferation and migration. Thus, our results demonstrate that the PI3K/Akt/mTOR axis is instrumental in FN transcription and alternative splicing, which regulates cell behavior.

  14. Circulating FGF21 proteolytic processing mediated by fibroblast activation protein

    PubMed Central

    Zhen, Eugene Y.; Jin, Zhaoyan; Ackermann, Bradley L.; Thomas, Melissa K.; Gutierrez, Jesus A.

    2015-01-01

    Fibroblast growth factor 21 (FGF21), a hormone implicated in the regulation of glucose homoeostasis, insulin sensitivity, lipid metabolism and body weight, is considered to be a promising therapeutic target for the treatment of metabolic disorders. Despite observations that FGF21 is rapidly proteolysed in circulation rending it potentially inactive, little is known regarding mechanisms by which FGF21 protein levels are regulated. We systematically investigated human FGF21 protein processing using mass spectrometry. In agreement with previous reports, circulating human FGF21 was found to be cleaved primarily after three proline residues at positions 2, 4 and 171. The extent of FGF21 processing was quantified in a small cohort of healthy human volunteers. Relative abundance of FGF21 proteins cleaved after Pro-2, Pro-4 and Pro-171 ranged from 16 to 30%, 10 to 25% and 10 to 34%, respectively. Dipeptidyl peptidase IV (DPP-IV) was found to be the primary protease responsible for N-terminal cleavages after residues Pro-2 and Pro-4. Importantly, fibroblast activation protein (FAP) was implicated as the protease responsible for C-terminal cleavage after Pro-171, rendering the protein inactive. The requirement of FAP for FGF21 proteolysis at the C-terminus was independently demonstrated by in vitro digestion, immunodepletion of FAP in human plasma, administration of an FAP-specific inhibitor and by human FGF21 protein processing patterns in FAP knockout mouse plasma. The discovery that FAP is responsible for FGF21 inactivation extends the FGF21 signalling pathway and may enable novel approaches to augment FGF21 actions for therapeutic applications. PMID:26635356

  15. Gene expression signatures in motor neurone disease fibroblasts reveal dysregulation of metabolism, hypoxia-response and RNA processing functions

    PubMed Central

    Raman, R; Allen, S P; Goodall, E F; Kramer, S; Ponger, L-L; Heath, P R; Milo, M; Hollinger, H C; Walsh, T; Highley, J R; Olpin, S; McDermott, C J; Shaw, P J; Kirby, J

    2015-01-01

    Aims Amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS) are two syndromic variants within the motor neurone disease spectrum. As PLS and most ALS cases are sporadic (SALS), this limits the availability of cellular models for investigating pathogenic mechanisms and therapeutic targets. The aim of this study was to use gene expression profiling to evaluate fibroblasts as cellular models for SALS and PLS, to establish whether dysregulated biological processes recapitulate those seen in the central nervous system and to elucidate pathways that distinguish the clinically defined variants of SALS and PLS. Methods Microarray analysis was performed on fibroblast RNA and differentially expressed genes identified. Genes in enriched biological pathways were validated by quantitative PCR and functional assays performed to establish the effect of altered RNA levels on the cellular processes. Results Gene expression profiling demonstrated that whilst there were many differentially expressed genes in common between SALS and PLS fibroblasts, there were many more expressed specifically in the SALS fibroblasts, including those involved in RNA processing and the stress response. Functional analysis of the fibroblasts confirmed a significant decrease in miRNA production and a reduced response to hypoxia in SALS fibroblasts. Furthermore, metabolic gene changes seen in SALS, many of which were also evident in PLS fibroblasts, resulted in dysfunctional cellular respiration. Conclusions The data demonstrate that fibroblasts can act as cellular models for ALS and PLS, by establishing the transcriptional changes in known pathogenic pathways that confer subsequent functional effects and potentially highlight targets for therapeutic intervention. PMID:24750211

  16. Epstein-Barr virus infection induces aberrant TLR activation pathway and fibroblast-myofibroblast conversion in scleroderma.

    PubMed

    Farina, Antonella; Cirone, Mara; York, Michael; Lenna, Stefania; Padilla, Cristina; McLaughlin, Sarah; Faggioni, Alberto; Lafyatis, Robert; Trojanowska, Maria; Farina, Giuseppina A

    2014-04-01

    Scleroderma (SSc) is a complex and heterogeneous connective tissue disease mainly characterized by autoimmunity, vascular damage, and fibrosis that mostly involve the skin and lungs. Epstein-Barr virus (EBV) is a lymphotropic γ-herpesvirus that has co-evolved with human species, infecting >95% of the adult population worldwide, and has been a leading candidate in triggering several autoimmune diseases. Here we show that EBV establishes infection in the majority of fibroblasts and endothelial cells in the skin of SSc patients, characterized by the expression of the EBV noncoding small RNAs (EBERs) and the increased expression of immediate-early lytic and latency mRNAs and proteins. We report that EBV is able to persistently infect human SSc fibroblasts in vitro, inducing an aberrant innate immune response in infected cells. EBV-Toll-like receptor (TLR) aberrant activation induces the expression of selected IFN-regulatory factors (IRFs), IFN-stimulated genes (ISGs), transforming growth factor-β1 (TGFβ1), and several markers of fibroblast activation, such as smooth muscle actin and Endothelin-1, and all of these genes play a key role in determining the profibrotic phenotype in SSc fibroblasts. These findings imply that EBV infection occurring in mesenchymal, endothelial, and immune cells of SSc patients may underlie the main pathological features of SSc including autoimmunity, vasculopathy, and fibrosis, and provide a unified disease mechanism represented by EBV reactivation. PMID:24129067

  17. Expression of fibroblast specific protein-1 in pleural tuberculosis and its clinical biological significance

    PubMed Central

    2014-01-01

    Background Fibroblast specific protein-1 (S100A4) is related with many fibrotic diseases, but its role in the pathogenesis of pleural fibrosis has not been fully elucidated. Then we aim to investigate the expression and effect of fibroblast specific protein-1 (S100A4) in pleural tuberculosis and, subsequently, pleural fibrosis. Methods The expression of S100A4 in pleura was examined in 30 patients with pleural tuberculosis and 5 control (disease-free) patients by immunohistochemistry using the streptavidin-peroxidase (S-P) conjugated method. Results The expression of S100A4 in pleura was mainly distributed in the nucleus and cytoplasm of fibroblasts and vascular endothelial cells, and the positive rate was 90.0% (27 out of 30 patients with pleural tuberculosis). There were no expressions of S100A4 in the control group. In the pleura of all 30 patients with pleural tuberculosis, S100A4 had a higher expression in the two- to eight-week duration of the disease. Conclusions S100A4 plays an important role in the phenotypic transformation of pleural mesothelial cells and the development of pleural fibrosis. PMID:24885536

  18. Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

    SciTech Connect

    Malpass, Gloria E.; Arimilli, Subhashini; Prasad, G.L.; Howlett, Allyn C.

    2014-09-01

    Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1 h or 5 h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1 h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1 h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5 h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1 h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5 h), which was increased by nicotine but suppressed by other components of STE. Within 2 h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research. - Highlights: • Tobacco product preparations (TPPs) alter gene expression in dermal fibroblasts. • Some immediate early genes critical to the inflammatory process are affected. • Different TPPs produce differential responses in certain pro-inflammatory genes.

  19. Esophageal Squamous Cell Carcinoma Cells Modulate Chemokine Expression and Hyaluronan Synthesis in Fibroblasts.

    PubMed

    Kretschmer, Inga; Freudenberger, Till; Twarock, Sören; Yamaguchi, Yu; Grandoch, Maria; Fischer, Jens W

    2016-02-19

    The aim of this study was to characterize the interaction of KYSE-410, an esophageal squamous cell carcinoma cell line, and fibroblasts with respect to the extracellular matrix component hyaluronan (HA) and chemokine expression. KYSE-410 cells induced the mRNA expression of HA synthase 2 (Has2) in normal skin fibroblasts (SF) only in direct co-cultures. Parallel to Has2 mRNA, Has2 antisense RNA (Has2os2) was up-regulated in co-cultures. Knockdown of LEF1, a downstream target of Wnt signaling, abrogated Has2 and Has2os2 induction. After knockdown of Has2 in SF, significantly less α-smooth muscle actin expression was detected in co-cultures. Moreover, it was investigated whether the phenotype of KYSE-410 was affected in co-culture with SF and whether Has2 knockdown in SF had an impact on KYSE-410 cells in co-culture. However, no effects on epithelial-mesenchymal transition markers, proliferation, and migration were detected. In addition to Has2 mRNA, the chemokine CCL5 was up-regulated and CCL11 was down-regulated in SF in co-culture. Furthermore, co-cultures of KYSE-410 cells and cancer-associated fibroblasts (CAF) were investigated. Similar to SF, Has2 and Ccl5 were up-regulated and Ccl11 was down-regulated in CAF in co-culture. Importantly and in contrast to SF, inhibiting HA synthesis by 4-methylumbelliferone abrogated the effect of co-culture on Ccl5 in CAF. Moreover, HA was found to promote adhesion of CD4(+) but not CD8(+) cells to xenogaft tumor tissues. In conclusion, direct co-culture of esophageal squamous cell carcinoma and fibroblasts induced stromal HA synthesis via Wnt/LEF1 and altered the chemokine profile of stromal fibroblasts, which in turn may affect the tumor immune response. PMID:26699196

  20. Moutan Cortex Radicis inhibits inflammatory changes of gene expression in lipopolysaccharide-stimulated gingival fibroblasts.

    PubMed

    Yun, Cheol-Sang; Choi, Yeong-Gon; Jeong, Mi-Young; Lee, Je-Hyun; Lim, Sabina

    2013-07-01

    Moutan Cortex Radicis (MCR), the root bark of Paeonia suffruticosa Andrews (Paeoniaceae), is found in the traditional Chinese medicinal formulae which were used to treat periodontal diseases. This study investigated the changes in gene expression by MCR treatment when stimulated with lipopolysaccharide (LPS) in cultured human gingival fibroblasts (HGFs). A genome-wide expression GeneChip was used for the gene array analysis, and real-time reverse transcription polymerase chain reaction (RT-PCR) analysis was also performed to confirm the gene expression. It was shown that 42 of the 643 genes up-regulated by LPS, when compared to the control, were down-regulated by the MCR treatment. Of these 42 genes, the inflammation and immune response-related genes were especially noted, which indicates that MCR inhibits the induction of inflammation by LPS stimulation. In addition, 33 of the 519 genes down-regulated by LPS, when compared to the control, were up-regulated by the MCR treatment. The expression patterns of some representative genes by real-time RT-PCR correlated with those of the genes shown in the microarray. In addition, the MCR extract contained paeonol and paeoniflorin, which are known to have the anti-inflammatory effect as the major phenolic components of MCR. This study showed that the MCR extract could comprehensively inhibit a wide variety of activations of inflammation-related genes, which may be due to paeonol and paeoniflorin. It is, thus, suggested that MCR may be applied to alleviate the inflammation of periodontal diseases. PMID:23086154

  1. Human FGF-21 Is a Substrate of Fibroblast Activation Protein

    PubMed Central

    Coppage, Andrew L.; Heard, Kathryn R.; DiMare, Matthew T.; Liu, Yuxin; Wu, Wengen; Lai, Jack H.; Bachovchin, William W.

    2016-01-01

    FGF-21 is a key regulator of metabolism and potential drug candidate for the treatment of type II diabetes and other metabolic disorders. However, the half-life of active, circulating, human FGF-21 has recently been shown to be limited in mice and monkeys by a proteolytic cleavage between P171 and S172. Here, we show that fibroblast activation protein is the enzyme responsible for this proteolysis by demonstrating that purified FAP cleaves human FGF-21 at this site in vitro, and that an FAP-specific inhibitor, ARI-3099, blocks the activity in mouse, monkey and human plasma and prolongs the half-life of circulating human FGF-21 in mice. Mouse FGF-21, however, lacks the FAP cleavage site and is not cleaved by FAP. These findings indicate FAP may function in the regulation of metabolism and that FAP inhibitors may prove useful in the treatment of diabetes and metabolic disorders in humans, but pre-clinical proof of concept studies in rodents will be problematic. PMID:26962859

  2. Human FGF-21 Is a Substrate of Fibroblast Activation Protein.

    PubMed

    Coppage, Andrew L; Heard, Kathryn R; DiMare, Matthew T; Liu, Yuxin; Wu, Wengen; Lai, Jack H; Bachovchin, William W

    2016-01-01

    FGF-21 is a key regulator of metabolism and potential drug candidate for the treatment of type II diabetes and other metabolic disorders. However, the half-life of active, circulating, human FGF-21 has recently been shown to be limited in mice and monkeys by a proteolytic cleavage between P171 and S172. Here, we show that fibroblast activation protein is the enzyme responsible for this proteolysis by demonstrating that purified FAP cleaves human FGF-21 at this site in vitro, and that an FAP-specific inhibitor, ARI-3099, blocks the activity in mouse, monkey and human plasma and prolongs the half-life of circulating human FGF-21 in mice. Mouse FGF-21, however, lacks the FAP cleavage site and is not cleaved by FAP. These findings indicate FAP may function in the regulation of metabolism and that FAP inhibitors may prove useful in the treatment of diabetes and metabolic disorders in humans, but pre-clinical proof of concept studies in rodents will be problematic. PMID:26962859

  3. Expression of fibrocyte markers by keloid fibroblasts: an insight into fibrosis during burn wound healing - a preliminary study

    PubMed Central

    Mathangi Ramakrishnan, K.; Meenakshi Janakiraman, M.; Babu, M.

    2012-01-01

    Summary In extensive burns it becomes difficult for fibroblasts to migrate from the periphery of the healthy tissue and colonize the injured area. Even under such circumstances healing takes place, and this is attributed to the differentiation of circulating fibrocytes which enter the wound site. This normal cell type is identified in keloid fibroblasts: it expresses fibrocyte markers and secretes extra cellular matrix proteins. In-vitro collagen contraction assay reveals that fibrocytes contract collagen gels with an efficacy similar to normal fibroblasts. The contribution of fibrocytes to the formation of keloid fibroblasts in post-burn healing is discussed. PMID:23467263

  4. Increased expression of matrix metalloproteinases in vivo in scleritis tissue and in vitro in cultured human scleral fibroblasts.

    PubMed Central

    Di Girolamo, N.; Lloyd, A.; McCluskey, P.; Filipic, M.; Wakefield, D.

    1997-01-01

    Scleritis is a sight-threatening inflammatory disorder of the eye characterized by the degradation of scleral matrix. Matrix metalloproteinases (MMPs) are ubiquitous proteolytic enzymes important in physiological and pathological processes, the activity of which is stringently controlled by the action of a family of natural antagonists, the tissue inhibitors of matrix metalloproteinases (TIMPs). We hypothesized that enhanced expression of MMPs, without the negative regulatory influence of TIMPs, may be a key feature of tissue destruction in inflammatory eye diseases, such as scleritis. The aim of this study was to localize and characterize cells expressing MMPs and TIMPs in sclera affected by necrotizing scleritis and, in a parallel study, to establish whether cytokines modulate MMP expression in cultured human scleral fibroblasts. In situ hybridization and immunohistochemical analyses indicated that resident scleral fibroblasts as well as inflammatory cells such as macrophages and T lymphocytes express stromelysin, gelatinase B, and TIMP-1 in necrotizing scleritis tissue. In addition, cytoplasmic immunoreactivity for tumor necrosis factor-alpha, an inducer of MMPs, was detected in infiltrating inflammatory cells. Cultured scleral fibroblasts stimulated with the combination of interleukin-1 alpha plus tumor necrosis factor-alpha increased TIMP-1 mRNA twofold above constitutive levels. By contrast, these cytokines induced a sevenfold increase in the steady-state levels of stromelysin mRNA. Using Western blotting, stromelysin and TIMP-1 protein production paralleled mRNA induction in cytokine-stimulated human scleral fibroblasts. Culture supernatants harvested from cytokine-stimulated human scleral fibroblasts were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis gelatin substrate zymography. Our results revealed a prominent 92-kd gelatinolytic band corresponding to gelatinase B, which was inducible with interleukin-1 alpha. These data provide

  5. Apoptosis signal-regulating kinase 1 is mediated in TNF-α-induced CCL2 expression in human synovial fibroblasts.

    PubMed

    Tsou, Hsi-Kai; Chen, Hsien-Te; Chang, Chia-Hao; Yang, Wan-Yu; Tang, Chih-Hsin

    2012-11-01

    Tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine with a critical role in osteoarthritis (OA), was primarily produced by monocytes/macrophages and plays a crucial role in the inflammatory response. Here, we investigated the intracellular signaling pathways involved in TNF-α-induced monocyte chemoattractant protein 1 (MCP-1)/CCL2 expression in human synovial fibroblast cells. Stimulation of synovial fibroblasts (OASF) with TNF-α induced concentration- and time-dependent increases in CCL2 expression. TNF-α-mediated CCL2 production was attenuated by TNFR1 monoclonal antibody (Ab). Pretreatment with an apoptosis signal-regulating kinase 1 (ASK1) inhibitor (thioredoxin), JNK inhibitor (SP600125), p38 inhibitor (SB203580), or AP-1 inhibitor (curcumin or tanshinone IIA) also blocked the potentiating action of TNF-α. Stimulation of cells with TNF-α enhanced ASK1, JNK, and p38 activation. Treatment of OASF with TNF-α also increased the accumulation of phosphorylated c-Jun in the nucleus, AP-1-luciferase activity, and c-Jun binding to the AP-1 element on the CCL2 promoter. TNF-α-mediated AP-1-luciferase activity and c-Jun binding to the AP-1 element were inhibited by TNFR1 Ab, thioredoxin, SP600125, and SB203580. Our results suggest that the interaction between TNF-α and TNFR1 increases CCL2 expression in human synovial fibroblasts via the ASK1, JNK/p38, c-Jun, and AP-1 signaling pathway. PMID:22711527

  6. Expression of mutant and wild-type TIMP3 in primary gingival fibroblasts from Sorsby's fundus dystrophy patients.

    PubMed

    Arris, Christine E; Bevitt, Debra J; Mohamed, Jeseem; Li, Zheng; Langton, Kevin P; Barker, Michael D; Clarke, Michael P; McKie, Norman

    2003-05-20

    Gingival fibroblast cell lines were derived from Sorsby's fundus dystrophy (SFD) patients carrying the S181C TIMP3 and the E139X TIMP3 mutations. These cell lines were grown in culture to study expression of the wild-type and mutant tissue inhibitor of metalloproteinase 3 (TIMP3) alleles from a normal diploid cell type. Firstly, patient cells were found to co-express the wild-type and mutant TIMP3 alleles, S181C TIMP3 or E139X TIMP3, at the mRNA level using restriction fragment length polymorphism (RFLP) analysis. A SpeI RFLP for E139X TIMP3 is described. Low levels of endogenous TIMP3 protein expression were elevated using the natural polysaccharide calcium pentosan polysulfate (CaPPs) in combination with the cytokine IL-1alpha. Immunoblotting detected protein expression from both wild-type and mutant alleles, S181C TIMP3 or E139X TIMP3. S181C TIMP3 from these cells was found to dimerise and retain MMP2 inhibitory activity. To facilitate studies of the E139X TIMP3 protein, the allele was expressed using HighFive insect cells. In this cell type, the E139X TIMP3 was synthesised as a mixture of monomer and dimer. Both monomeric and dimeric E139X TIMP3 protein retained MMP2 inhibitory activity in gelatin zymography. Expression of mutant E139X or S181C TIMP3 protein from a normal diploid patient-derived fibroblast cell had no effect on either MMP2 or MMP9 expression or activation whilst transcribed from their normal promoter context. PMID:12757930

  7. Microvesicles shed by oligodendroglioma cells and rheumatoid synovial fibroblasts contain aggrecanase activity.

    PubMed

    Lo Cicero, Alessandra; Majkowska, Iwona; Nagase, Hideaki; Di Liegro, Italia; Troeberg, Linda

    2012-05-01

    Membrane microvesicle shedding is an active process and occurs in viable cells with no signs of apoptosis or necrosis. We report here that microvesicles shed by oligodendroglioma cells contain an 'aggrecanase' activity, cleaving aggrecan at sites previously identified as targets for adamalysin metalloproteinases with disintegrin and thrombospondin domains (ADAMTSs). Degradation was inhibited by EDTA, the metalloproteinase inhibitor GM6001 and by tissue inhibitor of metalloproteinases (TIMP)-3, but not by TIMP-1 or TIMP-2. This inhibitor profile indicates that the shed microvesicles contain aggrecanolytic ADAMTS(s) or related TIMP-3-sensitive metalloproteinase(s). The oligodendroglioma cells were shown to express the three most active aggrecanases, namely Adamts1, Adamts4 and Adamts5, suggesting that one or more of these enzymes may be responsible for the microvesicle activity. Microvesicles shed by rheumatoid synovial fibroblasts similarly degraded aggrecan in a TIMP-3-sensitive manner. Our findings raise the novel possibility that microvesicles may assist oligodendroglioma and rheumatoid synovial fibroblasts to invade through aggrecan-rich extracellular matrices. PMID:22406378

  8. Differential β3 Integrin Expression Regulates the Response of Human Lung and Cardiac Fibroblasts to Extracellular Matrix and Its Components

    PubMed Central

    Merna, Nick; Fung, Kelsey M.; Wang, Jean J.; King, Cristi R.; Hansen, Kirk C.; Christman, Karen L.

    2015-01-01

    Extracellular matrix (ECM) derived from whole organ decellularization has been successfully used in a variety of tissue engineering applications. ECM contains a complex mixture of functional and structural molecules that are ideally suited for the tissue from which the ECM is harvested. However, decellularization disrupts the structural properties and protein composition of the ECM, which may impact function when cells such as the fibroblast are reintroduced during recellularization. We hypothesized that the ECM structure and composition, fibroblast source, and integrin expression would influence the fibroblast phenotype. Human cardiac fibroblasts (HCFs) and normal human lung fibroblasts (NHLFs) were cultured on intact cardiac ECM, collagen gels, and coatings composed of cardiac ECM, lung ECM, and individual ECM components (collagen and fibronectin [FN]) for 48 h. COL1A expression of HCFs and NHLFs cultured on ECM and FN coatings decreased to <50% of that of untreated cells; COL1A expression for HCFs cultured on ECM coatings was one- to twofold higher than HCFs cultured on intact ECM. NHLFs cultured on ECM and FN coatings expressed 12- to 31-fold more alpha-smooth muscle actin (αSMA) than HCFs; the αSMA expression for HCFs and NHLFs cultured on ECM coatings was ∼2- to 5-fold higher than HCFs and NHLFs cultured on intact ECM. HCFs expressed significantly higher levels of β3 and β4 integrins when compared to NHLFs. Inhibition of the β3 integrin, but not β4, resulted in a 16- to 26-fold increase in αSMA expression in HCFs cultured on ECM coatings and FN. Our results demonstrate that β3 integrin expression depends on the source of the fibroblast and that its expression inhibits αSMA expression (and thus the myofibroblast phenotype). We conclude that the fibroblast source and integrin expression play important roles in regulating the fibroblast phenotype. PMID:25926101

  9. Differential β3 Integrin Expression Regulates the Response of Human Lung and Cardiac Fibroblasts to Extracellular Matrix and Its Components.

    PubMed

    Merna, Nick; Fung, Kelsey M; Wang, Jean J; King, Cristi R; Hansen, Kirk C; Christman, Karen L; George, Steven C

    2015-08-01

    Extracellular matrix (ECM) derived from whole organ decellularization has been successfully used in a variety of tissue engineering applications. ECM contains a complex mixture of functional and structural molecules that are ideally suited for the tissue from which the ECM is harvested. However, decellularization disrupts the structural properties and protein composition of the ECM, which may impact function when cells such as the fibroblast are reintroduced during recellularization. We hypothesized that the ECM structure and composition, fibroblast source, and integrin expression would influence the fibroblast phenotype. Human cardiac fibroblasts (HCFs) and normal human lung fibroblasts (NHLFs) were cultured on intact cardiac ECM, collagen gels, and coatings composed of cardiac ECM, lung ECM, and individual ECM components (collagen and fibronectin [FN]) for 48 h. COL1A expression of HCFs and NHLFs cultured on ECM and FN coatings decreased to <50% of that of untreated cells; COL1A expression for HCFs cultured on ECM coatings was one- to twofold higher than HCFs cultured on intact ECM. NHLFs cultured on ECM and FN coatings expressed 12- to 31-fold more alpha-smooth muscle actin (αSMA) than HCFs; the αSMA expression for HCFs and NHLFs cultured on ECM coatings was ∼2- to 5-fold higher than HCFs and NHLFs cultured on intact ECM. HCFs expressed significantly higher levels of β3 and β4 integrins when compared to NHLFs. Inhibition of the β3 integrin, but not β4, resulted in a 16- to 26-fold increase in αSMA expression in HCFs cultured on ECM coatings and FN. Our results demonstrate that β3 integrin expression depends on the source of the fibroblast and that its expression inhibits αSMA expression (and thus the myofibroblast phenotype). We conclude that the fibroblast source and integrin expression play important roles in regulating the fibroblast phenotype. PMID:25926101

  10. E2a-Pbx1 induces aberrant expression of tissue-specific and developmentally regulated genes when expressed in NIH 3T3 fibroblasts.

    PubMed Central

    Fu, X; Kamps, M P

    1997-01-01

    The E2a-Pbx1 oncoprotein contains the transactivation domain of E2a joined to the DNA-binding homeodomain (HD) of Pbx1. In mice, E2a-Pbx1 transforms T lymphoblasts and fibroblasts and blocks myeloblast differentiation. Pbx1 and E2a-Pbx1 bind DNA as heterodimers with other HD proteins whose expression is tissue specific. While the transactivation domain of E2a is required for all forms of transformation, DNA binding by the Pbx1 HD is essential for blocking myeloblast differentiation but dispensable for fibroblast or T-lymphoblast transformation. These properties suggest (i) that E2a-Pbx1 causes cellular transformation by activating gene transcription, (ii) that transcription of E2a-Pbx1 target genes is normally regulated by ubiquitous Pbx proteins and tissue-specific partners, and (iii) that DNA-binding mutants of E2a-Pbx1 activate a subset of all gene targets. To test these predictions, genes induced in NIH 3T3 fibroblasts by E2a-Pbx1 were identified and examined for tissue- and stage-specific expression and their differential abilities to be upregulated by E2a-Pbx1 in NIH 3T3 fibroblasts and myeloblasts and by a DNA-binding mutant of E2a-Pbx1 in NIH 3T3 cells. Of 12 RNAs induced by E2a-Pbx1, 4 encoded known proteins (a J-C region of the immunoglobulin kappa light chain, natriuretic peptide receptor C, mitochondrial fumarase, and the 3',5'-cyclic nucleotide phosphodiesterase, PDE1A) and 5 encoded new proteins related to angiogenin, ion channels, villin, epidermal growth factor repeat proteins, and the human 2.19 gene product. Expression of many of these genes was tissue specific or developmentally regulated, and most were not expressed in fibroblasts, indicating that E2a-Pbx1 can induce ectopic expression of genes associated with lineage-specific differentiation. PMID:9032278

  11. Normal Expression of a Rearranged and Mutated c-myc Oncogene after Transfection into Fibroblasts

    NASA Astrophysics Data System (ADS)

    Richman, Adam; Hayday, Adrian

    1989-10-01

    Expression of the c-myc oncogene is deregulated in a variety of malignancies. Rearrangement and mutation of the c-myc locus is a characteristic feature of human Burkitt's lymphoma. Whether deregulation is solely a result of mutation of c-myc or whether it is influenced by the transformed B cell context has not been determined. A translocated and mutated allele of c-myc was stably transfected into fibroblasts. The rearranged allele was expressed indistinguishably from a normal c-myc gene: it had serum-regulated expression, was transcribed with normal promoter preference, and was strongly attenuated. Thus mutations by themselves are insufficient to deregulate c-myc transcription.

  12. Disentangling the multifactorial contributions of fibronectin, collagen and cyclic strain on MMP expression and extracellular matrix remodeling by fibroblasts.

    PubMed

    Zhang, Yang; Lin, Zhe; Foolen, Jasper; Schoen, Ingmar; Santoro, Alberto; Zenobi-Wong, Marcy; Vogel, Viola

    2014-11-01

    Early wound healing is associated with fibroblasts assembling a provisional fibronectin-rich extracellular matrix (ECM), which is subsequently remodeled and interlaced by type I collagen. This exposes fibroblasts to time-variant sets of matrices during different stages of wound healing. Our goal was thus to gain insight into the ECM-driven functional regulation of human foreskin fibroblasts (HFFs) being either anchored to a fibronectin (Fn) or to a collagen-decorated matrix, in the absence or presence of cyclic mechanical strain. While the cells reoriented in response to the onset of uniaxial cyclic strain, cells assembled exogenously added Fn with a preferential Fn-fiber alignment along their new orientation. Exposure of HFFs to exogenous Fn resulted in an increase in matrix metalloproteinase (MMP) expression levels, i.e. MMP-15 (RT-qPCR), and MMP-9 activity (zymography), while subsequent exposure to collagen slightly reduced MMP-15 expression and MMP-9 activity compared to Fn-exposure alone. Cyclic strain upregulated Fn fibrillogenesis and actin stress fiber formation, but had comparatively little effect on MMP activity. We thus propose that the appearance of collagen might start to steer HFFs towards homeostasis, as it decreased both MMP secretion and the tension of Fn matrix fibrils as assessed by Fluorescence Resonance Energy Transfer. These results suggest that HFFs might have a high ECM remodeling or repair capacity in contact with Fn alone (early event), which is reduced in the presence of Col1 (later event), thereby down-tuning HFF activity, a processes which would be required in a tissue repair process to finally reach tissue homeostasis. PMID:25217861

  13. Suppression of autocrine and paracrine functions of basic fibroblast growth factor by stable expression of perlecan antisense cDNA.

    PubMed Central

    Aviezer, D; Iozzo, R V; Noonan, D M; Yayon, A

    1997-01-01

    Heparan sulfate proteoglycans (HSPG) play a critical role in the formation of distinct fibroblast growth factor (FGF)-HS complexes, augmenting high-affinity binding and receptor activation. Perlecan, a secreted HSPG abundant in proliferating cells, is capable of inducing FGF-receptor interactions in vitro and angiogenesis in vivo. Stable and specific reduction of perlecan levels in mouse NIH 3T3 fibroblasts and human metastatic melanoma cells has been achieved by expression of antisense cDNA corresponding to the N-terminal and HS attachment domains of perlecan. Long-term perlecan downregulation is evidenced by reduced levels of perlecan mRNA and core protein as indicated by Northern blot analysis, immunoblots, and immunohistochemistry, using DNA probes and antibodies specific to mouse or human perlecan. The response of antisense perlecan-expressing cells to increasing concentrations of basic FGF (bFGF) is dramatically reduced in comparison to that in wild-type or vector-transfected cells, as measured by thymidine incorporation and rate of proliferation. Furthermore, receptor binding and affinity labeling of antisense perlecan-transfected cells with 125I-bFGF is markedly inhibited, indicating that eliminating perlecan expression results in reduced high-affinity bFGF binding. Both the binding and mitogenic response of antisense-perlecan-expressing clones to bFGF can be rescued by exogenous heparin or perlecan. These results support the notion that perlecan is a major accessory receptor for bFGF in mouse fibroblasts and human melanomas and point to the possible use of perlecan antisense constructs as specific modulators of bFGF-mediated responses. PMID:9121441

  14. Proteins and endotoxin in house dust mite extracts modulate cytokine secretion and gene expression by dermal fibroblasts.

    PubMed

    Rockwood, Jananie; Morgan, Marjorie S; Arlian, Larry G

    2013-11-01

    House dust mite extracts used for diagnostic tests and immunotherapy contain bioreactive molecules including proteins and endotoxin. These extracts can influence the cytokine secretion and adhesion molecule expression by cells in the skin and lung airways. The aim of this study was to determine the role of proteins and endotoxin in mite extracts in modulating gene expression and cytokine secretion by human dermal fibroblasts. Cultured normal human dermal fibroblasts were stimulated with whole mite extracts, mite extracts boiled to denature proteins, or mite extracts treated with polymyxin B to inactivate lipopolysaccharide. Gene expression and secretion of interleukin-6 (IL-6), IL-8, and monocyte chemoattractant protein-1 (MCP-1) were determined after 6 h of stimulation. Whole Dermatophagoides farinae, D. pteronyssinus and Euroglyphus maynei extracts induced dose-dependent IL-6 and IL-8 secretion. In addition, D. farinae and E. maynei induced secretion of MCP-1. Dermatophagoides farinae and E. maynei also induced parallel cytokine gene expression. Cells stimulated with boiled D. farinae extract showed moderate to marked reductions in IL-6 and IL-8 secretion. In contrast, boiled D. pteronyssinus and E. maynei extracts induced equal or greater cytokine secretions than untreated extracts. The stimulating properties were reduced for all three extracts following treatment with polymyxin B. Our data suggest that both endotoxin and proteins in mite extracts modulate the secretion of cytokines by dermal fibroblasts. The biological activities of D. farinae, D. pteronyssinus, and E. maynei extracts are not equivalent. There appears to be a lipopolysaccharide-binding protein in some mite extracts. PMID:23640713

  15. Activation of a muscle-specific actin gene promoter in serum-stimulated fibroblasts.

    PubMed Central

    Stoflet, E S; Schmidt, L J; Elder, P K; Korf, G M; Foster, D N; Strauch, A R; Getz, M J

    1992-01-01

    Treatment of AKR-2B mouse fibroblasts with serum growth factors or inhibitors of protein synthesis, such as cycloheximide, results in a stimulation of cytoskeletal beta-actin transcription but has no effect on transcription of muscle-specific isotypes, such as the vascular smooth muscle (VSM) alpha-actin gene. Deletion mapping and site-specific mutagenesis studies demonstrated that a single "CArG" element of the general form CC(A/T)6GG was necessary and possibly sufficient to impart serum and cycloheximide-inducibility to the beta-actin promoter. Although the VSM alpha-actin promoter exhibits at least three similar sequence elements, it remained refractory to serum and cycloheximide induction. However, deletion of a 33 base pair sequence between -191 and -224 relative to the transcription start site resulted in the transcriptional activation of this muscle-specific promoter in rapidly growing or serum-stimulated fibroblasts. Although the activity of this truncated promoter was potentiated by cycloheximide in a manner indistinguishable from that of the beta-actin promoter, this was dependent on a more complex array of interacting elements. These included at least one CArG box and a putative upstream activating element closely associated with the -191 to -224 inhibitory sequences. These results demonstrate that the expression of a muscle-specific actin gene in fibroblasts is suppressed by a cis-acting negative control element and that in the absence of this element, the promoter is responsive to growth factor-induced signal transduction pathways. Images PMID:1421567

  16. Efficient inhibition of fibroblast proliferation and collagen expression by ERK2 siRNAs

    SciTech Connect

    Li, Fengfeng; Fan, Cunyi; Cheng, Tao; Jiang, Chaoyin; Zeng, Bingfang

    2009-05-01

    Transforming growth factor-{beta}1 and fibroblast growth factor-2 play very important roles in fibroblast proliferation and collagen expression. These processes lead to the formation of joint adhesions through the SMAD and MAPK pathways, in which ERK2 is supposed to be crucial. Based on these assumptions, lentivirus (LV)-mediated small interfering RNAs (siRNAs) targeting ERK2 were used to suppress the proliferation and collagen expression of rat joint adhesion tissue fibroblasts (RJATFs). Among four siRNAs examined, siRNA1 caused an 84% reduction in ERK2 expression (p < 0.01) and was selected as the most efficient siRNA for use in this study. In subsequent experiments, significant downregulation of types I and III collagen were observed by quantitative RT-PCR and Western blot analyses. MTT assays and flow cytometry revealed marked inhibition of RJATF proliferation, but no apoptosis. In conclusion, LV-mediated ERK2 siRNAs may represent novel therapies or drug targets for preventing joint adhesion formation.

  17. Co-Culture of Tumor Spheroids and Fibroblasts in a Collagen Matrix-Incorporated Microfluidic Chip Mimics Reciprocal Activation in Solid Tumor Microenvironment.

    PubMed

    Jeong, Su-Yeong; Lee, Ji-Hyun; Shin, Yoojin; Chung, Seok; Kuh, Hyo-Jeong

    2016-01-01

    Multicellular 3D culture and interaction with stromal components are considered essential elements in establishing a 'more clinically relevant' tumor model. Matrix-embedded 3D cultures using a microfluidic chip platform can recapitulate the microscale interaction within tumor microenvironments. As a major component of tumor microenvironment, cancer-associated fibroblasts (CAFs) play a role in cancer progression and drug resistance. Here, we present a microfluidic chip-based tumor tissue culture model that integrates 3D tumor spheroids (TSs) with CAF in proximity within a hydrogel scaffold. HT-29 human colorectal carcinoma cells grew into 3D TSs and the growth was stimulated when co-cultured with fibroblasts as shown by 1.5-folds increase of % changes in diameter over 5 days. TS cultured for 6 days showed a reduced expression of Ki-67 along with increased expression of fibronectin when co-cultured with fibroblasts compared to mono-cultured TSs. Fibroblasts were activated under co-culture conditions, as demonstrated by increases in α-SMA expression and migratory activity. When exposed to paclitaxel, a survival advantage was observed in TSs co-cultured with activated fibroblasts. Overall, we demonstrated the reciprocal interaction between TSs and fibroblasts in our 7-channel microfluidic chip. The co-culture of 3D TS-CAF in a collagen matrix-incorporated microfluidic chip may be useful to study the tumor microenvironment and for evaluation of drug screening and evaluation. PMID:27391808

  18. Co-Culture of Tumor Spheroids and Fibroblasts in a Collagen Matrix-Incorporated Microfluidic Chip Mimics Reciprocal Activation in Solid Tumor Microenvironment

    PubMed Central

    Jeong, Su-Yeong; Lee, Ji-Hyun; Shin, Yoojin; Chung, Seok; Kuh, Hyo-Jeong

    2016-01-01

    Multicellular 3D culture and interaction with stromal components are considered essential elements in establishing a ‘more clinically relevant’ tumor model. Matrix-embedded 3D cultures using a microfluidic chip platform can recapitulate the microscale interaction within tumor microenvironments. As a major component of tumor microenvironment, cancer-associated fibroblasts (CAFs) play a role in cancer progression and drug resistance. Here, we present a microfluidic chip-based tumor tissue culture model that integrates 3D tumor spheroids (TSs) with CAF in proximity within a hydrogel scaffold. HT-29 human colorectal carcinoma cells grew into 3D TSs and the growth was stimulated when co-cultured with fibroblasts as shown by 1.5-folds increase of % changes in diameter over 5 days. TS cultured for 6 days showed a reduced expression of Ki-67 along with increased expression of fibronectin when co-cultured with fibroblasts compared to mono-cultured TSs. Fibroblasts were activated under co-culture conditions, as demonstrated by increases in α-SMA expression and migratory activity. When exposed to paclitaxel, a survival advantage was observed in TSs co-cultured with activated fibroblasts. Overall, we demonstrated the reciprocal interaction between TSs and fibroblasts in our 7-channel microfluidic chip. The co-culture of 3D TS-CAF in a collagen matrix-incorporated microfluidic chip may be useful to study the tumor microenvironment and for evaluation of drug screening and evaluation. PMID:27391808

  19. Glucocorticoid coordinate regulation of type I procollagen gene expression and procollagen DNA-binding proteins in chick skin fibroblasts

    SciTech Connect

    Cockayne, D.; Cutroneo, K.R.

    1988-04-19

    Nuclei were isolated from control and dexamethasone-treated (2 h) embryonic chick skin fibroblasts and transcribed in vitro. Nuclei isolated from dexamethasone-treated fibroblasts transcribed less pro..cap alpha..1(I) and pro..cap alpha..2(I) mRNAs but not ..beta..-actin mRNA. Fibroblasts receiving dexamethasone and (5,6-/sup 3/H)uridine also demonstrated decreased synthesis of nuclear type I procollagen mRNAs but not ..beta..-actin mRNA. In fibroblasts treated with cycloheximide the newly synthesized nuclear type I procollagen mRNA species were markedly decreased. An enhanced inhibitory effect was observed when fibroblasts were treated with cycloheximide plus dexamethasone. Since the studies above demonstrate that active protein synthesis is required to maintain the constitutive expression of the type I procollagen genes, the authors determined if glucocorticoids regulate DNA-binding proteins with sequence specificity for the ..cap alpha..2(I) procollagen gene. Nuclear protein blots were probed with the /sup 32/P-end-labeled pBR322 vector DNA and /sup 32/P-end-labeled ..cap alpha..2(I) procollagen promoter containing DNA. Nonhistone proteins remained bound to labeled DNA at stringency washes of 0.05 and 0.1 M NaCl. As the ionic strength was increased to 0.2 and 0.3 M NaCl, the nonhistone-protein DNA binding was preferentially lost. Only the low molecular weight proteins remained bound to labeled DNA at the highest ionic strength, indicating nonspecific binding of these nuclear proteins. Dexamethasone treatment resulted in an increase of binding of nonhistone proteins to vector- and promoter-labeled DNAs over that observed in control fibroblasts at stringency washes of 0.05 and 0.1 M NaCl and to a lesser extent at 0.2 M NaCl. The binding specificities of nonhistone proteins for the ..cap alpha..2(I) procollagen promoter containing DNA were calculated.

  20. Expression and function of fibroblast growth factor (FGF) 9 in hepatic stellate cells and its role in toxic liver injury

    SciTech Connect

    Antoine, Marianne; Wirz, Werner; Tag, Carmen G.; Gressner, Axel M.; Marvituna, Meltem; Wycislo, Mathias; Hellerbrand, Claus; Kiefer, Paul . E-mail: paul.kiefer@klinik.uni-regensburg.de

    2007-09-21

    Hepatic injury and regeneration of the liver are associated with activation of hepatic stellate cells (HSC). Fibroblast growth factors (FGFs) and their receptors are important regulators of repair in various tissues. HSC express FGFR3IIIc as well as FGFGR4 and different spliced FGFR1IIIc and FGFR2IIIc isoforms which differ in the presence or absence of the acid box and of the first Ig-like domain. Expression of FGF9, known to be capable to activate the HSC FGFR2/3-isoforms, was increased in HSC in liver slice cultures after exposition to carbon tetrachloride, as an acute liver injury model. FGF9 significantly stimulated 3-H thymidine incorporation of hepatocytes, but failed to induce DNA synthesis in HSC despite the fact that FGF9 induced a sustained activation of extracellular signal-related kinases (ERK) 1/2. FGF9 induced an increased phosphorylation of Tyr436 of the fibroblast growth factor receptor substrate (FRS) 2, while phosphorylation of Tyr196 which is required for efficient Grb2 recruitment remained unchanged. Our findings suggest that HSC FGF9 provide a paracrine mitogenic signal to hepatocytes during acute liver injury, while the autocrine FGF9 signaling appears to be not sufficient to induce cell proliferation.

  1. MAD2B-mediated SnoN downregulation is implicated in fibroblast activation and tubulointerstitial fibrosis.

    PubMed

    Tang, Hui; Su, Hua; Fan, Di; Ye, Chen; Lei, Chun-Tao; Jiang, Hua-Jun; Gao, Pan; He, Fang-Fang; Zhang, Chun

    2016-07-01

    MAD2B, an anaphase-promoting complex/cyclosome (APC/C) inhibitor and a small subunit of DNA polymerase ζ, is indispensible for mitotic checkpoint control and DNA repair. Previously, we established that MAD2B is expressed in glomerular and tubulointerstitial compartments and participates in high glucose-induced podocyte injury. However, its role in other renal diseases remains elusive. In the present study, we aim to illustrate the potential role of MAD2B in the pathogenesis of renal fibrosis. By immunofluorescence and Western blotting, we found MAD2B expression is obviously increased in tubulointerstitial fibrosis (TIF) patients and unilateral ureteral obstruction (UUO) mice. It is widely accepted that resident fibroblasts are the major source of collagen-producing myofibroblasts during TIF. Therefore, we evaluated the level of MAD2B in fibroblasts (NRK-49F) exposed to transforming growth factor (TGF)-β1 by immunoblotting and revealed that MAD2B is upregulated in a time-dependent manner. Intriguingly, SnoN, a transcriptional repressor of the TGF-β1/Smad signaling pathway, is decreased in TGF-β1-treated fibroblasts as well as the kidney cortex from TIF patients and UUO mice. Either in vitro or in vivo, local genetic depletion of MAD2B by lentiviral transfection could preserve SnoN abundance and suppress Smad3 phosphorylation, which finally dampens fibroblast activation, ECM accumulation, and alleviates the severity of TIF. However, the ubiquitin ligase APC/C is not involved in the MAD2B-mediated SnoN decline, although this process is ubiquitination dependent. In conclusion, our observation proposes that besides cell cycle management, MAD2B has a profibrotic role during fibroblast activation and TIF by suppressing SnoN expression. Targeting the MAD2B-SnoN pathway is a promising intervention for TIF. PMID:27122545

  2. Expression of a Secreted Fibroblast Growth Factor Binding Protein-1 (FGFBP1) in Angioproliferative Kaposi Sarcoma

    PubMed Central

    Ray, Patricio E; Al-Attar, Ali; Liu, Xue-Hui; Das, Jharna R; Tassi, Elena; Wellstein, Anton

    2014-01-01

    Objective Kaposi’s sarcoma (KS) is an angioproliferative disease frequently seen in patients with the acquired immunodeficiency syndrome (AIDS). Previous studies suggest that the HIV-1 protein Tat and Fibroblast Growth Factor 2 (FGF-2) have synergistic angiogenic effects in AIDS-KS tumors. However, the mechanisms by which FGF-2 is released and activated in KS tumors are not clearly defined. We carried out this study to determine whether an FGF-binding protein (FGFBP1 or BP1) that enhances the angiogenic activity of FGF-2 is expressed in AIDS-KS tumors, and to define whether BP1, FGF-2, and HIV-Tat protein-protein interactions could play a potential clinically role in the pathogenesis of AIDS-KS. Methods BP1 was localized in AIDS-KS lesions by immunohistochemistry and in situ hybridization studies. The binding of radiolabeled FGF-2 to His-tagged BP1 or the FGF-receptor 1 was assessed in the presence and absence of HIV-Tat and other viral proteins. Mice carrying tetracycline-regulated BP1 transgene mice were used to determine whether activation of BP1 during wound healing induces KS-like lesions. Results BP1 expression was detected in AIDS-KS tumor keratinocytes, spindle cells, and infiltrating mononuclear cells. In addition, HIV-Tat competed for the binding of FGF-2 to immobilized BP1, but does not affect the interactions of FGF-2 with its high affinity receptor (FGFR-1). In contrast, two other HIV-proteins, Nef and gp120, did not affect the binding of FGF-2 to BP1 or to FGFR-1. Finally, up-regulation of BP1 expression in tetracycline-regulated –conditional BP1 transgenic mice subjected to skin wounds, induced KS-like skin lesions. Conclusion Taking into consideration the results of previous studies showing that both HIV-Tat and BP1 enhance the mitogenic and angiogenic activity of locally-stored FGF-2, both in vitro and in vivo, our findings suggest a novel mechanism by which the release and activity of FGFs can be modulated in AIDS-KS tumors by HIV-Tat as well

  3. Characteristic Gene Expression Profiles of Human Fibroblasts and Breast Cancer Cells in a Newly Developed Bilateral Coculture System

    PubMed Central

    Ueno, Takayuki; Utsumi, Jun; Toi, Masakazu; Shimizu, Kazuharu

    2015-01-01

    The microenvironment of cancer cells has been implicated in cancer development and progression. Cancer-associated fibroblast constitutes a major stromal component of the microenvironment. To analyze interaction between cancer cells and fibroblasts, we have developed a new bilateral coculture system using a two-sided microporous collagen membrane. Human normal skin fibroblasts were cocultured with three different human breast cancer cell lines: MCF-7, SK-BR-3, and HCC1937. After coculture, mRNA was extracted separately from cancer cells and fibroblasts and applied to transcriptomic analysis with microarray. Top 500 commonly up- or downregulated genes were characterized by enrichment functional analysis using MetaCore Functional Analysis. Most of the genes upregulated in cancer cells were downregulated in fibroblasts while most of the genes downregulated in cancer cells were upregulated in fibroblasts, indicating that changing patterns of mRNA expression were reciprocal between cancer cells and fibroblasts. In coculture, breast cancer cells commonly increased genes related to mitotic response and TCA pathway while fibroblasts increased genes related to carbohydrate metabolism including glycolysis, glycogenesis, and glucose transport, indicating that fibroblasts support cancer cell proliferation by supplying energy sources. We propose that the bilateral coculture system using collagen membrane is useful to study interactions between cancer cells and stromal cells by mimicking in vivo tumor microenvironment. PMID:26171396

  4. Fibroblast growth factor receptor 1 is principally responsible for fibroblast growth factor 2-induced catabolic activities in human articular chondrocytes

    PubMed Central

    2011-01-01

    Introduction Cartilage degeneration driven by catabolic stimuli is a critical pathophysiological process in osteoarthritis (OA). We have defined fibroblast growth factor 2 (FGF-2) as a degenerative mediator in adult human articular chondrocytes. Biological effects mediated by FGF-2 include inhibition of proteoglycan production, up-regulation of matrix metalloproteinase-13 (MMP-13), and stimulation of other catabolic factors. In this study, we identified the specific receptor responsible for the catabolic functions of FGF-2, and established a pathophysiological connection between the FGF-2 receptor and OA. Methods Primary human articular chondrocytes were cultured in monolayer (24 hours) or alginate beads (21 days), and stimulated with FGF-2 or FGF18, in the presence or absence of FGFR1 (FGF receptor 1) inhibitor. Proteoglycan accumulation and chondrocyte proliferation were assessed by dimethylmethylene blue (DMMB) assay and DNA assay, respectively. Expression of FGFRs (FGFR1 to FGFR4) was assessed by flow cytometry, immunoblotting, and quantitative real-time PCR (qPCR). The distinctive roles of FGFR1 and FGFR3 after stimulation with FGF-2 were evaluated using either pharmacological inhibitors or FGFR small interfering RNA (siRNA). Luciferase reporter gene assays were used to quantify the effects of FGF-2 and FGFR1 inhibitor on MMP-13 promoter activity. Results Chondrocyte proliferation was significantly enhanced in the presence of FGF-2 stimulation, which was inhibited by the pharmacological inhibitor of FGFR1. Proteoglycan accumulation was reduced by 50% in the presence of FGF-2, and this reduction was successfully rescued by FGFR1 inhibitor. FGFR1 inhibitors also fully reversed the up-regulation of MMP-13 expression and promoter activity stimulated by FGF-2. Blockade of FGFR1 signaling by either chemical inhibitors or siRNA targeting FGFR1 rather than FGFR3 abrogated the up-regulation of matrix metalloproteinases 13 (MMP-13) and a disintegrin and

  5. Antiproliferative, Apoptotic, and Autophagic Activity of Ranibizumab, Bevacizumab, Pegaptanib, and Aflibercept on Fibroblasts: Implication for Choroidal Neovascularization

    PubMed Central

    Lytvynchuk, Lyubomyr; Sergienko, Andrii; Lavrenchuk, Galina; Petrovski, Goran

    2015-01-01

    Purpose. Choroidal neovascularization (CNV) is one of the most common complications of retinal diseases accompanied by elevated secretion of vascular endothelial growth factor (VEGF). Intravitreal anti-VEGFs (ranibizumab, bevacizumab, pegaptanib, and aflibercept) can suppress neovascularization, decrease vascular permeability and CNV size, and, thereby, improve visual function. The antiproliferative, apoptotic, and autophagic effect of anti-VEGF drugs on fibroblasts found in CNVs has not been yet explored. Methods. Concentration-dependent cellular effects of the four anti-VEGFs were examined in L929 fibroblasts over a 5-day period. The cell survival, mitotic and polykaryocytic indices, the level of apoptosis and autophagy, and the cellular growth kinetics were all assessed. Results. The anti-VEGFs could inhibit the survival, mitotic activity, and proliferation as well as increase the cellular heterogeneity, apoptosis, and autophagy of the fibroblasts in a dose-dependent manner. Cellular growth kinetics showed ranibizumab to be less aggressive, but three other anti-VEGFs showed higher antiproliferative and apoptotic activity and expressed negative cellular growth kinetics. Conclusions. The antiproliferative, apoptotic, and autophagic activity of anti-VEGFs upon fibroblasts may explain the cellular response and the etiology of CNV involution in vivo and serve as a good study model for CNV in vitro. PMID:26491557

  6. KCa3.1 mediates activation of fibroblasts in diabetic renal interstitial fibrosis

    PubMed Central

    Huang, Chunling; Shen, Sylvie; Ma, Qing; Gill, Anthony; Pollock, Carol A.; Chen, Xin-Ming

    2014-01-01

    Background Fibroblast activation plays a critical role in diabetic nephropathy (DN). The Ca2+-activated K+ channel KCa3.1 mediates cellular proliferation of many cell types including fibroblasts. KCa3.1 has been reported to be a potential molecular target for pharmacological intervention in a diverse array of clinical conditions. However, the role of KCa3.1 in the activation of myofibroblasts in DN is unknown. These studies assessed the effect of KCa3.1 blockade on renal injury in experimental diabetes. Methods As TGF-β1 plays a central role in the activation of fibroblasts to myofibroblasts in renal interstitial fibrosis, human primary renal interstitial fibroblasts were incubated with TGF-β1 +/− the selective inhibitor of KCa3.1, TRAM34, for 48 h. Two streptozotocin-induced diabetic mouse models were used in this study: wild-type KCa3.1+/+ and KCa3.1−/− mice, and secondly eNOS−/− mice treated with or without a selective inhibitor of KCa3.1 (TRAM34). Then, markers of fibroblast activation and fibrosis were determined. Results Blockade of KCa3.1 inhibited the upregulation of type I collagen, fibronectin, α-smooth muscle actin, vimentin and fibroblast-specific protein-1 in renal fibroblasts exposed to TGF-β1 and in kidneys from diabetic mice. TRAM34 reduced TGF-β1-induced phosphorylation of Smad2/3 and ERK1/2 but not P38 and JNK MAPK in interstitial fibroblasts. Conclusions These results suggest that blockade of KCa3.1 attenuates diabetic renal interstitial fibrogenesis through inhibiting activation of fibroblasts and phosphorylation of Smad2/3 and ERK1/2. Therefore, therapeutic interventions to prevent or ameliorate DN through targeted inhibition of KCa3.1 deserve further consideration. PMID:24166472

  7. Acacetin inhibits expression of matrix metalloproteinases via a MAPK-dependent mechanism in fibroblast-like synoviocytes.

    PubMed

    Chen, Wei-Ping; Yang, Zhi-Gao; Hu, Peng-Fei; Bao, Jia-Peng; Wu, Li-Dong

    2015-08-01

    It is well known that rheumatoid arthritis (RA) is an autoimmune joint disease in which fibroblast-like synoviocytes (FLSs) play a pivotal role. In this study, we investigated the anti-arthritic properties of acacetin in FLSs. The expression of matrix metalloproteinase (MMP)-1, MMP-3 and MMP-13 were investigated by quantitative RT-PCR and western blot at gene and protein levels. At the same time, the phosphorylation of mitogen-activated protein kinases (MAPK) was investigated. The DNA-binding activity of NF-κB was investigated by electrophoretic mobility shift assay. We found that acacetin inhibits p38 and JNK phosphorylation and reduces MMP-1, MMP-3 and MMP-13 expression in interleukin-1β-induced FLSs. Our results suggest that acacetin has antiarthritic effects in FLSs. Thus, acacetin should be further studied for the treatment of arthritis. PMID:25856795

  8. Transforming Growth Factor-β1 Downregulates Vascular Endothelial Growth Factor-D Expression in Human Lung Fibroblasts via the Jun NH2-Terminal Kinase Signaling Pathway

    PubMed Central

    Cui, Ye; Osorio, Juan C; Risquez, Cristobal; Wang, Hao; Shi, Ying; Gochuico, Bernadette R; Morse, Danielle; Rosas, Ivan O; El-Chemaly, Souheil

    2014-01-01

    Vascular endothelial growth factor (VEGF)-D, a member of the VEGF family, induces both angiogenesis and lymphangiogenesis by activating VEGF receptor-2 (VEGFR-2) and VEGFR-3 on the surface of endothelial cells. Transforming growth factor (TGF)-β1 has been shown to stimulate VEGF-A expression in human lung fibroblast via the Smad3 signaling pathway and to induce VEGF-C in human proximal tubular epithelial cells. However, the effects of TGF-β1 on VEGF-D regulation are unknown. To investigate the regulation of VEGF-D, human lung fibroblasts were studied under pro-fibrotic conditions in vitro and in idiopathic pulmonary fibrosis (IPF) lung tissue. We demonstrate that TGF-β1 downregulates VEGF-D expression in a dose- and time-dependent manner in human lung fibroblasts. This TGF-β1 effect can be abolished by inhibitors of TGF-β type I receptor kinase and Jun NH2-terminal kinase (JNK), but not by Smad3 knockdown. In addition, VEGF-D knockdown in human lung fibroblasts induces G1/S transition and promotes cell proliferation. Importantly, VEGF-D protein expression is decreased in lung homogenates from IPF patients compared with control lung. In IPF lung sections, fibroblastic foci show very weak VEGF-D immunoreactivity, whereas VEGF-D is abundantly expressed within alveolar interstitial cells in control lung. Taken together, our data identify a novel mechanism for downstream signal transduction induced by TGF-β1 in lung fibroblasts, through which they may mediate tissue remodeling in IPF. PMID:24515257

  9. Ca2+-calcineurin signaling is involved in norepinephrine-induced cardiac fibroblasts activation

    PubMed Central

    Tian, Chun-Jing; Pang, Xiao

    2015-01-01

    Cardiac fibroblasts (CFs) activation plays a vital role in cardiac fibrosis. There are some studies demonstrate that norepinephrine (NE, an α1-adrenoceptor agonist) induced CFs proliferation. But whether Ca2+-calcineurin, a signaling concerned with growth and differentiation in various cell types, is participated in NE-induced CFs activation is unclear. In present study, we determined NE-induced CFs proliferation and differentiation, synthesis of collagen, and calcineurin (CaN) activity, and the effects of phentolamine (Phen, an α1-adrenoceptor antagonist), verapamil (Ver, a calcium channel blocker) and cyclosporine A (CsA, an inhibitor of CaN) on NE-induced CFs activation. The results showed that NE induced CFs proliferation and differentiation, increased α-SMA protein expression, increased collagen I, collagen III and fibronectin production, promoted ECM expression, activated CaN and increased CaN protein expression, which were inhibited by Phen, Ver and CsA. In vivo, more collagen deposition could be observed and total collagen volume fraction (CVF) was significantly increased in NE group. Phen, Ver and CsA decreased NE-induced collagen deposition, reduced cardiac fibrosis. Thus, our results demonstrate that Ca2+/CaN is involved in NE-induced CFs proliferation and collagen synthesis. PMID:26191219

  10. Tetrandrine inhibits migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes through down-regulating the expressions of Rac1, Cdc42, and RhoA GTPases and activation of the PI3K/Akt and JNK signaling pathways.

    PubMed

    Lv, Qi; Zhu, Xian-Yang; Xia, Yu-Feng; Dai, Yue; Wei, Zhi-Feng

    2015-11-01

    Tetrandrine (Tet), the main active constituent of Stephania tetrandra root, has been demonstrated to alleviate adjuvant-induced arthritis in rats. The present study was designed to investigate the effects of Tet on the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and explore the underlying mechanisms. By using cultures of primary FLS isolated from synoviums of RA patients and cell line MH7A, Tet (0.3, 1 μmol·L(-1)) was proven to significantly impede migration and invasion of RA-FLS, but not cell proliferation. Tet also greatly reduced the activation and expressions of matrix degrading enzymes MMP-2/9, the expression of F-actin and the activation of FAK, which controlled the morphologic changes in migration process of FLS. To identify the key signaling pathways by which Tet exerts anti-migration effect, the specific inhibitors of multiple signaling pathways LY294002, Triciribine, SP600125, U0126, SB203580, and PDTC (against PI3K, Akt, JNK, ERK, p38 MAPK and NF-κB-p65, respectively) were used. Among them, LY294002, Triciribine, and SP600125 were shown to obviously inhibit the migration of MH7A cells. Consistently, Tet was able to down-regulate the activation of Akt and JNK as demonstrated by Western blotting assay. Moreover, Tet could reduce the expressions of migration-related proteins Rho GTPases Rac1, Cdc42, and RhoA in MH7A cells. In conclusion, Tet can impede the migration and invasion of RA-FLS, which provides a plausible explanation for its protective effect on RA. The underlying mechanisms involve the reduction of the expressions of Rac1, Cdc42, and RhoA, inhibition of the activation of Akt and JNK, and subsequent down-regulation of activation and/or expressions of MMP-2/9, F-actin, and FAK. PMID:26614458

  11. Changes in tension regulates proliferation and migration of fibroblasts by remodeling expression of ECM proteins

    PubMed Central

    Jiang, Minmin; Qiu, Juhui; Zhang, Lingling; Lü, Dongyuan; Long, Mian; Chen, Li; Luo, Xiangdong

    2016-01-01

    Wound healing is a complicated but highly organized process in which cell migration and proliferation are actively involved. However, the process by which mechanical stretch regulates the proliferation and migration of human skin fibroblasts (HFs) and keratinocytes is poorly understood. Using a house built mechanical stretch device, we examined the HFs extracellular matrix (ECM) components changes under non-stretch, static stretch or cyclic stretch conditions. We further investigated the changes in ECM component protein expression levels in keratinocytes and analyzed the effects of individual ECM component on keratinocyte proliferation and migration. Particularly, the roles of calcium/calmodulin-dependent serine protein kinase (CASK) in the HF proliferation under cyclic stretch were investigated. Cyclic stretch suppressed HF proliferation compared with HFs without stretch or with static stretch. Cyclic stretch also led to a significant reduction in the levels of collagen I and a marked increase of fibronectin in HFs ECM. By contrast, collagen I levels increased and fibronectin levels decreased in response to non-stretch and static stretch conditions. After cyclic stretch, the proliferation of keratinocytes was inhibited by the cyclic stretch-induced ECM in HFs. The inoculation of keratinocytes with single ECM component suggested that collagen I was more capable of inducing cell proliferation than fibronectin, while it had less impact on cell migration compared with fibronectin. Furthermore, cyclic stretch induced by proliferation inhibition was associated with altered integrin β1-CASK signal pathway. The present results demonstrated the existence of HF-ECM-keratinocyte ‘cross-talk’ in cutaneous tissues. Thus, the integrin β1-CASK signal pathway in HFs may be involved in the outside-in signal transduction of extracellular stretch and the altered ECM component expression. PMID:27588075

  12. Comparative study of histamine H4 receptor expression in human dermal fibroblasts.

    PubMed

    Ikawa, Yoshiko; Shiba, Kayoko; Ohki, Emi; Mutoh, Nanami; Suzuki, Masahiko; Sato, Hiromi; Ueno, Koichi

    2008-10-01

    The histamine H4 receptor (H4R) is the newest receptor identified of four histamine receptors. Its expression in numerous immune and inflammatory organs has been implicated in relation to immune systems and allergic diseases. In the present study, we demonstrate the expression of H4R in human dermal fibroblasts and investigate changes in its expression level when stimulated by histamine, phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), dexamethasone and indomethacin. Histamine and PMA showed no effects on H4R expression. LPS and indomethacin up-regulated H4R mRNA expression, and 20 microM dexamethasone increased H4R protein levels. These results indicate a good prospective for this new receptor in the development of effective treatments of inflammatory diseases and pruritus or for the appropriate prevention of toxicities. PMID:18827451

  13. Effects of donor fibroblasts expressing OCT4 on bovine embryos generated by somatic cell nuclear transfer.

    PubMed

    Goissis, Marcelo D; Suhr, Steven T; Cibelli, Jose B

    2013-02-01

    The production of healthy, live, cloned animals by somatic cell nuclear transfer (SCNT) has been hampered by low efficiencies. Significant epigenetic changes must take place to ensure proper chromatin remodeling in SCNT. We hypothesized that exogenous expression of OCT4 in donor fibroblasts prior to its fusion with enucleated oocytes would facilitate SCNT reprogramming. We infected bovine adult fibroblasts with retroviral vectors containing yellow fluorescent protein (YFP) only, or the OCT4 gene fused to YFP (YO). We found that development to the blastocyst stage was not different between NT-YFP and NT-YO groups. NT-YFP embryos had the fewest trophoblast cells, measured by numbers of CDX2-positive cells. Fibroblasts expressing OCT4 had reduced levels of histone 3 lysine 9 or 27 trimethylation (H3K9me3 and H3K27me3, respectively). NT-YO blastocysts displayed higher H3K9me3 levels than IVF and NT-YFP embryos; however, they did not have different H3K27me3 levels. Levels of XIST mRNA expression in NT-YO and NT-YF were higher when compared to in vitro-fertilized blastocysts. We observed no differences in the expression of SOX2, NANOG, and CDX2. Although overexpression of OCT4 in donor cells increased H3K9me3 and did not reduce XIST gene expression, we show that a single transcription factor can affect the number of trophectoderm cells in bovine SCNT embryos. PMID:23276226

  14. The expression profiles of fibroblast growth factor 9 and its receptors in developing mice testes.

    PubMed

    Lai, Meng-Shao; Wang, Chia-Yih; Yang, Shang-Hsun; Wu, Chia-Ching; Sun, H Sunny; Tsai, Shaw-Jenq; Chuang, Jih-Ing; Chen, Yung-Chia; Huang, Bu-Miin

    2016-04-01

    An expressional lack of fibroblast growth factor 9 (FGF9) would cause male-to-female sex reversal in the mouse, implying the essential role of FGF9 in testicular organogenesis and maturation. However, the temporal expression of FGF9 and its receptors during testicular development remains elusive. In this study, immunohistochemistry was used to identify the localization of FGF9 and its receptors at different embryonic and postnatal stages in mice testes. Results showed that FGF9 continuously expressed in the testis during development. FGF9 had highest expression in the interstitial region at 17-18 d post coitum (dpc) and in the spermatocytes, spermatids and Leydig cell on postnatal days (pnd) 35-65. Regarding receptor expression, FGFR1 and FGFR4 were evenly expressed in the whole testis during the embryonic and postnatal stages. However, FGFR2 and FGFR3 were widely expressed during the embryonic testis development with higher FGFR2 expression in seminiferous tubules at 16-18 dpc and higher FGFR3 expression in interstitial region at 17-18 dpc. In postnatal stage, FGFR2 extensively expressed with higher expression at spermatids and Leydig cells on 35-65 pnd and FGFR3 widely expressed in the whole testis. Taken together, these results strongly suggest that FGF9 is correlated with the temporal expression profiles of FGFR2 and FGFR3 and possibly associated with testis development. PMID:27078042

  15. Youngiasides A and C Isolated from Youngia denticulatum Inhibit UVB-Induced MMP Expression and Promote Type I Procollagen Production via Repression of MAPK/AP-1/NF-κB and Activation of AMPK/Nrf2 in HaCaT Cells and Human Dermal Fibroblasts.

    PubMed

    Kim, Myungsuk; Park, Young Gyun; Lee, Hee-Ju; Lim, Sue Ji; Nho, Chu Won

    2015-06-10

    This study investigated the effects of youngiaside A (YA), youngiaside C (YC), and Youngia denticulatum extract (YDE) on extrinsic aging and assessed its molecular mechanisms in UVB-irradiated HaCaT keratinocytes and human dermal fibroblasts (HDFs). The results showed that YA, YC, and YDE decreased matrix metalloproteinase (MMP) expression and production in HaCaT cell and HDFs and increased collagen expression and production in HDFs. In addition, YA, YC, and YDE significantly increased antioxidant enzyme expression, thereby down-regulating UVB-induced reactive oxygen species (ROS) production and ROS-induced mitogen-activated protein kinase (MAPK) and activator protein-1 (AP-1) signaling in HaCaT cells. Furthermore, YA, YC, and YDE reduced phosphorylation of IκBα and IKKα/β, blocked nuclear factor-κB (NF-κB) p65 nuclear translocation, and strongly suppressed pro-inflammatory mediators. Finally, YA, YC, and YDE augmented UVB-induced adenosine monophosphate activated protein kinase (AMPK) phosphorylation and YA and YC did not inhibit MMP-1 production in AMPK inhibitor or nuclear factor-erythroid 2-related factor-2 (Nrf2) siRNA-treated HaCaT cells. The results suggest that these compounds could be potential therapeutic agents for prevention and treatment of skin photoaging. PMID:25994852

  16. Ablation of the mitochondrial complex IV assembly protein Surf1 leads to increased expression of the UPR(MT) and increased resistance to oxidative stress in primary cultures of fibroblasts.

    PubMed

    Pharaoh, Gavin; Pulliam, Daniel; Hill, Shauna; Sataranatarajan, Kavithalakshmi; Van Remmen, Holly

    2016-08-01

    Mice deficient in the electron transport chain (ETC) complex IV assembly protein SURF1 have reduced assembly and activity of cytochrome c oxidase that is associated with an upregulation of components of the mitochondrial unfolded protein response (UPR(MT)) and increased mitochondrial number. We hypothesized that the upregulation of proteins associated with the UPR(MT) in response to reduced cytochrome c oxidase activity in Surf1(-/-) mice might contribute to increased stress resistance. To test this hypothesis we asked whether primary cultures of fibroblasts from Surf1(-/-) mice exhibit enhanced resistance to stressors compared to wild-type fibroblasts. Here we show that primary dermal fibroblasts isolated from Surf1(-/-) mice have increased expression of UPR(MT) components ClpP and Hsp60, and increased expression of Lon protease. Fibroblasts from Surf1(-/-) mice are significantly more resistant to cell death caused by oxidative stress induced by paraquat or tert-Butyl hydroperoxide compared to cells from wild-type mice. In contrast, Surf1(-/-) fibroblasts show no difference in sensitivity to hydrogen peroxide stress. The enhanced cell survival in response to paraquat or tert-Butyl hydroperoxide in Surf1(-/-) fibroblasts compared to wild-type fibroblasts is associated with induced expression of Lon, ClpP, and Hsp60, increased maximal respiration, and increased reserve capacity as measured using the Seahorse Extracellular Flux Analyzer. Overall these data support a protective role for the activation of the UPR(MT) in cell survival. PMID:27208630

  17. Fibroblast growth factor receptor 3 effects on proliferation and telomerase activity in sheep growth plate chondrocytes

    PubMed Central

    2012-01-01

    Background Fibroblast growth factor receptor 3 (FGFR3) inhibits growth-plate chondrocyte proliferation and limits bone elongation. Gain-of-function FGFR3 mutations cause dwarfism, reduced telomerase activity and shorter telomeres in growth plate chondroyctes suggesting that FGFR3 reduces proliferative capacity, inhibits telomerase, and enhances senescence. Thyroid hormone (T3) plays a role in cellular maturation of growth plate chondrocytes and a known target of T3 is FGFR3. The present study addressed whether reduced FGFR3 expression enhanced telomerase activity, mRNA expression of telomerase reverse transcriptase (TERT) and RNA component of telomerase (TR), and chondrocyte proliferation, and whether the stimulation of FGFR3 by T3 evoked the opposite response. Results Sheep growth-plate proliferative zone chondrocytes were cultured and transfected with siRNA to reduce FGFR3 expression; FGFR3 siRNA reduced chondrocyte FGFR3 mRNA and protein resulting in greater proliferation and increased TERT mRNA expression and telomerase activity (p < 0.05). Chondrocytes treated with T3 significantly enhanced FGFR3 mRNA and protein expression and reduced telomerase activity (p < 0.05); TERT and TR were not significantly reduced. The action of T3 at the growth plate may be partially mediated through the FGFR3 pathway. Conclusions The results suggest that FGFR3 inhibits chondrocyte proliferation by down-regulating TERT expression and reducing telomerase activity indicating an important role for telomerase in sustaining chondrocyte proliferative capacity during bone elongation. PMID:23216972

  18. Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

    SciTech Connect

    Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François

    2015-02-27

    During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β{sub 1} (TGF-β{sub 1})-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β{sub 1} at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β{sub 1} is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β{sub 1}.

  19. Celastrol inhibits IL-1β-induced inflammation in orbital fibroblasts through the suppression of NF-κB activity.

    PubMed

    Li, Hong; Yuan, Yifei; Zhang, Yali; He, Qianwen; Xu, Rongjuan; Ge, Fangfang; Wu, Chen

    2016-09-01

    Graves' disease is an autoimmune disease of the thyroid gland, which is characterized by hyperthyroidism, diffuse goiter and Graves' ophthalmopathy (GO). Although several therapeutic strategies for the treatment of GO have been developed, the effectiveness and the safety profile of these therapies remain to be fully elucidated. Therefore, examination of novel GO therapies remains an urgent requirement. Celastrol, a triterpenoid isolated from traditional Chinese medicine, is a promising drug for the treatment of various inflammatory and autoimmune diseases. CCK‑8 and apoptosis assays were performed to investigate cytotoxicity of celastrol and effect on apoptosis on orbital fibroblasts. Reverse transcription‑polymerase chain reaction, western blotting and ELISAs were performed to examine the effect of celastrol on interleukin (IL)‑1β‑induced inflammation in orbital fibroblasts from patients with GO. The results demonstrated that celastrol significantly attenuated the expression of IL‑6, IL‑8, cyclooxygenase (COX)‑2 and intercellular adhesion molecule‑1 (ICAM‑1), and inhibited IL‑1β‑induced increases in the expression of IL‑6, IL‑8, ICAM‑1 and COX‑2. The levels of prostaglandin E2 in orbital fibroblasts induced by IL‑1β were also suppressed by celastrol. Further investigation revealed that celastrol suppressed the IL‑1β‑induced inflammatory responses in orbital fibroblasts through inhibiting the activation of nuclear factor (NF)‑κB. Taken together, these results suggested that celastrol attenuated the IL‑1β‑induced pro‑inflammatory pathway in orbital fibroblasts from patients with GO, which was associated with the suppression of NF-κB activation. PMID:27484716

  20. Inhibition of zymosan-induced cytokine and chemokine expression in human corneal fibroblasts by triptolide

    PubMed Central

    Liu, Yang; Li, Jing; Liu, Ye; Wang, Ping; Jia, Hui

    2016-01-01

    AIM To investigate the effects of triptolide on proinflammatory cytokine and chemokine expression induced by the fungal component zymosan in cultured human corneal fibroblasts (HCFs). METHODS HCFs were cultured in the absence or presence of zymosan or triptolide. The release of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) into culture supernatants was measured with enzyme-linked immunosorbent assays. The cellular abundance of the mRNAs for these proteins was determined by reverse transcription and real-time polymerase chain reaction analysis. The phosphorylation of mitogen-activated protein kinases (MAPKs) and the endogenous nuclear factor-κB (NF-κB) inhibitor IκB-α was examined by immunoblot analysis. The release of lactate dehydrogenase (LDH) activity from HCFs was measured with a colorimetric assay. RESULTS Triptolide inhibited the zymosan-induced release of IL-6, IL-8, and MCP-1 from HCFs in a concentration- and time-dependent manner. It also inhibited the zymosan-induced up-regulation of IL-6, IL-8, and MCP-1 mRNA abundance in these cells. Furthermore, triptolide attenuated zymosan-induced phosphorylation of the MAPKs extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 as well as the phosphorylation and degradation of IκB-α. Triptolide did not exhibit cytotoxicity for HCFs. CONCLUSION Triptolide inhibited proinflammatory cytokine and chemokine production by HCFs exposed to zymosan, with this action likely being mediated by suppression of MAPK and NF-κB signaling pathways. This compound might thus be expected to limit the infiltration of inflammatory cells into the cornea associated with fungal infection. PMID:26949603

  1. Killing of Kaposi's sarcoma-associated herpesvirus-infected fibroblasts during latent infection by activated natural killer cells

    PubMed Central

    Matthews, Nick C; Goodier, Martin R; Robey, Rebecca C; Bower, Mark; Gotch, Frances M

    2011-01-01

    Abstract Kaposi's sarcoma-associated herpesvirus (KSHV) establishes life-long infection by evading clearance by the host immune system. In de novo infection and lytic replication, KSHV escapes cytotoxic T cells and NK cells through downregulation of MHC class-I and ICAM-1 molecules and associated antigens involved in forming and sustaining the immunological synapse. However, the efficacy of such mechanisms in the context of the predominantly latent KSHV infection reported in Kaposi's sarcoma (KS) lesions is unclear. Using primary dermal fibroblasts in a novel in vitro model of chronic latent KSHV infection, we generated target cells with viral loads similar to those in spindle cells extracted from KS lesions. We show that latently KSHV-infected fibroblasts had normal levels of MHC-class I, ICAM-1, HLA-E and NKG2D ligand expression, were resistant to NK-cell natural cytotoxicity and were highly susceptible to killing by cytokine-activated immunocompetent NK cells. KSHV-infected fibroblasts expressed normal levels of IFN-γR1 and responded to exogenous IFN-γ by upregulating MHC class I, ICAM-1 and HLA-E and resisting activated NK-cell killing. These data demonstrate that physiologically relevant levels of latent KSHV infection in primary cells cause limited activation of resting NK cells and confer little specific resistance to control by activated NK cells. PMID:21509779

  2. Activated FXR Inhibits Leptin Signaling and Counteracts Tumor-promoting Activities of Cancer-Associated Fibroblasts in Breast Malignancy

    PubMed Central

    Giordano, Cinzia; Barone, Ines; Vircillo, Valentina; Panza, Salvatore; Malivindi, Rocco; Gelsomino, Luca; Pellegrino, Michele; Rago, Vittoria; Mauro, Loredana; Lanzino, Marilena; Panno, Maria Luisa; Bonofiglio, Daniela; Catalano, Stefania; Andò, Sebastiano

    2016-01-01

    Cancer-associated fibroblasts (CAFs), the principal components of the tumor stroma, play a central role in cancer development and progression. As an important regulator of the crosstalk between breast cancer cells and CAFs, the cytokine leptin has been associated to breast carcinogenesis. The nuclear Farnesoid X Receptor-(FXR) seems to exert an oncosuppressive role in different tumors, including breast cancer. Herein, we demonstrated, for the first time, that the synthetic FXR agonist GW4064, inhibiting leptin signaling, affects the tumor-promoting activities of CAFs in breast malignancy. GW4064 inhibited growth, motility and invasiveness induced by leptin as well as by CAF-conditioned media in different breast cancer cell lines. These effects rely on the ability of activated FXR to increase the expression of the suppressor of the cytokine signaling 3 (SOCS3) leading to inhibition of leptin-activated signaling and downregulation of leptin-target genes. In vivo xenograft studies, using MCF-7 cells alone or co-injected with CAFs, showed that GW4064 administration markedly reduced tumor growth. Interestingly, GW4064-treated tumors exhibited decreased levels of leptin-regulated proteins along with a strong staining intensity for SOCS3. Thus, FXR ligands might represent an emerging potential anti-cancer therapy able to block the tumor supportive role of activated fibroblasts within the breast microenvironment. PMID:26899873

  3. Activated FXR Inhibits Leptin Signaling and Counteracts Tumor-promoting Activities of Cancer-Associated Fibroblasts in Breast Malignancy.

    PubMed

    Giordano, Cinzia; Barone, Ines; Vircillo, Valentina; Panza, Salvatore; Malivindi, Rocco; Gelsomino, Luca; Pellegrino, Michele; Rago, Vittoria; Mauro, Loredana; Lanzino, Marilena; Panno, Maria Luisa; Bonofiglio, Daniela; Catalano, Stefania; Andò, Sebastiano

    2016-01-01

    Cancer-associated fibroblasts (CAFs), the principal components of the tumor stroma, play a central role in cancer development and progression. As an important regulator of the crosstalk between breast cancer cells and CAFs, the cytokine leptin has been associated to breast carcinogenesis. The nuclear Farnesoid X Receptor-(FXR) seems to exert an oncosuppressive role in different tumors, including breast cancer. Herein, we demonstrated, for the first time, that the synthetic FXR agonist GW4064, inhibiting leptin signaling, affects the tumor-promoting activities of CAFs in breast malignancy. GW4064 inhibited growth, motility and invasiveness induced by leptin as well as by CAF-conditioned media in different breast cancer cell lines. These effects rely on the ability of activated FXR to increase the expression of the suppressor of the cytokine signaling 3 (SOCS3) leading to inhibition of leptin-activated signaling and downregulation of leptin-target genes. In vivo xenograft studies, using MCF-7 cells alone or co-injected with CAFs, showed that GW4064 administration markedly reduced tumor growth. Interestingly, GW4064-treated tumors exhibited decreased levels of leptin-regulated proteins along with a strong staining intensity for SOCS3. Thus, FXR ligands might represent an emerging potential anti-cancer therapy able to block the tumor supportive role of activated fibroblasts within the breast microenvironment. PMID:26899873

  4. Stromal fibroblast activation and their potential association with uterine fibroids (Review)

    PubMed Central

    ZHENG, LI-HUA; CAI, FENG-FENG; GE, ISABELL; BISKUP, EWELINA; CHENG, ZHONG-PING

    2014-01-01

    Uterine fibroids are the most common type of benign, gynecologic neoplasm and are the primary indication for performance of a hysterectomy, accounting for >200,000 hysterectomies annually in the USA. At present, females are younger and exhibit larger leiomyomas at the time of diagnosis. Cancer-associated fibroblasts in tumor microenvironments have emerged as an important target for cancer therapy. Repeated stimulation by infectious or non-infectious agents in the uterine tissues, including inflammation, mechanical forces or hypoxia, stimulate the resident fibroblasts to undergo specific activation and, thus, are significant in tumorigenesis. Furthermore, complex signaling pathways regulate the mechanisms of fibroblastic activation. The current review focuses on the molecular mechanisms of fibroblastic activation and the potential association with uterine leiomyoma pathogenesis, enabling an integrated pathogenic analysis for review of the therapeutic options. PMID:25013460

  5. Identification of selective and potent inhibitors of fibroblast activation protein and prolyl oligopeptidase.

    PubMed

    Poplawski, Sarah E; Lai, Jack H; Li, Youhua; Jin, Zhiping; Liu, Yuxin; Wu, Wengen; Wu, Yong; Zhou, Yuhong; Sudmeier, James L; Sanford, David G; Bachovchin, William W

    2013-05-01

    Fibroblast activation protein (FAP) is a serine protease selectively expressed on reactive stromal fibroblasts of epithelial carcinomas. It is widely believed to play a role in tumor invasion and metastasis and therefore to represent a potential new drug target for cancer. Investigation into its biological function, however, has been hampered by the current unavailability of selective inhibitors. The challenge has been in identifying inhibitors that are selective for FAP over both the dipeptidyl peptidases (DPPs), with which it shares exopeptidase specificity, and prolyl oligopeptidase (PREP), with which it shares endopeptidase specificity. Here, we report the first potent FAP inhibitor with selectivity over both the DPPs and PREP, N-(pyridine-4-carbonyl)-d-Ala-boroPro (ARI-3099, 6). We also report a similarly potent and selective PREP inhibitor, N-(pyridine-3-carbonyl)-Val-boroPro (ARI-3531, 22). Both are boronic acid based inhibitors, demonstrating that high selectivity can be achieved using this electrophile. The inhibitors are stable, easy to synthesize, and should prove to be useful in helping to elucidate the biological functions of these two unique and interesting enzymes, as well as their potential as drug targets. PMID:23594271

  6. Identification of Selective and Potent Inhibitors of Fibroblast Activation Protein and Prolyl Oligopeptidase

    PubMed Central

    Poplawski, Sarah E.; Lai, Jack H.; Li, Youhua; Jin, Zhiping; Liu, Yuxin; Wu, Wengen; Wu, Yong; Zhou, Yuhong; Sudmeier, James L.; Sanford, David G.; Bachovchin, William W.

    2014-01-01

    Fibroblast activation protein (FAP) is a serine protease selectively expressed on reactive stromal fibroblasts of epithelial carcinomas. It is widely believed to play a role in tumor invasion and metastasis and therefore to represent a potential new drug target for cancer. Investigation into its biological function, however, has been hampered by the current unavailability of selective inhibitors. The challenge has been in identifying inhibitors that are selective for FAP over both the dipeptidyl peptidases (DPPs), with which it shares exopeptidase specificity, and prolyl oligopeptidase (PREP), with which it shares endopeptidase specificity. Here, we report the first potent FAP inhibitor with selectivity over both the DPPs and PREP, N-(pyridine-4-carbonyl)-d-Ala-boroPro (ARI-3099, 6). We also report a similarly potent and selective PREP inhibitor, N-(pyridine-3-carbonyl)-Val-boroPro (ARI-3531, 22). Both are boronic acid based inhibitors, demonstrating that high selectivity can be achieved using this electrophile. The inhibitors are stable, easy to synthesize, and should prove to be useful in helping to elucidate the biological functions of these two unique and interesting enzymes, as well as their potential as drug targets. PMID:23594271

  7. Retrovirus-induced interference with collagen I gene expression in Mov13 fibroblasts is maintained in the absence of DNA methylation.

    PubMed Central

    Chan, H; Hartung, S; Breindl, M

    1991-01-01

    We have studied the role of DNA methylation in repression of the murine alpha 1 type I collagen (COL1A1) gene in Mov13 fibroblasts. In Mov13 mice, a retroviral provirus has inserted into the first intron of the COL1A1 gene and blocks its expression at the level of transcriptional initiation. We found that regulatory sequences in the COL1A1 promoter region that are involved in the tissue-specific regulation of the gene are unmethylated in collagen-expressing wild-type fibroblasts and methylated in Mov13 fibroblasts, confirming and extending earlier observations. To directly assess the role of DNA methylation in the repression of COL1A1 gene transcription, we treated Mov13 fibroblasts with the demethylating agent 5-azacytidine. This treatment resulted in a demethylation of the COL1A1 regulatory sequences but failed to activate transcription of the COL1A1 gene. Moreover, the 5-azacytidine treatment induced a transcription-competent chromatin structure in the retroviral sequences but not in the COL1A1 promoter. In DNA transfection and microinjection experiments, we found that the provirus interfered with transcriptional activity of the COL1A1 promoter in Mov13 fibroblasts but not in Xenopus laevis oocytes. In contrast, the wild-type COL1A1 promoter was transcriptionally active in Mov13 fibroblasts. These experiments showed that the COL1A1 promoter is potentially transcriptionally active in the presence of proviral sequences and that Mov13 fibroblasts contain the trans-acting factors required for efficient COL1A1 gene expression. Our results indicate that the provirus insertion in Mov13 can inactivate COL1A1 gene expression at several levels. It prevents the developmentally regulated establishment of a transcription-competent methylation pattern and chromatin structure of the COL1A1 domain and, in the absence of DNA methylation, appears to suppress the COL1A1 promoter in a cell-specific manner, presumably by assuming a dominant chromatin structure that may be

  8. Uncoordinate regulation of collagenase, stromelysin, and tissue inhibitor of metalloproteinases genes by prostaglandin E2: selective enhancement of collagenase gene expression in human dermal fibroblasts in culture.

    PubMed

    Mauviel, A; Halcin, C; Vasiloudes, P; Parks, W C; Kurkinen, M; Uitto, J

    1994-04-01

    The degradative effects of interleukin-1 (IL-1) on the extracellular matrix of connective tissue are mediated primarily by metalloproteinases and prostaglandins. Clinical observations suggest that these effects can be prevented, to some extent, by the use of non-steroidal anti-inflammatory drugs. We have examined the role of prostaglandin E2 (PGE2) in IL-1-induced gene expression by human skin fibroblasts in culture. Incubation of confluent fibroblast cultures with varying concentrations (0.01-1.0 microgram/ml) of PGE2 led to a dose-dependent elevation of collagenase mRNA steady-state levels, the promoter activity, and the secretion of the protein, whereas relatively little effect was observed on stromelysin and TIMP gene expression. Exogenous PGE2 had no additive or synergistic effect with IL-1 on collagenase gene expression. Furthermore, commonly used non-steroidal anti-inflammatory drugs (indomethacin, acetyl salicylic acid and ibuprofen), at doses which block prostaglandin synthesis in cultured fibroblasts, failed to counteract IL-1-induced collagenase and stromelysin gene expression, nor did they affect TIMP expression. Although the effects of PGE2 did not potentiate those of IL-1 on collagenase gene expression in vitro, one could speculate that massive production of PGE2 by connective tissue cells in vivo in response to inflammatory mediators such as IL-1 or tumor necrosis factor-alpha, could lead to sustained expression of collagenase in connective tissue cells after clearance of the growth factors. PMID:8014195

  9. The Orphan Nuclear Receptor ERRγ Regulates Hepatic CB1 Receptor-Mediated Fibroblast Growth Factor 21 Gene Expression

    PubMed Central

    Jung, Yoon Seok; Lee, Ji-Min; Kim, Don-Kyu; Lee, Yong-Soo; Kim, Ki-Sun; Kim, Yong-Hoon; Kim, Jina; Lee, Myung-Shik; Lee, In-Kyu; Kim, Seong Heon; Cho, Sung Jin; Jeong, Won-Il; Lee, Chul-Ho; Harris, Robert A.; Choi, Hueng-Sik

    2016-01-01

    Background Fibroblast growth factor 21 (FGF21), a stress inducible hepatokine, is synthesized in the liver and plays important roles in glucose and lipid metabolism. However, the mechanism of hepatic cannabinoid type 1 (CB1) receptor-mediated induction of FGF21 gene expression is largely unknown. Results Activation of the hepatic CB1 receptor by arachidonyl-2’-chloroethylamide (ACEA), a CB1 receptor selective agonist, significantly increased FGF21 gene expression. Overexpression of estrogen-related receptor (ERR) γ increased FGF21 gene expression and secretion both in hepatocytes and mice, whereas knockdown of ERRγ decreased ACEA-mediated FGF21 gene expression and secretion. Moreover, ERRγ, but not ERRα and ERRβ, induced FGF21 gene promoter activity. In addition, deletion and mutation analysis of the FGF21 promoter identified a putative ERRγ-binding motif (AGGTGC, a near-consensus response element). A chromatin immunoprecipitation assay revealed direct binding of ERRγ to the FGF21 gene promoter. Finally, GSK5182, an ERRγ inverse agonist, significantly inhibited hepatic CB1 receptor-mediated FGF21 gene expression and secretion. Conclusion Based on our data, we conclude that ERRγ plays a key role in hepatic CB1 receptor-mediated induction of FGF21 gene expression and secretion. PMID:27455076

  10. In vitro activation of human fibroblasts by retrieved titanium alloy wear debris.

    PubMed

    Manlapaz, M; Maloney, W J; Smith, R L

    1996-05-01

    Titanium-aluminum-vanadium wear particles isolated from the soft-issue membrane of a failed total hip arthroplasty were added to human fibroblasts in cell culture. The cellular response to particle challenge was determined by assaying for levels of interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, prostaglandin E2, basic fibroblast growth factor, platelet-derived growth factor-AB, and transforming growth factor-beta. Collagenase and gelatinase activities were analyzed by zymography and [3H]collagen degradation. Cell viability was assessed by measuring the uptake of [3H]thymidine. Over the range of particle concentrations tested, cell viability, as demonstrated by [3H]thymidine uptake, remained unaffected. Fibroblasts exhibited a dose-dependent release of interleukin-6 in response to exposure to titanium-aluminum-vanadium particles. At 6 and 48 hours, the highest concentration of titanium alloy particles (0.189% [vol/vol]) resulted in 7-fold and 16-fold increases in interleukin-6 release, respectively, when compared with negative controls. Neither interleukin-1 beta nor tumor necrosis factor-alpha was detected in the culture medium at any particle concentration tested for both dermal and foreskin fibroblasts. The pattern of prostaglandin E2 release by fibroblasts mirrored the pattern of interleukin-6 release. Fibroblasts exposed to the highest concentration of titanium alloy particles showed an increase in collagenase activity, starting at 12 hours. When medium samples were treated with amino phenylmercuric acetate to activate latent enzymes, a statistically significant increase in collagenase activity was observed as early as 6 hours (p < 0.001). Substrate gel analysis of medium from fibroblasts stimulated by high particle concentrations also showed an increase in gelatinolytic activity when compared with unstimulated controls. Analysis of medium samples for growth factors showed an increase in basic fibroblast growth factor at low particle