Dataset Information

Scleroderma keratinocytes promote fibroblast activation independent of transforming growth factor beta.

ABSTRACT: Objectives:SSc is a devastating disease that results in fibrosis of the skin and other organs. Fibroblasts are a key driver of the fibrotic process through deposition of extracellular matrix. The mechanisms by which fibroblasts are induced to become pro-fibrotic remain unclear. Thus, we examined the ability of SSc keratinocytes to promote fibroblast activation and the source of this effect. Methods:Keratinocytes were isolated from skin biopsies of 9 lcSSc, 10 dcSSc and 13 control patients. Conditioned media was saved from the cultures. Normal fresh primary fibroblasts were exposed to healthy control and SSc keratinocyte conditioned media in the presence or absence of neutralizing antibodies for TGF-?. Gene expression was assessed by microarrays and real-time PCR. Immunocytochemistry was performed for ?-smooth muscle actin (?-SMA), collagen type 1 (COL1A1) and CCL5 expression. Results:SSc keratinocyte conditioned media promoted fibroblast activation, characterized by increased ?-SMA and COL1A1 mRNA and protein expression. This effect was independent of TGF-?. Microarray analysis identified upregulation of nuclear factor ?B (NF-?B) and downregulation of peroxisome proliferator-activated receptor ? (PPAR-?) pathways in both SSc subtypes. Scleroderma keratinocytes exhibited increased expression of NF-?B-regulated cytokines and chemokines and lesional skin staining confirmed upregulation of CCL5 in basal keratinocytes. Conclusion:Scleroderma keratinocytes promote the activation of fibroblasts in a TGF-?-independent manner and demonstrate an imbalance in NF-?B1 and PPAR-? expression leading to increased cytokine and CCL5 production. Further study of keratinocyte mediators of fibrosis, including CCL5, may provide novel targets for skin fibrosis therapy.

SUBMITTER: McCoy SS

PROVIDER: S-EPMC5850806 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Scleroderma keratinocytes promote fibroblast activation independent of transforming growth factor beta.

McCoy Sara S SS Reed Tamra J TJ Berthier Celine C CC Tsou Pei-Suen PS Liu Jianhua J Gudjonsson Johann E JE Khanna Dinesh D Kahlenberg J Michelle JM

Rheumatology (Oxford, England) 20171101 11

<h4>Objectives</h4>SSc is a devastating disease that results in fibrosis of the skin and other organs. Fibroblasts are a key driver of the fibrotic process through deposition of extracellular matrix. The mechanisms by which fibroblasts are induced to become pro-fibrotic remain unclear. Thus, we examined the ability of SSc keratinocytes to promote fibroblast activation and the source of this effect.<h4>Methods</h4>Keratinocytes were isolated from skin biopsies of 9 lcSSc, 10 dcSSc and 13 control ...[more]

PMID: 28968684

Similar Datasets

Project description:ObjectiveAlthough transforming growth factor β (TGFβ) is recognized as being a key trigger of fibroblast activation in systemic sclerosis (SSc), prominent innate immunity suggests that additional pathways contribute to disease persistence. Toll-like receptor 9 (TLR9) is implicated in autoimmunity and fibrosis; however, the expression, mechanism of action, and pathogenic role of TLR9 signaling in SSc remain uncharacterized. The aim of this study was to explore the expression, activity, and potential pathogenic role of TLR9 in the context of skin fibrosis in SSc and in mouse models of experimental fibrosis.MethodsExpression and localization of TLR9 were evaluated in SSc skin biopsy specimens and explanted skin fibroblasts. Fibrotic responses elicited by type A CpG oligonucleotide and mitochondrial DNA (mtDNA) were examined in human skin fibroblasts by a combination of real-time quantitative polymerase chain reaction, Western blot analysis, transient transfection, immunofluorescence microscopy, and functional assays. Expression of TLR9 was examined in 2 distinct mouse models of experimental fibrosis.ResultsSkin biopsy specimens obtained from 2 independent cohorts of SSc patients showed up-regulation of TLR9, and myofibroblasts were the major cellular source. Moreover, SSc skin biopsy specimens showed evidence of TLR9 pathway activation. CpG induced robust TLR9-dependent fibrotic responses in explanted normal fibroblasts that could be blocked by bortezomib and were mediated through the action of endogenous TGFβ. Mice with experimental fibrosis showed a time-dependent increase in TLR9 localized primarily to myofibroblasts in the dermis.ConclusionIn isolated fibroblasts, TLR9 elicits fibrotic responses mediated via endogenous TGFβ. In patients with SSc, mtDNA and other damage-associated TLR9 ligands in the skin might trigger localized activation of TLR9 signaling, TGFβ production, and consequent fibroblast activation. Disrupting this fibrotic process with inhibitors targeting TLR9 or its downstream signaling pathways might therefore represent a novel approach to SSc therapy.

Project description:IntroductionThe objective of this study was to model the effects of transforming growth factor beta (TGF-beta) and platelet-derived growth factor (PDGF), both present in rheumatoid arthritis (RA) synovia, on the behavior of fibroblast-like synoviocytes (FLS) in response to pro-inflammatory cytokine (interleukin (IL)1beta, tumor necrosis factor-alpha (TNFalpha)) challenge.MethodsGene and protein expression by fibroblast-like synoviocytes in vitro was studied by quantitative Polymerase Chain Reaction (qPCR), ELISA and multiplex bead cytokine assays. Intracellular signaling pathway activation was determined by Western blot for phospho-kinases and the use of specific inhibitors.ResultsIn combination, TGF-beta and PDGF (2GF) synergistically augmented TNFalpha- or IL1beta-induced matrix metalloproteinase 3 (MMP3), IL6, IL8, and macrophage inflammatory protein 1 alpha (MIP1alpha) secretion by FLS. Other FLS-derived mediators remained unaffected. Individually, neither growth factor significantly potentiated TNFalpha or IL1beta-induced MMP3 secretion, and only slightly enhanced IL6. The effect of 2GF on TNFalpha-induced gene expression was transcriptionally mediated; blocked by imatinib mesylate; and occurred even if 2GF was added as much as four hours prior to TNFalpha. In addition, a 15-minute pulse of 2GF four hours prior to TNFalpha stimulation yielded a synergistic response. The extracellular-signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K) signaling pathways were induced for at least four hours by 2GF, as demonstrated by persistently upregulated levels of phospho-Akt and phospho-ERK. However, pharmacologic inhibitor studies demonstrated that the potentiating action of 2GF was dependent on PI3 kinase only, and not on ERK.ConclusionsThe combination of PDGF and TGF-beta dramatically potentiates FLS response to cytokines in a receptor-mediated and PI3 kinase-dependent fashion. These data suggest that 2GF contribute to synovitis by directing synovial fibroblasts toward a more aggressive phenotype in response to TNFalpha. Therefore, inhibition of growth factor signaling may constitute a complementary therapeutic approach to cytokine-targeted treatments for RA.

Project description:The tumor microenvironment contains various components, including cancer cells, tumor vessels, and cancer-associated fibroblasts, the latter of which are comprised of tumor-promoting myofibroblasts and tumor-suppressing fibroblasts. Multiple lines of evidence indicate that transforming growth factor-β (TGF-β) induces the formation of myofibroblasts and other types of mesenchymal (non-myofibroblastic) cells from endothelial cells. Recent reports show that fibroblast growth factor 2 (FGF2) modulates TGF-β-induced mesenchymal transition of endothelial cells, but the molecular mechanisms behind the signals that control transcriptional networks during the formation of different groups of fibroblasts remain largely unclear. Here, we studied the roles of FGF2 during the regulation of TGF-β-induced mesenchymal transition of tumor endothelial cells (TECs). We demonstrated that auto/paracrine FGF signals in TECs inhibit TGF-β-induced endothelial-to-myofibroblast transition (End-MyoT), leading to suppressed formation of contractile myofibroblast cells, but on the other hand can also collaborate with TGF-β in promoting the formation of active fibroblastic cells which have migratory and proliferative properties. FGF2 modulated TGF-β-induced formation of myofibroblastic and non-myofibroblastic cells from TECs via transcriptional regulation of various mesenchymal markers and growth factors. Furthermore, we observed that TECs treated with TGF-β were more competent in promoting in vivo tumor growth than TECs treated with TGF-β and FGF2. Mechanistically, we showed that Elk1 mediated FGF2-induced inhibition of End-MyoT via inhibition of TGF-β-induced transcriptional activation of α-smooth muscle actin promoter by myocardin-related transcription factor-A. Our data suggest that TGF-β and FGF2 oppose and cooperate with each other during the formation of myofibroblastic and non-myofibroblastic cells from TECs, which in turn determines the characteristics of mesenchymal cells in the tumor microenvironment.

Dataset Information

Scleroderma keratinocytes promote fibroblast activation independent of transforming growth factor beta.

Publications

Scleroderma keratinocytes promote fibroblast activation independent of transforming growth factor beta.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets