Effect of Protein Kinase C delta (PKC-δ) Inhibition on the Transcriptome of Normal and Systemic Sclerosis Human Dermal Fibroblasts In Vitro

Previous studies demonstrated that protein kinase C- δ (PKC-δ) inhibition with the selective inhibitor, rottlerin, resulted in potent downregulation of type I collagen expression and production in normal human dermal fibroblasts and abrogated the exaggerated type I collagen production and expression in fibroblasts cultured from affected skin from patients with the fibrosing disorder systemic sclerosis (SSc). To elucidate the mechanisms involved in the ability of PKC-δ to regulate collagen production in fibroblasts, we examined the effects of PKC-δ inhibition on the transcriptome of normal and SSc human dermal fibroblasts. Normal and SSc human dermal fibroblasts were incubated with rottlerin (5 µM), and their gene expression was analyzed by microarrays. Pathway analysis and gene ontology analysis of differentially expressed genes in each comparison were performed. Identification of significantly overrepresented transcriptional regulatory elements (TREs) was performed using the Promoter Analysis and Interaction Network Toolset (PAINT) program. PKC-δ activity was also inhibited using RNA interference (siRNA) and by treating fibroblasts with a specific PKC-δ inhibitory cell permeable peptide. Differential gene expression of 20 genes was confirmed using real time PCR. PKC-δ inhibition caused a profound change in the transcriptome of normal and SSc human dermal fibroblasts in vitro. Pathway and gene ontology analysis identified multiple cellular and organismal pathways affected by PKC-δ inhibition. Furthermore, both pathway and PAINT analyses indicated that the transcription factor NFκB played an important role in the transcriptome changes induced by PKC-δ inhibition. Multiple genes involved in the degradation of the extracellular matrix components were significantly reduced in SSc fibroblasts and their expression was increased by PKC-δ inhibition. These results indicate that isoform-specific inhibition of PKC-δ profibrotic effects may represent a novel therapeutic approach for SSc and other fibrotic diseases

Human Skin Culture as an Ex Vivo Model for Assessing the Fibrotic Effects of Insulin-Like Growth Factor Binding Proteins

Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology. A hallmark of SSc is fibrosis of the skin and internal organs. We recently demonstrated increased expression of IGFBP-3 and IGFBP-5 in primary cultures of fibroblasts from the skin of patients with SSc. In vitro, IGFBP-3 and IGFBP-5 induced a fibrotic phenotype and IGFBP-5 triggered dermal fibrosis in mice. To assess the ability of IGFBPs to trigger fibrosis, we used an ex vivo human skin organ culture model. Our findings demonstrate that IGFBP-3 and IGFBP-5, but not IGFBP-4, increase dermal and collagen bundle thickness in human skin explants, resulting in substantial dermal fibrosis and thickening. These fibrotic effects were sustained for at least two weeks. Our findings demonstrate that human skin ex vivo is an appropriate model to assess the effects of fibrosis-inducing factors such as IGFBPs, and for evaluating the efficacy of inhibitors/therapies to halt the progression of fibrosis and potentially reverse it

Frontiers of antifibrotic therapy in systemic sclerosis

Although fibrosis is becoming increasingly recognized as a major cause of morbidity and mortality in modern societies, targeted anti-fibrotic therapies are still not approved for most fibrotic disorders. However, intense research over the last decade has improved our understanding of the underlying pathogenesis of fibrotic diseases. We now appreciate fibrosis as the consequence of a persistent tissue repair responses, which, in contrast to normal wound healing, fails to be effectively terminated. Profibrotic mediators released from infiltrating leukocytes, activated endothelial cells and degranulated platelets may predominantly drive fibroblast activation and collagen release in early stages, whereas endogenous activation of fibroblasts due epigenetic modifications and biomechanical or physical factors such as stiffening of the extracellular matrix and hypoxia may play pivotal role for disease progression in later stages. In the present review, we discuss novel insights into the pathogenesis of fibrotic diseases using systemic sclerosis (SSc) as example for an idiopathic, multisystem disorder. We set a strong translational focus and predominantly discuss approaches with very high potential for rapid transfer from bench-to-bedside. We highlight the molecular basis for ongoing clinical trials in SSc and also provide an outlook on upcoming trials. This article is protected by copyright. All rights reserved

Syndecan-2 is a novel target of insulin-like growth factor binding protein-3 and is over-expressed in fibrosis

Extracellular matrix deposition and tissue scarring characterize the process of fibrosis. Transforming growth factor beta (TGFβ) and Insulin-like growth factor binding protein-3 (IGFBP-3) have been implicated in the pathogenesis of fibrosis in various tissues by inducing mesenchymal cell proliferation and extracellular matrix deposition. We identified Syndecan-2 (SDC2) as a gene induced by TGFβ in an IGFBP-3-dependent manner. TGFβ induction of SDC2 mRNA and protein required IGFBP-3. IGFBP-3 independently induced production of SDC2 in primary fibroblasts. Using an ex-vivo model of human skin in organ culture expressing IGFBP-3, we demonstrate that IGFBP-3 induces SDC2 ex vivo in human tissue. We also identified Mitogen-activated protein kinase-interacting kinase (Mknk2) as a gene induced by IGFBP-3. IGFBP-3 triggered Mknk2 phosphorylation resulting in its activation. Mknk2 independently induced SDC2 in human skin. Since IGFBP-3 is over-expressed in fibrotic tissues, we examined SDC2 levels in skin and lung tissues of patients with systemic sclerosis (SSc) and lung tissues of patients with idiopathic pulmonary fibrosis (IPF). SDC2 levels were increased in fibrotic dermal and lung tissues of patients with SSc and in lung tissues of patients with IPF. This is the first report describing elevated levels of SDC2 in fibrosis. Increased SDC2 expression is due, at least in part, to the activity of two pro-fibrotic factors, TGFβ and IGFBP-3. © 2012 Ruiz et al

A central role for G9a and EZH2 in the epigenetic silencing of cyclooxygenase-2 in idiopathic pulmonary fibrosis

Selective silencing of the cyclooxygenase-2 (COX-2) gene with the loss of the antifibrotic mediator PGE2 contributes to the fibrotic process in idiopathic pulmonary fibrosis (IPF). This study explored the role of G9a- and EZH2-mediated methylation of histone H3 lysine 9 (H3K9me3) and 27 (H3K27me3) in COX-2 silencing in IPF. Chromatin immunoprecipitation (ChIP) and Re-ChIP assays demonstrated marked increases in H3K9me3, H3K27me3 and DNA methylation, together with their respective modifying enzymes G9a, EZH2 and DNA methyltransferases (Dnmts) and respective binding proteins heterochromatin protein 1 (HP1), polycomb protein complex 1 (PRC1) and MeCP2, at the COX-2 promoter in lung fibroblasts from IPF patients (F-IPF) compared with fibroblasts from non-fibrotic lungs (F-NL). HP1, EZH2 and MeCP2 in turn were associated with additional repressive chromatin modifiers in F-IPF. G9a and EZH2 inhibitors and siRNAs and Dnmt1 inhibitor markedly reduced H3K9me3 (49-79%), H3K27me3 (44-81%) and DNA methylation (61-97%) at the COX-2 promoter. This was correlated with increased histone H3 and H4 acetylation, resulting in COX-2 mRNA and protein re-expression in F-IPF. Our results support a central role for G9a- and EZH2-mediated histone hypermethylation and a model of bidirectional, mutually reinforcing and interdependent crosstalk between histone hypermethylation and DNA methylation in COX-2 epigenetic silencing in IPF

The K+ Channel KCa3.1 as a Novel Target for Idiopathic Pulmonary Fibrosis

Background\ud \ud Idiopathic pulmonary fibrosis (IPF) is a common, progressive and invariably lethal interstitial lung disease with no effective therapy. We hypothesised that KCa3.1 K+ channel-dependent cell processes contribute to IPF pathophysiology.\ud Methods\ud \ud KCa3.1 expression in primary human lung myofibroblasts was examined using RT-PCR, western blot, immunofluorescence and patch-clamp electrophysiology. The role of KCa3.1 channels in myofibroblast proliferation, wound healing, collagen secretion and contraction was examined using two specific and distinct KCa3.1 blockers (TRAM-34 and ICA-17043 [Senicapoc]).\ud Results\ud \ud Both healthy non fibrotic control and IPF-derived human lung myofibroblasts expressed KCa3.1 channel mRNA and protein. KCa3.1 ion currents were elicited more frequently and were larger in IPF-derived myofibroblasts compared to controls. KCa3.1 currents were increased in myofibroblasts by TGFβ1 and basic FGF. KCa3.1 was expressed strongly in IPF tissue. KCa3.1 pharmacological blockade attenuated human myofibroblast proliferation, wound healing, collagen secretion and contractility in vitro, and this was associated with inhibition of TGFβ1-dependent increases in intracellular free Ca2+.\ud Conclusions\ud \ud KCa3.1 activity promotes pro-fibrotic human lung myofibroblast function. Blocking KCa3.1 may offer a novel approach to treating IPF with the potential for rapid translation to the clinic

The Membrane-Associated Adaptor Protein DOK5 Is Upregulated in Systemic Sclerosis and Associated with IGFBP-5-Induced Fibrosis

Systemic sclerosis (SSc) is characterized by excessive fibrosis of the skin and internal organs due to fibroblast proliferation and excessive production of extracellular matrix (ECM). We have shown that insulin-like growth factor binding protein (IGFBP)-5 plays an important role in the development of fibrosis in vitro, ex vivo, and in vivo. We identified a membrane-associated adaptor protein, downstream of tyrosine kinase/docking protein (DOK)5, as an IGFBP-5-regulated target gene using gene expression profiling of primary fibroblasts expressing IGFBP-5. DOK5 is a tyrosine kinase substrate associated with intracellular signaling. Our objective was to determine the role of DOK5 in the pathogenesis of SSc and specifically in IGFBP-5-induced fibrosis. DOK5 mRNA and protein levels were increased in vitro by endogenous and exogenous IGFBP-5 in primary human fibroblasts. DOK5 upregulation required activation of the mitogen-activated protein kinase (MAPK) signaling cascade. Further, IGFBP-5 triggered nuclear translocation of DOK5. DOK5 protein levels were also increased in vivo in mouse skin and lung by IGFBP-5. To determine the effect of DOK5 on fibrosis, DOK5 was expressed ex vivo in human skin in organ culture. Expression of DOK5 in human skin resulted in a significant increase in dermal thickness. Lastly, levels of DOK5 were compared in primary fibroblasts and lung tissues of patients with SSc and healthy donors. Both DOK5 mRNA and protein levels were significantly increased in fibroblasts and skin tissues of patients with SSc compared with those of healthy controls, as well as in lung tissues of SSc patients. Our findings suggest that IGFBP-5 induces its pro-fibrotic effects, at least in part, via DOK5. Furthermore, IGFBP-5 and DOK5 are both increased in SSc fibroblasts and tissues and may thus be acting in concert to promote fibrosis

TGF-β Isoform Specific Regulation of Airway Inflammation and Remodelling in a Murine Model of Asthma

The TGF-β family of mediators are thought to play important roles in the regulation of inflammation and airway remodelling in asthma. All three mammalian isoforms of TGF-β, TGF-β1–3, are expressed in the airways and TGF-β1 and -β2 are increased in asthma. However, there is little information on the specific roles of individual TGF-β isoforms. In this study we assess the roles of TGF-β1 and TGF-β2 in the regulation of allergen-induced airway inflammation and remodelling associated with asthma, using a validated murine model of ovalbumin sensitization and challenge, and isoform specific TGF-β neutralising antibodies. Antibodies to both isoforms inhibited TGF-β mediated Smad signalling. Anti-TGF-β1 and anti-TGF-β2 inhibited ovalbumin-induced sub-epithelial collagen deposition but anti-TGF-β1 also specifically regulated airway and fibroblast decorin deposition by TGF-β1. Neither antibody affected the allergen-induced increase in sub-epithelial fibroblast-like cells. Anti- TGF-β1 also specifically inhibited ovalbumin-induced increases in monocyte/macrophage recruitment. Whereas, both TGF-β1 and TGF-β2 were involved in regulating allergen-induced increases in eosinophil and lymphocyte numbers. These data show that TGF-β1 and TGF-β2 exhibit a combination of specific and shared roles in the regulation of allergen-induced airway inflammation and remodelling. They also provide evidence in support of the potential for therapeutic regulation of specific subsets of cells and extracellular matrix proteins associated with inflammation and remodelling in airway diseases such as asthma and COPD, as well as other fibroproliferative diseases

PDGF Promotes Dermal Fibroblast Activation via a Novel Mechanism Mediated by Signaling Through MCHR1

Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy and excessive fibrosis of the skin and internal organs. To this day, no effective treatments to prevent the progression of fibrosis exist, and SSc patients have disabilities and reduced life expectancy. The need to better understand pathways that drive SSc and to find therapeutic targets is urgent. RNA sequencing data from SSc dermal fibroblasts suggested that melanin-concentrating hormone receptor 1 (MCHR1), one of the G protein-coupled receptors regulating emotion and energy metabolism, is abnormally deregulated in SSc. Platelet-derived growth factor (PDGF)-BB stimulation upregulated MCHR1 mRNA and protein levels in normal human dermal fibroblasts (NHDF), and MCHR1 silencing prevented the PDGF-BB-induced expression of the profibrotic factors transforming growth factor beta 1 (TGFβ1) and connective tissue growth factor (CTGF). PDGF-BB bound MCHR1 in membrane fractions of NHDF, and the binding was confirmed using surface plasmon resonance (SPR). MCHR1 inhibition blocked PDGF-BB modulation of intracellular cyclic adenosine monophosphate (cAMP). MCHR1 silencing in NHDF reduced PDGF-BB signaling. In summary, MCHR1 promoted the fibrotic response in NHDF through modulation of TGFβ1 and CTGF production, intracellular cAMP levels, and PDGF-BB-induced signaling pathways, suggesting that MCHR1 plays an important role in mediating the response to PDGF-BB and in the pathogenesis of SSc. Inhibition of MCHR1 should be considered as a novel therapeutic strategy in SSc-associated fibrosis