Distinct PKC isoforms mediate cell survival and DNA synthesis in thrombin-induced myofibroblasts

ArticleinAJP Lung Cellular and Molecular Physiology 288(1):L190-201 · February 2005with9 Reads
DOI: 10.1152/ajplung.00448.2003 · Source: PubMed
Thrombin activates protease-activated receptor (PAR)-1 and induces a myofibroblast phenotype in normal lung fibroblasts that resembles the phenotype of scleroderma lung fibroblasts. We now demonstrate that PAR-1 expression is dramatically increased in lung tissue from scleroderma patients, where it is associated with inflammatory and fibroproliferative foci. We also observe that thrombin induces resistance to apoptosis in normal lung fibroblasts, and this process is regulated by protein kinase C (PKC)-epsilon but not by PKC-alpha. Overexpression of a constitutively active (c-a) form of PAR-1 or PKC-epsilon significantly inhibits Fas ligand-induced apoptosis in lung fibroblasts, whereas scleroderma lung fibroblasts are resistant to apoptosis de novo. Thrombin translocates p21Cip1/WAF1, a signaling molecule downstream of PKC, from the nucleus to cytoplasm in normal lung fibroblasts mimicking the localization of p21Cip1/WAF1 in scleroderma lung fibroblasts. Overexpression of c-a PKC-alpha or PKC-epsilon results in accumulation of p21Cip1/WAF1 in the cytoplasm. Depletion of PKC-alpha or inhibition of mitogen-activated protein kinase (MAPK) blocks thrombin-induced DNA synthesis in lung fibroblasts. Inhibition of PKC by calphostin or PKC-alpha, but not PKC-epsilon, by antisense oligonucleotides prevents thrombin-induced MAPK phosphorylation and accumulation of G(1) phase regulatory protein cyclin D1, suggesting that PKC-alpha, MAPK, and cyclin D1 mediate lung fibroblast proliferation. These data demonstrate that two distinct PKC isoforms mediate thrombin-induced resistance to apoptosis and proliferation and suggest that p21Cip1/WAF1 promotes both phenomena.
    • "Fibroblasts express the protease activated receptor PAR1 that enables fibroblast responsiveness to activated thrombin. PAR1 receptor expression is upregulated in IPF (Howell et al., 2005) and in lung tissue of SSc patients (Bogatkevich et al., 2005). Further, PAR1 knockout mice resist bleomycin-induced lung fibrosis (Howell et al., 2005). "
    [Show abstract] [Hide abstract] ABSTRACT: Fibroblasts are the most common cell type of the connective tissues found throughout the body and the principal source of the extensive extracellular matrix (ECM) characteristic of these tissues. They are also the central mediators of the pathological fibrotic accumulation of ECM and the cellular proliferation and differentiation that occurs in response to prolonged tissue injury and chronic inflammation. The transformation of the fibroblast cell lineage involves classical developmental signaling programs and includes a surprisingly diverse range of precursor cell types-most notably, myofibroblasts that are the apex of the fibrotic phenotype. Myofibroblasts display exaggerated ECM production; constitutively secrete and are hypersensitive to chemical signals such as cytokines, chemokines, and growth factors; and are endowed with a contractile apparatus allowing them to manipulate the ECM fibers physically to close open wounds. In addition to ECM production, fibroblasts have multiple concomitant biological roles, such as in wound healing, inflammation, and angiogenesis, which are each interwoven with the process of fibrosis. We now recognize many common fibroblast-related features across various physiological and pathological protracted processes. Indeed, a new appreciation has emerged for the role of non-cancerous fibroblast interactions with tumors in cancer progression. Although the predominant current clinical treatments of fibrosis involve non-specific immunosuppressive and anti-proliferative drugs, a variety of potential therapies under investigation specifically target fibroblast biology.
    Full-text · Article · May 2014
    • "There is increasing evidence that VEGF-mediated activation of PKC can also induce protection against damaging insults (e.g. radiation, inflammatory diseases, hyperoxia) [16,262728 . Our phospho-PKC antibody detected two PKC species that were activated in response to Vegf165, migrating at ranges between 80 and 90 kDa, respectively (Figure 9C-E). "
    [Show abstract] [Hide abstract] ABSTRACT: Previous work in our laboratory demonstrated that hyperoxia suppressed the expression of vascular endothelial growth factor (VEGF) by the embryonic lung, leading to increased epithelial cell apoptosis and failure of explant airway growth and branching that was rescued by the addition of Vegf165. The aims of this study were to determine protective pathways by which VEGF isoforms attenuate hyperoxic lung growth retardation and to identify the target cell for VEGF action. Timed pregnant CD-1 or fetal liver kinase (FLK1)-eGFP lung explants cultured in 3% or 50% oxygen were treated +/- Vegf121, VEGF164/Vegf165 or VEGF188 in the presence or absence of anti-rat neuropilin-1 (NRP1) antibody or GO6983 (protein kinase C (PKC) pan-inhibitor) and lung growth and branching quantified. Immunofluorescence studies were performed to determine apoptosis index and location of FLK1 phosphorylation and western blot studies of lung explants were performed to define the signaling pathways that mediate the protective effects of VEGF. Heparin-binding VEGF isoforms (VEGF164/Vegf165 and VEGF188) but not Vegf121 selectively reduced epithelial apoptosis and partially rescued lung bud branching and growth. These protective effects required NRP1-dependent FLK1 activation in endothelial cells. Analysis of downstream signaling pathways demonstrated that the VEGF-mediated anti-apoptotic effects were dependent on PKC activation. Vegf165 activates FLK1-NRP1 signaling in endothelial cells, leading to a PKC-dependent paracrine signal that in turn inhibits epithelial cell apoptosis.
    Full-text · Article · Mar 2014
    • "Activation of fibroblasts by one or more of the coagulation proteinases via proteinase-activated receptors (PARs) has been studied extensively in vitro over the last fifteen years in relation to the ability to promote fibroblast proliferation and extracellular matrix production. Thrombin is a known mitogen for human lung fibroblasts and inhibits fibroblast apoptosis [311,313,319320321322323. Thrombin stimulated mitogen-activated protein (MAP) kinase activation and DNA synthesis in normal mouse fibroblasts, and these responses were absent in fibroblasts from thrombin receptor knockout mice [319]. "
    [Show abstract] [Hide abstract] ABSTRACT: Pulmonary fibrosis is a chronic lung disease characterized by excessive accumulation of extracellular matrix (ECM) and remodeling of the lung architecture. Idiopathic pulmonary fibrosis is considered the most common and severe form of the disease, with a median survival of approximately three years and no proven effective therapy. Despite the fact that effective treatments are absent and the precise mechanisms that drive fibrosis in most patients remain incompletely understood, an extensive body of scientific literature regarding pulmonary fibrosis has accumulated over the past 35 years. In this review, we discuss three broad areas which have been explored that may be responsible for the combination of altered lung fibroblasts, loss of alveolar epithelial cells, and excessive accumulation of ECM: inflammation and immune mechanisms, oxidative stress and oxidative signaling, and procoagulant mechanisms. We discuss each of these processes separately to facilitate clarity, but certainly significant interplay will occur amongst these pathways in patients with this disease.
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