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

Abstract

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 in fibrosis: novel roles and mediators

“…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).…”

Direct thrombin inhibition with dabigatran attenuates pressure overload-induced cardiac fibrosis and dysfunction in mice

“…Thrombin, a multifunctional serine protease, exerts pro-inflammatory and pro-fibrotic cellular effects in normal tissues (7) and in settings of pathological conditions such as liver fibrosis (8), pulmonary fibrosis (9), renal fibrosis (10), atherosclerotic plaque formation (11) and tumor angiogenesis (12–16). Accumulating evidence suggests a role for thrombin in cardiac hypertrophy/fibrosis, either through its proinflammatory and profibrotic functions, or by signaling via the cell-surface protease-activated receptors (PAR) (17, 18).…”

“…Additionally increased intercellular calcium, extracellular signal-regulated kinase (ERK) 1/2, DNA synthesis and cellular proliferation have all been reported (21). In the profibrotic lung, ERK1/2 signaling, induced by PAR-1 expression, has been shown to be critical in the formation of pulmonary fibrosis, where PAR-1 is significantly increased in patients with severe lung disease (12, 14, 15). Additionally, increased PAR-1 expression has been observed in the hearts of patients with ischemic and idiopathic-dilated cardiomyopathy, and in animals with chronic heart failure (22).…”

Heparin binding VEGF isoforms attenuate hyperoxic embryonic lung growth retardation via a FLK1-neuropilin-1-PKC dependent pathway

“…There is increasing evidence that VEGF-mediated activation of PKC can also induce protection against damaging insults (e.g. radiation, inflammatory diseases, hyperoxia) [16,26-28]. 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).…”