Xiaoqiu Liu

cAMP inhibits transforming growth factor-β-stimulated collagen synthesis via inhibition of extracellular signal-regulated kinase 1/2 and smad signaling in cardiac fibroblasts

Cardiac fibroblasts produce and degrade extracellular matrix and are critical in regulating cardiac remodeling and hypertrophy. Cytokines such as transforming growth factor-β (TGF-β) play a fundamental role in the development of tissue fibrosis by stimulating matrix deposition and other profibrotic responses, but less is known about pathways that might inhibit fibrosis. Increased cAMP formation inhibits myofibroblast differentiation and collagen production by cardiac fibroblasts, but the mechanism of this inhibition is not known. We sought to characterize the signaling pathways by which cAMP-elevating agents alter collagen expression and myofibroblast differentiation. Treatment with 10 μM forskolin or isoproterenol increased cAMP production and cAMP response element binding protein (CREB) phosphorylation in cardiac fibroblasts and inhibited serum- or TGF-β-stimulated collagen synthesis by 37% or more. These same cAMP-elevating agents blunted TGF-β-stimulated expression of collagen I, collagen III, and α-smooth muscle actin. Forskolin or isoproterenol treatment blocked the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) induced by TGF-β despite the fact that these cAMP-elevating agents stimulated ERK1/2 activation on their own. cAMP-elevating agents also attenuated the activation of c-Jun NH2-terminal kinase and reduced binding of the transcriptional coactivator CREB-binding protein 1 to transcriptional complexes containing Smad2, Smad3, and Smad4. Pharmacological inhibition of ERK completely blocked TGF-β-stimulated collagen gene expression, but expression of an active mutant of MEK was additive with TGF-β treatment. Thus, cAMP-elevating agents inhibit the profibrotic effects of TGF-β in cardiac fibroblasts largely through inhibiting ERK1/2 phosphorylation but also by reducing Smad-mediated recruitment of transcriptional coactivators.

Thy-1 interaction with Fas in lipid rafts regulates fibroblast apoptosis and lung injury resolution.

Thy-1-negative lung fibroblasts are resistant to apoptosis. The mechanisms governing this process and its relevance to fibrotic remodeling remain poorly understood. By using either sorted or transfected lung fibroblasts, we found that Thy-1 expression is associated with downregulation of anti-apoptotic molecules Bcl-2 and Bcl-xL, as well as increased levels of cleaved caspase-9. Addition of rhFasL and staurosporine, well-known apoptosis inducers, caused significantly increased cleaved caspase-3, -8, and PARP in Thy-1-transfected cells. Furthermore, rhFasL induced Fas translocation into lipid rafts and its colocalization with Thy-1. These in vitro results indicate that Thy-1, in a manner dependent upon its glycophosphatidylinositol anchor and lipid raft localization, regulates apoptosis in lung fibroblasts via Fas-, Bcl-, and caspase-dependent pathways. In vivo, Thy-1 deficient (Thy1−/−) mice displayed persistence of myofibroblasts in the resolution phase of bleomycin-induced fibrosis, associated with accumulation of collagen and failure of lung fibrosis resolution. Apoptosis of myofibroblasts is decreased in Thy1−/− mice in the resolution phase. Collectively, these findings provide new evidence regarding the role and mechanisms of Thy-1 in initiating myofibroblast apoptosis that heralds the termination of the reparative response to bleomycin-induced lung injury. Understanding the mechanisms regulating fibroblast survival/apoptosis should lead to novel therapeutic interventions for lung fibrosis.