TGF-β1 prevents simulated ischemia/reperfusion-induced cardiac fibroblast apoptosis by activation of both canonical and non-canonical signaling pathways

Abstract

Ischemia/reperfusion injury is a major cause of myocardial death. In the heart, cardiac fibroblasts play a critical role in healing post myocardial infarction. TGF-β1 has shown cardioprotective effects in cardiac damage; however, if TGF-β1 can prevent cardiac fibroblast death triggered by ischemia/reperfusion is unknown. Therefore, we test this hypothesis, and whether the canonical and/or non-canonical TGF-β1 signaling pathways are involved in this protective effect. Cultured rat cardiac fibroblasts were subjected to simulated ischemia/reperfusion. Cell viability was analyzed by trypan blue exclusion and propidium iodide by flow cytometry. The processing of procaspases 8, 9 and 3 to their active forms was assessed by Western blot, whereas subG1 population was evaluated by flow cytometry. Levels of total and phosphorylated forms of ERK1/2, Akt and Smad2/3 were determined by Western blot. The role of these signaling pathways on the protective effect of TGF-β1 was studied using specific chemical inhibitors. Simulated ischemia over 8h triggers a significant cardiac fibroblast death, which increased by reperfusion, with apoptosis actively involved. These effects were only prevented by the addition of TGF-β1 during reperfusion. TGF-β1 pretreatment increased the levels of phosphorylated forms of ERK1/2, Akt and Smad2/3. The inhibition of ERK1/2, Akt and Smad3 also blocked the preventive effects of TGF-β1 on cardiac fibroblast apoptosis induced by simulated ischemia/reperfusion. Overall, our data suggest that TGF-β1 prevents cardiac fibroblast apoptosis induced by simulated ischemia-reperfusion through the canonical (Smad3) and non canonical (ERK1/2 and Akt) signaling pathways.

TGF-β1 Increases Resistance of NIH/3T3 Fibroblasts Toward Apoptosis Through Activation of Smad2/3 and Erk1/2 Pathways

“…38,40,43,44 Our findings extend these observations to FasL, a direct ligand for death receptor-induced apoptosis in NIH/3T3 fibroblasts. The role of Erk pathways in apoptosis has been previously demonstrated using NIH/3T3 cells overexpressing Bad, which resulted in inactivation of Erk pathways and reduced phosphorylated Erk compared to cells without Bad transfection that translated into enhanced sensitivity to ceramideand serum deprivation-mediated apoptosis.…”

“…1,2,11,13,18,22,23 There is also increasing evidence that signaling pathways downstream of TGF-β1 receptors --both canonical Smad-dependent and noncanonical Smad-independent --are involved in mediating the effects of TGF-β1, including its antiapoptotic effect. 9,21,[33][34][35][36][37][38][39][40] Our findings in NIH/3T3 fibroblasts indicate that inhibition of a single pathway only partially reverses the antiapoptotic effect of TGF-β1, suggesting that both Smad-dependent and Erk1/2 pathways complement each other in suppression of apoptosis. The underlying basis for that is not completely understood and requires further investigation.…”

The cytokines of pulmonary fibrosis: Much learned, much more to learn

“…Both canonical and noncanonical TGF-b signaling pathways are inhibited by Smad6 and/ or Smad7 through a negative feedback loop [119]. Non-canonical signaling occurs through several other intracellular signaling molecules, including but not limited to endoglin/Smad1 [114,115,[120][121][122][123]; TRAF6 [119,[124][125][126]; ERK1/2 [115,121,[127][128][129][130], p38 [131,132] and JunB [133] MAPK; c-Abl [134]; mTOR [134]; PI3K/ AKT [115,128,132,135]; and NF-jB [136]. Much attention has additionally been directed recently to the Wnt/b-catenin pathway, which appears to promote proliferation and migration of lung fibroblasts and profibrotic gene expression [137,138], control TGF-b production and profibrotic activity [139,140], and facilitate EMT through direct interaction between Smad3 and b-catenin [141].…”

Transcriptional control of cardiac fibroblast plasticity

“…Resolution of fibrosis typically culminates in fibroblast apoptosis, however, a subset of CFs are resistant to apoptosis and remain within the scar [176–178]. A recent study revealed that P53+COL1A2+ cells express the endothelial cell (EC) marker VE-cadherin 3 days after IR injury [21].…”