Abstract 620: The Cardiac Hormone BNP Inhibits Cardiac Fibroblast Proliferation via the Clearance Receptor and Modulation of p21 and Cyclin D1
Background: Previous studies have reported that genetic disruption of BNP results in cardiac fibrosis independent of cardiac load. Others and we have also reported that BNP inhibits collagen synthesis and cell proliferation, while stimulating collagen degradation through matrix metalloproteinase activation. Here we address cellular and molecular mechanisms of BNP-induced inhibition of cardiac fibroblast (CF) proliferation, a hallmark of cardiac fibrosis, by investigating BNP receptor activation and anti-proliferative actions. We also assessed for the first time the action of BNP on the cell cycle and mechanisms by which CF cell growth may be modulated by BNP.
Methods: Proliferation was assessed in human CFs by BrdU using the Roche Proliferation kit. CFs were treated with BNP (10−6M) with or without the NPR-A/B receptor antagonist HS-142–1 (10−6M) or the NPR-C receptor antagonist cANP (10−6M). BrdU incorporation was measured by absorbance on a spectrophotometer. Cell cycle protein levels were determined by western blot.
Results: Here we found that CF proliferation was suppressed (23%, p = 0.008) by BNP. Co-incubation with the NPRA/B antagonist HS-142–1 potentiated proliferation (38%, p= 0.0003) while co-incubation with the NPR-C antagonist cANP abolished this action, suggesting a non-cGMP mechanism via the NPR-C receptor. In addition, an inhibitor of protein kinase A and not protein kinase G mimicked the actions of the NPR-C antagonist. Interestingly, NPR-C displayed intense immunohistochemical staining in the nuclear region of dividing CFs compared to non-dividing cells. Analysis of cell cycle protein responses to BNP revealed that p21 expression was increased while Cyclin D1 expression was reduced. In contrast, Cyclin D3 and cdk6 were unchanged consistent with BNP disruption of the G1 phase of cell cycle progression.
Conclusions: Our findings underscore the anti-proliferative actions of BNP in cardiac fibroblasts. These studies suggest that anti-proliferative properties of BNP in cardiac fibroblasts may involve a non-cGMP mechanism that disrupts the cell cycle via upregulation of p21 and suppression of Cyclin D1 following binding to the natriuretic peptide clearance receptor.