Cavin-1 modulates BMP/Smad signaling through the interaction of Caveolin-1 with BMPRII in pulmonary artery endothelial cells. (14th October 2021) - Tomita, S
Abstract: Background: Pulmonary hypertension (PH) is a progressive disease associated with poor outcomes. Caveolin-1 (Cav1) and Cavin-1 are components of caveolae, and Cav1 is identified as a related gene of pulmonary arterial hypertension (PAH). Gene mutations of bone morphogenetic protein type II receptor (BMPRII) is the most common cause of PAH. BMPRII is localized in caveolae and associates with Cav1. However, the role of the Caveolin-Cavin system on the BMP/Smad signaling and the PAH progression has not been well-known. Purpose: Our study aims to investigate the relationship between Caveolin-Cavin system and BMP/Smad signaling pathway in pulmonary artery endothelial cells (PAECs). [Methods] Cav1 knockout mice were used to assess PH, and caveolae in PAECs were observed by electron microscope. After knocking down Cav1 and/or Cavin-1 in human PAECs (hPAECs) using siRNA, we evaluated the phosphorylation of Smad by Western blotting. Apoptosis was explored by flow cytometry. To assess the interaction between Cav1 and BMPRII, and the effect of Cavin-1 for this interaction and BMP/Smad signaling, we performed immunoprecipitation, Co-immunostaining, Proximal Ligation Assay (PLA), GST pulldown assay, and Western blotting. Results: As in previous reports, Cav1 knockout mice exhibited PH with pulmonary vascular remodeling and right ventricular hypertrophy and PAECs isolated from Cav1 knockout mice showed caveolae disappearance. Cav1 knockdown in hPAECs reduced BMPRII at the plasma membrane and Smad 1/5/9 phosphorylation. Cav1 knockdown also significantly increased hypoxia-induced apoptosis in hPAECs. Co-immunostaining revealed that Cav1 was associated with BMPRII at the membrane of hPAECs. Cavin-1 inhibited the interaction of BMPRII with Cav1 and reduced BMPRII localization on the membrane of hPAECs. GST pulldown assay revealed that Cavin-1 and BMPRII were associated with Cav1 through the scaffolding domain in Cav1. These findings suggest that Cavin-1 and BMPRII are competitively associated with Cav1. Cavin-1 knockdown improved the interaction between Cav1 and BMPRII and inhibited both BMPRII reduction at the plasma membrane and Smad 1/5/9 dephosphorylation. Conclusions: Cavin-1 affects the interaction of Cav1 with BMPRII at the plasma membrane and modulates BMP/Smad signaling in PAECs. The binding of Cavin-1 to Cav1 enhances the interaction between BMPR2 and Cav1, resulting in stabilization of BMPRII localization at the plasma membrane in PAECs and prevention of BMP/Smad signaling attenuation, which is important for PAH development. Funding Acknowledgement: Type of funding sources: None.