Aberrant Caveolin-1-Mediated Smad Signaling and Proliferation Identified by Analysis of Adenine 474 Deletion Mutation (c.474delA) in Patient Fibroblasts: A New Perspective in the Mechanism of Pulmonary Hypertension

Abstract

ABSTRACT A heterozygous caveolin-1 c.474delA mutation has been identified in a family with heritable pulmonary arterial hypertension (PAH). This frameshift mutation leads to a ca- veolin-1 protein that contains all known functional domains but has a change in only the final 20 amino acids of the C-terminus. Here we studied how this mutation alters caveolin-1 func- tion, using patient-derived fibroblasts. Transmission electron microscopy showed that fibro- blasts carrying the c.474delA mutation form typical caveolae. Expression of mutated caveo- lin-1 in caveolin-1–null mouse fibroblasts failed to induce formation of caveolae due to retention of the mutated protein in the endoplasmic reticulum. However, coexpression of wild-type caveolin-1 with mutated caveolin-1 restored the ability to form caveolae. Impor- tantly, fibroblasts carrying the mutation showed twofold increase in proliferation rate associ- ated with hyperphosphorylation of Smad1/5/8. This mutation impaired the antiproliferative function of caveolin-1. Inhibition of type I TGFβ receptors ALK1/2/3/6 responsible for phos- phorylation of Smad1/5/8 reduced the hyperproliferation seen in c.474delA fibroblasts. These results demonstrate the critical role of the final 20 amino acids of caveolin-1 in modu- lating fibroblast proliferation by dampening Smad signaling and suggest that augmented Smad signaling and fibroblast hyperproliferation are contributing factors in the pathogenesis of PAH in patients with caveolin-1 c.474delA mutation.