Functional Characterization of a Novel Caveolin‐1 Adenine 474 Deletion (c.474delA Mutation) in TGFβ Signaling and Caveolae Formation
Authors: Yuan Yang , Marsboom Glenn , Chen Zhenlong , Minshall Richard D. , Rehman Jalees , Malik Asrar B. 
Published: November 2023

A caveolin‐1 c.474delA (Cav1*) mutation has recently been identified in a family with pulmonary arterial hypertension. This frameshift mutation leads to a protein that contains all known functional domains of caveolin‐1 and only has a change in its final 20 amino acids. It is unknown how this mutation close to the C‐terminal of Cav1 alters its functionality. We studied this mutated protein in human fibroblasts carrying a heterozygous Cav1* mutation and transfected Cav1‐null mouse fibroblasts to study the function of Cav1* in the absence of WT Cav1.

Human fibroblasts carrying the heterozygous Cav1* mutation only had half the number of caveolae compared to WT human fibroblasts as shown with transmission electron microscopy (P<0.01). The caveolae size distribution was very similar between both groups (mean diameter 75.3 ± 0.5 nm in control fibroblasts vs. 76.0 ± 0.7 nm in Cav1* fibroblasts, >600 caveolae were studied/group) and Cav1 oligomerization was also normal in Cav1* fibroblasts. Cholera toxin subunit B (CtxB) uptake at baseline was identical and was inhibited by more than 50% after cholesterol depletion with 5mM methyl‐β‐cyclodextrin in both groups. This suggested that endocytosis was normal despite the decreased caveolae number.

Expression of mutated Cav1* in Cav1‐null mouse fibroblasts failed to induce formation of caveolae due to retention of the protein in the endoplasmic reticulum. Adding WT Cav1 induced punctate staining starting at 30% WT Cav1 and rescued trafficking of mutant Cav1* to the cytoplasmic membrane. WT Cav1 and mutant Cav1* co‐localized in punctae and immunoprecipitation showed a direct interaction between WT Cav1 and mutant Cav1*.

At the functional level, our most striking observation was a 2‐fold increased proliferation rate of Cav1* human fibroblasts as shown with flow cytometry (P<0.001), while the production of collagen and secretion of MMP9 were increased by 30%. Fibroblast proliferation is enhanced by TGFβ signaling while TGFβ receptors interact with Cav1. Therefore, we next examined TGFβ signaling in human Cav1* fibroblasts. We found hyperphosphorylation of Smad1/5/8 in Cav1* human fibroblasts, while Smad2/3 phosphorylation was unaltered. Phosphorylation of Smad1/5/8 occurs through the ALK1/2/3/6 receptors and to determine whether ALK1/2/3/6 activity regulated hyper‐proliferation of Cav1* fibroblasts, we used LDN193189, an inhibitor of these receptors. LDN193189 significantly reduced proliferation of Cav1* fibroblasts but not wild type cells (P< 0.001)

Cav1 has been shown to inhibit a large number of receptors/proteins at baseline including eNOS and VEGFR‐2 and we hypothesize that a relative lack of caveolae leads to the increased ALK1/2/3/6 activation and subsequent proliferation of Cav1* fibroblasts. These results demonstrate the importance of the final 20 amino acids of Cav1 and provide a detailed functional characterization of this novel Cav1 mutation.

Support or Funding Information

This work was supported in part by a Parker B. Francis fellowship and American Heart Scientist Development grant 15SDG23250002 (GM), NIH‐R01‐GM094220 (JR) and NIH‐R01‐HL045638, R01‐HL090152, R01‐HL118068, P01‐HL060678, P01‐HL077806 (ABM).