Caveolin-1 rs1997623 Single Nucleotide Polymorphism Creates a New Binding Site for the Early B-Cell Factor 1 That Instigates Adipose Tissue CAV1 Protein Overexpression

Abstract

Caveolin-1 (CAV1) is implicated in the pathophysiology of diabetes and obesity. Previously, we demonstrated an association between the CAV1 rs1997623 C > A variant and metabolic syndrome (MetS). Here, we decipher the functional role of rs1997623 in CAV1 gene regulation. A cohort of 38 patients participated in this study. The quantitative MetS scores (siMS) of the participants were computed. CAV1 transcript and protein expression were tested in subcutaneous adipose tissue using RT-PCR and immunohistochemistry. Chromatin immunoprecipitation assays were performed using primary preadipocytes isolated from individuals with different CAV1 rs1997623 genotypes (AA, AC, and CC). The regulatory region flanking the variant was cloned into a luciferase reporter plasmid and expressed in human preadipocytes. Additional knockdown and overexpression assays were carried out. We show a significant correlation between siMS and CAV1 transcript levels and protein levels in human adipose tissue collected from an Arab cohort. We found that the CAV1 rs1997623 A allele generates a transcriptionally active locus and a new transcription factor binding site for early B-cell factor 1 (EBF1), which enhanced CAV1 expression. Our in vivo and in vitro combined study implicates, for the first time, EBF1 in regulating CAV1 expression in individuals harboring the rs1997623 C > A variant.

Adipose Tissue Caveolin-1 Upregulation in Obesity Involves TNF-α/NF-κB Mediated Signaling

“…Peripheral blood samples (~10 mL each) were obtained from the participants after an overnight fast; the samples were collected in vacutainer EDTA tubes, and plasma was collected by centrifugation, aliquoted, and stored at −80 °C until assayed [ 25 ]. Fasting blood glucose (FBG) levels and lipid profile, including total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglycerides, were measured using the Siemens Dimension RXL Chemistry analyzer (Diamond Diagnostics, Holliston, MA, USA).…”

“…To remove the background, the normalized activity of the luciferase reporter vector alone was subtracted from its activity in the presence of TNF-α. Relative luciferase units (RLUs) represent luciferase activity normalized to Renilla activity [ 25 , 30 ].…”