Identification of a molecular signaling gene-gene regulatory network between GWAS susceptibility genes ADTRP and MIA3/TANGO1 for coronary artery disease

Abstract

Coronary artery disease (CAD) is the leading cause of death worldwide. GWAS have identified >50 genomic loci for CAD, including ADTRP and MIA3/TANGO1. However, it is important to determine whether the GWAS genes form a molecular network. In this study, we have uncovered a novel molecular network between ADTRP and MIA3/TANGO1 for the pathogenesis of CAD. We showed that knockdown of ADTRP expression markedly down-regulated expression of MIA3/TANGO1. Mechanistically, ADTRP positively regulates expression of PIK3R3 encoding the regulatory subunit 3 of PI3K, which leads to activation of AKT, resulting in up-regulation of MIA3/TANGO1. Both ADTRP and MIA3/TANGO1 are involved in endothelial cell (EC) functions relevant to atherosclerosis. Knockdown of ADTRP expression by siRNA promoted oxidized-LDL-mediated monocyte adhesion to ECs and transendothelial migration of monocytes, inhibited EC proliferation and migration, and increased apoptosis, which was reversed by expression of constitutively active AKT1 and MIA3/TANGO1 overexpression, while the over-expression of ADTRP in ECs blunted these processes. Knockdown of MIA3/TANGO1 expression also promoted monocyte adhesion to ECs and transendothelial migration of monocytes, and vice versa for overexpression of MIA3/TANGO1. We found that ADTRP negatively regulates the levels of collagen VII and ApoB in HepG2 and endothelial cells, which are downstream regulatory targets of MIA3/TANGOI. In conclusion, we have uncovered a novel molecular signaling pathway for the pathogenesis of CAD, which involves a novel gene-gene regulatory network. We show that ADTRP positively regulates PIK3R3 expression, which leads to activation of AKT and up-regulation of MIA3/TANGO1, thereby regulating endothelial cell functions directly relevant to atherosclerosis.

Androgen inhibits key atherosclerotic processes by directly activating ADTRP transcription

“…An assay for monocyte adhesion to ECs was carried out as described [18,37]. In brief, EAhy926 endothelial cells were transfected with siRNA or plasmid DNA as described above.…”

“…Transmigration of monocytes across endothelial cells was analyzed by a Transwell assay using the Boyden chamber method as described [17,18,37]. In brief, HL-60cells (3 × 10 5 ) were loaded into 0.4 µm pore size Transwell membranes within a Boyden chamber (Corning), which were plated with confluent EAhy926 endothelial cells transfected with siRNA or plasmid DNA.…”

“…Real-time RT-PCR analysis was performed as described previously by us [17,18,38]. The primer sequences used are as follows:

ADTRP Forward: 5′-GCCGCATCCTATGGCTCTACTTTG-3′.

ADTRP Reverse: 5′-CAAGTAGGTAGATGCTGGCGATGA-3′.

MIA3 Forward: 5′-TACAAGCGGAGAATTGAAGAAATGG-3′.

MIA3 Reverse: 5′-GCCAGTTTTCATGAGCTTTCTTCT-3′.

GAPDH Forward: 5′-ATGGGGAAGGTGAAGGTCG-3′.

GAPDH Reverse: 5′-GGGGTCATTGATGGCAACAATA-3′.

PIK3R3 Forward: 5′-ATGTACAATACGGTGTGGAGTATG-3′.

PIK3R3 Reverse: 5′-GCTGGAGGATCCATTTCAAT-3′.

…”

“…Recently, we reported that ADTRP regulates cell cycle progression, proliferation, and apoptosis by global regulation of gene expression [17]. Moreover, we also reported that ADTRP regulates the levels of MIA3/TANGO1 (another CAD and MI-associated gene identified by GWAS), collagen VII and ApoB by positively regulating the expression of PIK3R3 and activation of AKT [18]. The ADTRP-MIA3-collagen VII/ApoB network was associated with monocyte adhesion to endothelial cells and transmigration of monocytes across the endothelial cell layer, the two cellular processes directly relevant to atherosclerosis [18].…”

“…Moreover, we also reported that ADTRP regulates the levels of MIA3/TANGO1 (another CAD and MI-associated gene identified by GWAS), collagen VII and ApoB by positively regulating the expression of PIK3R3 and activation of AKT [18]. The ADTRP-MIA3-collagen VII/ApoB network was associated with monocyte adhesion to endothelial cells and transmigration of monocytes across the endothelial cell layer, the two cellular processes directly relevant to atherosclerosis [18]. Chinetti-Gbaguidi et al recently showed that ADTRP is expressed in human macrophages and atherosclerotic lesions, and its expression is regulated by the peroxisome proliferator-activated receptor (PPAR)γ in human primary macrophages [19].…”