Serum amyloid A and toll-like receptor 2 regulate vascular smooth muscle cell cholesterol trafficking and differentiation
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Vascular smooth muscle cells (SMCs) regulate vessel contraction but during diseases including atherosclerosis, SMCs undergo functional changes that contribute to pathology. Chronic inflammation in the vasculature exacerbates disease progression. Acute phase serum amyloid A (SAA) is up-regulated during inflammation and expressed in atherosclerotic lesions. Previous work in our laboratory demonstrated that SAA activates secretory phospholipase A2 group IIA (sPLA2), whose products impact cellular cholesterol homeostasis. It was hypothesized that SAA promotes cholesterol trafficking from the plasma membrane to the endoplasmic reticulum (ER) in an sPLA2-dependent manner. SAA induced SMC cholesterol accumulation in the ER. Levels of plasma membrane cholesterol decreased, confirming that cholesterol moved from the plasma membrane to the ER. Another family member, (cytosolic phospholipase A2, group IV), was also required for SAA-induced sPLA2 activation and cholesterol mobilization. SAA activated neutral sphingomyelinase and blocking this activity inhibited cholesterol trafficking. These studies show that SAA activated sPLA2 which activated neutral sphingomyelinase. As a result, sphingomyelin was cleaved, which liberated cholesterol for movement to the ER. Additional studies demonstrated that SAA repressed expression of SMC contractile markers including Acta2 and Myh11. Toll-like receptor 2 (TLR2) is an SAA receptor implicated in atherogenesis and it was hypothesized that TLR2 plays a role in SAA-mediated phenotype/gene changes. The TLR2 ligands, FSL and Pam3CSK4, down-regulated SMC contractile marker expression. Knockdown of TLR2 demonstrated that SAA-mediated phenotype modulation was TLR2-dependent. SAA, FSL, and Pam3CSK4 also induced mRNA expression of pro-inflammatory and adhesion genes, changes inhibited by TLR2 knockdown. SAA repressed activity of the αSMA promoter, demonstrating transcriptional regulation. Myocardin, a transcription factor required to drive expression of SMC contractile genes, was down-regulated by SAA and FSL. Myocardin overexpression abrogated SAA- and FSL-mediated repression of the αSMA and SM22α promoters. These studies demonstrate that SAA promoted a phenotypic switch through activation of TLR2 and down-regulation of myocardin expression. Taken together, novel SAA- and TLR2-mediated mechanisms of cholesterol trafficking and phenotypic modulation in SMCs are shown. Importantly, this work uncovers previously unknown effects of TLR2 signaling on vascular SMCs and provides a context by which TLR2 activation and lesion-associated SAA may promote atherosclerosis.