Adenylyl cyclase 1 as a major isoform to generate cAMP signaling for apoA-1-mediated cholesterol efflux pathway

Wanze Tang,Weilie Ma,Hang Ding,Margarita Lin,Le Xiang,Guorong Lin,Zhizhen Zhang

doi:10.1194/jlr.m082297

Abstract

HDL apoA-1-mediated cholesterol efflux pathway requires multiple cellular proteins and signal transduction processes, including adenylyl cyclase (AC)/cAMP signaling. Due to the existence of multiple transmembrane AC isoforms, it was not known how many AC isoforms are expressed and which ones are essential for cholesterol efflux in macrophage foam cells. These questions were investigated in THP-1 macrophages in this study. Quantitative RT-PCR detected mRNAs for all nine transmembrane AC isoforms, but only the mRNA and protein of the AC1 isoform were consistently upregulated by cholesterol loading and apoA-1. AC1 shRNA interference decreased AC1 mRNA and protein levels, resulting in reduction of apoA-1-mediated cAMP production and cholesterol efflux, while the intracellular cholesterol levels remained high. Confocal microscopy showed that apoA-1 promoted translocation of cholesterol and formation of cholesterol-apoA-1 complexes (protrusions) on the cholesterol-loaded macrophage surface. AC1 shRNA-interfered macrophages showed no translocation of cholesterol to the cell surface. AC1 shRNA interference also disrupted cellular localization of the intracellular cholesterol indicator protein adipophillin, and the expression as well as surface translocation of ABCA1. Together, our results show that AC1 is a major isoform for apoA-1-activated cAMP signaling to promote cholesterol transport and exocytosis to the surface of THP-1 macrophage foam cells.

Highlights

HDL apoA-1-mediated cholesterol efflux pathway requires multiple cellular proteins and signal transduction processes, including adenylyl cyclase (AC)/cyclic AMP (cAMP) signaling
AC isoform expressions have been reported in macrophages from liver and lung [21], it is known how many AC isoforms are expressed in macrophage foam cells, the principal components in atherosclerotic lesions, and which AC isoform(s) is required for apoA-1 mediated cholesterol efflux in foam cells is unknown
Real-time quantitative RT-PCR (qRT-PCR) expression profiling showed that mRNAs of all transmembrane adenylyl cyclases were detected in THP-1 macrophages

Summary

Introduction

HDL apoA-1-mediated cholesterol efflux pathway requires multiple cellular proteins and signal transduction processes, including adenylyl cyclase (AC)/cAMP signaling. Signal transduction is one of the most complex events, which includes the activation of adenylyl cyclase and cAMP generation [9, 10], activation of Cdc 42 [11], G protein [12], Rac GTPase [13], Janus kinase 2 [14], c-Jun N-terminal kinases and p38 MAP kinase [11], protein kinase A (PKA) [10], protein kinase C [15], and phospholipase C and D [16], as well as intracellular calcium release [17] How exactly these proteins and signal mediators facilitate cholesterol removal from cells remains ambiguous. AC isoform expressions have been reported in macrophages from liver and lung [21], it is known how many AC isoforms are expressed in macrophage foam cells, the principal components in atherosclerotic lesions, and which AC isoform(s) is required for apoA-1 mediated cholesterol efflux in foam cells is unknown We investigated these questions by using quantitative (q)RT-PCR screening, shRNA interference, biochemical analysis, and confocal microscopy in this study. We report that AC1 is a major isoform activated by apoA-1 to promote cholesterol efflux from THP-1 macrophage foam cells

Methods

Results

Conclusion