Sulfation of 25-hydroxycholesterol by SULT2B1b decreases cellular lipids via the LXR/SREBP-1c signaling pathway in human aortic endothelial cells

Abstract

Objective: 25-Hydroxycholesterol (25HC) and its sulfated metabolite, 25-hydroxycholesterol-3-sulfate (25HC3S), regulate certain aspects of lipid metabolism in opposite ways. Hence, the enzyme for the biosynthesis of 25HC3S, oxysterol sulfotransferase (SULT2B1b), may play a crucial role in regulating lipid metabolism. We evaluate the effect of 25HC sulfation on lipid metabolism by overexpressing the gene encoding SULT2B1b in human aortic endothelial cells (HAECs) in culture.

Methods and results: The human SULT2B1b gene was successfully overexpressed in HAECs following infection using a recombinant adenovirus. HPLC analysis demonstrated that more than 50% of (3)H-25HC was sulfated in 24h following overexpression of the SULT2B1b gene. In the presence of 25HC, SULT2B1b overexpression significantly decreased mRNA and protein levels of LXR, ABCA1, SREBP-1c, ACC-1, and FAS, which are key regulators of lipid biosynthesis and transport; and subsequently reduced cellular lipid levels. Overexpression of the gene encoding SULT2B1b gave similar results as adding exogenous 25HC3S. However, in the absence of 25HC or in the presence of T0901317, synthetic liver oxysterol receptor (LXR) agonist, SULT2B1b overexpression had no effect on the regulation of key genes involved in lipid metabolism.

Conclusions: Our data indicate that sulfation of 25HC by SULT2B1b plays an important role in the maintenance of intracellular lipid homeostasis via the LXR/SREBP-1c signaling pathway in HAECs.

Figure 1

Determination of SULT2B1b overexpression on the metabolism of 25HC in HAECs. HAECs were infected with Ad-Control or Ad-Sult2B1b at MOI = 10 pfu/cell for 24h, then the medium was changed to serum-free media containing 1μCi ³H-25HC or ³H-25HC3S. (A) The relative percent of counts in the media were detected at the different times as indicated. (B) The efficiency of 25HC sulfation in the cells was measured after folch partitioning with chloroform-methanol (2:1, v/v). (C and D) The standard controls of ³H-25HC and ³H-25HC3S were analyzed by HPLC. (E and F) HPLC analysis of the cells with Ad-control or Ad-SULT2B1b infection in the presence of ³H-25HC. The data represent one of three separate experiments. * P<0.05 vs. 25HC or Ad-Sult 2h; ** P<0.01 vs. 25HC; ## P<0.01 vs. Ad-Con.

Figure 2

Effect of SULT2B1b overexpression on lipids metabolism in the presence of 25HC in HAECs. HAECs were infected with Ad-control or Ad-SULT2B1b at the indicated MOI and treated with 25HC (3μM) for another 48h. Total intracellular neutral lipids were extracted by adding 10 volumes of chloroform/methanol mixture (2:1, v/v). (A-D) Intracellular triglycerides (TG), free fatty acids (FFA), total cholesterol (TC) and free cholesterol (FC) were analyzed as described in Methods. (E and F) TLC analysis of the newly synthesized lipids by using ¹⁴C-acetic acid and quantification by liquid-scintillation counting. The data represent one of two experiments performed in duplicate. * P<0.05 vs. Con, ** P<0.01 vs. Con, # P<0.05 vs. 25HC, ## P<0.01 vs. 25HC.

Figure 3

Effect of SULT2B1b overexpressoin on LXR and its target genes. (A) Western blot analysis of nuclear extracts and cytosol proteins with specific antibodies against LXRalpha, ABCA1, SREBP-1 and SREBP-2 after infection with adenovirus in the presence of 25HC. (B-E) Real-time PCR analysis of LXRalpha, ABCA1, SREBP-1c and SREBP-2 mRNA levels, respectively. The data of western blot represent one of three separate experiments; and the mRNA data represent one of two experiments performed in duplicate. * P<0.05 vs. Con, ** P<0.01 vs. Con, # P<0.05 vs. 25HC, ## P<0.01 vs. 25HC, & P<0.05 vs. Ethanol, && P<0.01 vs. Ethanol.

Figure 4

Effect of SULT2B1b overexpression on SREBPs target genes. (A) Western blot analysis of total proteins in HAECs with specific antibodies against ACC1, FAS, and HMGR after infection with adenovirus in the presence of 25HC. (B-E) Real-time PCR analysis of ACC1, FAS, HMGR, and LDLR mRNA levels. The data of western blot represent one of three separate experiments; and the mRNA data represent one of two experiments performed in duplicate. * P<0.05 vs. Con, ** P<0.01 vs. Con, # P<0.05 vs. 25HC, ## P<0.01 vs. 25HC, & P<0.05 vs. Ethanol, && P<0.01 vs. Ethanol.

Figure 5

Effect of SUL2B1b overexpression depended on the presence of 25HC in HAECs. HAECs were infected with Ad-Control or Ad-SULT2B1b in the presence or absence of 25HC (3μM) or with T0901317 (1μM) for another 24h. (A-B) Western blot and real-time PCR analysis of SREBP-1 protein and mRNA expression. (C-D) Intracellular triglycerides and total cholesterol were analyzed as described in Methods. The data of western blot represent one of three separate experiments; and the data of mRNA and lipids represent one of two experiments performed in duplicate.* P<0.05 vs. Con, # P<0.05 vs. NA, ## P<0.01 vs. NA.