Lanosterol metabolism and sterol regulatory element binding protein (SREBP) expression in male germ cell maturation

Expression of genes involved in cholesterol biosynthesis in male germ cells is insensitive to the negative cholesterol feedback regulation, in contrast to cholesterol level-sensitive/sterol regulatory element binding protein (SREBP)-dependent gene regulation in somatic cells. The role of sterol regulatory element binding proteins in spermatogenic cells was an enigma until recently, when a soluble, 55 kDa cholesterol-insensitive form of SREBP2 (SREBP2gc) was discovered [Mol. Cell. Endocrinol. 22 (2002) 8478], being translated from a germ cell-specific SREBP2 mRNA. Our RT-PCR results also show that SREBP2 as well as SREBP1c mRNAs are detectable in prepubertal and postpubertal male germ cells while SREBP1a is not detected. Surprisingly, three SREBP2 immunoreactive proteins (72, 63 and 55 kDa), that are not present in mouse liver nuclei, reside in testis nuclei of prepubertal and adult mice. The 55 kDa protein is likely SREBP2gc, the other two isoforms are novel. HPLC measurements in liver and testes of fasted prepubertal and postpubertal mice showed no significant difference in cholesterol level. However, FF-MAS and lanosterol/testis-meiosis activating sterol (T-MAS) intermediates that are detectable mainly in testes, increase in fasted postpubertal mice which coincides well with the elevated level of 68 kDa SREBP2. Similar to SREBP2gc, the two novel SREBP2 immunoreactive proteins seem to be insensitive to the level of cholesterol.     

Liver X receptor alpha interferes with SREBP1c-mediated Abcd2 expression. Novel cross-talk in gene regulation

The peroxisomal ATP binding cassette (ABC) transporter adrenoleukodystrophy-related protein, encoded by ABCD2, displays functional redundancy with the X-linked adrenoleukodystrophy-associated protein, making ABCD2 up-regulation of therapeutic value. Cholesterol lowering activates human ABCD2 in cultured cells. To investigate in vivo regulation by sterols, we first characterized a sterol regulatory element (SRE) in the murine Abcd2 promoter that is directly bound by SRE-binding proteins (SREBPs). Intriguingly, this element overlaps with a direct repeat 4, which serves as binding site for liver X receptor (LXR)/retinoid X receptor heterodimers, suggesting novel cross-talk between SREBP and LXR/retinoid X receptor in gene regulation. Using fasting-refeeding and cholesterol loading, SREBP accessibility to the SRE/direct repeat 4 was tested. Results suggest that adipose Abcd2 is induced by SREBP1c, whereas hepatic Abcd2 expression is down-regulated by concurrent activation of LXRalpha and SREBP1c. In cell culture, SREBP1c-mediated Abcd2 induction is counteracted by ligand-activated LXRalpha. Finally, hepatic Abcd2 expression in LXRalpha,beta-deficient mice is inducible to levels vastly exceeding wild type. Together, we identify LXRalpha as negative modulator of Abcd2, acting through a novel regulatory mechanism involving overlapping SREBP and LXRalpha binding sites.     

Different sterol regulatory element-binding protein-1 isoforms utilize distinct co-regulatory factors to activate the promoter for fatty acid synthase

Sterol regulatory element-binding proteins (SREBPs) activate genes of cholesterol and fatty acid metabolism. In each case, a ubiquitous co-regulatory factor that binds to a neighboring recognition site is also required for efficient promoter activation. It is likely that gene- and pathway-specific regulation by the separate SREBP isoforms is dependent on subtle differences in how the individual proteins function with specific co-regulators to activate gene expression. In the studies reported here we extend these observations significantly by demonstrating that SREBPs are involved in both sterol regulation and carbohydrate activation of the FAS promoter. We also demonstrate that the previously implicated Sp1 site is largely dispensable for sterol regulation in established cultured cells, whereas a CCAAT-binding factor/nuclear factor Y is critically important. In contrast, carbohydrate activation of the FAS promoter in primary hepatocytes is dependent upon SREBP and both the Sp1 and CCAAT-binding factor/nuclear factor Y sites. Because 1c is the predominant SREBP isoform expressed in hepatocytes and 1a is more abundant in sterol depleted established cell lines, this suggests that the different SREBP isoforms utilize distinct co-regulatory factors to activate target gene expression.     

Genetic deletion of low density lipoprotein receptor impairs sterol-induced mouse macrophage ABCA1 expression. A new SREBP1-dependent mechanism

Low density lipoprotein receptor (LDLR) mutations cause familial hypercholesterolemia and early atherosclerosis. ABCA1 facilitates free cholesterol efflux from peripheral tissues. We investigated the effects of LDLR deletion (LDLR(-/-)) on ABCA1 expression. LDLR(-/-) macrophages had reduced basal levels of ABCA1, ABCG1, and cholesterol efflux. A high fat diet increased cholesterol in LDLR(-/-) macrophages but not wild type cells. A liver X receptor (LXR) agonist induced expression of ABCA1, ABCG1, and cholesterol efflux in both LDLR(-/-) and wild type macrophages, whereas expression of LXRalpha or LXRbeta was similar. Interestingly, oxidized LDL induced more ABCA1 in wild type macrophages than LDLR(-/-) cells. LDL induced ABCA1 expression in wild type cells but inhibited it in LDLR(-/-) macrophages in a concentration-dependent manner. However, lipoproteins regulated ABCG1 expression similarly in LDLR(-/-) and wild type macrophages. Cholesterol or oxysterols induced ABCA1 expression in wild type macrophages but had little or inhibitory effects on ABCA1 expression in LDLR(-/-) macrophages. Active sterol regulatory element-binding protein 1a (SREBP1a) inhibited ABCA1 promoter activity in an LXRE-dependent manner and decreased both macrophage ABCA1 expression and cholesterol efflux. Expression of ABCA1 in animal tissues was inversely correlated to active SREBP1. Oxysterols inactivated SREBP1 in wild type macrophages but not in LDLR(-/-) cells. Oxysterol synergized with nonsteroid LXR ligand induced ABCA1 expression in wild type macrophages but blocked induction in LDLR(-/-) cells. Taken together, our studies suggest that LDLR is critical in the regulation of cholesterol efflux and ABCA1 expression in macrophage. Lack of the LDLR impairs sterol-induced macrophage ABCA1 expression by a sterol regulatory element-binding protein 1-dependent mechanism that can result in reduced cholesterol efflux and lipid accumulation in macrophages under hypercholesterolemic conditions.