Abstract
Activation of genes containing SRE-1 (sterol regulatory element 1) sequences is known to be under the regulation of sterols through modulation of the proteolytic maturation of SREBPs (SRE-1-binding proteins). Previous work has demonstrated SREBP-mediated transcriptional activation of genes encoding enzymes of sterol and fatty acid biosynthesis. Because synthesis of both sterols and C18 fatty acids are required for cell growth, in the absence of exogenous supplements of these lipids, we examined the hypothesis that fatty acid can also be regulatory in SREBP maturation. Our data indicate that C18 fatty acids can potentiate the biological activities of a typical, regulatory sterol: 25-hydroxycholesterol. Inhibition of C18 fatty acid synthesis in cells cultured in serum-free medium renders them resistant to killing by 25-hydroxycholesterol. Repression of expression of reporter constructs driven by promoters bearing SRE-1 element(s) by 25-hydroxycholesterol is increased by C18 fatty acid supplementation. C18 fatty acids also increase the inhibitory effect of 25-hydroxycholesterol on proteolytic maturation and nuclear localization of SREBPs. Furthermore, we also show that C18 fatty acid supplementation can enhance the inhibitory effect of 25-hydroxycholesterol on sterol and fatty acid biosynthesis. These results demonstrate that maximal down-regulation of SREBP maturation and the consequent repression of SRE-1 promoters occurs in response to both a regulatory sterol and fatty acid.
Highlights
Oxygenated sterols, such as 25-hydroxycholesterol, are down-regulators of cholesterol biosynthesis [1]. 25-Hydroxycholesterol down-regulates the rate-limiting step in this pathway, 3-hydroxy-3-methyl-glutaryl (HMG)1 coenzyme A reductase, through several mechanisms and has a pleiotropic effect on transcription of all the genes of the cholesterol biosynthetic pathway that have been studied to date [2]
The mutants characterized to date, fall into two classes [3]: class 1 mutants are sterol-resistant because they produce a truncated SREBP-2 that serves as a constitutive, mature transcription factor, and class 2 mutants are defective in a protein called SCAP (SREBP-cleavage activating protein), which appears to transduce the oxysterol-mediated inhibition of SREBP proteolysis
We have previously shown that a somatic cell mutant defective in elongation of palmitate is resistant to the cytotoxic effects of 25-hydroxycholesterol in lipid-free medium [15].we reasoned that if C18 fatty acid synthesis was necessary for 25-hydroxycholesterol cytotoxicity, Hexadecynoic acid (HDA) treatment might render cells resistant to 25-hydroxycholesterol
Summary
Materials—IgG-2A4 and IgG-7D4 cells, which produce monoclonal antibodies SREBP-1 and SREBP-2, respectively, were purchased from American Type Culture Collection (catalog numbers CRL-2121 and CRL-2198). On day 1, the cells were rinsed with phosphatebuffered saline (PBS) twice and fed either Nutridoma-SP (1% in Ham’s F12) or Nutridoma-SP containing fatty acids and sterols as described in the figure legends. Following a 16-h incubation, the medium was removed, and the cells were rinsed twice with PBS. The cells were cultured for 16 h in F12 medium containing 5% twice-delipidized [15] fetal calf serum (F12DIPE5) in the presence or absence of 1 g/ml 25-hydroxycholesterol and supplemented with either 0.03% FAF-BSA or 10 M stearate and 10 M oleate complexed to 0.03% FAF-BSA. The protein concentration of each dish was determined by solublizing the lipid extracted cells with 0.1 N NaOH and performing a BCA protein assay (Pierce) using BSA as standard
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