Regulation of low density lipoprotein receptor gene expression in HepG2 and Caco2 cells by palmitate, oleate, and 25-hydroxycholesterol.
Our in vivo studies in mice have shown that LDL-receptor gene expression is regulated differently in both liver and intestine by dietary cholesterol and dietary saturated fat. While dietary cholesterol serves to regulate at transcriptional levels, dietary fatty acids do not. To study the mechanism of regulation of LDL-receptor by saturated fat and cholesterol at the cellular level, where any secondary effects of long-term feeding in vivo are minimized we used the cultured hepatoma and colon carcinoma cells, HepG2 and Caco2. LDL-receptor activity was determined by 125I-labeled LDL binding and uptake, LDL-receptor protein by Western blotting, LDL-receptor mRNA by RNase protection assay, and relative rates of LDL-receptor mRNA transcription by nuclear 'run-off' assay. Incubation of cells in lipoprotein-deficient serum (LPDS) for 48 h progressively induced LDL-receptor activity and LDL-receptor protein by 5- to 6-fold in HepG2 cells and 2- to 3-fold in Caco2 cells. Absolute levels of LDL-receptor mRNA and relative rates of LDL-receptor mRNA transcription also increased in parallel to the LDL-receptor activity and protein levels in both cell lines. These data suggest that LPDS induced the LDL-receptor gene by transcriptional mechanism. The suppressive effect of 25-hydroxycholesterol on LDL-receptor regulation was studied by incubating HepG2 and Caco2 cells grown either in 10% FCS or 10% LPDS for 24 h and then for 0-24 h with various doses of 25-hydroxycholesterol. In HepG2 cells, LDL-receptor activity and protein mass progressively decreased to 50% of zero time controls over 24 h. LDL-receptor mRNA levels and relative rates of transcription decreased in parallel. In Caco2 cells, 25-hydrocholesterol lowered LDL-receptor activity, mRNA, and transcription by approximately 35%. To examine the effects of palmitate on LDL-receptor regulation, palmitate was complexed with albumin. Palmitate decreased LDL-receptor activity by 25% in HepG2 cells without altering LDL-receptor mass, mRNA levels, or rates of mRNA transcription. Similarly, in Caco2 cells, palmitate decreased LDL-receptor activity and protein mass 30% of controls, but did not change LDL-receptor mRNA levels and/or rates of transcription. The combination of palmitate (0.8 mM) and 25-hydroxycholesterol (2.5-5 micrograms/ml) suppressed LDL-receptor activity by 65% in HepG2 cells and by 52% in Caco2 cells. However, LDL-receptor mRNA decreased by approximately 50% in HepG2 cells and 30-40% in Caco2 cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Srivastava
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RA
Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
Ito
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H
Hess
M
M
Srivastava
N
N
Schonfeld
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G
D001769:Blood; D018938:Caco-2 Cells; D006528:Carcinoma, Hepatocellular; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D006888:Hydroxycholesterols / Q000008:administration & dosage / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008113:Liver Neoplasms; D019301:Oleic Acid; D009829:Oleic Acids / Q000008:administration & dosage / Q000494:pharmacology*; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000008:administration & dosage / Q000494:pharmacology*; D011973:Receptors, LDL / Q000235:genetics*; D012710:Serum Albumin, Bovine; D014407:Tumor Cells, Cultured
D006888:Hydroxycholesterols; D009829:Oleic Acids; D010169:Palmitic Acids; D011973:Receptors, LDL; D012710:Serum Albumin, Bovine; D019301:Oleic Acid; D019308:Palmitic Acid; C007997:25-hydroxycholesterol