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 expressi on is regulated differently in both liver and intestine by dietary cho lesterol and dietary saturated fat. While dietary cholesterol serves t o regulate at transcriptional levels, dietary fatty acids do not. To s tudy 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 det ermined by I-125-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 prot ein by 5- to 6-fold in HepG2 cells and 2- to 3-fold in Caco2 cells. Ab solute levels of LDL-receptor mRNA and relative rates of LDL-receptor mRNA transcription also increased in parallel to the LDL-receptor acti vity and protein levels in both cell lines. These data suggest that LP DS induced the LDL-receptor gene by transcriptional mechanism. The sup pressive effect of 25-hydroxycholesterol on LDL-receptor regulation wa s 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-hydr oxycholesterol. In HepG2 cells, LDL-receptor activity and protein mass progressively decreased to 50% of zero time controls over 24 h. LDL-r eceptor mRNA levels and relative rates of transcription decreased in p arallel. In Caco2 cells, 25-hydroxycholesterol lowered LDL-receptor ac tivity, mRNA, and transcription by similar to 35%. To examine the effe cts of palmitate on LDL-receptor regulation, palmitate was complexed w ith albumin. Palmitate decreased LDL-receptor activity by 25% in HepG2 cells without altering LDL-receptor mass, mRNA levels, or rates of mR NA transcription. Similarly, in Caco2 cells, palmitate decreased LDL-r eceptor activity and protein mass 30% of controls, but did not change LDL-receptor mRNA levels and/or rates of transcription. The combinatio n of palmitate (0.8 mM) and 25-hydroxycholesterol (2.5-5 mu g/ml) supp ressed LDL-receptor activity by 65% in HepG2 cells and by 52% in Caco2 cells. However, LDL-receptor mRNA decreased by similar to 50% in HepG 2 cells and 30-40% in Caco2 cells. Thus, there were further decreases in LDL-receptor activity and mRNA levels when palmitate and 25-hydroxy cholesterol were present together in the media as compared to 25-hydro xycholesterol alone. Oleate did not affect LDL-receptor activity. Thus , a) exogenously added 25-hydroxycholesterol regulates LDL-receptor ge ne expression by transcriptional mechanism, the amplitude of regulatio n being greater in HepG2 than in Caco2 cells; b) exogenously added pal mitate regulates LDL-receptor gene expression by posttranscriptional m echanism, possibly at the posttranslational level; and c) a combinatio n of cholesterol and palmitate have additive effects on the regulation of the LDL-receptor gene in part via posttranscriptional and in part via transcriptional mechanisms.