Comparison of expression and regulation of the high‐density lipoprotein receptor SR‐BI and the low‐density lipoprotein receptor in human adrenocortical carcinoma NCI‐H295 cells

Abstract

In rodents, cholesterol for adrenal steroidogenesis is derived mainly from high-density lipoproteins (HDL) via the HDL receptor, scavenger receptor-BI (SR-BI). In humans cholesterol for steroidogenesis is considered to be derived from the low-density lipoprotein (LDL) receptor pathway, and the contribution of SR-BI to that is unknown. In the present study SR-BI expression and regulation by steroidogenic stimuli was analysed in human adrenocortical cells and compared with LDL receptor expression. In addition, the functional contribution of both receptors for cholesteryl ester delivery to human adrenocortical cells was compared. Northern blot and reverse transcription±PCR amplification and sequence analysis demonstrated the presence of SR-BI mRNA in foetal and adult human adrenal cortex. Furthermore, SR-BI mRNA was expressed to similar levels in human primary adrenocortical and adrenocortical carcinoma NCI-H295 cells, indicating its presence in the steroid-producing cells. Treatment of NCI-H295 cells with 8Br-cAMP, a stimulator of glucocorticoid synthesis via the protein kinase A second messenger signal transduction pathway, resulted in an increase of both SR-BI and LDL receptor mRNA levels in a time-and dose-dependent manner. The induction of SR-BI and LDL receptor by cAMP was independent of ongoing protein synthesis and occurred at the transcriptional level. Ligand blot experiments indicated that a protein of similar size to SR-BI is the major HDL-binding protein in NCI-H295 cells. Western blot analysis demonstrated that cAMP treatment increased the levels of LDL receptor and, to a lesser extent, SR-BI protein in NCI-H295 cells. Binding and uptake of cholesterol was quantitatively smaller from HDL than from LDL, both in basal as well as in cAMPstimulated cells. Scatchard analysis under basal conditions indicated that NCI-H295 cells express twice as many specific binding sites for LDL than for HDL. Dissociation constant values (K d ; in nm) were approximately five times higher for HDL than for LDL, indicating a lower affinity of HDL compared with LDL. The combined effects of these two parameters and the low cholesteryl ester content of HDL subfraction 3 (HDL 3 ) contributes to a lower cholesteryl ester uptake from HDL than from LDL by the NCI-H295 cells. In conclusion, both the SR-BI and LDL receptor genes are expressed in the human adrenal cortex and coordinately regulated by activators of glucocorticoid synthesis. In contrast to rodents, in human adrenocortical cells the HDL pathway of cholesterol delivery appears to be of lesser importance than the LDL pathway. Nevertheless, the SR-BI pathway may become of major importance in conditions of functional defects in the LDL receptor pathway.

Apolipoprotein AII Enrichment of HDL Enhances Their Affinity for Class B Type I Scavenger Receptor but Inhibits Specific Cholesteryl Ester Uptake

“…27 In a recent study, we demonstrated upregulation of SR-BI expression and CE uptake by cAMP in these cells. 18 In the present study, we show in Figure 1 that reconstituted HDLs containing apoAI as the sole apolipoprotein bind to NCI-H295R cells. In cross-linking experiments, we also demonstrate that SR-BI is probably the major binding site for apoAI in these cells.…”

“…18 In the present study, we have established that in these cells SR-BI is the major apoAI binding protein and that these cells can be used to study the different ligands of this receptor. We have focused on the respective role of the 2 major HDL proteins, apoAI and apoAII, regarding binding to SR-BI and the consequences for CE uptake.…”

Cloning, expression, and promoter analysis of hepatic lipase derived from human hyperplastic adrenals

“…For comparison, we also used oligonucleotides specific for human SR-B1 in the RT-PCR. A product of the expected size (610 bp) was readily obtained, confirming previous reports that SR-B1 is expressed in this cell line (29).…”

Prediabetic and diabetic in vivo modification of circulating low-density lipoprotein attenuates its stimulatory effect on adrenal aldosterone and cortisol secretion

“…Lipoprotein receptor expression and AngII-mediated regulation of lipoprotein metabolism in H295R adrenocortical cells have been studied previously (Martin et al 1999, Cherradi et al 2001, Pilon et al 2003. Cherradi et al (2001) and Pilon et al (2003) reported that AngII stimulated SR-BI, but not LDL receptor expression.…”