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

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 der ived from the low-density lipoprotein (LDL) receptor pathway, and the contr ibution of SR-BI to that is unknown. In the present study SR-BI expression and regulation by steroidogenic stimuli was analysed in human adrenocortica l cells and compared with LDL receptor expression. In addition, the functio nal 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 adrenocorti cal carcinoma NCI-H295 cells, indicating its presence in the steroid-produc ing cells. Treatment of NCI-H295 cells with 8Br-cAMP, a stimulator of gluco corticoid synthesis via the protein kinase A second messenger signal transd uction 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 LD L receptor by cAMP was independent of ongoing protein synthesis and occurre d at the transcriptional level. Ligand blot experiments indicated that a pr otein of similar size to SR-BI is the major HDL-binding protein in NCI-H295 cells. Western blot analysis demonstrated that cAMP treatment increased th e 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 H DL than from LDL, both in basal as well as in cAMP-stimulated cells. Scatch ard analysis under basal conditions indicated that NCI-H295 cells express t wice as many specific binding sites for LDL than for HDL. Dissociation cons tant 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 H DL subfraction 3 (HDL3) contributes to a lower cholesteryl ester uptake fro m HDL than from LDL by the NCI-H295 cells. In conclusion, both the SR-BI an d LDL receptor genes are expressed in the human adrenal cortex and coordina tely regulated by activators of glucocorticoid synthesis. In contrast to ro dents, in human adrenocortical cells the HDL pathway of cholesterol deliver y appears to be of lesser importance than the LDL pathway. Nevertheless, th e SR-BI pathway may become of major importance in conditions of functional defects in the LDL receptor pathway.