Cholesterol sulphate affects production of steroid hormones by reducing steroidogenic acute regulatory protein level in adrenocortical cells.
Steroidogenic acute regulatory (StAR) protein plays a crucial role in the intramitochondrial movement of cholesterol, where P450 side chain cleavage enzyme resides. Cholesterol sulphate (CS), which is present ubiquitously in mammalian tissues, is not only a precursor of sulphated adrenal steroids but also an inhibitor of cholesterol biosynthesis. This study was designed to examine the biological roles of CS in steroidogenesis in adrenocortical cells. Human adrenocortical carcinoma H295R cells were cultured with various amounts of CS. To evaluate steroid hormone synthesis, pregnenolone production in cells was assayed. The amount of pregnenolone produced by H295R cells in culture medium, to which over 50 mug/ml CS was added, was significantly (P<0.05) decreased compared with that produced by control cells. Western blot analysis was performed to determine StAR protein level using whole cell extracts from cells. StAR protein level decreased when the concentration of CS in the medium was 50 mug/ml, whereas the level of glyceraldehyde-3-phosphate dehydrogenase did not change. To examine the mechanism by which StAR gene expression is controlled, we performed RT-PCR and measured promoter activity in cells transfected with pGL(2) StAR reporter constructs. StAR mRNA level and promoter activity were decreased in cells. The decrease in StAR protein level is a result of the low StAR gene expression level. In conclusion, CS affects the production of steroid hormones by reducing StAR protein level in adrenocortical cells.
Sugawara
Teruo
T
Departments of Biochemistry and Obstetrics and Gynecology, Hokkaido Graduate School of Medicine, Kita-ku, Kita 15, Nishi 7, Sapporo 060-8638, Japan. terusuga@med.hokudai.ac.jp
Nomura
Eiji
E
Hoshi
Nobuhiko
N
D000302:Adrenal Cortex / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002788:Cholesterol Esters / Q000378:metabolism* / Q000494:pharmacology; D003470:Culture Media / Q000737:chemistry; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D006728:Hormones / Q000096:biosynthesis*; D006801:Humans; D010750:Phosphoproteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D011284:Pregnenolone / Q000032:analysis / Q000096:biosynthesis; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013256:Steroids / Q000096:biosynthesis*; D014162:Transfection; D000098682:Steroidogenic Acute Regulatory Protein
D002788:Cholesterol Esters; D003470:Culture Media; D006728:Hormones; D010750:Phosphoproteins; D012333:RNA, Messenger; D013256:Steroids; D000098682:Steroidogenic Acute Regulatory Protein; D011284:Pregnenolone; C007045:cholesteryl sulfate