Regulation of oxysterol 7alpha-hydroxylase (CYP7B1) in primary cultures of rat hepatocytes.
Conversion of cholesterol into 7alpha-hydroxylated bile acids is a principal pathway of cholesterol disposal. Cholesterol 7alpha-hydroxylase (CYP7A1) is the initial and rate-determining enzyme in the "classic" pathway of bile acid synthesis. An "alternative" pathway of bile acid synthesis is initiated by sterol 27-hydroxylase (CYP27) with subsequent 7alpha-hydroxylation of 27-hydroxycholesterol by oxysterol 7alpha-hydroxylase (CYP7B1). The regulation of CYP7B1, possibly a rate-determining enzyme in the alternative pathway, has not been thoroughly studied. The aims of this study were to (1) study the regulation of liver CYP7B1 by bile acids, cholesterol, adenosine 3', 5'-cyclic monophosphate (cAMP), and phorbol myristate acetate (PMA) in primary rat hepatocytes and (2) determine the effect of CYP7B1 overexpression on rates of bile acid synthesis. The effects of different bile acids (3-150 micromol/L), cAMP (50 micromol/L), PMA (100 nmol/L; protein kinase C stimulator), cholesterol (200 micromol/L), and squalestatin (1 micromol/L; cholesterol synthesis inhibitor) on CYP7B1 expression in primary rat hepatocytes were studied. Taurocholic acid and taurodeoxycholic acid decreased CYP7B1 activity by 45% +/- 10% and 36% +/- 7%, respectively. Tauroursodeoxycholic acid and taurochenodeoxycholic acid did not alter CYP7B1 activity. Inhibition of cholesterol synthesis with squalestatin decreased CYP7B1 activity by 35%, whereas addition of cholesterol increased activity by 39%. Both PMA and cAMP decreased CYP7B1 activity by 60% and 34%, respectively, in a time-dependent fashion. Changes in CYP7B1 messenger RNA (mRNA) levels correlated with changes in specific activities. Overexpression of CYP7B1 led to a marked increase in CYP7B1 mRNA levels and specific activity but no change in rates of bile acid synthesis. In conclusion, in the rat, CYP7B1 specific activity is highly regulated but does not seem to be rate limiting for bile acid synthesis.
Pandak
William M
WM
Department of Medicine, Veterans Affairs Medical Center and Virginia Commonwealth University, Richmond, VA 23249, USA. william.pandak@med.va.gov
Hylemon
Phillip B
PB
Ren
Shunlin
S
Marques
Dalila
D
Gil
Gregorio
G
Redford
Kaye
K
Mallonee
Darrell
D
Vlahcevic
Z Rano
ZR
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D002478:Cells, Cultured; D002756:Cholagogues and Choleretics / Q000494:pharmacology; D002784:Cholesterol / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D003907:Dexamethasone / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D005938:Glucocorticoids / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology / Q000201:enzymology*; D008297:Male; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013250:Steroid Hydroxylases / Q000235:genetics*; D013656:Taurocholic Acid / Q000494:pharmacology; D013657:Taurodeoxycholic Acid / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013974:Thyroxine / Q000494:pharmacology
D002273:Carcinogens; D002756:Cholagogues and Choleretics; D005938:Glucocorticoids; D012333:RNA, Messenger; D013657:Taurodeoxycholic Acid; D013656:Taurocholic Acid; D003994:Bucladesine; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D013250:Steroid Hydroxylases; C115867:oxysterol 7-alpha-hydroxylase; D013755:Tetradecanoylphorbol Acetate; D013974:Thyroxine