J. Biochem, 1994, Vol. 115, No. 2 230-237
© 1994 Japanese Biochemical Society
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Rat Hepatic 3
-Hydroxysteroid Dehydrogenase: Expression of cDNA and Physiological Function in Bile Acid Biosynthetic Pathway
Department of Biochemistry, Hiroshima University School of Dentistry Kasumi 1-2-3, Minami-ku, Hiroshima 734
3
-Hydroxysteroid dehydrogenase (3-HSD) [EC 1.1.1.213
[EC]
]2 plays important multifunctional roles in metabolizing steroid hormones, polycyclic aromatic hydrocarbons, and prostaglandins and also in transforming the steroid nucleus for the biosynthesis of bile acids from cholesterol in liver. To gain insight into the details and physiological functions of 3-HSD in the bile acid biosynthetic pathway, cDNA clones of 3-HSD were isolated from rat liver 
phage cDNA libraries by using specific antibodies to 3-HSD purified from rat liver. Transfection of the 3-HSD cDNA in Simian COS7 cells resulted in the expression of an immunoreactive protein to the antibodies against the purified enzyme, and the transfected cells exhibited activities for not only 7-hydroxy-5ß-cholestan-3-one, the intermediate of bile acid biosynthesis, but also steroid hormones and 9,10-phenanthrenequinone. Northern blot analysis on poly(A)+ RNA by selective use of different cDNA fragments of the 5‘-untranslated region, the coding region, and the 3'-untranslated region as probes revealed three hybridizable bands, 3.6, 2.7, and 2.5 kb, in liver and four bands, 3.6, 2.7, 2.5, and 1.8 kb, in ovary. Of these, the 2.7- and 1.8-kb bands were predominant in liver and ovary, respectively. Northern hybridization analysis also revealed that the coding region of the various sizes of mRNA seemed to be common. Southern blot analysis of genomic DNA by the selective use of the cDNA fragments as probes indicated that the various mRNA species were derived from a single gene, probably due to an alternative splicing mechanism. Inhibition experiments with the specific antibodies and immunoblotting studies indicated that the enzymes expressed in liver and ovary were similar in size and catalytic properties, and immunochemically indistinguishable. Northern blotting of poly(A)+ RNA in liver using the cDNA fragment of the coding region as a probe showed that the major 2.7-kb mRNA level at 10:00 a.m. was approximately twofold higher in female rat than in male rat, whereas the mRNA level at 10:00 p.m. was nearly identical in both female and male rat liver. The activity for 7-hydroxy-5,ß-cholestan-3-one and the immunoreactive protein level did not show a significant sex difference although their levels were slightly higher at night than in daytime. Among various treatments tested, dexamethazone and 6-n-propyl-2-thiouracil modulated the levels of mRNA for 3-HSD, indicating that glucocorticoid and thyroid hormone may play a role in the regulation of 3-HSD expression.
1Present address: Miyazaki Medical College, Kiyotake-machi, Miyazaki 889–19.
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