Reciprocal regulation of sex-dependent expression of testosterone 15 alpha-hydroxylase (P-450(15 alpha)) in liver and kidney of male mice by androgen. Evidence for a single gene.
Testosterone 15 alpha-hydroxylase activities and its mRNA levels are higher in kidneys than in livers from male 129/J mice. Castration of 129/J male mice resulted in repression of P-450(15 alpha) in kidney, but increased it in liver. Two types of cDNA (p15 alpha-29 (Type I) and -15 (Type II)) encoding P-450(15 alpha) were previously cloned from 129/J female livers (Burkhart, B.A., Harada, N., and Negishi, M. (1985) J. Biol. Chem. 260, 15357-15361). With the use of p15 alpha-29 as a probe, Type I and II P-450(15 alpha) cDNAs were isolated from libraries of 129/J kidney poly(A)+ RNA. The nucleotide sequences of the cDNAs showed that Type I and II cDNAs from liver and kidney were identical and shared 98.3% similarity. The deduced amino acid sequence from a full-length Type I cDNA indicated that Type I P-450(15 alpha) consists of 494 amino acids with a molecular weight of 56,594. Nine amino acid substitutions were found in the Type II clone in 432 amino acids overlapping Type I. Type I cDNA clones accounted for approximately 90% P-450(15 alpha) clones isolated from a male kidney library, whereas approximately 90% of cDNA clones in a female kidney library were Type II. Liver cDNA libraries from males and females contained similar ratios of Type I and II. Effects of castration on Type I and II mRNAs were determined by Southern hybridization of a 32P-labeled ClaI-ClaI fragment from p15 alpha-29 to cDNAs synthesized from kidney and liver poly(A)+ RNAs prepared from sham-operated, castrated 129/J mice. The double-stranded cDNAs were digested with ClaI and PstI prior to gel electrophoresis to create the diagnostic restriction fragments specific for Type I or II. Castration resulted in decreased levels of Type I mRNA in male kidney. In male liver, only Type I mRNA rose significantly in response to castration. Testosterone administration returned the Type I mRNA to normal levels in castrated mice. It therefore appears that the high levels of P-450(15 alpha) in male kidney were due to androgen-dependent induction of Type I mRNA. Both Types I and II were repressed in male liver, which results in decreased levels of P-450(15 alpha). Androgen was responsible for the repression and expression of Type I in liver and kidney, but not Type II.
Squires
E J
EJ
Laboratory of Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Negishi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D002369:Castration; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D000072467:Cytochrome P450 Family 2; D005260:Female; D007668:Kidney / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D012727:Sex Characteristics; D013250:Steroid Hydroxylases / Q000235:genetics* / Q000378:metabolism
D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C460223:Cyp2a4 protein, mouse; D000072467:Cytochrome P450 Family 2; C050356:steroid 15-alpha-hydroxylase