Gene family of male-specific testosterone 16 alpha-hydroxylase (C-P-450(16 alpha)) in mice. Organization, differential regulation, and chromosome localization

Three genes in the testosterone 16 alpha-hydroxylase (C-P-450(16 alpha)) family, ca, cb, and cc, are characterized. The sizes of the genes are approximately 4.5 to 5.2 kilobase pairs, and all three consist of nine exons with junctions at identical sites. Gene ca is identified as the male-specific, androgen-dependent C-P-450(16 alpha) gene in adult mice, since the exonic sequence matched 100% to the cDNA, pc16 alpha-2 (Wong, G., Kawajiri, K., and Negishi, M. (1987) Biochemistry 26, 8683-8690). Gene cb and cc are organized in tandem within 18-kilobase pair DNA. Their encoded P-450s contain an approximate 94% nucleotide sequence similarity to the C-P-450(16 alpha). The high similarity in gene nucleotide sequences, including the introns and flanking regions, suggests a combination of an ancestral gene duplication and gene conversion as a mechanism for evolution of the C-P-450(16 alpha) family. Gene ca shows male-specific expression in mouse kidney as well as in liver; gene cb, neither sex-specific nor androgen-dependent, is seen only in liver; gene cc is not expressed in either adult mouse liver or kidney. Expression of these three genes is not detected in adult mouse lung. It appears, therefore, that the C-P-450(16 alpha) gene family includes a large number of genes whose expressions are differentially regulated. Southern hybridization of C-P-450(16 alpha) cDNA to genomic DNAs from mouse-hamster somatic hybrid cells localizes tentatively this gene family on mouse chromosome 15. The recombination frequency in BXD recombinant inbred mice suggests that the C-P-450(16 alpha) gene family is approximately 16M from the 55-kDa locus.     

Functional characterization of two cytochrome P-450s within the mouse, male-specific steroid 16 alpha-hydroxylase gene family: expression in mammalian cells and chimeric proteins

Two cDNAs, pc16 alpha-2 and pc16 alpha-25, which encode P-450s from within the mouse, male-specific steroid 16 alpha-hydroxylase (C-P-450(16 alpha)) gene family, were transfected into COS-1 cells in order to study catalytic activities of the expressed P-450s. pc16 alpha-2 was shown previously to encode the growth hormone dependent and androgen-dependent C-P-450(16 alpha) in adult male mice (Wong et al., 1987). The sequence of pc16 alpha-25-encoded P-450 (P-450cb) was identical with gene cb within the C-P-450(16 alpha) family. There was 94% and 87% nucleotide and amino acid sequence identity, respectively, between P-450cb and C-P-450(16 alpha). We expressed both P-450s by transfecting their cDNAs into COS-1 cells and found that steroid 16 alpha-hydroxylase activity was catalyzed by C-P-450(16 alpha) but not by P-450cb. In addition to testosterone, progesterone and estradiol were hydroxylated specifically at the 16 alpha-position by the expressed C-P-450(16 alpha). The results indicated that a broad steroid substrate specificity with high regio- and stereoselectivity at that position was a characteristic of C-P-450(16 alpha). We constructed and expressed chimeras between the two P-450s and found that the presence of about two-thirds of the C-P-450(16 alpha) molecule from its C-terminus was necessary for the chimeric cytochrome to maintain steroid 16 alpha-hydroxylase activity.     

Gene family of male-specific testosterone 16 alpha-hydroxylase (C-P-450(16) alpha) in mouse liver: cDNA sequences, neonatal imprinting, and reversible regulation by androgen

The cDNA clone p16 alpha-1 for the male-specific isozyme (C-P-450(16) alpha)1 of testosterone 16 alpha-hydroxylase in livers of 129/J mice [Harada, N., & Negishi, M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 2024-2028] and two additional full-length cDNAs overlapping with p16 alpha-1 (p16 alpha-2 and p16 alpha-16) were sequenced. p16 alpha-2 contained a single open reading frame of 1512 nucleotides, consisting of 71 base pairs of the 5'-noncoding region and 63 base pairs of the 3'-noncoding region with an additional poly(A) tract. From this DNA sequence, C-P-450(16) alpha was deduced to contain 504 amino acids with a calculated molecular mass of 56,948 daltons. p16 alpha-1 showed a nucleotide sequence identical with that of p16 alpha-2 but lacked nine amino acid residues from the N-terminus. Another cDNA clone, p16 alpha-16, also exhibited the same coding sequence with the exception of a 142 base pair deletion spanning from nucleotide 853 to nucleotide 994 of p16 alpha-2. This deletion seems to be a whole exon of this gene, resulting in a shift of reading frame and an early termination codon at 10 amino acid residues from the deletion. The expected translation product of this mRNA is calculated to be 294 amino acids and 33,300 daltons. The putative poly(A) addition signal AATAAA is present for all three clones, but there are polymorphisms in the start sites of polyadenylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat hepatic cytochrome P-450 isoenzyme 2c. Identification as a male-specific, developmentally induced steroid 16 alpha-hydroxylase and comparison to a female-specific cytochrome P-450 isoenzyme

Rat hepatic cytochrome P-450 isoenzyme 2c, purified to homogeneity from uninduced, adult rat liver (Waxman, D.J., Ko, A., and Walsh, C. (1983) J. Biol. Chem. 258, 11937-11947), was shown to exhibit a unique NH2-terminal amino acid sequence as well as distinctive peptide maps and immunochemical properties when compared to seven other purified rat liver P-450 isoenzymes. P-450 2c was an efficient monooxygenase catalyst with several xenobiotic substrates; P-450 2c also catalyzed 16 alpha- and 2 alpha-hydroxylations of testosterone, androst-4-ene-3,17-dione and progesterone (total turnover = 7-9 min-1 P-450(-1) at 25 microM steroid substrate) with the ratio of 2 alpha to 16 alpha hydroxylation varying from less than or equal to 0.02 to 1.6 depending on the steroid's C-17 substituent. Six different microsomal steroid hydroxylase activities characteristic of purified P-450 2c and sensitive to specific inhibition by anti-P-450 2c antibody were induced at puberty in male but not female rat liver. Microsomal steroid hydroxylations catalyzed by other P-450 isoenzymes exhibited age and sex dependencies distinct from those of the P-450 2c-mediated activities. Immunochemical analyses confirmed that this sex dependence and developmental induction reflected alterations in P-450 2c polypeptide levels. Attempts to chromatographically detect P-450 2c in either immature male or adult female microsomes were unsuccessful and led to purification of P-450 2d (female), a catalytically distinct and female-specific form. Peptide mapping and immunochemical analyses suggested significant structural homologies between the two sex-specific isoenzymes, P-450 2c and P-450 2d (female). A significant suppression of P-450 2c levels (up to 70-80%) was observed upon administration of several classical P-450 inducers. These studies establish that P-450 2c corresponds to the male-specific and developmentally-induced steroid 16 alpha-hydroxylase of rat liver and suggest that the expression of P-450 2c versus P-450 2d (female) may provide a biochemical basis for the sex differences characteristic of rat liver xenobiotic metabolism.     

Characterization of testosterone 16 alpha-hydroxylase (I-P-450(16) alpha) induced by phenobarbital in mice

Cytochrome P-450 (I-P-450(16) alpha), which is associated with phenobarbital-induced testosterone 16 alpha-hydroxylation activity, was purified from livers of phenobarbital-treated female 129/J mice on the basis of the specific hydroxylation activity in fractions eluted from columns of octylamino-Sepharose 4B, hydroxylapatite, DEAE-Bio-Gel A, and isobutyl-Sepharose 4B. The specific cytochrome P-450 content of the purified I-P-450(16) alpha fraction was 12.4 nmol/mg of protein, and it had an apparent molecular weight of 54K. The specific activity of reconstituted testosterone 16 alpha-hydroxylation activity with the purified I-P-450(16) alpha fraction was 6-8 nmol min-1 (nmol of cytochrome P-450)-1. Rabbit antibody raised against the purified I-P-450(16) alpha fraction inhibited nearly 100% of the 16 alpha-hydroxylation activity in liver microsomes of phenobarbital-treated female 129/J mice but did not affect hepatic microsomal 16 alpha-hydroxylation activity of untreated male and female 129/J mice at all. In hepatic microsomes of phenobarbital-treated male 129/J mice, 70% of the 16 alpha-hydroxylation activity, at most, was catalyzed by I-P-450(16) alpha, and the residual 30% of the activity was catalyzed by C-P-450(16) alpha. The increase of I-P-450(16) alpha by phenobarbital was due to de novo synthesis of I-P-450(16) alpha, and this induction was not sexually regulated in 129/J mice. Anti-C-P-450(16) alpha [Harada, N., & Negishi, M. (1984) J. Biol. Chem. 259, 12285-12290] did not inhibit the 16 alpha-hydroxylation catalyzed by I-P-450(16) alpha; thus, I-P-450(16) alpha and C-P-450(16) alpha are immunochemically distinct isozymes of testosterone 16 alpha-hydroxylase.     

Hormonal regulation of levels of the messenger RNA encoding hepatic P450 2c (IIC11), a constitutive male-specific form of cytochrome P450.

A cDNA clone for rat hepatic cytochrome P450 2c (gene product IIC11) was isolated and used to study the sex specificity, expression during development, and hormonal regulation of the mRNA encoding this protein in rat liver. P450 2c mRNA levels were about 16-fold higher in males than in females and were only slightly increased in male rats after administration of phenobarbital, a drug that dramatically raises the levels of mRNAs encoding several other members of the P450 II family. In contrast to the mRNA encoding P450 f (gene product IIC7), which increases gradually over the first 6 weeks of life, P450 2c mRNA showed a dramatic increase at puberty, between 4.5-5.5 weeks of life. The roles of sex steroids and GH in controlling this male-specific, developmentally regulated mRNA were then examined. A dependence on adult androgen was demonstrated by the 2- to 4-fold decrease in P-450 2c mRNA levels after castration of adult male rats and their restoration to normal by administration of the synthetic androgen methyltrienolone. Prolonged treatment (15 days) of ovariectomized female rats with this androgen also increased the levels of P450 2c mRNA and its encoded testosterone 16 alpha-hydroxylase to those of intact males. In male rats treated with estradiol valerate, mRNAs for P450 2c and alpha 2u-globulin, a major male-specific hepatic secretory protein that is under complex hormonal control, fell to negligible levels. None of these hormonal perturbations had a detectable effect on the levels of PB-1 (gene product IIC6) mRNA, which is not expressed in a sex-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of alpha-transinducing factor (VP16) in the induction of alpha genes within the context of viral genomes.

In herpes simplex virus 1, the five alpha genes are induced by alpha-transinducing factor (alpha TIF; VP16), a virion protein, acting in concert with Oct-1 and other cellular proteins on a cis-acting site in the promoter domain of alpha genes. Because alpha TIF is an essential virion protein, its function as an inducer can best be evaluated only by mutating the cis-acting site. Earlier we reported on a series of 17 mutations in and around the cis-acting site of a 275-bp alpha 27 promoter fused to a reporter gene and recombined into the viral genome. These recombinant viruses were tested in Vero cells in the presence of cycloheximide, and we demonstrated that mutations in the sequence required for Oct-1 binding abolished transactivation whereas mutations in the alpha TIF-dependent GARAT sequence decreased but did not abolish transactivation. We now report that (i) in limited-passage human embryonic lung cells, alpha gene expression from promoters mutated in the GARAT sequences is often higher and more variable than in Vero cells, (ii) in the absence of cycloheximide, the mutant viruses show less significant impairment of reporter gene expression, (iii) Oct-1 can bind either to the overlapping octamer element or to various TAATGARAT sequences with differing degrees of binding strength and these relative binding levels correlate well with levels of gene expression observed in infected cells, (iv) in the cis-acting site upstream of the alpha 4 gene, no degenerate overlapping Oct-1 sequence exists, and therefore in this instance Oct-1 must be binding directly to the TAATGARAT sequence, (v) extension of the alpha 27 promoter by an additional 1,334 bp results in much higher expression of the reporter gene as a result of additional upstream cis-acting sites, and (vi) obliteration of the most proximal Oct-1 binding element within the 275-bp promoter dramatically reduces gene expression even in the presence of the additional upstream cis-acting sites.     

Genetic regulation of estrogen-dependent repression of female-specific testosterone 16 alpha-hydroxylase (I-P-450(16 alpha) in male mouse liver: murine Ripr locus

The genetic basis for repression of I-P-450(16 alpha) in livers of male mice was examined in 129/J and BALB/cJ mice. Castration of adult male BALB/cJ but not 129/J mice resulted in derepression of I-P-450(16 alpha) at its mRNA and activity levels. It was further found that the patterns of derepression in (129/J x BALB/cJ) F1 and F2 offspring indicated that the derepression of I-P-450(16 alpha) is inherited as an autosomal additive trait. The distribution of derepression among castrated recombinant inbred strains (9 X A) indicated a close link of a locus repressing I-P-450(16 alpha) in male mice to the Rip locus on chromosome 7. Rip was previously defined as a locus that regulates specific expression of I-P-450(16 alpha) in livers of female mice [Noshiro, M., Lakso, M., Kawajiri, K., & Negishi, M. (1988) Biochemistry (preceding paper in this issue)]. Other tested inbred mice (A/HeJ, C57BL/6J, C3H/HeJ, and DBA/2J) showed the derepression of I-P-450(16 alpha) by castration, such as BALB/cJ. We propose Ripr (repression of an action of Rip locus in male mice) as the name of the locus by which repression of I-P-450(16 alpha) is regulated in male mice. Treatment of castrated male BALB/cJ mice by testosterone propionate, estradiol valerate, or diethylstilbestrol repressed I-P-450(16 alpha) to the levels seen in normal BALB/cJ male mice. Dihydrotestosterone, however, had little effect in repressing I-P-450(16 alpha) in castrated mice. The results suggested that estrogen rather than androgen is a repressor of I-P-450(16 alpha) in livers of male mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adult male-specific and neonatally programmed rat hepatic P-450 forms RLM2 and 2a are not dependent on pulsatile plasma growth hormone for expression

Rat hepatic cytochrome P-450 form RLM2 is a testosterone 15 alpha-hydroxylase reported to be male-specific on the basis of purification studies (Jansson, I., Mole, J., and Schenkman, J. B. (1985) J. Biol. Chem. 260, 7084-7093). The sex dependence, developmental regulation, xenobiotic induction, and hormonal control of P-450 RLM2 expression were studied using P-450 form-specific immunochemical and catalytic assays. Polyclonal antibodies raised to rat hepatic P-450 3 (P-450 gene IIA1) were found to cross-react strongly with P-450 RLM2, but not with 10 other rat P-450 forms, suggesting that P-450 3 and P-450 RLM2 are highly conserved in primary structure. Western blotting of liver microsomes under conditions where P-450s 3 and RLM2 are resolved electrophoretically revealed that P-450 RLM2 is markedly induced at puberty in male rats, with no protein detected (less than or equal to 5% of adult male levels) in adult females or immature animals of either sex. A similar developmental dependence was observed for hepatic microsomal testosterone 15 alpha-hydroxylase activity, which was found to be catalyzed primarily by P-450 RLM2. P-450 RLM2 was resistant to induction by several xenobiotics and in the case of phenobarbital and beta-naphthoflavone, was suppressed by 50-60%. Studies on the steroid hormonal regulation of P-450 RLM2 revealed that its adult male-specific expression is imprinted (programmed) in response to neonatal testosterone exposure. Ovariectomy studies demonstrated that suppression by estrogen does not contribute significantly to the absence of P-450 RLM2 in adult female rats. Although the male-specific developmental induction of P-450 RLM2 in response to neonatal testosterone is strikingly similar to that of P-450 2c (testosterone 2 alpha/16 alpha-hydroxylase; gene IIC11), P-450 RLM2 expression is not dependent on the pulsatile pituitary growth hormone secretion required for P-450 2c synthesis. Rather, hypophysectomy of adult male rats increased P-450 RLM2 and its associated testosterone 15 alpha-hydroxylase activity by 50-100%.(ABSTRACT TRUNCATED AT 400 WORDS)