Adult male rodents have a pulsatile profile of growth hormone (GH) release, whereas female rodents have a relatively steady-state pattern with uniform, albeit lower levels of GH. The expression of a number of sexually differentiated hepatic proteins is primarily determined by these plasma GH profiles and only secondarily regulated by gonadal hormones. An important subset of these sexually dimorphic proteins is cytochrome P450s. CYP3A10/6 beta-hydroxylase is a cytochrome P450 that catalyzes the 6 beta-hydroxylation of lithocholic acid. CYP3A10/6 beta-hydroxylase is expressed only in male hamsters; however, mimicking the male GH secretion pattern in females induces expression of the gene to male levels. Using chimeric CYP3A10/6 beta-hydroxylase promoter/luciferase reporter genes transfected into hamster primary hepatocytes, we have shown a GH-mediated induction of promoter activity. A combination of 5'-deletion constructs, heterologous promoter constructs, and specific mutagenesis was used to localize the DNA element involved in the GH-mediated regulation of CYP3A10/6 beta-hydroxylase promoter activity, which resembles a STAT binding site. Footprint and gel shift analyses confirmed that the expression of the protein binding to this site is regulated by GH and that the DNA-protein complex can be partially supershifted by anti-STAT-5 antibodies. This protein is 50% more abundant in male than in female hamster livers, is absent in hypophysectomized female livers, and is restored when hypophysectomized females are injected with GH in a manner that masculinizes female hamsters in terms of CYP3A10/6 beta-hydroxylase expression. The system characterized and described here is ideally suited for dissecting the molecular details governing the sexually dimorphic expression of liver-specific genes.

Cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase) is the rate-limiting enzyme in bile acid biosynthesis. It is subject to a feedback control, whereby high levels of bile acids suppress its activity, and cholesterol exerts a positive control. It has been suggested that posttranscriptional control plays a major part in that regulation. We have studied the mechanisms by which cholesterol and bile acids regulate expression of the 7 alpha-hydroxylase gene and found it to be solely at the transcriptional level by using two different approaches. First, using a tissue culture system, we localized a liver-specific enhancer located 7 kb upstream of the transcriptional initiation site. We also showed that low-density lipoprotein mediates transcriptional activation of chimeric genes, containing either the 7 alpha-hydroxylase or the albumin enhancer in front of the 7 alpha-hydroxylase proximal promoter, to the same extent as the in vivo cholesterol-mediated regulation of 7 alpha-hydroxylase mRNA. In a second approach, using transgenic mice, we have found that expression of an albumin enhancer-7 alpha-hydroxylase-lacZ fusion gene is restricted to the liver and is regulated by cholesterol and bile acids in a manner quantitatively similar to that of the endogenous gene. We also found, that a liver-specific enhancer is necessary for expression of the rat 7 alpha-hydroxylase gene, in agreement with the tissue culture experiments. Together, these results demonstrate that cholesterol and bile acids regulate the expression of the 7 alpha-hydroxylase gene solely at the transcriptional level.

CYP 3A10 is a hamster liver cytochrome P-450 (P450) that encodes lithocholic acid 6 beta-hydroxylase, an enzyme that plays an important role in the detoxification of the cholestatic secondary bile acid lithocholate. Western-blot analysis revealed that the expression of CYP 3A10 protein is male-specific in hamster liver microsomes, a finding that is consistent with earlier analysis of CYP 3A10 mRNA. Since it has not been established whether the specificities of bile acid hydroxylase P450s, such as CYP 3A10, are restricted to their anionic bile acid substrates, we investigated the role of CYP 3A10 in the metabolism of a series of neutral steroid hormones using cDNA directed-expression in COS cells. The steroid hormones examined, testosterone, androstenedione and progesterone, were each metabolized by the expressed CYP 3A10, with 6 beta-hydroxylation corresponding to a major activity in all three instances. CYP 3A10-dependent steroid hydroxylation was increased substantially when the microsomes were prepared from COS cells co-transfected with NADPH:P450 reductase cDNA. In this case, the expressed P450 actively catalysed the 6 beta-hydroxylation of testosterone (288 +/- 23 pmol of product formed/min per mg of COS-cell microsomal protein), androstenedione (107 +/- 19 pmol/min per mg) and progesterone (150 +/- 7 pmol/min per mg). Other major CYP 3A10-mediated steroid hydroxylase activities included androstenedione 16 alpha-hydroxylation, progesterone 16 alpha- and 21-hydroxylation, and the formation of several unidentified products. CYP 3A10 exhibited similar Vmax. values for the 6 beta-hydroxylation of androstenedione and lithocholic acid (132 and 164 pmol/min per mg respectively), but metabolized the bile acid with a 3-fold lower Km (25 microM, as against 75 microM for androstenedione). Together, these studies establish that the substrate specificity of the bile acid hydroxylase CYP 3A10 is not restricted to bile acids, and further suggest that CYP 3A10 can play a physiologically important role in the metabolism of two classes of endogenous P450 substrates:steroid hormones and bile acids.

N-Heparan sulfate sulfotransferase catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to the nitrogen of glucosamine in heparan sulfate. The enzyme has been previously purified to apparent homogeneity from rat liver (Brandan, E., and Hirschberg, C. B. (1988) J. Biol. Chem. 263, 2417-2422). We have now cloned the rat liver enzyme using the following strategy: (a) the amino acid sequence was obtained from tryptic peptides of the purified protein, (b) mixed oligonucleotides were generated based on the sequence of the tryptic peptides, (c) a polymerase chain reaction fragment was obtained using mixed oligonucleotide interprimer amplification of cDNA, and (d) this fragment was used to screen rat liver lambda gt 10 and lambda ZAP libraries. Three clones were obtained, one of which seems to contain the complete coding sequence of the N-heparan sulfate sulfotransferase (N-HSST). Evidence that the cDNA clone corresponds to the previously purified and characterized N-HSST was the following: (a) the predicted sequence of the N-HSST contains all of the 11 tryptic peptides obtained from the purified protein, (b) when a cDNA containing the sequence coding for the N-HSST was introduced in a eukaryotic expression vector and transfected in COS-1 cells, the enzyme activity was expressed 9-fold over controls, and (c) the characteristic of the predicted protein fits with the purified protein in terms of molecular weight, membrane localization, and its being an N-linked glycoprotein. The size of the longest cDNA isolated is 4.1 kilobases, which is in close agreement with the 4.2-kilobase size of one of the mRNA observed in Northern analyses. In addition, messages of 7.0 and 8.5 kilobases were also observed, suggesting that a large portion is untranslated. The latter messages were the major mRNA species detected.

We have isolated a hamster liver cDNA whose expression is induced upon feeding hamsters with a cholic acid-rich diet. It was identified as a cytochrome P450 family 3 protein, by sequence homology, and named CYP3A10. The activity of CYP3A10 was determined by transient expression of its cDNA in transfected COS cells and was found to hydroxylate lithocholic acid at position 6 beta. CYP3A10 RNA is 50-fold higher in males than in female hamsters. In males, it appears to be regulated by age with expression highest after puberty. Shortly after weaning (28 days), cholic acid feeding of male hamsters elevates the level of message over that of hamsters fed with normal laboratory chow. Females do not exhibit regulation by cholic acid. In hamster liver, murideoxycholic acid, the 6 beta-metabolite of lithocholic acid, is the major hydroxylated product of lithocholic acid. Lithocholic acid 6 beta-hydroxylase (6 beta-hydroxylase) activity is greatly diminished in hamster female liver microsomes as would be expected due to the lack of CYP3A10 mRNA in females. Additionally, male liver microsomal 6 beta-hydroxylase activity was increased by cholic acid feeding, consistent with the cholic acid-mediated induction of its RNA. These results indicate that, in male hamsters, 6 beta-hydroxylation is the major pathway for detoxification of lithocholate and that, likely, CYP3A10 is responsible for that activity.

Using differential hybridization techniques we have isolated a hamster cDNA encoding a cholesterol-regulated protein. By sequence homology we concluded that the isolated cDNA encodes alpha 1-inhibitor III (alpha 1 I3), a protein of the alpha-macroglobulin (alpha M) family. When hamsters were fed diets rich in cholesterol, cholic acid, or chenodeoxycholic acid, the amount of alpha 1I3 RNA was reduced between 5- and 10-fold. Drugs that lower plasma cholesterol levels, such as colestipol and mevinolin, increased alpha 1I3 RNA between 2- and 3-fold. Additionally, plasma alpha 1I3 protein levels, as measured by immunoblotting techniques using an anti-human alpha 2M antibody, correlate well with alpha 1I3 RNA levels in those hamsters. Plasma alpha 1I3 protein was inversely proportional to plasma cholesterol levels in those hamsters. The observed suppression of alpha 1I3 expression by cholesterol mimics the cholesterol-mediated regulation of other genes that maintain cholesterol homeostasis, such as 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and low density lipoprotein receptor. We hypothesize that alpha 1I3 may play a role in the onset of atherosclerosis and may provide a link between cholesterol and the clotting system. Furthermore, the availability of another sterol-regulated gene, like alpha 1I3, should help elucidate the molecular mechanisms of cholesterol-mediated regulation of gene transcription.