Androgenic regulation of hepatic gene expression

Androgen-dependent synthesis of alpha 2u globulin in the rat liver has been used in our laboratory as a model for studying the effect of sex hormones on hepatic gene expression. alpha 2u Globulin is a group of low molecular weight (Mr approximately 18,000) male specific urinary proteins synthesized and secreted by hepatocytes. In the male rat hepatic synthesis of alpha 2u globulin begins at puberty (approximately 40 days), reaches a peak level (approximately 20 mg/day) at about 75 days and declines during old age. Androgens can induce alpha 2u globulin in ovariectomized female rats in vivo and in the liver perfusion system in vitro. However, both prepubertal and senescent (greater than 800 days) male rats not only do not produce alpha 2u globulin but are also refractory to androgen administration. alpha 2u Globulin is coded by a multigene family comprising about 20-30 gene copies per haploid genome. All of these gene copies seem to express translationally active mRNAs giving rise to individual isoforms of alpha 2u globulin. Appearance and disappearance of the cytoplasmic androgen-binding protein (CAB) correlates with the androgen responsiveness of hepatocytes. Photoaffinity labeling of the hepatic cytosol shows that the biologically active binding protein, found in the cytosol of the mature male rat liver, has a molecular weight of 31 kDa. A molecular transition of the 31-kDa CAB to a biologically inactive 29-kDa form may be the basis of hepatic androgen insensitivity during prepuberty and senescence.     

Glucocorticoid induction of hepatic alpha2u-globulin synthesis and messenger RNA level in castrated male rats in vivo.

alpha2u-Globulin is a male rat liver protein of Mr = 20,000 which is synthesized in the liver of adult male rats, secreted into the serum, and excreted in the urine. Its function is unknown. The hepatic synthesis of this protein is under complex hormonal control. We had previously shown that castration of male rats diminishes hepatic alpha2u-globulin synthesis and the level of its mRNA, and that administration of androgen to these castrated animals results in the reinduction of the synthesis of this protein and the level of its mRNA. We now report that alpha2u-globulin synthesis and the level of its mRNA can be fully reinduced in castrated males by administration of glucocorticoid alone. This induction is much more rapid than the androgenic induction and is inhibited by the glucocorticoid antagonist progesterone. Administration of glucocorticoid to intact male animals does not induce alpha2u-globulin synthesis above normal levels; however, if alpha2u-globulin synthesis has been depressed in intact male rats by pretreatment with estrogen or cyproterone acetate, the level of this protein can be reinduced by administration of glucocorticoids. The implications for the control of alpha2u-globulin gene expression are discussed.     

Hormonal regulation of the hepatic messenger RNA levels for alpha2u globulin.

The messenger RNA rat alpha2u globulin has been identified and quantitated in a cell-free translational system derived from Krebs II ascites cells. Hepatic tissue of the mature male rats which normally produce alpha2u globulin was also found to contain a high level of alpha2u mRNA. Approximately 1.6 per cent of all poly(A) containing RNA of the adult male rat liver could be accounted for alpha2u messenger activity. Female rats do not produce alpha2u globulin and no alpha2u mRNA activity could be detected in the poly(A) containing RNA fraction obtained from the livers of these animals. However, androgen treatment to spayed female rats was found to induce the parallel appearance to both alpha2u globulin and its corresponding mRNA. Both hypophysectomy and adrenalectomy which are known to reduce the level of alpha2u globulin in the urine of male rats were found also to reduce the hepatic level of alpha2u mRNA. The results indicate that hormonal control of alpha2u globulin synthesis in rat liver is achieved primarily through regulation of its translatable mRNA level and that more than one hormone may participate in this regulation.     

Translational control of hepatic alpha2u globulin synthesis by growth hormone.

α_2u Globulin is a male rat liver protein under complex hormonal control which represents approximately 1% of hepatic protein synthesis in an adult male rat. Hypophysectomy completely abolishes the hepatic synthesis of this protein, and the reinduction of its synthesis can be effected by the simultaneous administration of glucocorticoid, androgen, thyroid hormone and pituitary growth hormone. Growth hormone is absolutely required for the synthesis of a normal level of α_2u globulin. It was found, however, that hepatic α_2u globulin mRNA can be raised to a normal level in hypophysectomized animals by administration of steroids and thyroid hormone alone; nevertheless, no detectable synthesis of the protein occurs in these animals. Administration of growth hormone to the hypophysectomized animals that had been pretreated with steroids and thyroid hormone results in the immediate synthesis of α_2u globulin protein with very little change in the level of α_2u globulin mRNA. In an intact male animal, α_2u globulin mRNA sequences are found to be preferentially associated with bound polysomes. By contrast, the untranslated α_2u globulin mRNA sequences that accumulate in the livers of hypophysectomized rats treated with steroids and thyroid hormone are found on free polysomes and in the supernatant (nonpolysomal) fractions. Administration of growth hormone to these animals effects a shift of α_2u globulin mRNA sequences to bound polysomes, concurrent with the induction of detectable synthesis of the protein. This effect of growth hormone in vivo can be mimicked by administration of high doses of insulin, indicating that this effect may be somatomedin-mediated. It thus appears that growth hormone induces hepatic α_2u globulin synthesis by way of a translational control mechanism.     

Tissue-specific control of alpha 2u globulin gene expression: constitutive synthesis in the submaxillary gland

Synthesis of alpha 2u globulin, previously thought to occur only in the male rat liver, has now been demonstrated in the submaxillary salivary gland. Unlike liver, submaxillary synthesis of alpha 2u globulin mRNA is constitutive--that is, independent of the endocrine state, age and sex. Liver and submaxillary alpha 2u globulin mRNAs are of similar size, and their 5' ends map to the same region of the gene. Isoelectric focusing of in vitro translation products revealed that submaxillary mRNA encodes a more acidic subset of alpha 2u globulins than does liver. Salivary alpha 2u globulin mRNA manifests 5% nucleotide divergence, encoding 20 amino acid substitutions, which specifies a more acidic polypeptide than its hepatic counterpart. Thus the liver and submaxillary gland synthesize alpha 2u globulin from different sets of genes that are subject to very different developmental and hormonal control.     

Synthesis and immunocytochemical localization of alpha 2u globulin in the duct cells of the rat submaxillary gland

Alpha 2u globulin, a protein of unknown function so far believed to be synthesized exclusively in the male liver under multihormonal control, is now shown to be localized by immunocytochemistry in the granular convoluted tubules of the adult male submaxillary gland. In addition, using Northern blot analysis, we have shown specific alpha 2u globulin mRNA sequences in the RNA extracted from the submaxillary gland. Thus, it is evident that the protein is being synthesized therein. Alpha 2u globulin was also detected in the submaxillary gland duct cells of adult female and immature animals of both sexes, all of which are known not to synthesize alpha 2u globulin in their livers. The present data have established that alpha 2u globulin is synthesized in the rat submaxillary gland and indicate that the control of alpha 2u globulin gene expression in the rat liver and in the submaxillary gland is different.     

Cloning of alpha 2u globulin cDNA using a high efficiency technique for the cloning of trace messenger RNAs

An extremely high-efficiency technique is described for cloning double-stranded (ds) cDNAs in Escherichia coli. The method, which uses two synthetic oligonucleotide linkers rather than one, results in approx. 200--500 recombinant clones per ng of ds cDNA. This technique was used to clone a cDNA comprising 95% of the full length of the mRNA of alpha 2u globulin, a male rat liver protein, which represents approx. 1% of hepatic messenger RNA. The cloned probe was applied to study the complex hormone controls of alpha 2u globulin mRNA in male and female rats.     

Differential gene expression of organic anion transporters in male and female rats.

Sex-related differential gene expression of organic anion transporters (rOAT1, rOAT2, and rOAT3) in rat brain, liver, and kidney was investigated. There were no sex differences in the expression of rOAT1 mRNA. rOAT2 mRNA was abundant in the liver and weakly expressed in the kidney of male rats; however, the OAT2 gene was strongly expressed in both organs of females. The abundance of rOAT2 mRNA markedly increased in castrated male rat kidney; however, treatment of castrated male rats with testosterone led to a decrease of rOAT2 mRNA. Expression of rOAT3 mRNA in intact female rats was found in the kidney and brain, whereas in males rOAT3 mRNA was also found in the liver. rOAT3 mRNA markedly decreased in the liver of castrated male rats but increased in testosterone-treated castrated male rats. Moreover, rOAT3 mRNA increased in the hypophysectomized female rat liver, indicating that rOAT3 is an inducible isoform. The present findings suggest that sex steroids play an important role in the expression and maintenance of OAT2/3 isoforms in the rat liver and kidney. Our results provide information on the differential gene expression of OAT isoforms with sex hormone dependency.     

Calorie restriction delays age-dependent loss in androgen responsiveness of the rat liver

We have shown that restricted calorie intake retards age-associated loss in androgen responsiveness of the rat liver. Sustained androgen receptivity delays age-dependent decline in the synthesis of the androgen-inducible alpha 2u globulin and derepression of the androgen-repressible senescence marker protein (SMP-2). Quantitation of mRNAs for alpha 2u globulin and SMP-2 in the liver of animals of various ages maintained on either ad libitum or restricted diets revealed that, although the 27-month-old ad libitum-fed rat had only 5% as much alpha 2u mRNA as the 6-month-old rat, the mRNA level was as high as 45% in the 27-month-old food-restricted rat. Conversely, the 27-month-old food-restricted rat had a much reduced amount (45%) of SMP-2 mRNA compared to the age-matched control that was allowed unlimited access to food. Furthermore, we have correlated the effect of dietary restriction on age-dependent changes in specific gene expression with the hepatic level of the immunoreactive cytoplasmic androgen-binding (CAB) protein. We observed that senescence in the male causes a substantial decrease in the circulating level of testosterone. However, dietary restriction does not retard the rate of decline in the plasma level of the male hormone during aging. These results indicate that age-dependent changes in the expression of androgen-responsive genes (alpha 2u globulin and SMP-2) reflect changing androgen sensitivity and that food restriction may directly influence the androgen receptivity of the liver.     

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)     

The senescence marker protein (SMP-2) of the rat liver: purification, immunochemical characterization and age-dependent regulation

In vitro translation of total rat hepatic mRNAs has identified a 31 kilodalton senescence marker protein (SMP-2) which is present in higher amounts in prepubertal and senescent males than in the post-pubertal adult male (more than 10-fold). SMP-2 is an androgen-repressible protein. The negative regulation of the SMP-2 gene activity by androgen accounts for its increased expression during the androgen insensitive states of the prepubertal and senescent livers, and its constitutive expression in the female liver. A combination of separation procedures including salt fractionation, chromatofocusing, ion-exchange chromatography and preparative gel electrophoresis have led to the purification of SMP-2 to apparent homogeneity. The purified protein showed the same electrophoretic mobility as the sex- and age-specific in vitro translation product of hepatic mRNAs. The polyclonal antibody to SMP-2 was produced in the rabbit. The antibody selectively reacted with the 31 kDa sex- and age-specific translation product of hepatic mRNAs. Western blot analysis of the liver cytosol confirms monospecificity of the antiserum, as well as age- and sex-dependent changes in the tissue level of SMP-2. Histochemical staining of liver sections with the antiserum reveals a preferential periportal localization of SMP-2 in the hepatocytes. This finding is in marked contrast to the androgen-inducible alpha 2u globulin which is preferentially synthesized and localized in the pericentral hepatocytes. Thus, the zonal distribution of SMP-2 correlates with polarized androgen sensitivity of the hepatocytes within the liver lobule.     

Early events in the steroidal regulation of alpha2mu globulin in rat liver. Evidence for both androgenic and estrogenic induction.

1. A double-antibody radioimmunoassay for alpha2mu globulin has been developed. With the help of this highly sensitive radoimmunoassay the early effects of both androgen and estrogen treatments on the hepatic synthesis of alpha2mu globulin in the rat have been investigated. 2. Results show that the earlier observation of the long lag period in the androgenic induction of alpha2mu globulin is more apparent than real. 3. Single injections of either 5alpha-dihydrotestosterone(5alpha-dihydro-17beta-hydroxy-4-androsten-3-one) or its physiological antagonist estradiol-17beta (1,3,5(10)-estratriene-3,17beta-idol) to castrated female rats resulted in the induction of alphamu globulin reaching maximum hepatic level of the protein between 6--9 h after the hormone administration. Administration of cycloheximide 15 prior to hormone treatment blocked both androgenic andestrogenic induction of alpha2mu globulin. 4. Daily pretreatments with 5alphamu-dihydrotestosterone increased the sensitivity of subsequent androgenic response to alpha2muglobulin synthesis. On the other hand, daily pretreatments with estradiol 17beta decreased and ultimately abolished the estrogenic induction of alpha2mu globulin. 5. The possible mechanism of both androgenic and estrogenic induction of alpha2mu globulin in rat liver mediated through a sex-hormone-binding protein with dual affinity for both dihydrotestosterone and estradiol has been suggested.     

Selective actions of growth hormone on rat liver estrogen binding proteins

Levels of hepatic estrogen receptor were 9.0 ± 2.4 fmoles/mg cytosol protein in intact females compared to 3.4 ± 2.2 in hypophysectomized females. Likewise, levels of receptor were 9.8 ± 1.5 fmoles/mg cytosol protein in intact males and 2.7 ± 1.8 in hypophysectomized males. Hypophysectomy abolished the sex differences in a second class of binding sites termed higher capacity lower affinity binding sites by increasing female levels and decreasing male levels. Treatment of hypophysectomized male or female rats with growth hormone (2 units/kg body wt, two times daily) restored normal levels of hepatic estrogen receptor. Administration of growth hormone to hypophysectomized rats did not reverse the effects of hypophysectomy on higher capacity lower affinity binding sites. These studies demonstrate that growth hormone exerts selective actions on different forms of hepatic estrogen binding proteins.     

Regulation of pro-gonadotropin-releasing hormone gene expression by sex steroids in the brain of male and female rats

The fine modulation of gonadotropin gene expression and secretion is well recognized to be regulated by sex steroids through their direct action both at the anterior pituitary level and on the pulsatile pattern of GnRH secretion at the hypothalamic level. Since the influence of sex steroids on hypothalamic GnRH mRNA levels remains to be elucidated, quantitative in situ hybridization was used to study the effect of sex steroids on cellular levels of pro-GnRH mRNA in adult rats of both sexes. The effects of 14-day gonadectomy as well as administration of 17 beta-estradiol (E2, 0.25 micrograms) or dihydrotestosterone (DHT, 100 micrograms) twice a day during 14 days to gonadectomized animals were evaluated. In addition, the effect of progesterone (P, 2 mg, twice daily) alone or in the presence of E2 was also studied in ovariectomized animals. Hybridization was performed using a 35S-labeled cDNA probe encoding rat pro-GnRH and the corresponding mRNA levels were assessed by counting the number of silver grains overlying labeled neurons. In male rats, castration induced a highly significant 65% increase (compared to intact rats) in the mean number of grains per neuron. Administration of E2 or DHT to castrated animals completely prevented the post castration rise in pro-GnRH mRNA levels. In female animals, the effect of ovariectomy was less striking than in the male, a 25% increase (P less than 0.001) being observed. Treatment with E2 or DHT also completely prevented the increase in pro-GnRH mRNA levels induced by ovariectomy. Moreover, treatment with P in ovariectomized animals markedly potentiated the inhibitory effect of E2 on pro-GnRH mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and androgen effects of 7 alpha,17 beta-dihydroxy-5 alpha-androstan-3-one, 5 alpha-androstan-3 alpha,7 alpha,17 beta-triol and 5 alpha-androstane-3 beta,7 alpha,17 beta-triol

The steroids 7 alpha,17 beta-dihydroxy-5 alpha-androstan-3-one (7 alpha-hydroxy-Dht), 5 alpha-androstan-3 alpha,7 alpha,17 beta-triol (7 alpha-hydroxy-3 alpha-A'DIOL) and 5 alpha-androstane-3 beta,7 alpha,17 beta-triol (7 alpha-hydroxy-3 beta-A'DIOL) have been synthetized from 7 alpha,17 beta-dihydroxy-4-androsten-3-one (7 alpha-hydroxy-testosterone). The effect of administering 7 alpha-hydroxy-Dht, 7 alpha-hydroxy-3 alpha-A'DIOL or 7 alpha-hydroxy-3 beta-A'DIOL on serum levels of LH, FSH and on ventral prostate and seminal vesicle weight were investigated in gonadectomized adult male rats. Each steroid was administered for seven days in a dose of 300 micrograms per day. No suppression of serum LH or FSH levels was recorded following injections of these 7 alpha-hydroxylated steroids to castrated rats, compared to castrated control rats receiving vehicle only. Administration of 7 alpha-hydroxy-Dht or 7 alpha-hydroxy-3 alpha-A'DIOL to castrated mature rats could maintain ventral prostate and seminal vesicle weights above that of castrated control rats. Administration of 7 alpha-hydroxy-3 beta-A'DIOL to castrated mature rats resulted in ventral prostate weights slightly above castrate control levels, while seminal vesicle weight in such rats were in the same range as castrated control rats. Intraperitoneal administration of testosterone or of 5 alpha-androstane-3 beta,17 beta-diol (3 beta-A'DIOL) to castrated rats maintained activity of the androgen dependent isoenzyme of acid phosphatase in the ventral prostate; 7 alpha-hydroxy-testosterone or 7 alpha-hydroxy-3 beta-A'DIOL showed, however, no effect on this enzymic activity.     

Sex differences in the accumulation of estrogen receptors in nuclei of liver cells and increase of angiotensinogen concentration in the blood of rats after administration of low doses of synthetic estrogens

The significance of sex differences in the level of estrogen receptors (ER) in hepatocytes for direct effects of estrogens in male and female rat livers was investigated. 4-5-fold increase in ER level and 20-30%-elevation in plasma angiotensinogen (AG) occurred after a single injection of 0.5 microgram of hexestrol (HE) in female and gonadectomized male rats. In male liver, where the cytosol ER content is two fold lower than that in female rats, nuclear ER level was shown to be very low and unchanged after HE injection, plasma AG also did not change. Injection of 0.5 microgram of ethinylestradiol produced similar effect. Injection of a greater dose of estrogen caused an enhancement in plasma AG level in males. The existence of sex differences in hepatic ER level seems to cause in some conditions different response of metabolic processes in male and female rat liver after estrogenization.