Studies on the mode of oestrogenic inhibition of hepatic synthesis of alpha2u-globulin and its corresponding messenger ribonucleic acid in rat liver.

1. The possible mechanism of the oestrogenic inhibition of the androgen-dependent synthesis of alpha2u-globulin in rat liver was explored by a correlative study of the amounts of alpha2u-globulin, its corresponding mRNA and circulating testosterone in oestrogen-treated male rats. 2. Daily treatments of mature male rats with oestradiol-17beta (10 microgram/100g body wt.) decreased and ultimately stopped the hepatic synthesis of alpha2u-globulin as determined by both hepatic and urinary concentrations of the protein. The oestrogen-mediated decrease in the hepatic synthesis of alpha2u-globulin was correlated with a decrease in the mRNA for this protein. 3. Withdrawal of oestrogen resulted in the recovery of alpha2u-globulin synthesis and an increase in mRNA for alpha2u-globulin. 4. At higher doses of oestradiol-17beta (50 microgram/100g body wt.), synthesis of alpha2u-globulin was totally suppressed. In addition, this treatment resulted in an extended period of androgen-insensitivity during which treatment with androgens induced synthesis of neither alpha2u-globulin nor its corresponding mtrna. 5. it is concluded that the oestrogenic inhibition of alpha2u-globulin synthesis is mediated by an oestrogen-dependent decrease in the hepatic content of translatable mRNA for alpha2u-globulin.     

Pretranslational regulation of alpha 2u-globulin in rat liver by growth hormone

Hypophysectomy is known to cause complete suppression of the hepatic synthesis alpha 2u-globulin. The effect of hypophysectomy on the synthesis of alpha 2u-globulin can be reversed by multiple hormone treatment. The role of pituitary growth hormone in the multihormonal regulation of alpha 2u-globulin in rat liver was examined in the hypophysectomized male rats with and without growth hormone supplementation. Daily treatment of hypophysectomized rats with 5 alpha-dihydrotestosterone, corticosterone, thyroxine, and growth hormone for 8 days caused about 80% recovery in the hepatic content of alpha 2u-globulin and its corresponding mRNA as determined by radioimmunoassay, in vitro translation, and liquid hybridization with a cloned cDNA probe. However, omission of growth hormone from the treatment regimen failed to raise hepatic alpha 2u-globulin and its mRNA to more than 5% of the normal control. The possible effect of growth hormone on the translation of the mRNA for alpha 2u-globulin was examined with cultured hepatocytes derived from growth hormone-deficient rats. Culture of these cells in the presence of growth hormone for 24 h did not turn on the synthesis of alpha 2u-globulin. These results indicate that growth hormone regulates the synthesis of alpha 2u-globulin by acting at a step antecedent to mRNA translation.     

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.     

Monoclonal antibodies to alpha 2u-globulin and immunocytofluorometric analysis of alpha 2u-globulin-synthesizing hepatocytes during androgenic induction and aging

Stable hybridomas generated by fusion of spleen cells from hyperimmunized mice and mouse myeloma cells were cloned to prepare monoclonal antibodies to alpha 2u-globulin, an androgen-dependent urinary protein of hepatic origin. One of these monoclonal antibodies was used as a probe for immunocytofluorometric analysis of alpha 2u-globulin producing hepatocytes during androgenic induction and aging through fluorescence-activated cell sorting (FACS). FACS patterns of hepatocytes from mature male rats that produce high levels of alpha 2u-globulin showed tow distinct peaks, arbitrarily designated as peak I (weakly fluorescent) and peak II (brightly fluorescent). In the mature male rat, peak II represented about 40% of the total hepatocytes, and the fluorescence intensity of this subpopulation decreased in direct correspondence with the gradual decline of alpha 2u-globulin synthesis during aging. Similarly the androgenic induction of this protein in ovariectomized female rats was associated with an increase in the fluorescence intensity of the hepatocyte subpopulation under peak II rather than an increase in the relative number of these cells. From these results we conclude that the androgen-dependent synthesis of alpha 2u-globulin and its alteration during aging are confined to a specific subpopulation of hepatocytes within the liver.     

Hormonal induction of alpha2u-globulin synthesis in isolated rat hepatocytes.

Hepatocytes freshly prepared with collagenase synthesize alpha 2u-globulin and other hepatic proteins in vitro at approximately the same rate throughout 30 h of incubation. The newly synthesized proteins are efficiently secreted into the medium throughout this period. That the secretion of proteins by hepatocytes is not due to cell leakage is shown by the fact that 30 micrometer colchicine prevents the appearance of labeled alpha2u-globulin and other proteins in the medium. Hepatocytes isolated from animals in different endocrine states synthesize alpha2u-globulin in vitro at rates consistent with the hormonal effects upon its in vivo biosynthesis. In vitro addition of androgens to hepatocytes isolated from castrated male rats evokes an elevation of general protein synthesis in these hepatocytes. Glucocorticoids, added in vitro, specifically induce and elevated rate of alpha2u-globulin synthesis.     

Interacting role of thyroxine and growth hormone in the hepatic synthesis of alpha 2u-globulin and its messenger RNA

Hypophysectomy completely abolishes and thyroidectomy results in a 90% reduction in the hepatic content of alpha 2u-globulin and its mRNA in the male rat. Thyroid hormone is also known to be required for the synthesis and secretion of pituitary growth hormone. In the hypothyroid rat either thyroxine or growth hormone was found to increase the activity and number of sequences of the mRNA for alpha 2u-globulin (measured by translational assay and hybridizational analysis with a cloned cDNA probe) to the euthyroid level. Treatment of hypophysectomized rats with a hormone combination containing growth hormone but not thyroxine increased the hepatic level of the mRNA for alpha 2u-globulin to that of normal animals. From these results we conclude that thyroxine indirectly influences the hepatic concentration of the mRNA for alpha 2u-globulin through its effect on pituitary growth hormone. Although administration of growth hormone to hypothyroid animals raised the hepatic concentration of alpha 2u-globulin mRNA to the euthyroid level, synthesis of alpha 2u-globulin remained low (50% of the normal). Complete recovery of alpha 2u-globulin synthesis required thyroxine. Therefore, in addition to an indirect effect on the hepatic level of alpha 2u-globulin mRNA, thyroxine also directly influences the synthesis of this protein. This direct effect of thyroxine on alpha 2u-globulin synthesis seems to be exerted at a step distal to the formation of mature mRNA.     

Synthesis and processing of the dimorphic forms of rat alpha 2u-globulin

alpha 2u-Globulin the androgen-dependent male rat urinary protein, can be resolved into two distinct molecular forms by SDS-polyacrylamide slab gel electrophoresis. These two forms designated as alpha 2u-A (M, 18,800) and alpha 2u (Mr 18,100) are found both in urine and in the liver cells. Translation of rat liver mRNA in the rabbit reticulocyte lysate produced two preprotein forms of alpha 2u-globulin, designated as alpha 2uA' (Mr 20,300) and alpha 2uB' (Mr 19,600). Cell-free translation of rat liver mRNA in the presence of dog pancreas microsomal membrane or in Xenopus oocytes produced the two processed forms of alpha 2u-globulin (alpha 2uA and alpha 2uB). Quantitation of alpha 2uA and alpha 2uB within the in vitro translation products of the hepatic mRNA from albino rats of Yale, Sprague-Dawley and Fischer strains showed genetic differences in the proportion of translatable mRNA for alpha 2uA and alpha 2uB. The ratio of alpha 2uA: alpha 2uB in the translation products of liver mRNA from Yale rats was found to be 1:2.5 while in the case of both Sprague-Dawley and Fischer rats, the ratio was 1:4. A small portion of the alpha 2uA and alpha 2uB synthesized in the cultured hepatocytes, in the Xenopus oocytes or in the membrane-supplemented cell-free system appeared as two additional forms, designated as alpha 2uA" (Mr 21,200) and alpha 2uB" (Mr 20,600). Unlike alpha 2uA and alpha 2uB both alpha 2uA" and alpha 2uB" were found to bind to Con A-Sepharose, suggesting their glycoprotein nature.     

Messenger RNA for alpha 2u-globulin of rat liver. Purification, partial characterization of the mRNA, and synthesis of a Hae III restriction fragment as its cDNA probe

The mRNA for the androgen-dependent hepatic protein, alpha 2u-globulin is normally present in the liver of mature male rats to the extent of about 1% of the total mRNA population. alpha 2u mRNA which was found to migrate as a 14 S band was purified about 18-fold through preparative urea-agarose gel electrophoresis. 32P-Labeled cDNA synthesized with this partially purified alpha 2u mRNA was used as substrate for two restriction endonucleases Hha I and Hae III. Digestion of the cDNA with Hha I failed to reduce its electrophoretic heterogeneity. However, Hae III digestion of the cDNA preparation greatly reduced the molecular complexity and produced several distinct cDNA bands. One of these Hae III fragments (Band A) containing 410 nucleotide residues was extracted from polyacrylamide gel and found to be complementary to alpha 2u mRNA. The identity of this cDNA fragment was established by its ability to inhibit selectively the translation of alpha 2u mRNA in the rabbit reticulocyte cell-free system and by its hybridization kinetics with poly(A)+ hepatic RNA from animals with different rates of alpha 2u synthesis. The relative R0t 1/2 values showed a direct correlation between mRNA sequences complementary to the cDNA fragment (A) and to both translatable alpha 2u mRNA and hepatic level of alpha 2u-globulin in adult male, female, and maturing male rats. Thus, the cDNA fragment containing 410 nucleotide residues generated by the restriction cleavage with Hae III can be used as a convenient probe for identification and quantitation of alpha 2u mRNA under different physiological and experimental conditions.     

Cytoplasmic androgen binding protein of rat liver: molecular characterization after photoaffinity labeling and functional correlation with the age-dependent synthesis of alpha 2u-globulin

The liver of the mature male rat contains a moderate affinity (Kd = 10(-8)M), low-capacity, cytoplasmic androgen binding protein (CAB) whose appearance during puberty and disappearance during senescence correlate with the androgen-dependent synthesis of alpha 2u-globulin. Molecular properties of CAB were examined by photoaffinity labeling with tritiated methyltrienolone (R-1881), a synthetic androgen, and by its localization within the hepatocytes which are competent to produce alpha 2u-globulin. Photoaffinity labeling of the liver cytosol derived from postpubertal male rats, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, showed a predominant androgen binding band corresponding to Mr 31,000. This 31-kilodalton (kDa) binding component was conspicuously absent in the liver of androgen-insensitive prepubertal and senescent male rats and in adult male rats treated with estradiol-17 beta. In addition, unlike the cytoplasmic extract, the nuclear lysate of the male rat hepatocytes did not contain the 31-kDa androgen binder. Disappearance of the 31-kDa androgen binding band from the cytosolic fraction of androgen-insensitive animals was associated with a concomitant appearance of a minor androgen binding component of apparent Mr 29,000. The livers of postpubertal male rats normally contain two subpopulations of hepatocytes, only one of which is highly active (competent) in alpha 2u-globulin synthesis. Separation of these two subpopulations through a fluorescence-activated cell sorter followed by whole cell labeling showed more than a 2-fold higher uptake of R-1881 by the competent cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transcortin and alpha 2u-globulin messenger RNA activities during turpentine-induced inflammation in the rat

Previously we have shown that the serum concentration of transcortin and alpha 2u-globulin markedly decreases during turpentine-induced inflammation. In the present study transcortin and alpha 2u-globulin mRNA from healthy rats and from animals with inflammation was translated in a rabbit reticulocyte lysate system. Female rats had higher levels of translatable transcortin mRNA than male animals and the level of mRNA for transcortin and alpha 2u-globulin decreased rapidly during inflammation. These results indicate that the sex difference in the serum level of transcortin and the changes in serum transcortin and alpha 2u-globulin during inflammation are mainly determined by differences in the mRNAs in the liver.     

Alpha 2u-globulin in modified sebaceous glands with pheromonal functions: localization of the protein and its mRNA in preputial, meibomian, and perianal glands

alpha 2u-Globulin, the principal urinary protein of the male rat, has extensive sequence homology with many lipid binding proteins. The highest concentration of alpha 2u-globulin is found in the preputial gland, a holocrine secretory organ with pheromonal function. Meibomian and perianal glands are two other modified sebaceous glands with holocrine secretory cycles and pleiomorphic peroxisomes capable of synthesizing pheromonal lipids. Immunocytochemical examination shows the presence of alpha 2u-globulin in the acinar cells of all three of these modified sebaceous glands. Whereas in the preputial gland all of the acinar cells exhibit immunoreactivity, in the meibomian and perianal glands only selective cells contain alpha 2u-globulin. In the case of the preputial gland, in addition to the acinar cells some stratified epithelial cells also were immunoreactive. In the perianal and meibomian glands, keratinocytes lining nearby hair shafts and select cells of accessory oil glands stained for alpha 2u-globulin. In situ hybridization with a cloned cRNA probe confirmed the immunocytochemical data. Presence of the alpha 2u-globulin mRNA in these glands was also established by Northern blot analysis. Immunoelectron microscopic examination of preputial alpha 2u-globulin showed the presence of this protein in secretory granules of various maturational stages. Immunolabeled alpha 2u was also found in attached vesicles containing protein and lipid inclusions. The lytic cells were not only loaded with alpha 2u-globulin but also contained sharp-edged, irregularly shaped electron-dense granules which stained heavily for this protein. Specific localization of alpha 2u-globulin and its mRNA in three pheromone-producing sebaceous glands and its structural homology with known lipid binding proteins indicate a pheromone carrier role of alpha 2u-globulin.     

Heterogeneity of alpha 2u-globulin gene products in different translational systems, plasma and urine

alpha 2u-Globulin, an androgen dependent rat urinary protein, displays considerable microheterogeneity. To explore whether this microheterogeneity of alpha 2u-globulin in male rat urine is related to the heterogeneity at the level of the genes encoding this protein, or whether it is due to post-translational processing we studied the alpha 2u-globulin mRNA translation products in rabbit reticulocyte and Xenopus oocytes. Comparison of the alpha 2u-globulin species produced in these two heterologous systems with those observed in plasma and urine indicates that the heterogeneity of this protein in urine is mainly due to heterogeneity at the level of the corresponding mRNAs.     

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)