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.     

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.     

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)     

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.     

Synthesis of surface immunoglobulin E receptor in Xenopus oocytes by translation of mRNA from rat basophilic leukemia cells

Immunoglobulin E-binding activity was expressed in Xenopus oocytes injected with mRNA from rat basophilic leukemia cells which possess abundant immunoglobulin E (IgE) receptor. Such activity was demonstrated with intact oocytes by their binding of 125I-labeled mouse monoclonal IgE. Binding activity was specific as shown by the total inhibition of 125I-IgE binding by unlabeled IgE but not by unlabeled IgG1. The relevance of the IgE-binding activity to the IgE receptor was also supported by the absence of this activity in oocytes injected with mRNA from cells lacking surface IgE receptors. mRNA coding for the IgE-binding activity was enriched in fractions sedimenting at 13.5 S in sucrose density gradients. From oocytes injected with rat basophilic leukemia mRNA, two major polypeptides were isolated by affinity purification on IgE immunoadsorbent. One (Mr = 31,000) is equivalent in size to the previously identified "receptor-associated protein;" the other (Mr = 40,000) is speculated to be a partially glycosylated or unglycosylated form of the alpha subunit of the IgE receptor. The binding of IgE-coated fluorescent microspheres by oocytes injected with rat basophilic leukemia mRNA demonstrated the surface expression of the IgE-binding proteins.     

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.     

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.     

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 of alpha 2-macroglobulin in rat hepatocytes and in a cell-free system

The biosynthesis and secretion of alpha 2-macroglobulin was studied in rat hepatocyte primary cultures. After immunoprecipitation of alpha 2-macroglobulin from a cell homogenate and the hepatocyte medium, two forms of alpha 2-macroglobulin with app. Mr of 176000 and 182000, respectively, were identified. A precursor-product relationship for the two alpha 2-macroglobulin forms was demonstrated by a pulse-chase experiment. The cellular form of alpha 2-macroglobulin could be deglycosylated by endoglucosaminidase H, whereas the medium form of alpha 2-macroglobulin remained unaffected. On the other hand, only the medium form of alpha 2-macroglobulin was found to be susceptible to neuraminidase. In vitro translation of rat liver poly(A)+ RNA resulted in a translation product of an app. Mr of 162000.     

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.     

Selenium Regulates Gene Expression for Estrogen Sulfotransferase and Alpha 2U-globulin in Rat Liver

Dietary intake of the essential trace element selenium (Se) regulates expression of genes for seleno-proteins and certain non-Se-containing proteins. However, these proteins do not account for all of Se's biological effects. The objective of this work was to identify additional genes whose expression is regulated by Se. Identification of these genes may reveal new functions for Se or define mechanisms for its biological effects. Weanling male Sprague-Dawley rats were fed a Torula yeast-based Se-deficient basal diet or the same diet supplemented with 0.5 mg Se/kg diet as sodium selenite for 13 weeks. Total RNA was used as template for RNA fingerprinting. Two differentially expressed cDNA fragments were identified and cloned. The first had 99% nucleotide identity with rat liver estrogen sulfotransferase (EST) isoform-6. The second had 99% nucleotide sequence identity with rat liver 2u-globulin. The mRNA levels for both were markedly reduced in Se deficiency. Laser densitometry showed that EST mRNA in Se deficiency was 7.3% of that in Se-adequate rat liver. The level of 2u-globulin mRNA in Se-deficient rat liver was only 12.6% of that in Se-adequate rat liver. These results indicate that dietary Se may play a role in steroid hormone metabolism in rat liver.     

Human Z alpha 1-antitrypsin accumulates intracellularly and stimulates lysosomal activity when synthesised in the Xenopus oocyte

Microinjection of human liver mRNA from a patient homozygous for alpha 1-antitrypsin deficiency (PiZZ) into Xenopus oocytes led to a 2--10-fold increase in lysosomal activity. Stimulation of lysosomal activity was not observed when mRNA from a normal human liver (alpha 1-antitrypsin PiMM), or water was injected into the oocyte. This lysosomal activity was oocyte derived and was not due to translation products of the human liver mRNA. Thus a protein that accumulates intracellularly in the secretory pathway is capable of stimulating lysosomal activity.     

Electrophoretic and immunochemical characterization of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenases of rat tissues.

The properties of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase from Sprague-Dawley rat liver cytosol have been re-examined in light of several reports which suggest that multiple forms of the enzyme may exist in this tissue. During enzyme purification, chromatography on DE-52 cellulose and chromatofocusing columns indicated the existence of only one form of the protein. Re-chromatography of the purified enzyme by either of these techniques failed to resolve the protein into additional forms. When the purified enzyme was subjected to SDS/polyacrylamide-gel electrophoresis a single band corresponding to Mr 34,000 was detected. Two-dimensional gels showed one predominant protein with a pI of 5.9. Using the homogeneous enzyme as antigen, high-titre polyclonal antibody was raised in rabbits. Western-blot analysis of cytosolic proteins prepared from male and female Sprague-Dawley rat liver indicated the presence of a single immunoreactive band with an Mr of 34,000 in both sexes. All of the 3 alpha-hydroxysteroid dehydrogenase activity present in rat liver cytosol could be immunotitrated with the antibody and the resulting titration curve was superimposable on the titration curve obtained with the purified enzyme. Western-blot analysis of cytosolic proteins prepared from livers of male Wistar and Fischer rats also revealed the presence of a single immunoreactive protein with an Mr of 34,000. These data indicate that, contrary to previous reports, only one form of the dehydrogenase may exist in liver cytosols prepared from a variety of rat strains. Although 3 alpha-hydroxysteroid dehydrogenase activity is known to be widely distributed in male Sprague-Dawley rat tissues, Western blots indicate that only the liver, lung, testis and small intestine contain immunoreactive protein with an Mr of 34,000. The levels of immunoreactive protein in these tissues follow the distribution of dihydrodiol dehydrogenase.     

Effect of physiological concentrations of insulin and antidiabetic drugs on RNA release from isolated liver nuclei

The addition of 10(-11) M insulin to a cell-free system from rat liver promotes the release of messengerlike RNA from isolated prelabeled nuclei. The stimulation was similar whether the nuclei were preincubated with insulin, or if insulin was added directly to the cell-free system with or without a protease inhibitor. Dot blot hybridization using cloned cDNA for alpha 2u-globulin mRNA showed that this was one of the messages whose release was enhanced by insulin. Nuclei isolated from rats treated with either of the antidiabetics tolbutamide or tolazamide showed no increase in RNA release in the presence of insulin over the concentration range 10(-5) - 10(-14) M. Furthermore, these nuclei did not release detectable levels of alpha 2u-globulin mRNA.