Purification and immunochemical characterization of the cytoplasmic androgen-binding protein of rat liver.

The cytoplasmic androgen-binding (CAB) protein of the male rat liver has been implicated to play a role in the androgen-dependent regulation of alpha 2u-globulin synthesis. The liver of the adult male rat contains about 50 fmol of specific high-affinity androgen-binding activity per milligram of total cytosolic protein. Photoaffinity labeling with [3H]R-1881 followed by SDS-polyacrylamide gel electrophoresis and autoradiography shows that the CAB is a 31-kilodalton protein. By means of DEAE-cellulose chromatography and preparative SDS-polyacrylamide gel electrophoresis, we have purified the CAB protein to electrophoretic homogeneity and have raised polyclonal rabbit antiserum that is monospecific to this protein. In the sucrose density gradient, the antiserum reacted with the androgen-binding component of the male liver cytosol prelabeled with tritiated dihydrotestosterone. Western blot analysis of the liver cytosol showed that the antiserum recognizes only the 31-kDa androgen-binding component. Such immunoblotting also showed that unlike the young adult, the androgen-insensitive states during prepuberty and senescence are associated with a marked reduction in the hepatic concentration of the immunoreactive CAB protein. No immuno-chemical cross-reactivity between CAB and another androgen-binding component of Mr 29K (which is associated with androgen insensitivity during prepuberty and senescence) was observed. The latter finding favors the possibility that 31- and 29-kDa androgen-binding components may have distinct sequence structure.

Hydrocarbon-induced hyaline droplet nephropathy in male rats during senescence.

Male rats administered unleaded gasoline rapidly develop nephropathy characterized by accumulation of hyaline droplets in cells of the proximal convoluted tubules (PCT). This acute response is implicated in development of renal carcinoma in male rats exposed chronically to wholly volatilized gasoline. A major constituent of hyaline droplets is alpha 2 mu-globulin, a protein of hepatic origin for which the rate of synthesis declines during aging. Little information, however, is presently available on possible age-dependent susceptibility of male rats to hydrocarbon-induced nephropathy. In kidneys of untreated male Fischer 344 rats the number of constitutive hyaline droplets declined progressively with increasing age. Electrophoresis of renal cortical homogenates revealed a protein with Mr about 18 X 10(3), probably alpha 2 mu-globulin, in young (3.5 months old) male rats and total absence of this protein in aged (26 months old) males. RIA confirmed that constitutive levels of renal and hepatic alpha 2 mu-globulin in old rats were less than 1.5% of those in young adults. Unleaded gasoline (0.4 ml/kg/day, po, 5 days) caused accumulation of hyaline droplets in renal PCT of 3.5-month-old males accompanied by a marked increase (about twofold) in the renal content of alpha 2 mu-globulin, whereas the same treatment was without effect in 26-month-old rats. Finally, in the renal cortex of young rats the activities of the lysosomal proteases cathepsin B and D were increased following gasoline administration, presumably in response to protein accumulation. However, in 26-month-old rats cathepsin B activity was unaffected, while cathepsin D was increased by gasoline administration. Thus, we conclude that animal age is an important determinant in the development of hydrocarbon-induced nephropathy and only rats which produce large amounts of alpha 2 mu-globulin are susceptible to development of this pathology.

Changes in transcriptional activity and matrix association of alpha 2u-globulin gene family in the rat liver during maturation and aging.

Hepatic synthesis of alpha 2u-globulin in the male rat begins at puberty (about 40 days), reaches a peak level at about 80 days, and ceases at about 750-800 days of age. The age-dependent changes in alpha 2u-globulin synthesis are correlated with both the steady-state level of the hepatic mRNA for this protein and the rate of transcription of the alpha 2u-globulin gene family. Transcriptional activation of the alpha 2u-globulin gene family at puberty and cessation of transcription at senescence correlate with the association and dissociation of this gene domain with the nuclear matrix. Unlike the alpha 2u-globulin gene, the albumin gene in the liver shows preferential association with the nuclear matrix throughout the life. From these results we conclude that the age-dependent changes in alpha 2u-globulin synthesis are due to the alteration in the rate of transcription of the alpha 2u-globulin gene, and that the association of this gene domain to the nuclear matrix is a prerequisite to its transcriptional activation.

Rapid androgenic stimulation of alpha 2u-globulin synthesis in the perfused rat liver.

Hepatic synthesis of alpha 2u-globulin and its mRNA in the male rat is dependent on androgen, glucocorticoid, T4, insulin, and GH. Some of these hormones may act directly on the liver, while others may influence alpha 2u-globulin synthesis through indirect physiological changes. In the present study the specific role of androgen in the synthesis of alpha 2u-globulin was examined in an in vitro liver perfusion system. The addition of 5 alpha-dihydrotestosterone to the medium perfused through livers from castrated rats resulted in a rapid increase (approximately 10-fold over the vehicle control within 120 min) in the circulating level of alpha 2u globulin. Labeling with [35S]-methionine showed that the androgen-mediated increase in the circulating level of alpha 2u-globulin is due to release of the newly synthesized protein. Quantification of alpha 2u-globulin mRNA in the perfused livers with and without androgen supplementation indicated that the increased mRNA level can only partially account for the elevation of the circulating level of this protein. From these results it is concluded that androgen can act directly on the liver to stimulate alpha 2u-globulin synthesis, and the hormone may influence more than one regulatory step.

Independent regulatory influence of growth hormone on the hepatic synthesis of alpha 2u-globulin.

Administration of GH through sc injections to hypophysectomized male rats induces the hepatic mRNA for alpha 2u-globulin (a male-specific urinary protein) from an undetectable level to 43.4% of the normal male level. The same treatment administered to hypophysectomized-gonadectomized rats and androgen-insensitive Tfm rats induces alpha 2u-globulin mRNA to a level of only 5-10% of that in the normal male. However, none of these types of animals shows an appreciable response when GH is administered continuously through osmotic minipumps. Perfusion of the livers derived from hypophysectomized male rats with the blood from hypothyroid rabbits (also deficient in GH) was used to examine in vitro effects of GH on alpha 2u-globulin synthesis. Supplementation of the perfusion medium with GH and T4 failed to induce alpha 2u-globulin within the perfusion period of 120 min. These results show that GH can influence alpha 2u-globulin synthesis independent of the androgen and that the mode of administration of GH plays an important role in its biological response.

Sex-independent synthesis of alpha 2u-globulin and its messenger ribonucleic acid in the rat preputial gland: biochemical and immunocytochemical analyses.

alpha 2u-Globulin is the principal urinary protein of the mature male rat. The major urinary source of this protein is the liver where it is synthesized and secreted by hepatocytes under hormonal regulation. High levels of alpha 2u-globulin and its messenger RNA (mRNA) are also present in the preputial gland of both male and female rats, and neither castration nor ovariectomy significantly alters the preputial concentration of this protein and its mRNA. Per unit mass of RNA and protein, the preputial gland as compared to liver contains about 3-fold higher level of alpha 2u-globulin mRNA and about 300-fold higher level of alpha 2u-globulin. Despite a 3-fold (300%) difference in the content of alpha 2u-globulin mRNA, nuclear run-off experiments show only a 30% higher rate of alpha 2u-globulin gene transcription in the preputial gland than in the liver. Immunocytochemical analyses reveal that the liver possesses two alpha 2u-globulin cell populations, one showing higher immunoreactivity than the other. In contrast, the preputial gland contains only one type of alpha 2u-globulin containing acinar cells, and a large amount of alpha 2u-globulin accumulates in the ductal lumen. From these results we conclude that the 300% higher level of alpha 2u-globulin mRNA in the preputial gland is not due to a corresponding difference in the rate of transcription of alpha 2u-globulin gene. Such a difference may represent tissue-specific regulation at a posttranscriptional level of mRNA metabolism. Furthermore, the huge difference in the alpha 2u-globulin content of the preputial gland and the liver is primarily due to the cellular and ductal accumulation of this protein in the preputial gland vs. its rapid secretion by the liver.

Accumulation of alpha 2u-globulin in the renal proximal tubules of male rats exposed to unleaded gasoline.

Saturated branched-chain aliphatic hydrocarbons, found in motor fuels, induce nephrotoxicity in male rats. Treatment of male rats with unleaded gasoline (0.04-2.0 ml/kg body wt, po) for 9 days increased markedly the number and size of hyaline (protein resorption) droplets in epithelial cells of the renal proximal convoluted tubules (PCT) and enhanced cellular exfoliation at high dose levels. No other treatment-related pathological effects were observed in the glomeruli, distal tubules, or medulla. The renal content of alpha 2u-globulin, a major urinary protein of male rats, was increased maximally by about 4.4-fold after gasoline administration (1.0 ml/kg, po, 9 days); no further increase was observed at higher doses. Immunoperoxidase staining of kidney tissue sections for alpha 2u-globulin revealed large accumulations of antigen localized in many of the PCT epithelial cells which contained hyaline droplets. The hepatic content of alpha 2u-globulin and its mRNA were not altered by gasoline administration. These data show, for the first time, that alpha 2u-globulin is accumulated in the kidneys of gasoline-intoxicated male rats and sequestered specifically in some of the hyaline droplets characteristic of gasoline-induced nephropathy. A hydrocarbon-induced defect in the renal lysosomal degradation of low-molecular-weight urinary proteins, rather than increased synthesis of these proteins, appears to cause hyaline droplet accumulation.

Cloning and expression of the rat liver cDNA for peroxisomal enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase in lambda GT11. Transcriptional regulation of enzyme activity by Wy-14643 in primary cultures of rat hepatocytes.

Proliferation of rat liver peroxisomes by the hypolipidemic drug Wy-14643 is associated with a concomitant induction of peroxisomal enzymes involved in the beta-oxidation of fatty acids. In order to explore the molecular mechanism of this induction process we have cloned the cDNA for the peroxisomal bifunctional enzyme enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase (ECH) in the lambda gt11 expression vector. The library was screened with the monospecific rabbit antiserum to ECH. Hybrid-selected-mRNA translation established that the immunoreactive clones contain the cDNA sequences of the ECH bifunctional enzyme. The cloned cDNA was used to define the early events associated with enzyme induction in primary cultures of rat hepatocytes. Dot-blot hybridization of the total hepatocyte RNA with the ECH cDNA probe showed that the ECH mRNA begins to rise at about 10-15 h following incubation with Wy-14643. At 24 h and 48 h of incubation the stimulation of the ECH mRNA over the vehicle-treated control reached 26-fold and 47-fold respectively. Run-off experiments in the isolated nuclei of hepatocytes showed no increase in the transcription rate of the ECH gene at 5 h after drug treatment and a 2-fold and 11-fold increase at 10 h and 20 h of drug treatment. From these results we conclude that the increase in ECH activity by Wy-14643 is due to an enhancement of the rate of transcription of the ECH gene. However, the relatively long lag period of about 10-15 h after exposure of hepatocytes to Wy-14643 suggests that the induction of the ECH mRNA may involve an indirect effect of the drug on the transcription of this gene.

Molecular cloning and characterization of cDNA for androgen-repressible rat liver protein, SMP-2.

SMP-2 is a rat liver protein whose synthesis is influenced by both androgens and aging. The steady-state level of its mRNA is repressed by the androgen. Compared to the adult male, SMP-2 mRNA is found in higher amounts in the prepubertal and senescent male rat livers which show relative androgen insensitivity. A cDNA library in the plasmid pBR322 was constructed from the female rat liver which contains a high level of SMP-2 mRNAs. Recombinant plasmids were screened by differential colony hybridization to 32P-labeled single-stranded cDNAs from adult female and adult male hepatic poly(A)+ RNAs. From a total of 3500 recombinant clones, 11 highly female specific clones were identified. From these female specific colonies the SMP-2 cDNA-containing plasmid (pSP11) was identified by its ability to select an mRNA species whose translation product is immunochemically and electrophoretically indistinguishable from SMP-2. This insert represents a 571-base pair portion of the SMP-2 cDNA. Rescreening of the library at a high colony density using the 32P-labeled cDNA insert of pSP11 identified several positive clones with larger inserts. Hybrid-selected mRNA translation again confirmed these clones to carry SMP-2 cDNA sequences. The plasmid pSP4a containing a 1040-base pair cDNA insert of SMP-2 was characterized by DNA sequence analysis. The size of the cDNA insert of pSP4a is close to the estimated size of the SMP-2 mRNA. The cDNA sequence provides an open reading frame of 282 amino acid residues. A comparison of the translated amino acid sequence with the protein sequences of NBRF-PIR, PSQNEW, and LOSALA data bases did not establish any sequence homology with known proteins. Northern blot analysis using the 32P-labeled cDNA insert of pSP4a confirms the androgenic repression of the SMP-2 mRNA.

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.

Partial reversal of alpha 2u globulin gene expression by thyroxine in the liver of diabetic rats.

Synthesis of alpha 2u globulin and its mRNA has been used as an index to monitor the effect of thyroxine on specific gene expression in the liver of hypoinsulinemic male rats. Administration of a physiological dose of thyroxine can partially reverse (to approximately 30% of the normal control) the marked reduction (more than 90%) in the hepatic levels of alpha 2u globulin and its mRNA during streptozotocin-induced diabetes. Estimation of newly synthesized alpha 2u globulin RNA transcripts from the native chromatin of isolated liver nuclei by "nuclear runoff experiments" showed that thyroxine can elevate the rate of transcription of alpha 2u globulin gene in the diabetic rat. Hypoinsulinemic diabetes is also found to be associated with an approximately 35% reduction in the thyroid hormone receptor level as compared to the normal control. The stimulatory effect of thyroxine on the synthesis of alpha 2u globulin and its mRNA was also evident in spontaneous diabetic Wistar "BB" rats. From these studies it can be concluded that severe hypoinsulinemia can cause a decrease in thyroid hormone action at the level of specific gene expression.

Estrogen induction of biotin-binding protein in immature chicks: kinetics, hormonal specificity and modulation.

Employing a specific radioimmunoassay for quantification, the kinetics of estrogen-induced elevation in the plasma concentration of biotin-binding protein (BBP) in immature male chicks was investigated. A single injection of the steroid hormone enhanced the plasma BBP content several-fold at 6 h, reaching peak levels around 48 h and declining thereafter. A 2-fold amplification of the response was evident during secondary stimulation with the hormone. The magnitude of the response was hormonal dose-dependent while the initial lag phase and the time of peak protein accumulation were unaltered within the hormonal doses tested. The circulatory half-life of the specific protein in normal and estrogenized birds was 10 h. Hyperthyroidism markedly decreased the hormonal response while the opposite effect was seen during hypothyroidism. The antiestrogens E- and Z-clomiphene citrate effectively blocked the protein induction whereas progesterone, either alone or in combination with estrogen, was ineffective in modulating the induction. Cycloheximide administration drastically inhibited the inductive response. The above observations clearly suggest that the genes corresponding to the two isofunctional proteins of chicken egg, viz. BBP and avidin, are differentially regulated.

Vitamin carrier proteins during embryonic development in birds and mammals.

Egg maturation in oviparous vertebrates involves the hepatic synthesis, secretion, and deposition in the developing oocyte of several maternal proteins with specific nutrient carrier function. Thus, in the chicken, adequate yolk deposition of riboflavin, thiamin, etc. is obligatorily mediated by carrier proteins specific to each vitamin. Like vitellogenin, these are oestrogen-inducible specific gene products. Despite differences in patterns of embryonic development in mammals vis-à-vis oviparous species, immunologically and biochemically similar maternal vitamin carriers participate in the transplacental transport and fetal accumulation of these vitamins during gestation in the rat. The rodent riboflavin and thiamin carrier proteins are also oestrogen-induced maternal proteins of hepatic origin. Their functional importance in fetal development was established by in vivo passive immunoneutralization of the endogenous proteins, which precipitated fetal wastage leading to pregnancy termination, due to curtailment of the vitamin supply to the fetuses. Similarly, active immunization of female rats with the vitamin carrier proteins led to early fetal resorption without interference with maternal health, cyclicity and fecundity. The discovery of similar gestation-specific carrier proteins in higher mammals and humans suggests that carrier-mediated vitamin delivery mechanisms ensuring embryonic growth have been conserved during evolution.

Pregnancy suppression by active immunization against gestation-specific riboflavin carrier protein.

A riboflavin carrier protein isolated from chickens cross-reacts with a gestation-specific rodent carrier for riboflavin. Active immunization of female rats of proved fertility with the purified chicken carrier protein completely yet reversibly suppressed early pregnancy without impairing implantation per se. Concurrently there were no discernible adverse effects on maternal health in terms of weight gain, vitamin status, and fertility.