Androgen regulated expression of the alpha 2u-globulin gene in pancreatic hepatocytes of rat.

Under a copper-deficient regimen, pancreatic cells in the adult rat can be found to undergo differentiation into hepatocytes. Pancreatic hepatocytes induced in male and female rats were examined for the expression of the androgen-inducible hepatic protein, alpha 2u-globulin. Alpha 2u-Globulin protein was demonstrable by immunoperoxidase method in all the pancreatic hepatocytes of male rats. Northern blot analysis confirmed the presence of 1.3 kb alpha 2u-globulin mRNA transcript in the pancreas of male rats with hepatocytes. Orchiectomy resulted in marked decrease of alpha 2u-globulin protein and its mRNA. Administration of dihydrotestosterone to castrated rats resulted in increased levels of alpha 2u-globulin mRNA and the amount of alpha 2u-globulin protein in the pancreatic hepatocytes. Unlike normal males, in intact and ovariectomized females alpha 2u-globulin was not detectable in pancreatic hepatocytes. These results indicate that similar to hepatic parenchymal cells pancreatic hepatocytes synthesize alpha 2u-globulin under androgenic regulation. Furthermore, unlike in liver where it is expressed predominantly in perivenular and midlobular hepatocytes, there is no localized difference in the expression of this gene in the transdifferentiated pancreatic hepatocytes.

Developmentally regulated male-specific transfactor(s) enable in vitro transcription of a cloned alpha 2u-globulin gene.

Selected members of the rat alpha 2u-globulin gene family are expressed in several tissues, manifesting characteristic developmental and endocrine transcriptional control. Studies are now underway to identify the responsible cis sequences and transacting factors. We recently reported that the cloned rat alpha 2u-globulin 207 gene manifests tissue-specific androgen-dependent expression; it is expressed in livers of male, but not female, transgenic mice. In the present study a portion of this gene (-639 to +1395) is used as an in vitro template in the presence of nuclear extracts derived from hepatic nuclei of prepubescent and mature male and female rats. alpha-Amanitin-sensitive in vitro transcription of the alpha 2u 207 gene by extracts derived from mature male rats is highly active and is at least 13 times as rapid as that with preparations from mature female or immature animals. In contrast, the rate of transcription of a control template, the adenoviral late promoter, is the same with all of these nuclear extracts. S1 nuclease analysis and the size of the transcript indicate that transcription is initiated in vitro at the same site as it is in vivo and that it continues to the 3' terminus of the alpha 2u-globulin template. Thus, cis sequences are present in this gene fragment which are controlled by developmentally regulated male transacting factors, enabling selective transcription of this alpha 2u-globulin gene. Mixing experiments indicate that this transcriptional control is positive.

Nuclear factors from expressing tissues interact in vitro with a rat alpha-2u globulin gene intron.

Members of the rat alpha 2u globulin gene family are expressed in a tissue-specific manner. Using a DNase I footprinting assay, we find that expressing tissues (liver, lachrymal, and salivary gland) contain nuclear proteins that interact specifically with two sites in the third intron of a cloned gene. These sequences are similar to a CCAAT-like sequence in the 5'-flanking region of the gene and the binding site for the transcription factor Sp1. Extracts of nonexpressing tissues (kidney, testis, brain, and spleen) do not show footprinting activity at these sites. Whereas hepatic expression of alpha 2u globulin is restricted to normal adult males, nuclear extracts of liver from all animals (adult and juvenile, male and female, intact and hypophysectomized) produce similar footprints. Footprints by extracts of lachrymal and salivary glands, tissues that express a subset of alpha 2u globulin genes distinct from the hepatic set, are also similar to the liver activity. The findings suggest that the third intron of an alpha 2u globulin gene contains sites that interact with tissue-specific factors to establish the potential for hormonal and developmental control of expression.

Tissue-specific and hormonally regulated expression of a rat alpha 2u globulin gene in transgenic mice.

To investigate the tissue-specific and hormonal regulation of the rat alpha 2u globulin gene family, we introduced one cloned member of the gene family into the mouse germ line and studied its expression in the resulting transgenic mice. Alpha 2u globulingene 207 was microinjected on a 7-kilobase DNA fragment, and four transgenic lines were analyzed. The transgene was expressed at very high levels, specifically in the liver and the preputial gland of adult male mice. The expression in male liver was first detected at puberty, and no expression was detected in female transgenic mice. This pattern of expression is similar to the expression of endogenous alpha 2u globulin genes in the rat but differs from the expression of the homologous mouse major urinary protein (MUP) gene family in that MUPs are synthesized in female liver and not in the male preputial gland. We conclude that these differences between rat alpha 2u globulin and mouse MUP gene expression are due to evolutionary differences in cis-acting regulatory elements. The expression of the alpha 2u globulin transgene in the liver was abolished by castration and fully restored after testosterone replacement. The expression could also be induced in the livers of female mice by treatment with either testosterone or dexamethasone, following ovariectomy and adrenalectomy. Therefore, the cis-acting elements responsible for regulation by these two hormones, as well as those responsible for tissue-specific expression, are closely linked to the alpha 2u globulin gene.

Glucocorticoid-regulated expression from the 5'-flanking region of the rat alpha 1-acid glycoprotein gene. Requirement for ongoing protein synthesis.

The induction of alpha 1-acid glycoprotein (AGP) mRNA by glucocorticoids in rat hepatoma cells requires ongoing protein synthesis. Here we show that the 5'-flanking region of the AGP gene confers glucocorticoid responsiveness on the expression of heterologous coding sequences. Moreover, the induction of beta-globin mRNA directed by the AGP promoter is inhibited by concurrent treatment with cycloheximide, thereby suggesting that the requirement for protein synthesis is mediated through 5'-flanking sequences. Analysis of the effects of varying durations of cycloheximide treatment indicates that both the endogenous AGP gene and the transfected AGP-beta-globin chimeric gene are induced normally by dexamethasone during the first 1-2h of concurrent treatment. In addition, pretreatment with cycloheximide yields a decrease in the subsequent induction of both beta-globin and AGP RNAs. These data support the notion that a pre-existing and labile protein, perhaps in concert with the glucocorticoid receptor, acts through the 5'-flanking region of the AGP gene to stimulate transcription from the AGP promoter.

Acute phase mediators and glucocorticoids elevate alpha 1-acid glycoprotein gene transcription.

Acute phase mediators and glucocorticoids increase the synthesis of the acute phase protein alpha 1-acid glycoprotein, also known as orosomucoid, by inducing the hepatic level of its mRNA. Concurrently the acute phase response depresses the hepatic synthesis of albumin and alpha 2u-globulin and their mRNA levels. Present transcriptional studies in isolated liver nuclei demonstrate that turpentine-induced acute phase mediators simultaneously enhance transcription of the alpha 1-acid glycoprotein gene and diminish transcription of albumin and alpha 2u-globulin genes; parallel alterations in the hepatic level of the corresponding mRNAs ensue. The present transcriptional studies also demonstrate that administration of dexamethasone to adrenalectomized rats dramatically elevates the rate of transcription of the alpha 1-acid glycoprotein gene as well as the alpha 2u-globulin and the albumin genes, leading to elevations in alpha 1-acid glycoprotein and alpha 2u-globulin hepatic mRNA levels. Thus, hepatic alpha 1-acid glycoprotein mRNA levels are predominantly regulated in vivo at the transcriptional level by glucocorticoids as well as by acute phase mediators.

Developmental and hormonal regulation of alpha 2u-globulin gene transcription.

Hepatic alpha 2u-globulin protein and RNA levels are under developmental and complex multihormonal control. The present studies directly evaluate the degree to which this regulation is transcriptional. alpha 2u-Globulin transcription was determined by measuring nuclear runoff RNA in vitro, and tissue alpha 2u-globulin mRNA levels were measured by dot blot hybridization. These studies reveal that (i) in male rats the transcriptional rate of the alpha 2u-globulin genes increases during postnatal development; (ii) no alpha 2u-globulin transcription is detectable in hepatic nuclei derived from hypophysectomized rats; (iii) growth hormone and glucocorticoid are both absolutely required, and glucocorticoid can replace androgen for alpha 2u-globulin gene transcription in the livers of hypophysectomized male rats; and (iv) chronic treatment of mature male rats with estrogen results in a progressive decrease in the hepatic transcription of alpha 2u-globulin genes. In all instances changes in the transcriptional rate of alpha 2u-globulin genes paralleled the tissue level of alpha 2u-globulin RNA. Thus transcriptional control predominates in regulating hepatic alpha 2u-globulin RNA levels.

Rat alpha 1-acid glycoprotein. Gene sequence and regulation by glucocorticoids in transfected L-cells.

We have cloned and sequenced the rat gene coding for the acute phase reactant protein alpha 1-acid glycoprotein in order to determine which sequences are necessary for its regulation by glucocorticoids and which sequences are responsible for the sensitivity of this regulation to protein synthesis inhibitors. The gene contains six exons, as determined from the alpha 1-acid glycoprotein cDNA sequence, and five introns. Primer extension and S1 nuclease experiments have shown that there are two transcriptional start sites 4 base pairs apart. After cotransfection into mouse L-cells, the gene retains its inducibility by glucocorticoids, indicating that the sequences required for induction are within or around the gene. The induction of alpha 1-acid glycoprotein levels in transfected L-cells is sensitive to protein synthesis inhibitors, implying that in addition to the glucocorticoid receptor another protein(s) is necessary for full induction.

Differential regulation of alpha 2u globulin gene expression in liver, lachrymal gland, and salivary gland.

The alpha 2u globulins, products of a highly homologous multigene family, are synthesized in the liver and submaxillary salivary glands of the rat. Although their precise function has not been ascertained, they are of interest because of the complex developmental and hormonal regulation of their tissue levels. We now report that alpha 2u globulin is synthesized in a third tissue of the rat, the extraorbital lachrymal gland. Immunocytochemical studies indicate that the distribution of alpha 2u globulin is more homogeneous in the lachrymal gland than in the liver or submaxillary gland. In situ hybridization to alpha 2u globulin RNA reveals specific signal only over the acinar cells of the lachrymal gland. Several different isoelectric forms of alpha 2u globulin are encoded by lachrymal gland mRNA. The major lachrymal and salivary gland isoforms are indistinguishable from one another, but more acidic than the hepatic isoforms. In addition, analysis of double-stranded cDNAs with a diagnostic restriction-enzyme pair detects no differences between the alpha 2u globulin mRNAs of lachrymal and salivary gland, but clearly distinguishes these from their hepatic counterparts. In spite of the similarity between the lachrymal and salivary gland alpha 2u globulin gene products, we find that the hormonal and developmental regulation of alpha 2u globulin expression differs markedly in these two tissues. In the liver, where a different subset of alpha 2u globulin genes is expressed, a third regulatory phenotype is observed.

alpha 2u-Globulin is present in the rat anterior pituitary.

The possibility that the pituitary gland may contain as yet undiscovered regulatory factors is intriguing. Recent reports have suggested the presence, in the anterior pituitary, or a number of proteins of extrapituitary origin. alpha 2u-Globulin, a rat serum and urinary protein, previously shown to be synthesized in the submaxillary gland and in the liver under anterior pituitary control, has now been localized by immunocytochemistry in the cytoplasm of some cells of the anterior pituitary. No alpha 2u-globulin could be detected in either the intermediate or posterior pituitary. The presence of alpha 2u-globulin was confirmed and quantitated by radioimmunoassay. Using RNA blot analysis and cloned alpha 2u-globulin cDNA probes, we could not detect alpha 2u-globulin mRNA sequences in pituitary RNA, indicating that alpha 2u-globulin is not synthesized therein. The presence of alpha 2u-globulin, presumably of circulatory origin, in certain anterior pituitary cells suggests that it may play a role in anterior pituitary function.

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.

Modifications in alpha 2u globulin gene structure, transcription, and mRNA translation in hepatomas.

alpha 2u globulin synthesis ceases when a liver cell becomes malignant. We have compared the structure and transcription of the alpha 2u globulin genes in Morris hepatomas 5123D and 7793 with that of normal hepatic genes using alpha 2u globulin cDNA as a hybridization probe. No alpha 2u globulin mRNA was detected in hepatoma 7793 by cell-free translation, Northern blot, or R0t analysis. In hepatoma 5123D, however, a small number of alpha 2u globulin RNA sequences were detected by Northern blot and R0t analysis, but this RNA was not detectably translationally active either in vivo or in an in vitro cell-free translational system. No structural differences between normal liver and the hepatoma alpha 2u globulin genes were observed by Southern blot analysis of genomic DNA digested with restriction endonucleases Pst I, Bam HI, or Ava I. However, a general demethylation of cytosine residues in the alpha 2u globulin genes of hepatomas has been found using the restriction enzyme Hha I and the isoschizomeric pair of restriction enzymes Msp I and Hpa II. Therefore, loss of the alpha 2u globulin phenotype in these hepatomas is accompanied by extensive demethylation of DNA sequences within and/or immediately flanking the alpha 2u globulin genes.

The role of growth hormone in alpha 2u globulin synthesis: a reexamination.

Hypophysectomy of adult male rats abolishes alpha 2u globulin synthesis; synthesis can be fully restored by daily administration of androgen, thyroid hormone, glucocorticoid and growth hormone for 12 days. It has been previously reported that growth hormone is not required to maintain alpha 2u globulin mRNA levels, and that growth hormone functions only translationally. We have reexamined the role of growth hormone in alpha 2u globulin synthesis using a cloned alpha 2u globulin cDNA probe. Measurement of alpha 2u globulin mRNA levels by hybridization and cell-free translation, and of alpha 2u globulin synthesis by pulse-labeling in vivo, demonstrates that growth hormone is required to maintain full, steady state hepatic levels of both alpha 2u globulin and its mRNA. Furthermore, the small amount of hepatic alpha 2u globulin mRNA that accumulates in the absence of growth hormone is efficiently translated in vivo. Thus we now find no evidence for growth-hormone-mediated translational control. We have also found that rats hypophysectomized prepubescently respond to the multihormonal restoration therapy to only 10% of the alpha 2u globulin mRNA levels observed in rats hypophysectomized as adults. This finding suggests that some unidentified pituitary factor(s) is required during puberty to potentiate normal alpha 2u globulin gene expression.

Cloning and sequence of several alpha 2u-globulin cDNAs.

We describe a simple cloning procedure for alpha 2u-globulin that requires neither enrichment of mRNA for cloning nor purification of a specific probe for screening recombinant colonies. Total adult male liver poly(A)+RNA was used as template for cloning, and the subsequent recombinant colonies were screened by comparing hybridization to radioactive cDNA probes prepared from hepatic male and female mRNA, respectively. Almost all of the selected "male-specific" clones were later shown to contain alpha 2u-globulin sequences. This cloned alpha 2u-globulin cDNA has been shown to specifically hybridize to male rat liver RNA, which, when isolated and translated in vitro, codes for a 21,000-dalton protein (pro-alpha 2u-globulin) immunologically identical to alpha 2u-globulin. When translation occurs in the presence of pancreatic microsomes this in vitro synthesized pro-alpha 2u-globulin is processed to the 19,000-dalton mature form of alpha 2u-globulin. The nucleotide sequence of the alpha 2u-globulin cDNA has been determined, thus elucidating the complete amino acid sequence of alpha 2u-globulin and most of the hydrophobic "leader" sequence of pro-alpha 2u-globulin. The amino acid sequence deduced from the cDNA is in agreement with the partial sequence that we previously determined by sequential Edman degradation of the purified protein. alpha 2u-Globulin cDNA clones contain within the 3'-untranslated region one or both of the two putative polyadenylylation/transcription termination sites (A-A-T-A-A-A and A-A-T-T-A-A-A). Either of these can be used, generating alpha 2u-globulin mRNA species of two lengths. A codon usage analysis of the cDNA showed that, although all six leucine codons are used for the 14 leucine residues in mature alpha 2u-globulin, the seven leucines in the partial leader sequence reported are all encoded by the same codon, CTG. The primary amino acid sequence contains a unique Asn-Gly-Ser sequence, likely to be in beta-turn conformation, as the probable site of glycosylation for this glycoprotein.

Covalent modification and repressed transcription of a gene in hepatoma cells.

When liver cells undergo malignant transformation, certain genes cease being expressed. We have studied the structure of one such gene, whose protein product we have designated hepatic protein 22 (hp22), which is not expressed in the two Morris hepatomas studied. We have prepared a chimeric clone of pBR322 containing cDNA sequences complementary to mRNA coding for this protein. By using this cloned cDNA, we have examined changes in expression of this gene and changes in the restriction pattern of the DNA isolated from normal liver and these hepatomas. In both hepatomas, studies using the isoschizomeric pair of restriction enzymes Msp I and Hpa II have indicated hypermethylation of a cytosine residue within or proximal to the hp22 gene. Other differences in the restriction pattern between normal liver and hepatoma DNA were also detected with EcoRI and Ava I. Thus, in the nontranscribed form of this gene, the DNA has undergone covalent modification, distinguishing these two hepatomas from each other and from normal liver.