Regulation of messenger ribonucleic acid levels for five urea cycle enzymes in cultured rat hepatocytes. Requirements for cyclic adenosine monophosphate, glucocorticoids, and ongoing protein synthesis

In adult rat liver, amounts of the urea cycle enzymes are regulated by diet, glucocorticoids, and cAMP. Rat hepatocytes cultured in chemically defined medium were used to precisely define the roles of glucocorticoids and cAMP in regulation of these enzymes at the pretranslational level. With the exception of ornithine transcarbamylase mRNA, cultured rat hepatocytes retain the capacity to express mRNAs for the urea cycle enzymes at the same level observed for liver of intact rats. In the absence of added hormones, mRNAs for argininosuccinate synthetase and argininosuccinate lyase remained at or above normal in vivo levels, while mRNAs for the other three enzymes declined to very low levels. Messenger RNAs for carbamyl phosphate synthetase I, argininosuccinate synthetase, argininosuccinate lyase, and arginase increased in response to either dexamethasone or 8-(4-chlorophenylthio) cAMP (CPT-cAMP). Half-maximal responses occurred at 2-3 nM dexamethasone and at 2-7 microM CPT-cAMP. Cycloheximide abolished the response to dexamethasone but not to CPT-cAMP, suggesting that dexamethasone induced expression of an intermediate gene product required for induction of these mRNAs. The effects of a combination of both hormones were additive for argininosuccinate lyase mRNA and synergistic for carbamyl phosphate synthetase I, argininosuccinate synthetase, and arginase mRNAs. Messenger RNA for ornithine transcarbamylase showed little or no response to any condition tested. Depending on the particular mRNA and hormonal condition tested, increases in mRNA levels ranged from 1.4- to 70-fold above control values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential, multihormonal regulation of the mouse major urinary protein gene family in the liver.

The hormonal requirements for the regulation of the major urinary protein (MUP) mRNA levels in mouse liver have been examined. Previous experiments have shown that administration of testosterone to female or castrated male mice increases MUP mRNA levels approximately fivefold to normal male levels. We have found that thyroxine and the peptide hormone, growth hormone, each had a pronounced effect on MUP mRNA levels. MUP mRNA was reduced 150-fold in growth-hormone-deficient mutant mice (little). The administration of growth hormone and thyroxine induced MUP mRNA approximately 150-fold, and when administered together, they induced MUP mRNA approximately 1,000-fold. testosterone administration. When administered separately to these mice, growth hormone and thyroxine induced with MUP mRNA approximately 150-fold, and when administered together, they induced MUP mRNA approximately 1,000-fold. Testicular feminized mice, which lack a functional major testosterone receptor protein, can also be induced to male levels by treatment with both growth hormone and thyroxine. In addition, we present evidence which indicates that growth hormone, thyroxine, and testosterone differentially regulate the levels of distinct MUP mRNA species.     

Hormonal induction of hepatic mitochondrial ornithine/citrulline transporter mRNA

The urea cycle, which involves enzymes located in both the mitochondrion and cytoplasm, requires transport of ornithine and citrulline across the mitochondrial membrane by the ornithine/citrulline antiporter ORNT1. Expression of the urea cycle enzymes can change dramatically in response to hormones, but it is not known whether ORNT1 expression also is hormonally regulated. This study therefore tested the hypothesis that ORNT1 mRNA levels in hepatocytes are induced by cAMP and glucocorticoid as are the urea cycle enzyme mRNAs. ORNT1 mRNA was rapidly induced by a cAMP analog and dexamethasone in cultured rat hepatocytes and there was a strong synergistic response to a combination of these agents. Ongoing protein synthesis was required for induction of ORNT1 mRNA by dexamethasone but not by cAMP, suggesting that the dexamethasone response required an accessory factor. Thus, hormonal regulation of ORNT1 mRNA in hepatocytes is coordinated with that of mRNAs encoding the urea cycle enzymes.     

Subfamilies of the mouse major urinary protein (MUP) multi-gene family: sequence analysis of cDNA clones and differential regulation in the liver

The mouse major urinary proteins (MUPs) are the products of a multi-gene family of 30-35 genes whose members exhibit diverse tissue specific, developmental, and hormonal controls. Three cDNA clones corresponding to liver MUP mRNAs have been sequenced. Two of the clones (p499, C57BL/6 and p1057, BALB/c) share strong homology whereas a third clone (p199, C57BL/6) has diverged considerably from the others at the nucleic acid (85% homology) and protein (68% homology) levels. The 5' regions of p499 and p199 which show the most sequence divergence were subcloned and shown to hybridize to different liver MUP mRNAs. The p499-5' sequence was expressed in all MUP expressing tissues (liver, lachrymal, submaxillary and mammary) whereas the p199-5' sequence was expressed primarily in the liver and lachrymal. Analysis of liver RNA from mice in different endocrine states indicates that the p499-5' sequence is strongly regulated by thyroxine administration whereas the p199-5' sequence is not. Both sequences appear to be regulated by growth hormone and testosterone. Southern blot analysis of mouse genomic DNA indicates that there are multiple genes homologous to each sequence.     

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.     

The gene family for major urinary proteins: Expression in several secretory tissues of the mouse

The major urinary proteins (MUPs) of the mouse are encoded by a multigene family located at the Mup a locus on chromosome 4. Previous investigations have shown that the MUPs are synthesized in the liver, secreted and then excreted in the urine. We have found significant levels of MUP mRNA in several secretory tissues: the liver and the submaxillary, lachrymal and mammary glands. There are striking differences in hormonal and developmental regulation of MUP gene expression in these tissues. Furthermore, each tissue appears to express a characteristic pattern of MUP mRNAs. In particular, the lachrymal glands appear to express an entirely different set of MUP mRNAs. These results are discussed in relation to the organization of the MUP gene cluster and a possible function of the MUPs.     

Nutritional and hormonal regulation of mRNA abundance for arginine biosynthetic enzymes in kidney

Argininosuccinate synthetase and argininosuccinate lyase catalyze the synthesis of arginine from citrulline in kidney and also serve as components of the urea cycle in liver of ureotelic animals. Dietary and hormonal regulation of mRNAs encoding these enzymes have been well studied in liver but not in kidney. Messenger RNAs for these enzymes are localized within the renal cortex. Starvation and extreme variations in dietary protein content (0% vs 60% casein) produced 2.6- to 3.5-fold increases in mRNA abundance for these two enzymes in rat kidney. Argininosuccinate lyase mRNA was not induced by dibutyryl cAMP, dexamethasone, or a combination of the two agents. In contrast, argininosuccinate synthetase mRNA was induced 2-fold by dibutyryl cAMP but was unresponsive to dexamethasone. Thus, diet and hormones regulate levels of these mRNAs in rat kidney, but the responses are both qualitatively and quantitatively distinct from the responses previously reported for rat liver.