Rapid upregulation of serum and glucocorticoid-regulated kinase (sgk) gene expression by corticosteroids in vivo.

The molecular mechanisms by which corticosteroids regulate epithelial sodium transport remain to be fully elucidated. Expression of the serum and glucocorticoid-regulated kinase (sgk) has recently been reported to be regulated acutely by corticosteroids in the amphibian A6 cell line and in cortical collecting tubule cells in vitro. In order to extend this observation to a mammalian system in vivo, the acute response of the sgk gene to a single parenteral dose of aldosterone or dexamethasone was examined in the rat kidney and distal colon. The sgk mRNA levels were significantly elevated by both steroids by 30 min in the distal colon, reaching a peak at 2 h. A more modest increase in sgk mRNA levels was also seen in the kidney in response to both steroids. In both tissues, sgk mRNA has a very short half-life. As for other corticosteroid-regulated genes, the response appears to be mediated by both the mineralocorticoid and glucocorticoid receptors. The response to aldosterone in the distal colon in the presence of cycloheximide was superinduced, strongly suggesting that this is a primary response. The responses to both adrenalectomy and carbenoxolone sodium treatment suggest that the observed responses to corticosteroids can occur in the physiological range of endogenous circulating corticosteroids. These studies provide strong evidence that sgk is an aldosterone-induced gene in vivo in a mammalian system.

Specificity in mineralocorticoid versus glucocorticoid action.

The mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) share considerable structural and functional homology. Overlapping effects on epithelial sodium transport are observed in vivo; in vitro, both are able to bind and transactivate through a common hormone response element. This has led several investigators to suggest that specificity is conferred primarily by prereceptor mechanisms, and we have addressed this question using both in vitro and in vivo approaches. Although the MR has been regarded as less transcriptionally active than the GR in vitro, significant differences are observed when epithelial rather than fibroblast cell lines are used. These differences are mediated by the N-termini of the receptors. Activation of intracellular signaling pathways differentially modulates MR- versus GR-mediated transactivation. Although these studies identify mechanisms by which specificity may be achieved, they do not prove that this occurs in vivo. Such studies have been limited by an absence of MR-regulated genes. Known candidate aldosterone-responsive genes have been examined in the rat distal colon; the time course and the specificity of the response to a single parenteral dose of corticosteroid has been characterized. The epithelial sodium channel beta and gamma subunit genes are both up-regulated within 60 minutes by either MR or GR activation. Similar responses are observed for the serum and glucocorticoid-regulated kinase and channel-inducing factor genes. All four genes show clear and rapid up-regulation of their mRNA levels by aldosterone, which is paralleled by GR-mediated up-regulation of expression. While they are indeed aldosterone-responsive genes, genes that are uniquely aldosterone-regulated remain to be identified.

Acute differential regulation by corticosteroids of epithelial sodium channel subunit and Nedd4 mRNA levels in the distal colon.

The molecular mechanisms by which corticosteroids affect fluid and electrolyte balance have yet to be fully elucidated. The apical amiloride-sensitive electrogenic epithelial sodium channel (ENaC) has been shown to have a central role in corticosteroid-mediated sodium transport in the distal colon. The acute response of the alpha-, beta- and gammaENaC subunit genes to a single parenteral dose of aldosterone or dexamethasone was examined in the rat distal colon in vivo. The response of the Nedd4 gene, whose product is involved in channel turnover, was also examined. Whilst the alphaENaC and Nedd4 genes showed no significant response to either steroid, both the beta- and gammaENaC mRNA levels were increased acutely by both aldosterone and dexamethasone. The gammaENaC mRNA appears to have a very short half-life. Use of the highly selective glucocorticoid receptor agonist RU28362 confirmed that the response was mediated by both the mineralocorticoid and glucocorticoid receptors.

Transcriptional control by corticosteroids of CHIF gene expression in the rat distal colon.

1. Previous studies have shown that levels of CHIF mRNA are increased in the distal colon of the rat in response to corticosteroids. We have recently reported that this response occurs within 2 h of a single dose of either dexamethasone or aldosterone and that the response is mediated via both the mineralocorticoid and glucocorticoid receptors. 2. In the present study we sought to further define the nature of the various transcripts detected by a CHIF coding region cRNA probe in northern blot analysis of corticosteroid-stimulated colonic RNA. The identification of an intronic sequence was used to synthesize an intron-specific cDNA probe to characterize the transcripts. 3. The presence of an intronic sequence in the originally published sequence was confirmed using coupled reverse transcriptase-polymerase chain reactions with primers spanning and within the intronic sequence. The intronic cDNA probe hybridized to the higher molecular weight transcripts detected by the cRNA probe. These transcripts are induced in response to both corticosteroids. 4. Taken together with our observations that the increase in CHIF mRNA levels in the distal colon in response to corticosteroids is not blocked by prior cycloheximide treatment, the increase in the levels of the primary transcript and partially spliced forms argues that this is a primary transcriptional response. This is the first clear demonstration of an aldosterone-induced gene in vivo in a mammalian system.

Acute regulation by corticosteroids of channel-inducing factor gene messenger ribonucleic acid in the distal colon.

The molecular mechanisms by which corticosteroids affect fluid and electrolyte balance are unclear. Though glucocorticoid-responsive genes have been identified, genes regulated by aldosterone have not. CHIF (channel-inducing factor gene) is a recently identified gene that is up-regulated in the distal colon by chronic corticosteroid exposure, is expressed in the kidney, and induces a K+-specific current in Xenopus oocytes. The predicted protein shows similarity to gammaNa.K-ATPase, phospholemman, and Mat-8; all seem to be involved in ion transport. CHIF thus presents as a potential aldosterone target gene. In this study, CHIF expression was examined in rats in the acute timeframe of 0.5-4 h after corticosteroid administration. CHIF messenger RNA showed up-regulation by both mineralocorticoid and glucocorticoid receptor agonists in the distal colon, which was not diminished by cycloheximide. Corticosteroid regulation was not observed in the kidney. Basal and induced expression was absent in the lung and in all gastrointestinal tissues except colon, with expression increasing proximal to distal. CHIF is the first gene to show acute regulation by aldosterone and thus encodes a candidate aldosterone-induced protein. In addition, gammaNa.K-ATPase gene expression was found to be very low in colon and significantly higher in kidney. Regulation by corticosteroids was not evident in either tissue.

De novo insertion of an intron into the mammalian sex determining gene, SRY.

Two theories have been proposed to explain the evolution of introns within eukaryotic genes. The introns early theory, or "exon theory of genes," proposes that introns are ancient and that recombination within introns provided new exon structure, and thus new genes. The introns late theory, or "insertional theory of introns," proposes that ancient genes existed as uninterrupted exons and that introns have been introduced during the course of evolution. There is still controversy as to how intron-exon structure evolved and whether the majority of introns are ancient or novel. Although there is extensive evidence in support of the introns early theory, phylogenetic comparisons of several genes indicate recent gain and loss of introns within these genes. However, no example has been shown of a protein coding gene, intronless in its ancestral form, which has acquired an intron in a derived form. The mammalian sex determining gene, SRY, is intronless in all mammals studied to date, as is the gene from which it recently evolved. However, we report here comparisons of genomic and cDNA sequences that now provide evidence of a de novo insertion of an intron into the SRY gene of dasyurid marsupials. This recently (approximately 45 million years ago) inserted sequence is not homologous with known transposable elements. Our data demonstrate that introns may be inserted as spliced units within a developmentally crucial gene without disrupting its function.

Widespread expression of the testis-determining gene SRY in a marsupial.

There is compelling evidence from mutation analysis and transgenesis that the SRY gene isolated from human and mouse encodes the testis-determining factor on the mammalian Y chromosome. However, how SRY achieves this function is unclear. Although marsupials have been separated from eutherian mammals for approximately 100 million years, homologues of SRY have been localised to the Y chromosome of two unrelated marsupial species, the tammar wallaby and the Darling Downs dunnart. Gonadal development is fundamentally similar in eutherian and marsupial mammals, but the timing of morphological events is different. Fetal Sry transcripts are confined to somatic cells of the male mouse genital ridge between 10.5-12.5 days post coitum, corresponding with the onset of testis differentiation. Analysis of Sry gene expression in the genital ridge of normal and germ cell-deficient fetal mice has established that this gene acts in the somatic cell lineage, and is presumed to induce the formation of Sertoli cells. This assumption can be tested more critically in the tammar, where the equivalent stages of testis differentiation are observed over a 7-day period. We have examined the relationship of SRY expression to testis differentiation in the tammar wallaby. We show the marsupial SRY gene cannot be exclusively coupled to Sertoli cell differentiation, as this gene is expressed in the male fetus from several days before genital ridge formation until 40 days after birth. SRY transcripts are also present in a variety of extra-gonadal tissues in the developing young and adult male, a pattern of SRY expression similar to that observed in humans. These data indicate that, in addition to a role in testis determination, SRY may have other functions [corrected].

Evolution of sex determination and the Y chromosome: SRY-related sequences in marsupials.

In mammals, testis determination is under the control of the testis-determining factor borne by the Y chromosome. SRY, a gene cloned from the sex-determining region of the human Y chromosome, has been equated with the testis-determining factor in man and mouse. We have used a human SRY probe to identify and clone related genes from the Y chromosome of two marsupial species. Comparisons of eutherian and metatherian Y-located SRY sequences suggest rapid evolution of these genes, especially outside the region encoding the DNA-binding HMG box. The SRY homologues, together with the mouse Ube1y homologues, are the first genes to be identified on the marsupial Y chromosome.

Expression in transgenic mice of class I histocompatibility antigens controlled by the metallothionein promoter.

To study the effects of increased expression of major histocompatibility complex class I molecules on the development of self-tolerance, transgenic mice were produced that expressed the H-2Kb gene under the control of the metallothionein promoter. Administration of zinc enhanced transgene expression in liver, kidney and exocrine pancreas. No evidence suggestive of an autoimmune response was found in transgene-expressing tissues in mice otherwise allogeneic to H-2Kb. Despite this lack of responsiveness in vivo, T cells could be stimulated in vitro to lyse H-2Kb-bearing target cells. No infiltration was detected in transgenic mice after irradiation and reconstitution with bone marrow cells. When spleen cells were used for reconstitution, however, dense lymphocytic infiltration was seen, particularly in the portal tracts of the liver, and this was accompanied by piecemeal necrosis and apoptosis of periportal hepatocytes. This aggressive response progressively diminished with time, and by 12 weeks after reconstitution many of the portal tracts were free of infiltration while the others showed no accompanying necrosis. The picture at this stage was similar to that seen in chronic persistent hepatitis. These results suggest that, in addition to negative selection in the thymus, peripheral mechanisms not involving clonal deletion or permanent clonal anergy can prevent immune responses to self molecules.