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Michael Wöltje, Beate Tschöke, Verena von Bülow, Ralf Westenfeld, Bernd Denecke, Steffen Gräber, and Willi Jahnen-Dechent

% liver-derived. Thus, Ahsg is a major hepatic serum protein. A thorough understanding of Ahsg gene regulation during inflammation and recovery is important for several reasons. A well-documented physiological function of Ahsg is the systemic inhibition of

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G Flouriot, B Ducouret, L Byrnes, and Y Valotaire

Estrogens modulate the expression of many liver-specific genes in oviparous species. For instance, expression of the estrogen receptor and vitellogenin genes is strongly up-regulated by estradiol in rainbow trout liver. Using hepatocyte primary cultures, we demonstrate that trout albumin (Alb) gene is also regulated by this hormone. Indeed, treatment of hepatocytes with 1 microM estradiol led, after 24 h, to a dramatic decrease in Alb mRNA level. To investigate the mechanism of this down-regulation, run-off experiments were performed and mRNA half-lives were determined in the presence and absence of estradiol. The results show that the down-regulation of Alb mRNA expression by estrogens occurs only at the transcriptional level.

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B He, TK Tong, FF Hiou-Tim, B Al-Akad, HM Kronenberg, and AC Karaplis

The type 1 parathyroid hormone receptor (PTHR1) binds, with equal affinity, two ligands with distinct biological functions: PTH, the major peptide hormone controlling calcium homeostasis, and the paracrine factor, PTH-related peptide (PTHrP), a local regulator of cellular proliferation and differentiation. To clarify the complexity of possible interactions between two distinct ligands, PTH and PTHrP, and their common receptor in the intact organism, and to identify as yet unrecognized roles for PTH in normal physiology, we have cloned and characterized the structural organization, nucleotide sequence and transcriptional regulation of the murine gene encoding PTH. One recombinant clone isolated from a mouse genomic library contained 14 kb of DNA, encompassing the entire Pth gene. The transcriptional unit spans 3.2 kb of genomic DNA and, analogous to the human PTH gene, it is interrupted by two introns. The deduced mRNA encodes the 115-amino acid precursor, preproPTH. Comparison of the murine preproPTH sequence with other mammalian forms of the protein shows it to be highly conserved and to share limited structural similarity to PTHrP at the amino-terminal region, a domain critical for binding and activation of their common receptor. Putative binding motifs for the transcription factors sex-determining region Y gene product, transcriptional repressor CDP, hepatic nuclear factor 3beta, GATA-binding factor 1, glucocorticoid receptor, SRY-related high mobility group box protein 5 and cAMP response element binding protein were identified in the 5' flanking region of the Pth gene. When placed upstream of a reporter gene, these sequences failed to confer transcriptional regulation in response to 1,25(OH)(2) vitamin D(3), but responded positively to the addition of isoproterenol and forskolin. Mutational analysis identified a cAMP-response element in the Pth promoter.

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HT Huynh, L Alpert, DW Laird, G Batist, L Chalifour, and MA Alaoui-Jamali

Androgens play an important role in prostate gland development and function, and have been implicated in prostate carcinogenesis. We report the regulation of the gap junctional intercellular communication gene connexin 43 (Cx43) by androgens in the prostate gland. In rat ventral prostate tissue, only trace levels of Cx43 mRNA were detected. Castration, however, resulted in a high increase in Cx43 mRNA and protein. Cx32 was unchanged. Castration-induced Cx43 mRNA and protein were abolished by administration of dihydrotestosterone (DHT). Following castration, prostate weights were approximately 16% of sham-treated controls. However, DHT replacement resulted in prostate weights which were not different from sham-treated controls. Under similar castration conditions, Cx43 induction coincided with pronounced apoptosis in the prostate gland cells, and DHT prevented the induction of apoptosis. Given the physiological role of gap junctions and androgens in the regulation of prostate tissue homeostasis, our observations are relevant to the understanding of androgen-dependent prostate carcinogenesis.

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Y Le Dréan, G Lazennec, L Kern, D Saligaut, F Pakdel, and Y Valotaire

ABSTRACT

We previously reported that the expression of the rainbow trout estrogen receptor (rtER) gene is markedly increased by estradiol (E2). In this paper, we have used transient transfection assays with reporter plasmids expressing chloramphenicol acetyl transferase (CAT), linked to 5′ flanking regions of the rtER gene promoter, to identify cis-elements responsible for E2 inducibility. Deletion analysis localized an estrogen-responsive element (ERE), at position +242, with one mutation on the first base compared with the consensus sequence. This element confers estrogen responsiveness to CAT reporter linked to both the herpes simplex virus thymidine kinase promoter and the homologous rtER promoter. Moreover, using a 0·2 kb fragment of the rtER promoter encompassing the ERE and the rtER DNA binding domain obtained from a bacterial expression system, DNase I footprinting experiments demonstrated a specific protection covering 20 bp (+240/+260) containing the ERE sequence. Based on these studies, we believe that this ERE sequence, identified in the rtER gene promoter, may be a major cis-acting element involved in the regulation of the gene by estrogen.

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A Nandy, S Jenatschke, B Hartung, K Milde-Langosch, AM Bamberger, and B Gellersen

The NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is a catabolic enzyme that controls the biological activities of prostaglandins by converting them into inactive keto-metabolites. Here we report the genomic organisation of the complete human PGDH gene and characterise its transcriptional regulation. The PGDH gene spans about 31 kb on chromosome 4 and contains 7 exons. Within 2.4 kb of the 5'-flanking sequence we identified two regions with clustered putative transcription factor binding sites. The distal promoter element PGDH-DE (positions-2152/-1944 relative to the start codon) contains binding sites for Ets and activating protein-1 (AP-1) flanked by two cAMP-responsive element-binding protein binding sites (CREB1, CREB2), whereas the proximal element PGDH-PE (-235/-153) includes an Ets and an AP-1 binding sequence. By electrophoretic mobility shift assay, no high affinity binding of Ets or AP-1 factors was observed with PGDH-PE, whereas we confirmed interaction of members of the Ets, AP-1 and CREB families of transcription factors with PGDH-DE. Transcriptional control of the PGDH promoter was assessed by transiently transfecting JEG-3 choriocarcinoma cells. A luciferase reporter gene construct containing the PGDH-PE was not induced by c-jun/c-fos in the absence or presence of co-expressed Ets-1. A construct carrying the PGDH-DE in front of the minimal homologous promoter was activated by co-transfection of expression vectors for AP-1 proteins. Mutation of the AP-1 or CREB2 site reduced the response to c-jun/c-fos, whereas mutation of the Ets site of the distal element reduced basal promoter activity. CREB activated the PGDH-DE construct through the CREB1 site. These results defined the distal element as an integrator of transcriptional regulation by AP-1, Ets and CREB proteins.

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I. Porsch Hällstöm, J.-Å. Gustafsson, and A. Blanck

ABSTRACT

Expression of the c-myc gene was studied in the livers of male and female Wistar rats. Furthermore, the effects on hepatic c-myc expression of neonatal and adult castration, with or without testosterone supplementation, as well as of continuous administration of GH to intact males, were analysed. Expression of c-myc was low in 6-day-old animals of both sexes, reached a maximum at 35 days of age and declined to the level of adult animals at 70 days. In prepubertal animals, expression was higher in females, but was higher in males after the onset of puberty, the postpubertal female rat liver exhibiting 50–70% of the expression in males.

Treatment of adult male rats with bovine GH in osmotic minipumps for 1 week reduced c-myc expression to the level of female rats. Castration, both neonatally and of adults, also feminized hepatic c-myc expression. Testosterone supplementation of the castrated animals increased the expression towards the level in sham-operated controls.

These results indicate that the c-myc gene is regulated by the hypothalamo-pituitary-liver axis via the sex-differentiated pattern of GH secretion, in analogy with other sex-differentiated hepatic functions, such as metabolism of steroids and xenobiotics. Neuroendocrine regulation of a gene such as c-myc, which is involved in the control of cell proliferation and differentiation, represents another aspect of the complex influence of GH on various somatic functions.

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P Balanathan, EM Ball, H Wang, SE Harris, AN Shelling, and GP Risbridger

Inhibin was first identified as a gonad-derived regulator of pituitary FSH; however, it has subsequently been shown to be a tumour suppressor in the gonad and adrenal glands. Whereas non-malignant regions of human primary prostate carcinomas express inhibin alpha-subunit (INHA), malignant tissues lack INHA transcript and protein, which is consistent with epigenetic regulation of the inhibin alpha-subunit gene (INHA) promoter. This study investigated whether methylation of the INHA promoter was responsible for inactivation of INHA transcription and translation in the prostate cancer cell lines, LNCaP, DU145 and PC3. Methylation of the promoter was revealed by bisulphite genomic sequencing and use of inhibitors of methylation and histone deacetylation resulted in reactivation of the INHA transcription and translation. Significant (P<0.05) downregulation of a luciferase reporter gene downstream from a methylated INHA promoter compared with unmethylated INHA promoter occurred in vitro. The data demonstrate that promoter methylation is associated with downregulation of the INHA gene in prostate cancer cell lines, which is consistent with its tumour suppressive role. Therefore INHA has a significant role in prostate tumorigenesis.

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C Breton, D Di Scala-Guenot, and HH Zingg

The differential, tissue-specific regulation of oxytocin (OT) binding sites allows the neurohypophysial nonapeptide OT to fulfill a dual role: to induce uterine contractions at parturition and to mediate milk ejection during lactation. Whereas uterine OT binding sites are up-regulated prior to parturition and are rapidly down-regulated thereafter, mammary gland OT binding sites gradually increase throughout gestation and remain up-regulated during the ensuing lactation period. Here, we structurally characterized OT receptor (OTR) mRNA in mammary gland and analyzed its expression during gestation and lactation and in response to steroid treatment. In mammary gland tissues, we found a 6.7 and a 5.4 kb OTR mRNA species, and both species were further analyzed by RACE (rapid amplification of cDNA ends). The 6.7 kb mRNA was found to be common to mammary gland and uterus and to extend 618 nucleotides beyond the published sequence of the rat OTR gene. The 5.4 kb mRNA species is unique to the mammary gland and terminates at a mammary gland-specific polyadenylation site that is not preceded by a classical polyadenylation signal. RT-PCR analysis did not provide any evidence for differences in the coding regions, suggesting that both uterine and mammary gland OTR mRNAs encode the same receptor protein. Furthermore, primer extension experiments showed that no differences exist in the specific transcriptional initiation sites of the OTR gene in the two tissues. During pregnancy, OTR mRNA per mammary gland increased approximately 150-fold and remained high during lactation, consistent with the previously identified regulation of OT binding sites and the role of OT during lactation. Whereas estrogen administration strongly induced the uterine OTR mRNA levels (>5-fold), mammary gland remained unaffected by steroid treatment. Moreover, tamoxifen had no effect on the mammary gland OTR mRNA level. In summary, our data demonstrate a differential control of OTR expression in uterus versus mammary gland and a mammary gland-specific OTR mRNA polyadenylation site. However, this differential control apparently does not involve the expression of different receptor genes nor the utilization of tissue-specific transcriptional initiation sites.

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L Laflamme, G Hamann, N Messier, S Maltais, and Langlois M-F

Thyroid hormone receptors (TRs) often modulate transcriptional activity of target genes by heterodimerization with the 9-cis retinoic acid receptor (RXR). On positive thyroid response elements (TREs), RXR favors binding of the TR-RXR complex to DNA and stimulates transcription. RXR action on negative TREs is unclear. Furthermore, the single half-site configuration of many negative TREs does not favor the binding of a classic TR-RXR heterodimer. In a comparative study using CV-1 cells (relatively RXR- and TR-deficient) and JEG-3 cells (relatively TR-deficient), we demonstrate the importance of RXR in the negative transcriptional regulation of genes of the hypothalamo-pituitary axis by tri-iodothyronine. While RXR has variable effects on ligand-independent activation produced by TRs, it was required for efficient ligand-dependent repression of the TRH gene for TRalpha1 and TRbeta1 and of the TSH genes by all TRs. Using different RXR constructs we also observed the importance of the C-terminus of RXR but not of the N-terminus nor the DNA-binding domain, in the potentiation of negative regulation. We thus suggest that, with regard to negative regulation of the TRH and TSH genes by thyroid hormones, RXR behaves more like a cofactor than a classic heterodimerization partner.