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G Tillmann

Publications and source records attributed to G Tillmann.

6 recordsLinked to original sources
PubMed2002

The affinity and activity of the multiple hormone response element in the proximal promoter of the human oxytocin gene.

In vivo there appears to be a marked association between oestrogen levels and the expression of the oxytocin (OT) gene in most tissues. Transfection and DNA-protein binding experiments using high levels of either oestrogen receptor (ER)alpha or ERbeta imply a direct interaction of these transcription factors with the multiple hormone response element (HRE) at approximately -160 from the transcription start site of the OT gene in most species. In an extensive set of experiments, we show, using both transfection and protein-DNA binding, that low to moderate amounts of either oestrogen receptor, while being able to interact directly with a classic oestrogen response element (ERE) fail to interact with the human OT -160 HRE. Instead, this element, similar to its bovine counterpart, has a high affinity for the orphan receptors steroidogenic factor 1 and chicken ovalbumin upstream promoter transcription factor. Second, the human and bovine OT promoter can be made artificially responsive towards oestrogen in a cotransfection system over-expressing ERalpha or ERbeta, but not in cells expressing natural levels of these steroid receptors. Interestingly, nuclear extracts from both ERalpha-positive MCF7 cells and ERalpha-negative MDA-MB231 cells both contain a transcription factor which binds specifically to both the hOT-HRE element and to a classic ERE, and which has orphan receptor-like binding properties rather than those of an oestrogen receptor. Together, these and other results suggest that oestrogen action in vivo on the OT gene in all species is more likely to involve a DNA-independent mechanism than classic direct interactions with dimeric oestrogen receptors.

Animals↗
PubMed2001

The structure and regulation of the oxytocin receptor.

The oxytocin receptor (OTR) is part of an ancient hormone system expressed in diverse phyla in relation to acute reproductive smooth muscle responses, such as egg-laying, birth, or milk letdown. The regulation of the OTR gene, while correlating with steroid levels in vivo, remains elusive. There appear to be both inhibitory and stimulatory influences acting upon a constitutive pattern of basal expression. We have found no evidence, however, for an effect of the sex steroids either directly on gene transcription, or on the receptor itself at the protein level. In the prostatic carcinoma cell line Du145, we have shown that up-regulation of the OTR gene transcription can be effected by cAMP. In an attempt to characterize the expression of the OTR protein in vivo, we have shown, using ligand-blotting, that the OTR can be expressed at different sizes in transfected cells and in myometrium. Also, in the myometrium at term, immunohistochemistry suggests that there is both an increase in OTR protein per cell, as well as in the number of smooth muscle cells expressing OTR, emphasizing that perinatal changes are the results of both individual gene activation events and gross cellular differentiation. The OTR is a valuable model system reflecting molecular changes in the perinatal period. When we understand how this important molecule is regulated, we will also be a long way towards understanding the mechanisms controlling myometrial contractility at birth. Experimental Physiology (2001) 86.2, 289-296.

Animals↗
PubMed2000

Acute regulation of the bovine gene for the steroidogenic acute regulatory protein in ovarian theca and adrenocortical cells.

Upregulation of the steroidogenic acute regulatory protein (StAR) is implicated in the rapid synthesis and secretion of steroidogenic cells to produce steroids in response to stimulation by trophic hormones of the gonadal and stress axes. In the present study, we have assessed the kinetics of both StAR gene transcription and protein biosynthesis in primary cell cultures of bovine adrenocortical and ovarian theca cells, under conditions of acute stimulation by corticotrophin (ACTH) and luteinizing hormone (LH), respectively. In both cell systems, detectable upregulation of StAR gene transcription occurred within 1-2 h, reaching maxima at 4 h (theca cells) or 6 h (adrenocortical cells). mRNA levels returned rapidly to baseline, by 12 h or 24 h, respectively. Specific StAR protein levels were assessed by western blotting using a novel antibody raised against a bovine StAR peptide, and showed a similar fast upregulation, albeit delayed by 1-2 h compared with the mRNA. The response of the cultured theca cells was more acute than that of the adrenocortical cells, possibly reflecting the propensity of the LH receptor to desensitize rapidly, unlike the ACTH receptor. The primary bovine theca cell cultures were also used for fully homologous transfection studies using various deletion promoter-reporter constructs of the bovine StAR gene. Kinetic analysis of the results indicated that the acute transcriptional response resides within the proximal (-315 bp) promoter region, which includes two putative responsive elements for the steroidogenic factor-1. More distal promoter regions may be involved in modulating the specificity of expression by combining enhancer and inhibitory functions.

Adrenal Cortex↗
PubMed1999

Cloning of bovine estrogen receptor beta (ERbeta): expression of novel deleted isoforms in reproductive tissues.

cDNAs coding for bovine estrogen receptor beta (ERbeta) isoforms were cloned from bovine granulosa cells using a combination of several RT-PCR strategies. The cloned full-length receptor contains an open reading frame of 474 amino acids encoding a protein with high homology to the ERbeta sequences from other species. A second isoform nearly totally lacking the ligand binding domain was cloned that is expressed to relatively high levels in reproductive tissues. Expression of both ERbeta isoforms is down-regulated in corpus luteum and endometrium during the luteal phase of the female cycle. In addition, in granulosa cells several ERbeta isoforms carrying major internal deletions were detected by RT-PCR and cloned. Transient transfection studies expressing the two major bovine ERbeta isoforms together with an ERE reporter construct show estrogen-dependent transactivation by the full-length isoform, whereas the isoform lacking the ligand binding domain did not show any transactivation.

Amino Acid Sequence↗
PubMed1998

The role of SF-1/Ad4BP in the control of the bovine gene for the steroidogenic acute regulatory (StAR) protein.

The bovine gene for the steroidogenic acute regulatory protein (StAR) was cloned and sequenced, including 2 kb of the upstream control region of the gene. The gene comprises seven exons arranged similarly to those of the human and mouse gene sequences. The sequence analysis identified three cis elements corresponding to the binding motif for the transcription factor SF-1/Ad4BP, at - 100, - 240 and - 1190 from the transcription start site. Electrophoretic mobility shift analysis (EMSA) using nuclear proteins from bovine corpus luteum and bovine adrenal as well as in vitro transcribed/translated SF-1/Ad4BP consistently showed that only the site at -1190 bound the transcription factor significantly. Very weak binding was detectable also at the - 240 site, but none at the -100 site. Heterologous transfection of StAR promoter deletion-reporter constructs into Hela cells cotransfected with an expression vector for bovine SF-1/Ad4BP, showed that this transcription factor can specifically act on the bovine StAR gene promoter, but preferentially in regions corresponding to the two proximal SF-1/Ad4BP elements at - 100 and - 240, though with only low relative effect. Furthermore, additional cotransfection of a construct expressing a constitutive protein kinase A catalytic subunit to mimic the effects of cAMP stimulation, led to a small SF-1/Ad4BP-dependent increase in reporter activity mediated only by the same proximal sites. Since the bovine StAR gene promoter does not appear to have a functional cAMP responsive element (CRE), either this effect is mediated in this system directly by SF-1/Ad4BP, or by other factors interacting with this transcription factor, but which do not involve CRE-mediated gene activation. Taken together, the results show that there is a discrepancy between the results of the EMSA experiments and those using transfection of promoter-reporter constructs, which needs to be resolved before a clear understanding of SF-1/Ad4BP-mediated regulation of the StAR gene is attained.

Animals↗