Biomedical subjects

Wilfried Rombauts

Publications and source records attributed to Wilfried Rombauts.

5 recordsLinked to original sources

PubMed2003

DNA recognition by the androgen receptor: evidence for an alternative DNA-dependent dimerization, and an active role of sequences flanking the response element on transactivation.

The androgen receptor has a subset of target DNA sequences, which are not recognized by any other steroid receptors. The androgen selectivity of these sequences was proposed to be the consequence of the ability of the androgen receptor to dimerize on direct repeats of 5'-TGTTCT-3'-like sequences. This is in contrast with the classical non-selective elements consisting of inverted repeats of the 5'-TGTTCT-3' elements separated by three nucleotides and which are recognized by other steroid receptors in addition to the androgen receptor. We demonstrate that while the DNA-binding domain of the oestrogen receptor is unable to dimerize on direct repeats, dimeric binding can be rescued by replacing the second Zn finger and part of the hinge region by the corresponding fragment of the androgen receptor, but not the glucocorticoid receptor. In this study, we investigate the androgen receptor binding to all natural androgen-selective response elements described so far. We show that a 12-amino acid C-terminal extension of the DNA-binding domain is required for high-affinity binding of the androgen receptor to all these elements. For one androgen-specific low-affinity binding site, the flanking sequences do not contribute to the in vitro affinity of the androgen receptor DNA-binding domain. Surprisingly, however, they control the transcriptional activity of the androgen receptor in transient transfection experiments. In conclusion, we give evidence that the alternative DNA-dependent dimerization of the androgen receptor on direct repeats is a general mechanism for androgen specificity in which the second Zn finger and hinge region are involved. In addition, the sequences flanking an androgen-response element can control the activity of the androgen receptor.

Animals↗

PubMed2002

Characterization of the two coactivator-interacting surfaces of the androgen receptor and their relative role in transcriptional control.

The androgen receptor interacts with the p160 coactivators via two surfaces, one in the ligand binding domain and one in the amino-terminal domain. The ligand binding domain interacts with the nuclear receptor signature motifs, whereas the amino-terminal domain has a high affinity for a specific glutamine-rich region in the p160s. We here describe the implication of two conserved motifs in the latter interaction. The amino-terminal domain of the androgen receptor is a very strong activation domain constituent of Tau5, which is mainly active in the absence of the ligand binding domain, and Tau1, which is only active in the presence of the ligand binding domain. Both domains are, however, implicated in the recruitment of the p160s. Mutation analysis of the p160s has shown that the relative contribution of the two recruitment mechanisms via the signature motifs or via the glutamine-rich region depend on the nature of the enhancers tested. We propose, therefore, that the androgen receptor-coactivator complex has several alternative conformations, depending partially on the context of the enhancer.

Amino Acid Motifs↗

PubMed2002

Both retinoids and androgens are required to maintain or promote functional differentiation in reaggregation cultures of human prostate epithelial cells.

BACKGROUND: Primary cultures and subcultures of prostate epithelial cells (PEC) proliferate markedly, but rapidly loose secretory differentiated function and androgen responsiveness. Here, we investigated whether differentiation could be restored or preserved by using three-dimensional reaggregation cultures treated with retinoids and/or androgens. METHODS: PEC were cultured as monolayers or as reaggregation cultures on a rotatory shaker. Reaggregation cultures were also developed from freshly isolated cells. Morphology was evaluated microscopically. Expression of cytokeratins (CKbasal for basal cells and CK18 for luminal cells), E-cadherin, alpha- and beta-catenin, androgen receptor (AR), and prostate specific antigen (PSA) was evaluated by immunohistochemistry and/or Western blotting. Differentiated function was further evaluated by measurements of PSA in the medium and by reverse transcriptase-polymerase chain reactions for AR, PSA, prostate specific membrane antigen, beta-microseminoprotein, and zinc-alpha 2-glycoprotein. Proliferation was evaluated by immunohistochemical staining for Ki-67. RESULTS: Monolayer cultures of PEC expressed CKbasal as well as CK18, a combination compatible with an intermediary amplifying population of epithelial cells. No expression of PSA could be detected, and all attempts to re-induce differentiation of PEC in classic two-dimensional culture systems failed. In reaggregation cultures of subcultured PEC, retinoids proved essential to maintain a compact three-dimensional structure. This effect was accompanied by increased levels of E-cadherin and of the catenins and by a shift in the cytokeratin expression pattern toward that typical for secretory differentiated cells (CK18 only). Even in the presence of androgens, however, PSA remained undetectable. Similar effects of retinoids were observed in reaggregation cultures of freshly prepared PEC, and in the latter cultures, the combination of androgens and retinoids maintained a low level of PSA secretion for at least 40 days. CONCLUSIONS: A combination of retinoids and androgens is able to preserve, for a prolonged period of time, some degree of secretory differentiation in freshly isolated PEC maintained in reaggregation culture. The same combination is unable to restore secretory differentiation in subcultured PEC.

Androgens↗

PubMed2002

Functional interplay between two response elements with distinct binding characteristics dictates androgen specificity of the mouse sex-limited protein enhancer.

Many of the aspects involved in steroid-specific transcriptional regulation are still unsolved to date. We describe here the detailed characterization of the mouse sex-limited protein enhancer as a paradigm for androgen-specific control of gene expression. By deletion analysis, we delineate the minimal enhancer region displaying androgen sensitivity and specificity. We also show that each of the three hormone response elements (HRE), which constitute this minimal enhancer region, is essential but not sufficient for its functionality. When investigated as isolated elements, HRE1 is inactive and HRE3 is a potent androgen response element as well as GRE. Only the non-canonical HRE2 (5-TGGTCAgccAGTTCT-3') is capable of conferring an androgen-specific transcriptional response to a heterologous promoter. This finding is correlated with the fact that HRE2 is recognized in binding assays in vitro by the DNA-binding domain (DBD) of the androgen but not the glucocorticoid receptor, while HRE3 is recognized by both DBDs. Differential binding of the androgen receptor to HRE2 in the context of the enhancer was analyzed in more detail in footprinting assays in vitro. In transient transfection experiments using chimeric receptors, the inability of the glucocorticoid receptor to transactivate via the slp-ARU as well as the isolated slp-HRE2 was rescued by the replacement of its DNA-binding domain with that of the androgen receptor. Our data suggest that the functional interplay between the weak, but highly androgen-specific HRE2 and the adjacent strong, but non-selective HRE3 is the major determinant in the generation of androgen specificity of transcriptional response via the sex-limited protein enhancer.

Androgens↗

PubMed2002

Comparative analysis of the influence of the high-mobility group box 1 protein on DNA binding and transcriptional activation by the androgen, glucocorticoid, progesterone and mineralocorticoid receptors.

We performed a comparative analysis of the effect of high-mobility group box protein 1 (HMGB1) on DNA binding by the DNA-binding domains (DBDs) of the androgen, glucocorticoid, progesterone and mineralocorticoid receptors. The affinity of the DBDs of the different receptors for the tyrosine aminotransferase glucocorticoid response element, a classical high-affinity binding element, was augmented up to 7-fold by HMGB1. We found no major differences in the effects of HMGB1 on DNA binding between the different steroid hormone receptors. In transient transfection assays, however, HMGB1 significantly enhances the activity of the glucocorticoid and progesterone receptors but not the androgen or mineralocorticoid receptor. We also investigated the effect of HMGB1 on the binding of the androgen receptor DBD to a subclass of directly repeated response elements that is recognized exclusively by the androgen receptor and not by the glucocorticoid, progesterone or mineralocorticoid receptor. Surprisingly, a deletion of 26 amino acid residues from the C-terminal extension of the androgen receptor DBD does not influence DNA binding but destroys its sensitivity to HMGB1. Deletion of the corresponding fragment in the DBDs of the glucocorticoid, progesterone and mineralocorticoid receptor destroyed their DNA binding. This 26-residue fragment is therefore essential for the influence of HMGB1 on DNA recognition by all steroid hormone receptors that were tested. However, it is dispensable for DNA binding by the androgen receptor.

Animals↗