Biomedical subjects

Annemie Haelens

Publications and source records attributed to Annemie Haelens.

8 recordsLinked to original sources

PubMed2005

Comparison of different androgen bioassays in the screening for environmental (anti)androgenic activity.

Endocrine disruptors pose a growing threat to human and wildlife health. Validated test systems are required to study the mechanisms by which chemicals may interfere with the endocrine system. In order to identify compounds with (anti)androgenic activity, we used several in vitro bioassays, based on different androgen receptor (AR) functions, including AR transactivation and interaction between the amino-terminal domain and the ligand-binding domain. The AR activity assay, based on activation of the transcription of an androgen-responsive reporter gene in the presence of androgen, proved to excel in terms of high fold induction range and low minimal detection limit. The EC50 value, defined as the concentration that leads to half the maximal response and reflecting the potency of the synthetic androgen R1881 (methyltrienolone), was found to be 250 pM, consistent with the high affinity of this ligand to the human AR. A number of environmental samples were tested in the bioassay. The bioassay also could be used to detect antiandrogenic activity, because known AR-antagonists were able to inhibit R1881-mediated transactivation.

Androgen Antagonists↗

PubMed2004

The hinge region of the androgen receptor plays a role in proteasome-mediated transcriptional activation.

To investigate the function of the hinge region in transcriptional activation by the androgen receptor, we compared the actions of the wild-type receptor with a mutant receptor, deleted of amino acids 628-646 of the hinge. The role of the proteasome on the expression and activity of these two proteins was investigated. The deletion mutant demonstrated a threefold increase in transcriptional activity when compared to the wild-type receptor protein. Furthermore, we found that hormone-dependent stabilization of the receptor protein was more enhanced for the deletion mutant. In addition, experiments using the proteasome inhibitor, MG132, demonstrated that the deletion mutant is more sensitive to proteasome-mediated degradation than the wild-type receptor. However, inhibition of the proteasome had a negative effect on the transcriptional activity of the deletion mutant. Taken together, our results suggest that the hinge region not only plays an important role in controlling the transactivation potential of the androgen receptor but also in determining the influence of the proteasome on androgen receptor-mediated transcriptional activation.

Animals↗

PubMed2003

Implications of a polyglutamine tract in the function of the human androgen receptor.

The androgen receptor (AR) is a ligand-dependent transcription factor and belongs to the nuclear receptor family. The AR gene contains a long polymorphic CAG repeat, coding for a polyglutamine tract. In the full size AR, the deletion of the polyglutamine tract results in an increase in the transactivation through canonical AREs. However, this effect is clearly dependent on the response elements, since it is not observed on selective elements. In our assays, a deletion of the repeat positively affected the interactions of the ligand-binding domain with the amino-terminal domain as well as the recruitment of the p160 coactivator SRC-1e to the amino-terminal domain of the AR. This is reflected by an enhanced coactivation of the AR by SRC-1e.

Animals↗

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↗

PubMed2003

Selective DNA recognition by the androgen receptor as a mechanism for hormone-specific regulation of gene expression.

The androgen receptor (AR) is a member of the highly conserved group of the class I steroid hormone receptors, a subgroup of the nuclear receptor superfamily of ligand-induced transcription factors. All class I receptors influence the expression of their target genes by binding three-nucleotide spaced partial palindromic repeats of the core 5'-TGTTCT-3' motif. The implication that all class I receptors activate transcription by binding similar DNA motifs, poses the problem of how the in vivo steroid-specificity of transcriptional control is achieved. The AR, however, is able to interact with DNA motifs that are divergent from the classical hormone response elements. We will describe this AR-specific DNA interaction in the context of the general mechanisms that dictate the sequence-specificity of DNA-binding and dimerization of the nuclear receptors. The androgen receptor is the only steroid hormone receptor that is able to interact with response elements that are essentially arranged as a direct repeat of the 5'-TGTTCT-3' monomer binding element. We propose that the DNA-binding domain of the AR can interact with these androgen-specific response elements in a head-to-tail conformation, similar to many other nuclear hormone receptors. The fact that subtle differences in the sequence of response elements can dictate androgen-specific responses is a new and intriguing finding. It creates new possibilities in the research on hormone-selective action and provides a new angle in the search for selective ligands or co-factors that might influence androgen receptor action via either type of DNA motif.

Amino Acid Sequence↗

PubMed2002

Dual function of an amino-terminal amphipatic helix in androgen receptor-mediated transactivation through specific and nonspecific response elements.

Steroid receptors are transcription factors that, upon binding to their response elements, regulate the expression of several target genes via direct protein interactions with transcriptional coactivators. For the androgen receptor, additional interactions between the amino- and carboxyl-terminal regions have been reported. The first amino acids of the amino-terminal domain are necessary for this amino/carboxyl-terminal interaction. Deletion of a FQNLF core sequence in this region blunts the interaction, as does a G21E mutation. We investigated the effect of the aforementioned mutations in the context of the full size androgen receptor on a series of selective and nonselective androgen response elements. Strikingly, the FQNLF deletion strongly reduced the androgen receptor capacity to transactivate through nonselective motifs but did not affect its activity on selective elements. Although the G21E mutation strongly impairs the amino/carboxyl-terminal interaction, it does not significantly influence androgen receptor activity on either selective or nonselective elements. Surprisingly, this mutation leads to an increased binding of the amino-terminal domain to the glutamine-rich region of the steroid receptor coactivator-1 of the p160 family. Taken together, these data suggest that the amino-terminal amino acids of the androgen receptor play a key role in determining its transcriptional activity by modulating the interaction with the ligand-binding domain as well as interaction with p160 coactivators.

Amino Acid Motifs↗

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↗