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F Claessens

Publications and source records attributed to F Claessens.

At least 19 recordsLinked to original sources
PubMed2006

The androgen receptor DNA-binding domain determines androgen selectivity of transcriptional response.

The AR (androgen receptor) is a hormone-dependent transcription factor that translates circulating androgen hormone levels into a physiological cellular response by directly regulating the expression of its target genes. It is the key molecule in e.g. the development and maintenance of the male sexual characteristics, spermatocyte production and prostate gland development and growth. It is also a major factor in the onset and maintenance of prostate cancer and a first target for pharmaceutical action against the further proliferation of prostate cancer cells. The AR is a member of the steroid hormone receptors, a group of steroid-inducible transcription factors sharing an identical consensus DNA-binding motif. The problem of how specificity in gene activation is achieved among the different members of this nuclear receptor subfamily is still unclear. In this report, we describe our investigations on how the AR can specifically activate its target genes, while the other steroid hormone receptors do not, despite having the same consensus monomeric DNA-binding motif. In this respect, we describe how the AR interacts with a newly identified class of steroid-response elements to which only the AR and not, for example, the glucocorticoid receptor can bind.

Aging↗
PubMed2004

Differential effect of small ubiquitin-like modifier (SUMO)-ylation of the androgen receptor in the control of cooperativity on selective versus canonical response elements.

The androgen receptor (AR) can be small ubiquitin-like modifier (SUMO)-ylated in its amino-terminal domain at lysines 385 and 511. This SUMO-ylation is responsive to several agonists, but is not induced by the pure antagonist hydroxyflutamide. We show that the main site of interaction of Ubc9, the SUMO-1 conjugating enzyme, resides in transcription activation unit 5. Overexpression of SUMO-1 represses the AR-mediated transcription, and this effect is abolished after mutating both SUMO-1 acceptor sites. On the other hand, the mutation of lysine 385 clearly affects the cooperativity of the receptor on multiple hormone response elements. Lysine 511 is not implicated in this function. Surprisingly, these effects on cooperativity clearly depend on the nature of the response elements. When selective androgen response elements, which are organized as direct repeats of 5'-TGTTCT-3'-like sequences, were tested, the lysine 385 mutation did not increase the androgen response. Point mutations changing the direct-repeat elements into inverted-repeat elements restored the effects of the lysine 385 mutation on cooperativity. In conclusion, SUMO-ylation of the AR might have a differential function in the control of cooperativity, depending on the conformation of the AR dimer bound to DNA.

Amino Acid Motifs↗
PubMed2004

Mechanisms of androgen receptor signalling via steroid receptor coactivator-1 in prostate.

The androgen receptor (AR) is a member of the nuclear receptor superfamily. These ligand-activated transcription factors usually contain two activation functions, a ligand-independent activation function 1(AF1) in the divergent N-terminal domain and a ligand-dependent AF2 in the more conserved C-terminal ligand-binding domain. To promote transcription from target promoters, DNA-bound nuclear receptors recruit coactivator proteins that promote transcription by modifying histones within nucleosomes, resulting in altered topology of chromatin to allow access of the basal transcriptional machinery, or stabilising the pre-initiation complex. It is well known that most coactivators interact with AF2 of many nuclear receptors via conserved, helical LxxLL motifs (where L is leucine and x is any amino acid). The AF2 of the AR is very weak, but we were able to demonstrate that its intrinsic ligand-dependent activity is potentiated by steroid receptor coactivator-1 (SRC1) and that this region interacts with coactivators via LxxLL motifs. However, a mutant SRC1 coactivator with no functional LxxLL motifs was still able to potentiate AR activity. We found that SRC1 can also be recruited to (and increase activity of) AF1 of the AR via a conserved, glutamine-rich region. Point mutations within this region abolish SRC1 interaction with AF1 and also abolish or severely impair its ability to potentiate AR activity on all promoters tested. Thus the AR interacts with SRC1 via two different regions and the AF1 interaction is functionally the more important, although the contribution of the two interactions varies in a promoter-dependent fashion. SRC1 then potentiates receptor activity via recruitment of CBP/p300, a histone acetyltranferase. This is important in the context of prostate cancer as SRC1 and other coactivators including CBP are coexpressed with AR in the luminal epithelial cells of the prostate, where over 90% of prostate tumours arise. There is a need for effective second-line prostate cancer therapy aimed at blocking the AR pathway when anti-androgen therapy has failed. Since there is growing evidence that nuclear receptor cofactors may be implicated in the progression of hormone-dependent tumours to hormone-independent states, novel targets could include the interaction of AR with coactivator proteins. We suggest that the N-terminal interaction would be a more specific and effective target in the case of prostate cancer than the LxxLL/AF2 interaction.

Acetyltransferases↗
PubMed2003

Anti-androgenic properties of Compound A, an analog of a non-steroidal plant compound.

We investigated the interactions between Compound A (CpdA), an analog of a hydroxyphenyl aziridine precursor found in an African shrub, and the androgen receptor (AR). CpdA represses androgen-induced activation of both specific and non-specific androgen DNA response elements. While a similar effect was obtained for the progesterone receptor (PR) via a non-specific hormone response element, CpdA had no effect on the actions of the glucocorticoid and mineralocorticoid receptors. CpdA represses the ligand-dependent interaction between the NH(2)- and COOH-terminal domains of the AR, similar to well-characterised anti-androgens. CpdA also interferes with the interaction of steroid receptor co-activator 1 (SRC1) with the activation domain AF2 but not with AF1. However, CpdA does not compete with androgen for binding to the AR. These results demonstrate that CpdA elicits anti-androgenic actions by a mechanism other than competitive binding for the AR.

Acetates↗
PubMed2003

The retinoblastoma protein-associated transcription repressor RBaK interacts with the androgen receptor and enhances its transcriptional activity.

In search of potential androgen receptor coregulators we performed a yeast two-hybrid screening using the androgen receptor ligand-binding domain as bait and a human prostate cDNA library as prey and found that the carboxy-terminal domain of retinoblastoma-associated Krüppel protein (RbaK), a member of the Krüppel zinc finger protein family, interacts in a ligand-dependent way with the ligand-binding domain of the androgen receptor. RBaK was recently identified as a transcriptional regulator that interacts with the retinoblastoma protein and thereby influences E2F regulated transcription. The interaction of RBaK with the androgen receptor was further documented using mammalian two-hybrid experiments, in vitro binding studies and coimmunoprecipitation. Finally, we demonstrated that both RBaK and the retinoblastoma protein coactivate androgen receptor-mediated transcription in cotransfection experiments. In conclusion, our data show that RBaK interacts with the androgen receptor and increases its transcriptional activity. Moreover, the double interaction of RBaK with the retinoblastoma protein and with the androgen receptor provides a novel link between the androgen receptor and the regulation of the cell cycle.

Animals↗
PubMed2001

Androgen-receptor-specific DNA binding to an element in the first exon of the human secretory component gene.

Androgens and glucocorticoids are steroid hormones, which exert their effects in vivo by binding and activating their cognate receptors. These intracellular receptors are transcription factors that can bind specific DNA sequences, called hormone response elements, located near the target genes. Although the androgen receptor (AR) and the glucocorticoid receptor (GR) bind the same consensus DNA sequence, androgen-specific responses can be achieved by non-conventional androgen response elements (AREs). Here we determine the specificity mechanism of such a selective element recently identified in the first exon of the human gene for secretory component (sc ARE). This sc ARE consists of two receptor-binding hexamers separated by three nucleotides. The DNA-binding domains of the AR and GR both bind the sc ARE, but, although the AR fragment dimerizes on the element, the GR fragment does not. Comparing the affinities of the DNA-binding domains for mutant forms of the sc ARE revealed that dimeric GR binding is actively excluded by the left hexamer and more precisely by the presence of a G residue at position -3, relative to the central spacer nucleotide. Inserting a G at this position changed a non-selective element into an androgen-selective one. We postulate that the AR recognizes the sc ARE as a direct repeat of two 5'-TGTTCT-3'-like core sequences instead of the classical inverted repeat. Direct repeat binding is not possible for the GR, thus explaining the selectivity of the sc ARE. This alternative dimerization by the AR on the sc ARE is also indicated by the DNA-binding characteristics of receptor fragments in which the dimerization interfaces were swapped. In addition, the flanking and spacer sequences seem to affect the functionality of the sc ARE.

Animals↗
PubMed2001

Tissue-specific androgen responses in primary cultures of lacrimal epithelial cells studied by adenoviral gene transfer.

The lacrimal gland secretes most of the water and many proteins present in tear fluid. The composition of the tear fluid is affected dramatically by androgens, an observation which has been linked to the fact that more than 90% of the patients with Sjögren syndrome are female. Although the presence of androgen receptors in the lacrimal gland has been established, the molecular biology of the protective effects of androgens remains largely unknown. Here, we report the use of primary cultures of the lacrimal gland which express endogenous proteins under androgen control, as a more homologous test system for tissue-specific transcription studies. Infection with recombinant adenoviral vectors was the most efficient method to introduce foreign gene constructs in these cultures. A thus introduced mouse mammary tumor virus promoter was inducible with androgens and this effect was independent of the sexual genotype of the infected cells. By use of two recombinant adenoviral vectors containing genomic fragments of the SC gene, which is androgen responsive in the lacrimal gland, we could demonstrate the functionality of the sc promoter as well as its androgen regulation in this culture system.

Adenoviridae↗
PubMed2000

Apparent coactivation due to interference of expression constructs with nuclear receptor expression.

Transient cotransfection in COS-7 cells, a standard approach to demonstrate coactivation, was used to study the coactivation properties of NuRIP183, a new nuclear receptor interacting protein of 183 kDa, isolated by a yeast two-hybrid screening. Transfection with a NuRIP183 expression construct strongly increased the ligand-dependent response of reporter constructs for several nuclear receptors when compared to transfection with the empty expression vector. A more detailed study, however, revealed major changes in the expression level of the nuclear receptors in cotransfection experiments, indicating that the observed changes in receptor activity were not due to coactivation but to differences in receptor concentration caused by interference from the cotransfected expression constructs with the expression of the receptor. Such interference, which is inversely related to the length of the insert, was observed, not only in COS-7 cells but also in CV-1 and MCF-7 cells, using different transfection techniques (FuGENE-6 and calcium phosphate) and different expression vectors (pSG5, pcDNA1.1 and pIRESneo). These data cast some doubt on coactivation of nuclear receptors based on similar cotransfection experiments without measurement of receptor concentration. Moreover, it is recommended to limit the amounts of (co)transfected expression plasmid and to avoid the use of empty expression plasmid as a control. Finally, one should be aware of similar misleading results in other experimental set-ups based on cotransfection.

Animals↗
PubMed2000

Differences in DNA binding characteristics of the androgen and glucocorticoid receptors can determine hormone-specific responses.

The basis for specificity of gene regulation by steroid hormone receptors remains an important problem in the study of steroid hormone action. One possible mechanism for steroid specificity is the difference in DNA binding characteristics of the receptors, although they share a high homology in their DNA-binding domains. Indeed, the androgen-specific expression of, for example, the probasin (PB) gene can be explained by the presence of an androgen response element (ARE) in its promoter (PB-ARE-2), specifically recognized by the androgen and not by the glucocorticoid receptor. Three residues in the DNA-binding domain of the AR were identified as main determinants for its high affinity for the PB-ARE-2. In addition, the direct repeat nature of this ARE seems to prohibit high affinity binding by the glucocorticoid receptor. This is confirmed by the fact that several imperfect direct repeats of the 5'-TGTTCT-3' core recognition sequence are recognized by the androgen receptor and not by the glucocorticoid receptor. Up to now, only differences between the androgen and glucocorticoid receptor in the transcription activation functions were invoked to explain the specificity of their genomic actions. In the present study, we describe the influence of the DNA-binding domain on the specificity of androgen action. The novelty of our working hypothesis resides in the demonstration of the capacity of the AR-DNA-binding domain to recognize elements with a direct repeat structure.

Amino Acid Sequence↗
PubMed2000

Change of specificity mutations in androgen-selective enhancers. Evidence for a role of differential DNA binding by the androgen receptor.

The androgen and glucocorticoid receptors recognize identical DNA motifs, leaving unanswered the question of how steroid specificity of transcriptional regulation is established in cells containing both receptors. Here, we provide evidence that subtle differences in low affinity DNA recognition might be a crucial element in the generation of steroid-specific responses. Here we identify simple hormone response elements in the mouse sex-limited protein enhancer and the human secretory component androgen response unit to be essential for the androgen specificity of both enhancers. We describe specific in vitro binding to these motifs by the DNA-binding domain of the androgen but not the glucocorticoid receptor. Both elements can be considered partial direct repeats of the 5'-TGTTCT-3' core binding motif. In addition, we show that specific point mutations in their left half-sites, essentially changing the nature of the repeats, strongly enhance the glucocorticoid sensitivity of the respective enhancers, whereas they have no effect on their androgen responsiveness. Accordingly, these mutations allow specific binding of the glucocorticoid receptor DNA-binding domain to both elements in vitro. With these experiments, we demonstrate that differential recognition by the androgen receptor of nonconventional steroid response elements is, at least in some cases, an important mechanism in androgen-specific transcriptional regulation.

Animals↗
PubMed1999

Differential DNA binding by the androgen and glucocorticoid receptors involves the second Zn-finger and a C-terminal extension of the DNA-binding domains.

The androgen and glucocorticoid hormones evoke specific in vivo responses by activating different sets of responsive genes. Although the consensus sequences of the glucocorticoid and androgen response elements are very similar, this in vivo specificity can in some cases be explained by differences in DNA recognition between both receptors. This has clearly been demonstrated for the androgen response element PB-ARE-2 described in the promoter of the rat probasin gene. Swapping of different fragments between the androgen- and glucocorticoid-receptor DNA-binding domains demonstrates that (i) the first Zn-finger module is not involved in this sequence selectivity and (ii) that residues in the second Zn-finger as well as a C-terminal extension of the DNA-binding domain from the androgen receptor are required. For specific and high-affinity binding to response elements, the DNA-binding domains of the androgen and glucocorticoid receptors need a different C-terminal extension. The glucocorticoid receptor requires 12 C-terminal amino acids for high affinity DNA binding, while the androgen receptor only involves four residues. However, for specific recognition of the PB-ARE-2, the androgen receptor also requires 12 C-terminal residues. Our data demonstrate that the mechanism by which the androgen receptor binds selectively to the PB-ARE-2 is different from that used by the glucocorticoid receptor to bind a consensus response element. We would like to suggest that the androgen receptor recognizes response elements as a direct repeat rather than the classical inverted repeat.

Amino Acid Sequence↗
PubMed1999

The first exon of the human sc gene contains an androgen responsive unit and an interferon regulatory factor element.

Secretory component (SC) plays a key role in the transport of IgA and IgM to the lumina of many glands. The gene is constitutively expressed, but can be modulated by hormonal and immunological stimuli. Recently, the promoter and the first exon of the human sc gene have been cloned. The first exon contains a putative androgen/glucocorticoid response element (ARE/GRE) and an Interferon Regulatory Factor Element (IRF-E). Here we show that the ARE/GRE can bind the DNA-binding domain (DBD) of both the androgen (AR) and glucocorticoid receptor (GR) with a preference for the AR-DBD. In transient transfection experiments, this element confers higher responsiveness to androgens than to glucocorticoids. The IRF-E can function as an IRF-2, but surprisingly not as an IRF-I responsive element. We postulate that these two regulatory elements play a key role in the complex regulation of the sc gene in vivo.

Androgens↗
PubMed1999

The AF1 and AF2 domains of the androgen receptor interact with distinct regions of SRC1.

The androgen receptor is unusual among nuclear receptors in that most, if not all, of its activity is mediated via the constitutive activation function in the N terminus. Here we demonstrate that p160 coactivators such as SRC1 (steroid receptor coactivator 1) interact directly with the N terminus in a ligand-independent manner via a conserved glutamine-rich region between residues 1053 and 1123. Although SRC1 is capable of interacting with the ligand-binding domain by means of LXXLL motifs, this interaction is not essential since an SRC1 mutant with no functional LXXLL motifs retains its ability to potentiate androgen receptor activity. In contrast, mutants lacking the glutamine-rich region are inactive, indicating that this region is both necessary and sufficient for recruitment of SRC1 to the androgen receptor. This recruitment is in direct contrast to the recruitment of SRC1 to the estrogen receptor, which requires interaction with the ligand-binding domain.

Adaptor Proteins, Signal Transducing↗
PubMed1999

The androgen receptor amino-terminal domain plays a key role in p160 coactivator-stimulated gene transcription.

Steroid receptors are conditional transcription factors that, upon binding to their response elements, regulate the expression of target genes via direct protein interactions with transcriptional coactivators. We have analyzed the functional interactions between the androgen receptor (AR) and 160-kDa nuclear receptor coactivators. Upon overexpression in mammalian cells, these coactivators enhance the transcriptional activity of both the amino-terminal domain (NTD) and the ligand-binding domain (LBD) of the AR. The coactivator activity for the LBD is strictly ligand-controlled and depends on the nature of the DNA-binding domain to which it is fused. We demonstrate that the NTD physically interacts with coactivators and with the LBD and that this interaction, like the functional interaction between the LBD and p160 coactivators, relies on the activation function 2 (AF2) core domain. The mutation of a highly conserved lysine residue in the predicted helix 3 of the LBD (K720A), however, blunts the functional interaction with coactivators but not with the NTD. Moreover, this mutation does not affect the transcriptional activity of the full-size AR. A mutation in the NTD of activation function AF1a (I182A/L183A), which dramatically impairs the activity of the AR, has no effect on the intrinsic transcriptional activity of the NTD but interferes with the cooperation between the NTD and the LBD. Finally, p160 proteins in which the three LXXLL motifs are mutated retain most of their coactivator activity for the full-size AR, although they are no longer functional for the isolated LBD. Together, these data suggest that in the native AR the efficient recruitment of coactivators requires a functional association of the NTD with the LBD and that the binding of coactivators occurs primarily through the NTD.

Amino Acid Sequence↗
PubMed1999

Interaction of the putative androgen receptor-specific coactivator ARA70/ELE1alpha with multiple steroid receptors and identification of an internally deleted ELE1beta isoform.

Steroid-regulated gene transcription requires the coordinate physical and functional interaction of hormone receptors, basal transcription factors, and transcriptional coactivators. In this context ARA70, previously called RFG and ELE1, has been described as a putative coactivator that specifically enhances the activity of the androgen receptor (AR) but not that of the glucocorticoid receptor (GR), the progesterone receptor, or the estrogen receptor (ER). Here we describe the cloning of the cDNA for ELE1/ARA70 by RT-PCR from RNA derived from different cell lines (HeLa, DU-145, and LNCaP). In accordance with the previously described sequence, we obtained a 1845-bp PCR product for the HeLa and the LNCaP RNA. Starting from T-47D RNA, however, an 860-bp PCR product was obtained. This shorter variant results from an internal 985-bp deletion and is called ELE1beta; accordingly, the longer isoform is referred to as ELE1alpha. The deduced amino acid sequence of ELE1alpha, but not that of ELE1beta, differs at specific positions from the one previously published by others, suggesting that these two proteins are encoded by different nonallelic genes. ELE1alpha is expressed in the three prostate-derived cell lines examined (PC-3, DU-145, and LNCaP), and this expression is not altered by androgen treatment. Of all rat tissues examined, ELE1alpha expression is highest in the testis. This is also the only tissue in which we could demonstrate ELE1beta expression. Both ELE1alpha and ELE1beta interact in vitro with the AR, but also with the GR and the ER, in a ligand-independent way. Overexpression of either ELE1 isoform in DU-145, HeLa, or COS cells had only minor effects on the transcriptional activity of the human AR. ELE1alpha has no intrinsic transcription activation domain or histone acetyltransferase activity, but it does interact with another histone acetyltransferase, p/CAF, and the basal transcription factor TFIIB. The interaction with the AR occurs through the ligand-binding domain and involves the region corresponding to the predicted helix 3. Mutation in this domain of leucine 712 to arginine greatly reduces the affinity of the AR for ELE1alpha but has only moderate effects on its transcriptional activity. Taken together, we have identified two isoforms of the putative coactivator ARA70/ELE1 that may act as a bridging factor between steroid receptors and components of the transcription initiation complex but which lack some fundamental properties of a classic nuclear receptor coactivator. Further experiments will be required to highlight the in vivo role of ELE1 in nuclear receptor functioning.

Acetyltransferases↗
PubMed1999

Androgen specificity of a response unit upstream of the human secretory component gene is mediated by differential receptor binding to an essential androgen response element.

The expression of secretory component (SC), the epithelial receptor for poly-immunoglobulins, is regulated in a highly tissue-specific manner. In several tissues, e.g. lacrimal gland and prostate, SC synthesis is enhanced by androgens at the transcriptional level. In this study, we describe the presence of an androgen response unit, located 3.3 kb upstream of the sc transcription initiation site and containing several 5'-TGTTCT-3'-like motifs. Although each of these elements is implicated in the enhancer function, one element, the ARE1.2 motif, is found to be the main interaction site for the androgen receptor as demonstrated in in vitro binding assays as well as in transient transfection assays. A high-affinity binding site for nuclear factor I, adjacent to this ARE, is also involved in the correct functioning of the sc upstream enhancer. The ARE1.2 motif consists of an imperfect direct repeat of two core binding elements with a three-nucleotide spacer and therefore constitutes a nonconventional ARE. We demonstrate that this element displays selectivity for the androgen receptor as opposed to glucocorticoid receptor both in in vitro binding assays and in transfection experiments. Mutational analysis suggests that the direct nature of the half-site repeat is responsible for this selectivity. We have thus determined a complex and androgen-specific response unit in the far upstream region of the human SC gene, which we believe to be involved in its androgen responsiveness in epithelial cells of different organs such as prostate and lacrimal gland. We were also able to demonstrate that the primary sequence of a single nonconventional ARE motif within the enhancer is responsible for its androgen specificity.

Androgens↗
PubMed1998

Primary rat lacrimal cells undergo acinar-like morphogenesis on reconstituted basement membrane and express secretory component under androgen stimulation.

Single cells or small cell clusters, isolated from the rat lacrimal gland, were incubated on reconstituted basement membrane (matrigel) in a well-defined serum-free medium. During the first days of culture, cells reassociated and reorganized in structures resembling acini. These multicellular structures, maintained in culture for 2 weeks, consisted of well-polarized cuboidal cells surrounding a central lumen and exhibiting apically located microvilli. Myoepithelial cells were observed at the periphery of the acinar structures. Both in the native lacrimal and in the cultured aggregates, epithelial cells displayed strong immunoreactivity for cytokeratin 8, while myoepithelial cells were immunoreactive for vimentin and alpha-smooth muscle isoactin. These data indicate that the cultured aggregates closely mimic the in vivo architecture of lacrimal glands both by morphology and immunohistochemistry. We further demonstrated the presence of an intact androgen receptor and the ability of the cultured aggregates to respond to androgens with increased secretion of the secretory component. Comparable androgen responses were observed in lacrimal gland cultures of 5-week-old male and female rats. In conclusion, we report a morphologically and functionally differentiated culture system of primary rat lacrimal cells, in which androgen-regulated gene expression was observed. This culture model provides a unique experimental paradigm for studying the effects of hormones, cytokines, and growth factors on the morphogenesis, growth, and functional differentiation of lacrimal glands.

Actins↗