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

Hofman, Kurt; Swinnen, Johannes V.; Claessens, Frank; Verhoeven, Guido; Heyns, Walter

doi:10.1016/s0303-7207(00)00311-7

Cited by 19 publications

(14 citation statements)

References 37 publications

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“…Ethanol (EtOH) was used as vehicle at a constant final concentration of 0.1%. Forty-eight hours (or the time indicated) after addition of the androgen, cells were collected in 500 l of passive lysis buffer (Promega) and assayed for luciferase activity as described by Hofman et al (12). Protein concentrations of the lysates were determined as follows: 40 l of the lysate was incubated on ice for 10 min with 0.5 ml 0.03% sodium deoxycholate.…”

Section: Methodsmentioning

confidence: 99%

E2F Activity Is Biphasically Regulated by Androgens in LNCaP Cells

Hofman

Swinnen

Verhoeven

et al. 2001

Biochemical and Biophysical Research Communications

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Section: Methodsmentioning

confidence: 99%

E2F Activity Is Biphasically Regulated by Androgens in LNCaP Cells

Hofman

Swinnen

Verhoeven

et al. 2001

Biochemical and Biophysical Research Communications

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“…Reporter activity was measured after treatment of cells with the prototypic MR and GR agonists, aldosterone and dexamethasone, respectively; both ligands were applied at a dose of 10 Ϫ6 M. Controls. As demonstrated recently, cotransfection of different expression vectors can influence the transactivation of the reporter gene through squelching phenomena and alteration of the concentrations of individual components; i.e., the resulting data may not necessarily reflect the influence of the factor being tested on receptor-mediated transcription (Hofman et al, 2000). Therefore, the expression levels of MR and GR were monitored by immunoblotting in the presence of transfected DAXX, FLASH, and FAF-1 cDNAs.…”

Section: Selection Of Potential Mr-interacting Partnersmentioning

confidence: 99%

DAXX, FLASH, and FAF-1 Modulate Mineralocorticoid and Glucocorticoid Receptor-Mediated Transcription in Hippocampal Cells—Toward a Basis for the Opposite Actions Elicited by Two Nuclear Receptors?

Obradovic¹,

Tirard²,

Némethy³

et al. 2004

Mol Pharmacol

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Mineralocorticoid (MR) and glucocorticoid (GR) receptors are two closely-related members of the steroid nuclear receptor family of transcription factors that bind common ligands in the brain (corticosterone and cortisol) and supposedly have identical hormone response elements. This raises the important question of how they can elicit differential biological actions in neurons in which they are often colocalized. One plausible explanation is that they differentially recruit proteins (coregulators or other receptor-interacting factors) through cell-specific interactions with regions that diverge between MR and GR to modulate target gene transcription in a receptor-specific manner. We therefore performed a yeast-two-hybrid screening of a human brain cDNA library with an AF1-containing region of the human MR as bait. This screening revealed several potential MR-interacting partners; among them were several clones bearing homology to DAXX, FLASH, and FAF-1, all previously implicated in apoptosis. Coexpression of candidate clones in a mouse hippocampal cell line confirmed these interactions in a mammalian neural cell environment as well. In transient transactivation assays, DAXX and FLASH influenced MR-and GRdriven transcription of the MMTV-Luc reporter similarly; in contrast, although FAF-1 did not transactivate GR, it did selectively stimulate MR-mediated transcription. Thus, the present findings, that 1) DAXX, FLASH, and FAF-1 modulate the transcriptional activities of MR and GR and that 2) FAF-1 selectively coactivates only MR, provide possible clues for how these closely related receptors might differentially influence neuronal function.

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“…Cotransfection experiments were performed using the Fugene-6 method (Roche) as previously described (Hofman et al 2000) with (per well) 100 ng reporter construct (C3 ARE-luciferase), 0·17 ng pSG5-AR and various amounts of pSG5 expression constructs as indicated in the figures. For the mammalian two-hybrid experiments, cells were transfected with (per well) 100 ng p(Gal4) 5 -TK-luciferase reporter construct, 0·67 ng pAB-Gal4 DBD-ARLBD construct and 0·67 ng pSNATCHII, pSNATCHIIRBaK(S) or pSNATCHII-RBaK(AS) construct respectively.…”

Section: Transfections Of Mammalian Cellsmentioning

confidence: 99%

“…In these experiments the effect of a sense RBaK expression construct (RBaK(S)) was compared with an antisense RBaK expression construct (RBaK(AS)) or to the pSG5 1 control. This construct contains an unrelated cDNA fragment (NuRIP-6) without coregulator properties but which has approximately the same size as the RBaK insert and therefore produces nearly the same suppression of the concentration of the AR (Hofman et al 2000). As shown in Fig.…”

Section: Influence Of Rbak On Ar-mediated Transcriptionmentioning

confidence: 99%

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

Hofman

Swinnen²,

Claessens³

et al. 2003

Journal of Molecular Endocrinology

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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.

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

Cited by 19 publications

References 37 publications

E2F Activity Is Biphasically Regulated by Androgens in LNCaP Cells

E2F Activity Is Biphasically Regulated by Androgens in LNCaP Cells

DAXX, FLASH, and FAF-1 Modulate Mineralocorticoid and Glucocorticoid Receptor-Mediated Transcription in Hippocampal Cells—Toward a Basis for the Opposite Actions Elicited by Two Nuclear Receptors?

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

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