Piia Aarnisalo

Ubc9 Interacts with the Androgen Receptor and Activates Receptor-dependent Transcription

Ubc9, a homologue of the class E2 ubiquitin-conjugating enzymes, has recently been shown to catalyze conjugation of a small ubiquitin-like molecule-1 (SUMO-1) to a variety of target proteins. SUMO-1 modifications have been implicated in the targeting of proteins to the nuclear envelope and certain intranuclear structures and in converting proteins resistant to ubiquitin-mediated degradation. In the present work, we find that Ubc9 interacts with the androgen receptor (AR), a member of the steroid receptor family of ligand-activated transcription factors. In transiently transfected COS-1 cells, AR-dependent but not basal transcription is enhanced by the coexpression of Ubc9. The N-terminal half of the AR hinge region containing the C-terminal part of the bipartite nuclear localization signal is essential for the interaction with Ubc9. Deletion of this part of the nuclear localization signal, which does not completely prevent the transfer of AR to the nucleus, abolishes the AR-Ubc9 interaction and attenuates the transcriptional response to cotransfected Ubc9. The C93S substitution of Ubc9, which prevents SUMO-1 conjugation by abrogating the formation of a thiolester bond between SUMO-1 and Ubc9, does not influence the capability of Ubc9 to stimulate AR-dependent transactivation, implying that Ubc9 is able to act as an AR coregulator in a fashion independent of its ability to catalyze SUMO-1 conjugation.

Coregulator Small Nuclear RING Finger Protein (SNURF) Enhances Sp1- and Steroid Receptor-mediated Transcription by Different Mechanisms

The small nuclear RING finger protein SNURF is not only a coactivator in steroid receptor-dependent transcription but also activates transcription from steroid-independent promoters. In this work, we show that SNURF, via the RING finger domain, enhances protein binding to Sp1 elements/GC boxes and interacts and cooperates with Sp1 in transcriptional activation. The activation of androgen receptor (AR) function requires regions other than the RING finger of SNURF, and SNURF does not influence binding of AR to cognate DNA elements. The zinc finger region (ZFR) together with the hinge region of AR are sufficient for contacting SNURF. The nuclear localization signal in the boundary between ZFR and the hinge region participates in the association of AR with SNURF, and a receptor mutant lacking the C-terminal part of the bipartite nuclear localization signal shows attenuated response to coexpressed SNURF. Some AR ZFR point mutations observed in patients with partial androgen insensitivity syndrome or male breast cancer impair the interaction of AR with SNURF and also render AR refractory to the transcription-activating effect of SNURF. Collectively, SNURF modulates the transcriptional activities of androgen receptor and Sp1 via different domains, and it may act as a functional link between steroid- and Sp1-regulated transcription.

Requirements for transcriptional regulation by the orphan nuclear receptor ERRgamma

Estrogen-related receptor gamma (ERRgamma) is an orphan nuclear receptor lacking identified natural ligands. We have addressed the requirements for ERRgamma-mediated gene regulation. ERRgamma transactivates constitutively reporter genes driven by ERR response elements (ERREs) or estrogen response elements (EREs). The activation depends on an intact DNA-binding domain (DBD) and activation function-2 (AF2). ERRgamma-mediated transactivation is further enhanced by peroxisome proliferator-activated receptor coactivator-1. Interestingly, ligand-binding domain (LBD) mutations predicted to either enlarge or diminish the putative ligand-binding pocket have no effect on the transcriptional activity implying that ERRgamma activity does not depend on any ligands. Antiestrogens 4OH-tamoxifen (4OHT) and 4-hydroxytoremifene (4OHtor) inhibit the ability of ERR to transactivate ERRE and ERE reporters. In contrast, ERRgamma activates transcription at AP-1 sites in the presence of 4OHT and 4OHtor. Thus, the transcriptional activity of ERRgamma seems not to require ligand binding but is modulated by binding of certain small synthetic ligands.

Transcription Activating and Repressing Functions of the Androgen Receptor Are Differentially Influenced by Mutations in the Deoxyribonucleic Acid-Binding Domain1

Despite the wide spectrum of androgen receptor (AR) mutants described in androgen insensitivity syndromes (AIS), their influence on transactivating and, in particular, transrepressing functions of AR are poorly defined. Rat AR mutants with substitutions in the DNA-binding domain, corresponding to several mutations in AIS patients, were examined for these activities. AR variants (G551V and C562G) with mutations in the first zinc finger (ZF) exhibited reduced DNA binding activity and attenuated transactivation. An R590Q substitution in the second ZF diminished transcriptional activity only from a promoter with a single androgen response element, whereas activation at multiple androgen response element sites was unaffected, despite the poor DNA-binding affinity of R590Q. Another substitution in the second ZF, A579T, yielded similar findings. In comparison to wild-type AR, G551V, and C562G variants had markedly reduced ability to repress an NF-kappaB/RelA-activated promoter but R590Q behaved like the native receptor. AP1 function was repressed not only by wild-type AR but also by the transactivating mutants A579T and R590Q as well as by the transcriptionally inactive mutants G551V and C562G. Furthermore, a Lys-to-Ala substitution in codon 563 of the first ZF switched AR into a ligand-dependent activator at AP1 sites but maintained the ability to repress NF-kappaB/RelA function. Taken together, DNA-binding domain mutations in AIS patients influence transcriptional activating and repressing functions of AR in a selective fashion, which probably contributes to the complexity in the presentation of the AIS phenotype.

Transcriptional activity of estrogen-related receptor γ (ERRγ) is stimulated by the phytoestrogen equol

Estrogen-related receptor γ (ERRγ) is an orphan nuclear receptor lacking identified natural ligands. The synthetic estrogen receptor ligands 4-hydroxytamoxifen and diethylstilbestrol have, however, been shown to bind to and abolish the constitutive transcriptional activity of ERRγ. Certain phytoestrogens were recently reported to act as agonists of the related ERRα. We investigated whether phytoestrogens also modulated the transcriptional activity of ERRγ. We analyzed a selection of phytoestrogens for their potential agonistic or antagonistic activity on ERRγ. In transiently transfected PC-3 and U2-OS cells equol stimulated the transcriptional activity of ERRγ and enhanced its interaction with the coactivator GRIP1. The agonistic effect of equol was abolished by 4-hydroxytamoxifen. Equol induced a conformational change in the ERRγ ligand-binding domain. Based on structural models of the ERRγ ligand-binding domain, we were able to introduce mutations that modulated the agonistic potential of equol. Finally, equol enhanced the growth inhibitory effect of ERRγ on the prostate cancer PC-3 cells. In conclusion, we have demonstrated that the phytoestrogen equol acts as an ERRγ agonist.

Cross-talk between NR4A orphan nuclear receptors and β-catenin signaling pathway in osteoblasts.

The canonical Wnt signaling pathway and its key mediator β-catenin are important regulators of osteoblast function. NR4A orphan nuclear receptors (Nurr1, NGFI-B, and Nor1) are expressed in osteoblasts and have been shown to regulate the expression of osteoblastic genes and osteoblastic differentiation. Recently, interplay between Nurr1 and the canonical Wnt signaling pathway was reported in 293F cells. We have studied the potential interplay between NR4A receptors and β-catenin in osteoblasts. NR4A receptors repressed β-catenin-mediated transactivation when cotransfected in U2-OS cells. In addition, Nurr1 inhibited β-catenin-mediated expression of Axin2 in MC3T3-E1 cells. The repression involved the DNA-binding domain of NR4A receptors. The repression of β-catenin did not result from reduced β-catenin expression or direct protein-protein interaction between β-catenin and NR4A receptors. β-Catenin was capable of inhibiting the transcriptional activity of NR4A receptors in U2-OS cells by a mechanism that involved the ligand-binding domain of NR4A receptors. As the canonical Wnt signaling pathway and β-catenin are crucial for the development and function of osteoblasts, the repressive effect of NR4A receptors on β-catenin is of potential biological and pathophysiological importance.

ERRα regulates osteoblastic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells

The orphan nuclear receptor estrogen-related receptor-alpha (ERRalpha) has been reported to have both a positive and a negative regulatory role in osteoblastic and adipocytic differentiation. We have studied the role of ERRalpha in osteoblastic and adipogenic differentiation of mesenchymal stem cells. Bone marrow mesenchymal stem cells were isolated from ERRalpha deficient mice and their differentiation capacities were compared to that of the wild-type cells. ERRalpha deficient cultures displayed reduced cellular proliferation, osteoblastic differentiation, and mineralization. In the complementary experiment, overexpression of ERRalpha in MC3T3-E1 cells increased the expression of osteoblastic markers and mineralization. Alterations in the expression of bone sialoprotein (BSP) may at least partially explain the effects on mineralization as BSP expression was reduced in ERRalpha deficient MSCs and enhanced upon ERRalpha overexpression in MC3T3-E1 cells. Furthermore, a luciferase reporter construct driven by the BSP promoter was efficiently transactivated by ERRalpha. Under adipogenic conditions, ERRalpha deficient cultures displayed reduced adipocytic differentiation. Our data thus propose a positive role for ERRalpha in osteoblastic and adipocytic differentiation. The variability in the results yielded in the different studies implies that ERRalpha may play different roles in bone under different physiological conditions.

FGF-8 stimulates the expression of NR4A orphan nuclear receptors in osteoblasts.

Nurr1, NGFI-B, and Nor1 form the NR4A subfamily of orphan nuclear receptors. The NR4A receptors are immediate early genes that can be rapidly induced in response to a variety of stimuli in many cell types, for example, in osteoblasts. Nurr1 regulates the differentiation of osteoblasts and the expression of several osteoblastic genes. Fibroblast growth factor 8b (FGF-8b) regulates osteoblastic differentiation. We show here that treatment of preosteoblastic MC3T3-E1 cells or mouse bone marrow mesenchymal cells with FGF-8b induces the expression of NR4A receptors rapidly and in a dose-dependent manner. This induction involves mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI-3K), and protein kinase C (PKC) pathways. FGF-8b stimulates the proliferation of MC3T3-E1 cells. This effect is enhanced by overexpression of Nurr1 and NGFI-B whereas it is abolished by a dominant negative Nurr1 variant. In conclusion, FGF-8b induces the expression of NR4A orphan nuclear receptors that are involved in mediating the growth promoting effect of FGF-8b in osteoblasts.

Corepressor interaction differentiates the permissive and non-permissive retinoid X receptor heterodimers

Nurr1 is an orphan nuclear receptor regulating transcription both as a monomer and as a heterodimer with retinoid X receptor (RXR). RXR-Nurr1 heterodimers are permissive RXR heterodimers as they activate transcription in response to RXR ligands. In contrast, heterodimers formed by RXR and retinoic acid receptor (RAR) are non-permissive as they activate transcription only upon RAR ligand binding. We studied the mechanism mediating permissiveness and non-permissiveness by creating receptor chimeras between Nurr1 and RAR. We show that the amino-terminal part of the Nurr1 ligand binding domain conveys permissiveness to RXR-Nurr1 heterodimers. This region is involved in interactions with the corepressors SMRT and NcoR. The corepressors were released from RXR-Nurr1 heterodimers by RXR ligand binding. In contrast, RXR ligand increased the interaction between RXR-RAR heterodimers and the corepressors. The corepressors were released only upon binding of RAR ligand. In conclusion, corepressor interaction differentiates the permissive RXR-Nurr1 heterodimers from the non-permissive RXR-RAR heterodimers.