Aberrant E2F activation by polyglutamine expansion of androgen receptor in SBMA neurotoxicity.

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disorder caused by a polyglutamine repeat (polyQ) expansion within the human androgen receptor (AR). Unlike other neurodegenerative diseases caused by abnormal polyQ expansion, the onset of SBMA depends on androgen binding to mutant human polyQ-AR proteins. This is also observed in Drosophila eyes ectopically expressing the polyQ-AR mutants. We have genetically screened mediators of androgen-induced neurodegeneration caused by polyQ-AR mutants in Drosophila eyes. We identified Rbf (Retinoblastoma-family protein), the Drosophila homologue of human Rb (Retinoblastoma protein), as a neuroprotective factor. Androgen-dependent association of Rbf or Rb with AR was remarkably potentiated by aberrant polyQ expansion. Such potentiated Rb association appeared to attenuate recruitment of histone deacetyltransferase 1 (HDAC1), a corepressor of E2F function. Either overexpression of Rbf or E2F deficiency in fly eyes reduced the neurotoxicity of the polyQ-AR mutants. Induction of E2F function by polyQ-AR-bound androgen was suppressed by Rb in human neuroblastoma cells. We conclude that abnormal expansion of polyQ may potentiate innate androgen-dependent association of AR with Rb. This appears to lead to androgen-dependent onset of SBMA through aberrant E2F transactivation caused by suppressed histone deacetylation.

Ligand-dependent interaction between estrogen receptor alpha and adenomatous polyposis coli.

Numerous independent clinical and experimental studies indicate that estrogens confer a protective effect against development of intestinal tumors, however the molecular mechanisms involved remain unclear. Physiological effects of estrogens are predominantly mediated by the action of nuclear estrogen receptors (ERs). A multifunctional protein adenomatous polyposis coli (APC) is a tumor suppressor and thought to act as a gatekeeper in colon tumorigenesis, as loss of function APC mutations trigger the development of colorectal cancer. Here we report that APC physically associates with ERa in the ligand-dependent manner. We have shown in the endogenous setting that the ligand-activated ERa recruits APC to the promoters in ER target genes and that increased levels of ER-dependent recruitment of APC enhances the ER transactivation through stimulation of histone acetylation. Found in majority of human colon tumors APC truncation mutants lost the ability to interact with ER. Thus, here we present the first evidence of a functional interaction between APC and ER that may be accounted for a tumor protective action of estrogens.

Activation of facultatively silenced Drosophila loci associates with increased acetylation of histone H2AvD.

H2A.Z is an evolutionarily highly conserved non-allelic variant of histone H2A. H2A.Z and its homologues have been shown to involve in both chromatin silencing and activation. Although much of our knowledge of H2A.Z biological activity has come from studies on its yeast homologue Htz1, H2A.Z appears to have more complex and diverse functions in higher eukaryotes. To investigate the involvement of H2AvD, a Drosophila homologue of mammalian H2A.Z, in mechanisms of conditional activation of facultatively silenced genes, we generated transgenic Drosophila lines expressing H2AvD fused at the C- or N-terminus with the green fluorescent protein (GFP). Using heat shock-induced gene activation and polytene chromosome puff formation as an in vivo model system, we analyzed effects of H2AvD termini modifications on transcription. We found that N-terminally fused GFP inhibited H2AvD acetylation and impaired heat shock-induced puff formation and hsp70 gene activation. Our data suggest that the N-terminal region of H2AvD plays a pivotal role in transcriptional activation and that induction of transiently silenced Drosophila loci associates with increased acetylation of H2AvD.

Vitamin D receptor B1 and exon 1d: functional and evolutionary analysis.

The vitamin D receptor (VDR) shares a conserved structural and functional organization with other nuclear receptor (NR) superfamily members. For many NRs, N-terminal variant isoforms that display distinct cell-, stage- and promoter-specific actions have been identified. The novel VDR isoform VDRB1, with a 50 amino acid N-terminal extension, is produced from low abundance transcripts that contain exon 1d of the human VDR locus. There is evidence for the conservation of this exon in other mammalian and avian species. The transactivation differences between VDRB1 and the original VDR, clarified here, provide insights into mechanisms that may contribute to functional differences and potentially distinct physiological roles for these two VDR isoforms.

PPAR-gamma Signaling Crosstalk in Mesenchymal Stem Cells.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear receptor (NR) superfamily of ligand-activated transcriptional factors. Among other functions, PPAR-gamma acts as a key regulator of the adipogenesis. Since several cytokines (IL-1, TNF-alpha, TGF-beta) had been known to inhibit adipocyte differentiation in mesenchymal stem cells (MSCs), we examined the effect of these cytokines on the transactivation function of PPAR-gamma. We found that the TNF-alpha/IL-1-activated TAK1/TAB1/NIK (NFkappaB-inducible kinase) signaling cascade inhibited both the adipogenesis and Tro-induced transactivation by PPAR-gamma by blocking the receptor binding to the cognate DNA response elements. Furthermore, it has been shown that the noncanonical Wnts are expressed in MSCs and that Wnt-5a was capable to inhibit transactivation by PPAR-gamma. Treatment with Wnt5a-activated NLK (nemo-like kinase) induced physical association of the endogenous NLK and H3K9 histone methyltransferase (SETDB1) protein complexes with PPAR-gamma. This resulted in histoneH3K9 tri-methylation at PPAR-gamma target gene promoters. Overall, our data show that cytokines and noncanonical Wnts play a crucial role in modulation of PPAR-gamma regulatory function in its target cells and tissues.

Molecular switching of osteoblastogenesis versus adipogenesis: implications for targeted therapies.

Osteoblasts and adipocytes differentiate from a common precursor, the pluripotent mesenchymal stem cell (MSC) found in bone marrow (BMSC) and adipose tissue (AD-MSC). Numerous transcription factors and multiple extracellular and intracellular signals regulating adipogenesis and osteoblastogenesis have been identified and analyzed. Significantly, inducers of differentiation towards one lineage may inhibit cell differentiation into an alternative lineage. For example, the canonical Wnt/beta-catenin pathway induces osteoblastogenesis and inhibits adipogenesis, whereas the peroxisome proliferator activated receptor-gamma (PPAR-gamma) is a prime inducer of adipogenesis and, as shown in recent studies, inhibits osteoblastogenesis. We have identified two signaling pathways that switch the cell fate decision from adipocytes to osteoblasts by suppressing the transactivation function of PPAR-gamma. In the first pathway, the TNF-alpha- or IL-1-induced TAK1/TAB1/NIK signaling cascade attenuates PPAR-gamma-mediated adipogenesis by inhibiting the binding of PPAR-gamma to the DNA response element. The second is the noncanonical Wnt pathway through the CaMKII-TAK1/TAB2-NLK (nemo-like kinase) signaling cascade. Specifically, Wnt-5a-induced phosphorylation of NLK triggers formation of a complex with the histone methyltransferase SETDB1 (SET domain, bifurcated 1) that represses PPAR-gamma transactivation through histone H3-K9 methylation at the target genes. Thus, two signaling cascades promote osteoblastic differentiation from MSC through two distinct modes of PPAR-gamma transrepression.

Wnt and PPARgamma signaling in osteoblastogenesis and adipogenesis.

Osteoblasts and adipocytes differentiate from a common pluripotent precursor, the mesenchymal stem cell (MSC). Studies have identified numerous transcription factors, and multiple extracellular and intracellular signaling pathways that regulate the closely linked processes of adipogenesis and osteoblastogenesis. Interestingly, inducers of differentiation along one lineage often inhibit differentiation along the other; for example, the transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) is a prime inducer of adipogenesis that inhibits osteoblastogenesis. The latest research has shown that inducers of osteoblastogenesis (such as bone morphogenetic protein 2 and Wnt ligands) use different mechanisms to suppress the transactivation function of PPARgamma during osteoblastogenesis from MSCs. Signaling via the canonical Wnt-beta-catenin pathway inhibits PPARgamma mRNA expression, whereas signaling via the noncanonical Wnt pathway results in activation of a histone methyltransferase SETDB1 that represses PPARgamma transactivation through histone H3K9 methylation of target genes. This article summarizes Wnt and PPARgamma signaling in MSCs and the crosstalk between these pathways, and speculates on future clinical application of this knowledge as the basis of novel approaches for regeneration therapy.

Corepressive action of CBP on androgen receptor transactivation in pericentric heterochromatin in a Drosophila experimental model system.

Ligand-bound nuclear receptors (NR) activate transcription of the target genes. This activation is coupled with histone modifications and chromatin remodeling through the function of various coregulators. However, the nature of the dependence of a NR coregulator action on the presence of the chromatin environment at the target genes is unclear. To address this issue, we have developed a modified position effect variegation experimental model system that includes an androgen-dependent reporter transgene inserted into either a pericentric heterochromatin region or a euchromatic region of Drosophila chromosome. Human androgen receptor (AR) and its constitutively active truncation mutant (AR AF-1) were transcriptionally functional in both chromosomal regions. Predictably, the level of AR-induced transactivation was lower in the pericentric heterochromatin. In genetic screening for AR AF-1 coregulators, Drosophila CREB binding protein (dCBP) was found to corepress AR transactivation at the pericentric region whereas it led to coactivation in the euchromatic area. Mutations of Sir2 acetylation sites or deletion of the CBP acetyltransferase domain abrogated dCBP corepressive action for AR at heterochromatic areas in vivo. Such a CBP corepressor function for AR was observed in the transcriptionally silent promoter of an AR target gene in cultured mammalian cells. Thus, our findings suggest that the action of NR coregulators may depend on the state of chromatin at the target loci.

Wnt/beta-catenin and estrogen signaling converge in vivo.

Wnt and estrogen signaling represent important regulatory pathways, each controlling a wide range of biological processes. While an increasing number of observations suggest potential convergence between these pathways, no direct evidence of their functional interaction has been reported. Using human colon and breast cancer cells, we found that estrogen receptor (ER) alpha- and beta-catenin precipitated within the same immunocomplexes, reciprocally enhanced the transactivation of cognate reporter genes, and were reciprocally recruited to cognate response elements in the promoters of endogenous target genes. Using transgenic Drosophila that ectopically expressed human ERalpha alone or together with metabolically stable beta-catenin/Armadillo mutants, we demonstrated genetic interaction between these signal transducers in vivo. Thus, we present here the first direct evidence of cross-talk between Wnt and estrogen signaling pathways via functional interaction between beta-catenin and ERalpha.