Multimeric complexes of the PML-retinoic acid receptor alpha fusion protein in acute promyelocytic leukemia cells and interference with retinoid and peroxisome-proliferator signaling pathways.

The t(15;17) chromosomal translocation, specific for acute promyelocytic leukemia (APL), fuses the PML gene to the retinoic acid receptor alpha (RAR alpha) gene, resulting in expression of a PML-RAR alpha hybrid protein. In this report, we analyzed the nature of PML-RAR alpha-containing complexes in nuclear protein extracts of t(15;17)-positive cells. We show that endogenous PML-RAR alpha can bind to DNA as a homodimer, in contrast to RAR alpha that requires the retinoid X receptor (RXR) dimerization partner. In addition, these cells contain oligomeric complexes of PML-RAR alpha and endogenous RXR. Treatment with retinoic acid results in a decrease of PML-RAR alpha protein levels and, as a consequence, of DNA binding by the different complexes. Using responsive elements from various hormone signaling pathways, we show that PML-RAR alpha homodimers have altered DNA-binding characteristics when compared to RAR alpha-RXR alpha heterodimers. In transfected Drosophila SL-3 cells that are devoid of endogenous retinoid receptors PML-RAR alpha inhibits transactivation by RAR alpha-RXR alpha heterodimers in a dominant fashion. In addition, we show that both normal retinoid receptors and the PML-RAR alpha hybrid bind and activate the peroxisome proliferator-activated receptor responsive element from the Acyl-CoA oxidase gene, indicating that retinoids and peroxisome proliferator receptors may share common target genes. These properties of PML-RAR alpha may contribute to the transformed phenotype of APL cells.     

Receptor interacting protein 140 as a thyroid hormone-dependent, negative co-regulator for the induction of cellular retinoic acid binding protein I gene

Over-expression of receptor interacting protein 140 (RIP140) suppressed thyroid hormone (T3) induction of cellular retinoic acid binding I protein (CRABPI) gene in P19 embryonal carcinoma cells. CRABPI induction by T3 is mediated by a direct-repeat four-element bound by T3 receptor (T3R) and retinoid receptor X (RXR). Three receptor-interacting domains (RIDs) in RIP140 mediate its interaction with T3R: one constitutive RID within the amino terminus, and two T3-dependent RIDs in the central portion and the carboxyl terminus. In co-immunoprecipitation and chromatin immunoprecipitation assays, RIP140 formed complexes with T3R/RXR in solution and on the endogenous target, the CRABPI promoter. T3 treatment resulted in elevated histone acetylation of the endogenous CRABPI gene promoter, but simultaneous expression of RIP140 resulted in significantly reduced histone acetylation of this promoter, primarily through the recruitment of HDAC4. This study presents the first evidence that over-expressed RIP140 acts as a T3-dependent negative co-regulator for T3 induction of the endogenous CRABPI gene in P19 cells.     

Retinoid X receptor-COUP-TF interactions modulate retinoic acid signaling.

We have recently described the properties of direct repeats (DRs) of the half-site AGGTCA as hormone response elements (HREs). According to our results, spacing the half sites by 3, 4, or 5 nucleotides determines specificity of response for vitamin D3, thyroid hormone, and retinoic acid receptors, respectively. This so-called 3-4-5 rule led to the prediction that remaining spacing options of 0, 1, and 2 might serve as targets for other nuclear receptors. A concurrent prediction is that receptors recognizing common sites might display more complex or combinatorial interactions. In exploring these predictions, we discovered that both the retinoid X receptor (RXR) and COUP-TF bind preferentially to a DR-1 motif. In vivo, RXR and COUP-TF display antagonistic action such that RXR-mediated activation is fully repressed by COUP-TF. In vitro studies reveal that COUP-TF and RXR form heterodimers on DR-1. Thus, these results support a general proposal in which the half-site spacing preferences may be used as a means to decipher potentially complex and interactive regulatory circuits.     

Sequences required for the transition from monomeric to homodimeric forms of thyroid hormone receptor alpha and v-erbA

Thyroid hormone receptor alpha (TRalpha) and the oncoprotein v-erbA (a mutated form of TRalpha incapable of binding T3) bind as heterodimers with retinoid X receptor (RXR) to DNA sequences with different orientations of AGGTCA half sites. v-erbA can also form homodimers, whereas, TRalpha1 homodimerizes poorly. Therefore, in order to obtain a better understanding for the distinct homodimerization properties between TRalpha1 and v-erbA, we created chimeras between these two receptors and tested their abilities to homodimerize on direct and everted repeats (DRs, ERs). We found that the enhanced homodimerization properties of v-erbA compared to TRalpha1 map to isoleucine at position 339 in conjunction with serine at position 351 and alanine at position 358. Our data indicate that the methyl group in isoleucine at position 339 plays an important role in v-erbA homodimerization, particularly on ER 6. Functional studies with I339V+S351P+A358T, a v-erbA mutant unable to homodimerize but still able to heterodimerize with RXR on ERs and DRs, indicate that v-erbA-RXR heterodimers mediate the dominant negative activity of v-erbA on DRs. However, the repressor activity of this mutant is weaker than that of the wild type v-erbA on ERs, suggesting that v-erbA homodimers rather than v-erbA-RXR heterodimers mediate the potent dominant negative activity of v-erbA on ERs.     

Myeloid differentiation mediated through retinoic acid receptor/retinoic X receptor (RXR) not RXR/RXR pathway

Retinoids, such as all-trans-retinoic acid and 9-cis-retinoic acid, are naturally occurring ligands of the nuclear retinoic acid receptors (RARs). In concert with binding of ligand, these receptors from heterodimers with the retinoic X receptor (RXR) and transactivate RAR/RXR-responsive genes. Retinoids can differentiate leukemic cell lines in vitro and induce clinically complete remissions in patients with acute promyelocytic leukemia. Synthetic ligands to the RAR and RXR receptors have been developed that selectively bind and activate RAR/RXR (TTAB) and RXR/RXR dimers (SR11217). We investigated the affect of these ligands, either alone or in combination, on in vitro growth and differentiation of cells from the HL-60, KG-1, THP-1, and WEHI-3 myeloid cell lines as well as on clonal growth of fresh myeloid leukemic blasts from patients. Clonal inhibition of proliferation of these cells was studied in soft agar cultures. Cells were plated in the presence of either one or a combination of retinoids at concentrations of 10(-5) to 10(-10) mol/L. TTAB inhibited 50% clonal growth at an effective dose (ED50) that was about 1,000-fold lower than the concentration of SR11217 required to achieve an ED50 for the same leukemic cells. Combination of both ligands at a variety of concentrations showed no synergistic effects. Superoxide production (nitroblue tetrazolium reduction) and CD11b expression as parameters of differentiation of HL-60 cells were also examined. Results paralleled those of clonal growth, with SR11217 being markedly less potent than TTAB. These results show that the ligand selective for RXR-homodimers has little effect on either inducing differentiation or inhibiting clonal growth of leukemic cells. The differentiating and antiproliferative effects of retinoids are mainly induced through RAR/RXR heterodimers, and development of therapeutic analogs should focus on this category of retinoids.     

Activation of liver X receptors and retinoid X receptors induces growth arrest and apoptosis in insulin-secreting cells

Liver X receptors (LXRs) form functional heterodimers with the retinoid X receptors (RXRs) and regulate cholesterol, lipid, and glucose metabolism. We demonstrated previously that activation of LXR modulates insulin secretion in MIN6 cells and pancreatic islets. In this study we investigated the effects of the LXR agonist T0901317 and the RXR agonist 9-cis-retinoic acid (9cRA) on cell proliferation and apoptosis in MIN6 cells. Whereas T0901317 showed no effect on proliferation of MIN6 cells, combination of T0901317 with 9cRA inhibited cell proliferation. Flow cytometry analysis of cell cycle demonstrated that activation of LXR/RXR prevented MIN6 cells from G1 to G2 phase progression. Combination of T0901317 and 9cRA increased apoptosis rate and caspase-3/7 activity in MIN6 cells. Moreover, T0901317 or its combination with 9cRA significantly increased the cell susceptibility to free fatty acid- and cytokine-induced apoptosis. Treatment of MIN6 cells with LXR and RXR agonists produced a strong increase in expression of mothers against decapentaplegic homolog 3, a protein known to inhibit cell cycle G1/S phase progression and induce apoptosis. In isolated rat islets, the effect of palmitic acid on caspase-3/7 activity was increased with T0901317 alone and even more with the combination of T0901317 and 9cRA. Thus, activation of LXR/RXR signaling inhibits cell proliferation and induces apoptosis in pancreatic beta-cells.     

Regulation of retinoid and thyroid hormone action through homodimeric and heterodimeric receptors

Biologic responses to retinoids and thyroid hormones are mediated by their intracellular receptor proteins. Many exciting advances have been made recently in understanding the molecular mechanism by which these receptor proteins operate. In contrast to the steroid hormone receptors that function predominantly as homodimers, thyroid hormone receptors (TRs)and retinoic acid receptors (RARs) require interaction with the retinoid X receptors (RXRs) for efficient DNA binding and transactivation. In addition, RXRs, in the presence of their specific ligands such as 9-cis RA, can form homodimers that recognize a subset of retinoic acid responsive elements (RAREs). The retinoid responses mediated by RXR homodimers and RAR-RXR heterodimers can be restricted by the COUP-TF orphan receptors that bind strongly to certain RAREs as homodimers. Thus, a complex network of receptor interaction has been unraveled that promises a better understanding of thyroid and retinoid hormone regulation of fundamental biologic processes and diseases.     

Retinoid-dependent pathways suppress myocardial cell hypertrophy.

Utilizing an in vitro model system of cardiac muscle cell hypertrophy, we have identified a retinoic acid (RA)-mediated pathway that suppresses the acquisition of specific features of the hypertrophic phenotype after exposure to the alpha-adrenergic receptor agonist phenylephrine. RA at physiological concentrations suppresses the increase in cell size and induction of a genetic marker for hypertrophy, the atrial natriuretic factor (ANF) gene. RA also suppresses endothelin 1 pathways for cardiac muscle cell hypertrophy, but it does not affect the increase in cell size and ANF expression induced by serum stimulation. A trans-activation analysis using a transient transfection assay reveals that neonatal rat ventricular myocardial cells express functional RA receptors of both the retinoic acid receptor and retinoid X receptor (RAR and RXR) subtypes. Using synthetic agonists of RA, which selectively bind to RXR or RAR, our data indicate that RAR/RXR heterodimers mediate suppression of alpha-adrenergic receptor-dependent hypertrophy. These results suggest the possibility that a pathway for suppression of hypertrophy may exist in vivo, which may have potential therapeutic value.     

Thyroid hormone-dependent gene expression in differentiated embryonic stem cells and embryonal carcinoma cells: identification of novel thyroid hormone target genes by deoxyribonucleic acid microarray analysis

T3 is required for normal early development, but relatively few T3-responsive target genes have been identified. In general, in vitro stem cell differentiation techniques stimulate a wide range of developmental programs, including thyroid hormone receptor (TR) pathways. We developed several in vitro stem cell models to more specifically identify TR-mediated gene expression in early development. We found that embryonic carcinoma (EC) cells have reduced T3 nuclear binding capacity and only modestly express the known T3 target genes, neurogranin (RC3) and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), in response to T3. Full T3 induction in transient transfection of EC cells was restored with cotransfection of a TR expression vector. We, therefore, performed gene expression profiles in wild-type embryonic stem (ES) cells compared with expression in cells with deficient (EC) or mutant TR (TRalpha P398H mutant ES cells), to identify T3 target genes. T3 stimulation of wild-type ES cells altered mRNA expression of 610 known genes (26% of those studied), although only approximately 60 genes (1%) met criteria for direct T3 stimulation based on the magnitude of induction and requirement for the presence of TR. We selected five candidate T3 target genes, neurexophilin 2, spermatid perinuclear RNA-binding protein (SPNR), kallikrein-binding protein (KBP), prostate-specific membrane antigen (PSMA), and synaptotagmin II, for more detailed study. T3 responsiveness of these genes was evaluated in both in vitro endogenous gene expression and in vivo mouse model systems. These genes identified in a novel stem cell system, including those induced and repressed in response to T3, may mediate thyroid hormone actions in early development.