Expression of adipogenesis markers in a murine stromal cell line treated with 15-deoxy Delta(12,14)-prostaglandin J2, interleukin-11, 9-cis retinoic acid and vitamin K2.

Recent studies have demonstrated that bone marrow stromal cells can undergo adipogenesis or osteoblastogenesis in vivo, and in vitro, and that peroxisome proliferator-activated receptor gamma (PPAR gamma) plays a central role in the control of adipocyte differentiation. In the present study, we treated a murine stromal cell line (TMS-14) with a cocktail of dexamethasone, insulin and glucose (DIG cocktail), which caused the cells to convert to fat-laden cells with adipocyte-like morphology. We also exposed TMS-14 cells to DIG cocktail followed by 15-deoxy Delta(12,14)-prostaglandin J2 (15d-PGJ2), a ligand of PPAR gamma, interleukin- 11 (IL-11), 9-cis retinoic acid (9-cis RA) and vitamin K2. 15d-PGJ2 enhanced DIG cocktail-induced adipogenesis, whereas IL-11, 9-cis RA and vitamin K2 each inhibited adipogenesis induced by DIG cocktail. The gene expressions of four adipogenesis markers, PPAR gamma 2, adipocyte P2 (aP2), adipocyte determination and differentiation factor 1 (ADD1), and fatty acid synthase (FAS) were enhanced by DIG cocktail and these expressions were more enhanced by 15d-PGJ2, in contrast they were attenuated by 9-cis RA. IL-11 also attenuated the adipogenesis markers except ADD1. Western blotting showed that 15d-PGJ2 enhanced the levels of PPAR gamma, C/EBP alpha and RXR alpha proteins, while IL-11 and 9-cis RA decreased the level of PPAR gamma protein, but not C/EBP alpha protein and vitamin K2 decreased the level of C/EBP alpha protein. We also tested the effect of 15d-PGJ2 on osteoblastogenesis, using TMS-12 cells, another stromal cell clone from the same mouse, which differentiate into osteoblasts spontaneously. 15d-PGJ2 did not affect osteoblastogenesis, as detected by von Kossa staining and Cbfa-1 gene expression. These data indicate that 15d-PGJ2 enhances the expression of both PPAR gamma and C/EBP alpha and as a result it stimulates adipogenesis in murine bone marrow cells.     

Regulation of the human endogenous retroviral Syncytin-1 and cell-cell fusion by the nuclear hormone receptors PPARγ/RXRα in placentogenesis

Cytotrophoblast (CT) cell fusion into a syncytiotrophoblast is obligatory for placentation and mediated by the human endogenous retrovirus (HERV)-W envelope gene Syncytin-1. Abnormal placentation is associated with preeclampsia (PE), HELLP and intrauterine growth restriction (IUGR). In placentogenesis, the MAP-kinase p38α regulates PPARγ/RXRα signaling and target genes, like leptin, resistin, ABCG2, and hCG. The aim of this study was to analyze PPARγ/RXRα signaling and target gene regulation using primary CT cultures, the trophoblastic cell line BeWo and placental tissues from patients with normal and abnormal placentation. CT from four different human control placentae and BeWo cells demonstrated that Syncytin-1, other signaling members and CT cell fusions were regulated with PPARγ/RXRα activators troglitazone and 9-cis retinoic acid, via protein kinase A and p38α inhibition. Significant discordant regulations between CTs and BeWo were found. Two PPARγ/RXRα-response-elements from upstream regulatory elements and the 5'LTR of HERV-W were confirmed with DNA-protein binding assays using nuclear extracts and recombinant PPARγ/RXRα proteins. These promoter elements were validated with luciferase assays in the presence of PPARγ/RXRα modulators. Furthermore, troglitazone or 9-cis retinoic acid treatment of siRNA-PPARγ and siRNA-RXRα transfected BeWo cells proved the requirement of these proteins for Syncytin-1 regulation. Thirty primary abnormal placentae from PE, HELLP and IUGR patients compared to 10 controls showed significant deregulation of leptin RNA and protein, p38α, phospho-p38α, PPARγ, ABCG2, INSL4 and Syncytin-1. Our study characterized PPARγ/RXRα signaling in human CT and cell fusions identifying Syncytin-1 as a new target gene. Based on these results, a disturbed PPARγ/RXRα pathway could contribute to pathological human pregnancies.     

9-cis and all-trans retinoic acid induce a similar phenotype in human teratocarcinoma cells

Abstract. Prior work has shown that all-trans retinoic acid (t-RA) treatment of the human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1) induces a neuronal phenotype and other cell lineages. This study sought to explore the potential of 9-cis retinoic acid (9-cis RA) as a differentiation-inducing agent of this multipotent cell. Findings reported here show that 9-cis RA induced a phenotype similar to t-RA treatment of NT2/D1 cells. This similarity extended to their effects on the nuclear receptors retinoic acid receptor-β (RAR-β) and retinoid X receptor-α (RXR-α). Both retinoids prominently augmented RAR-β expression and transactivated a reporter plasmid containing putative RAR response elements (RAREs) with direct repeats separated by five nucleotides (DR5). Both retinoids had no appreciable effect on RXR-α expression and both minimally transactivated a reporter plasmid containing putative RXR response elements (RXREs) with direct repeats separated by one nucleotide (DR1). These studies suggest that 9-cis RA and t-RA activate common events during retinoid-mediated NT2/D1 differentiation. This hypothesis was supported by the finding that NT2/D1 cells rendered refractory to t-RA (NTZ/D1-R1) were also resistant to 9-cis RA. To discover alterations that could confer retinoid-refractoriness, retinoid receptor expression was examined in NT2/D1-R1 cells. In contrast to NT2/D1, the NT2/D1-R1 cell was found to have reduced RXR-α expression at the level of total cellular RNA. These studies establish the effectiveness of 9-cis RA as a differentiation agent of human TC cells and demonstrate that retinoids with different nuclear receptor affinities can induce similar phenotypes in NT2/D1 cells. In addition, the findings in the retinoid resistant NT2/Dl-R1 cell implicate a role for specific retinoid receptors in this human TC differentiation program.     

9-cis-retinoic acid decreases the level of its cognate receptor, retinoid X receptor, through acceleration of the turnover.

In this study, we investigated the ligand-mediated regulation of retinoid X receptor (RXR) in two human cell lines (HepG2 and JEG-3 cells), which have been reported to express RXRalpha predominantly. Western blot analysis revealed that a treatment with 1 microM 9-cis-retinoic acid (9-cis RA) for 24 h decreased RXRalpha protein level to 72 +/- 9 and 75 +/- 7% in HepG2 and JEG-3 cells, respectively, when the levels in the non-treated cells were expressed as 100%. The decrease was not due to the changes in steady-state level of RXRalpha mRNA or its stability as revealed by Northern blot analysis. However, the 9-cis RA treatment decreased the half-life of RXRalpha protein as determined by pulse-chase analysis. It was thus demonstrated that 9-cis RA downregulates RXRalpha by increasing the turnover of the protein in the two cell lines. The ligand-dependent downregulation of RXRalpha protein may be important for several hormonal signalings, in which the receptors heterodimerize with RXR.     

9-cis and all-trans retinoic acid induce a similar phenotype in human teratocarcinoma cells

Abstract. Prior work has shown that all-trans retinoic acid (t-RA) treatment of the human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1) induces a neuronal phenotype and other cell lineages. This study sought to explore the potential of 9-cis retinoic acid (9-cis RA) as a differentiation-inducing agent of this multipotent cell. Findings reported here show that 9-cis RA induced a phenotype similar to t-RA treatment of NT2/D1 cells. This similarity extended to their effects on the nuclear receptors retinoic acid receptor-β (RAR-β) and retinoid X receptor-α (RXR-α). Both retinoids prominently augmented RAR-β expression and transactivated a reporter plasmid containing putative RAR response elements (RAREs) with direct repeats separated by five nucleotides (DR5). Both retinoids had no appreciable effect on RXR-α expression and both minimally transactivated a reporter plasmid containing putative RXR response elements (RXREs) with direct repeats separated by one nucleotide (DR1). These studies suggest that 9-cis RA and t-RA activate common events during retinoid-mediated NT2/D1 differentiation. This hypothesis was supported by the finding that NT2/D1 cells rendered refractory to t-RA (NT2/D1-R1) were also resistant to 9-cis RA. To discover alterations that could confer retinoid-refractoriness, retinoid receptor expression was examined in NT2/D1-R1 cells. In contrast to NT2/D1, the NT2/D1-R1 cell was found to have reduced RXR-α expression at the level of total cellular RNA. These studies establish the effectiveness of 9-cis RA as a differentiation agent of human TC cells and demonstrate that retinoids with different nuclear receptor affinities can induce similar phenotypes in NT2/D1 cells. In addition, the findings in the retinoid resistant NT2/D1-R1 cell implicate a role for specific retinoid receptors in this human TC differentiation program.     

Retinoid signalling and gene expression in neuroblastoma cells: RXR agonist and antagonist effects on CRABP-II and RARbeta expression

9-cis Retinoic acid (RA) induces gene expression in neuroblastoma cells more effectively and with different kinetics than other RA isomers, and could be acting in part through Retinoid X Receptors (RXRs). The aim of this study was to characterise the effects of an RXR agonist and RXR homodimer antagonist on the induction of cellular RA binding protein II (CRABP-II) and RA receptor-beta (RARbeta) in neuroblastoma cells in response to different retinoids. The RXR agonist, LDG1069, was as effective as all-trans RA in inducing gene expression, but less effective than 9-cis RA. The RXR-homodimer antagonist, LG100754, inhibited the induction of CRABP-II mRNA in SH-SY5Y neuroblastoma cells by 9-cis RA or the RXR-specific agonist LGD1069, but had no effect when used with all-trans RA. Conversely, LG100754 did not inhibit induction of RARbeta mRNA by 9-cis or all-trans RA, or by LGD1069. RAR- and RXR-specific ligands used together induced CRABP-II and RARbeta as effectively as 9-cis RA. These results demonstrate the value of combining RXR- and RAR-specific ligands to regulate RA-inducible gene expression. The possibility that RXR-homodimers mediate, in part, the induction of CRABP-II by 9-cis RA and RXR-specific ligands is discussed.