T-box binding protein type two (TBX2) is an immediate early gene target in retinoic-acid-treated B16 murine melanoma cells

Retinoic acid induces growth arrest and differentiation in B16 mouse melanoma cells. Using gene arrays, we identified several early response genes whose expression is altered by retinoic acid. One of the genes, tbx2, is a member of T-box nuclear binding proteins that are important morphogens in developing embryos. Increased TBX2 mRNA is seen within 2 h after addition of retinoic acid to B16 cells. The effect of retinoic acid on gene expression is direct since it does not require any new protein synthesis. We identified a degenerate retinoic acid response element (RARE) between -186 and -163 in the promoter region of the tbx2 gene. A synthetic oligonucleotide spanning this region was able to drive increased expression of a luciferase reporter gene in response to retinoic acid; however, this induction was lost when a point mutation was introduced into the RARE. This oligonucleotide also specifically bound RAR in nuclear extracts from B16 cells. TBX2 expression and its induction by retinoic acid was also observed in normal human and nonmalignant mouse melanocytes.     

Characterization of an inverted repeat with a zero spacer (IR0)-type retinoic acid response element from the mouse nuclear orphan receptor TR2-11 gene.

An inverted repeat with zero nucleotides in the spacer (IR0, 5'-GGGTCA CGAACT-3') element was localized in the proximal promoter region of the mouse TR2-11 gene, and characterized as a functional retinoic acid response element (RARE). In gel mobility shift assays, heterodimers of retinoic acid receptor alpha (RARalpha) and retinoid X receptor beta (RXRbeta) bound specifically to this element. Neither receptor alone was able to bind to this element efficiently. The dissociation constant (Kd) with respect to RAR-RXR binding was estimated to be 8 nM. The biological activity of this IR0 element was assessed in a heterologous reporter system. The IR0-containing reporter was induced by RA in COS-1 cells in the presence of exogenously provided RARalpha and RXRbeta. In addition, the IR0 oligomers could be bound by nuclear extracts isolated from COS-1 cells harboring the expression vectors for RARalpha and RXRbeta, but not by extracts isolated from control COS-1 cells. RA responsiveness of this IR0 was further confirmed in P19 cells that expressed endogenous RARs and RXRs. Collectively, these data demonstrated, for the first time, the presence of a natural RARE of the IR0 type, and suggested a potential cross-talk between nuclear orphan receptor TR2-11 and RAR-RXR families.     

Retinoic acid activates human inducible nitric oxide synthase gene through binding of RARalpha/RXRalpha heterodimer to a novel retinoic acid response element in the promoter

Human inducible nitric oxide synthase (hiNOS) catalyzes nitric oxide (NO) which has a significant effect on tumor suppression and cancer therapy. Here we revealed the detailed molecular mechanism involved in the regulation of hiNOS expression induced by retinoic acid (RA). We showed that RARalpha/RXRalpha heterodimer was important in hiNOS promoter activation, hiNOS protein expression, and NO production. Serial deletion and site-directed mutation analysis revealed two half-sites of retinoic acid response element (RARE) spaced by 5bp located at -172 to -156 in the hiNOS promoter. EMSA and ChIP assays demonstrated that RARalpha/RXRalpha directly bound to this RARE of hiNOS promoter. Our results suggested the identification of a novel RARE in the hiNOS promoter and the roles of the nuclear receptors (RARalpha/RXRalpha) in the induction of hiNOS by RA.     

羟基脲诱导前后HEL细胞内GATA转录因子与人β—类珠蛋白基因调控元件 …

HEL cells, a human erythroleukemia cell line, expressing mainly the gamma-globin genes, small amount of epsilon-globin gene, but not beta-globin gene. Our previous studies demonstrated that beta-globin gene could be expressed in HEL cells induced by hydroxyurea. However, the molecular mechenism is still unknown. Here the binding patterns of GATA factors (GATA-1 and GATA-2) to the regulatory elements of human beta-globin gene were examined with the nuclear extracts from hydroxyurea-induced and uninduced HEL cells. Our results showed in EMSA assay that GATA factors could bind to the core sequence of HS2(-10681 to -10971 bp), the 3' flanking sequence of HS2 core(-10323 to -10680 bp) and the promoter of human beta-globin gene(+20 to -112 bp). However, the binding patterns between hydroxyurea-induced and uninduced HEL cells were different. Furthermore, by using Western-blot analysis, our data showed that the amount of GATA-2 was decreased in hydroxyurea-induced HEL cells. In contrast to GATA-2, the amount of GATA-1 was increased in hydroxyurea-induced HEL cells. These results showed that the different members of GATA family might play different roles during the differentiation of erythrocytes. GATA-1 may stimulate the differentiation of HEL cells, while GATA-2 can probably inhibit the differentiation of HEL cells.     

Induction and Translocation of Tissue Transglutaminase Isoforms Increased Phosphorylation in Retinoic Acid Treated Erythroleukemia Cells

Tissue transglutaminase (TGC, TG2, 80 kDa) is inactive in cross-linking reactions and is converted in vitro and in vivo to the TG (55 kDa) active isoform (Fraij in J Cell Biochem 112:2469–2489, 2011). Two isoforms of human TGC were cloned from human erythroleukemia (HEL) cells induced with retinoic acid (RA) and termed TGH, 63 kDa (Fraij et al. in J Biol Chem 267:22616–22673, 1992) and TGH2, 37 kDa. The purified TGC isoforms exhibited GTPase activity and TGH and TGH2 showed higher activities than the native TGC protein. In all normal cells examined, TGC was found in membrane fractions several fold higher than the supernatant fractions; however, in the natural tumor cell line HEL the TGC cellular distribution was reversed. Although TGC is the major enzyme in normal human erythrocytes, its expression level was significantly decreased in HEL cells. RA treatment induced a sevenfold increase in the level of TGC protein in HEL cells and was accompanied by its translocation to cell membranes. When isolated membrane and supernatant fractions from normal human foreskin (CF3), normal human embryonic lung (WI-38), and HEL cells treated with or without RA were incubated with [³²P]-ATP at 37 °C for 1 h, more radio-labeled proteins were detected in the membrane fractions than the cytosolic fractions. More labeled protein bands were detected in RA treated HEL cells in comparison to control HEL cell extracts. Radio labeled proteins coimmunoprecipitated with the TGC isoforms in RA treated HEL membrane fractions thereby confirming that the radio-labeled material consists of endogenous proteins associated with TGC isoforms. Protein phosphorylation is related to the induction and translocation of the isoforms in RA treated cells. These results show that the TGC isoforms complexes with proteins in vivo and that the phosphorylation of these proteins is catalyzed directly by TGC kinase activity or indirectly by the TGC phosphorylation of other protein kinases.     

Regulation of the murine alpha(2)(I) collagen promoter by retinoic acid and retinoid X receptors

Retinoic acid decreases collagen production by hepatic stellate cells. This study investigated the effects of retinoic acid receptor beta (RARbeta) and retinoid X receptor alpha (RXRalpha) on the regulation of the alpha(2)(I) collagen promoter. Retinoic acid and the RARbeta and RXRalpha expression vectors suppressed the promoter in transfected stellate cells with maximal suppression obtained when combined. Mutation of the retinoic acid response element (RARE) at -879 to -874 (site 1) enhanced promoter activity and diminished but did not eliminate the suppression by RARbeta and RXRalpha. Mutation of another RARE site (site 2), at -930 to -911, resulted in low activity that was inhibited by retinoic acid. Mutation of the AP-2-binding site enhanced promoter activity that was inhibited by retinoic acid. This study shows that the suppressive effect of retinoic acid on the promoter is maximal with a combination of RARbeta and RXRalpha and occurs at more than one RARE site. The effect of retinoic acid is not mediated by AP-2.     

Retinoic acid receptor isoform RAR gamma 1: an antagonist of the transactivation of the RAR beta RARE in epithelial cell lines and normal human keratinocytes.

The diversity of isoforms of retinoic acid (RA) receptors (RARs) and of DNA sequences of retinoic acid-responsive elements (RAREs) suggests the existence of selectivities in the RAR/RARE recognition or in the subsequent gene modulation. Such selectivities might be particularly important for RAREs involved in positive feedback, eg. the RAR beta RARE. In the present work we found that in several epithelial cell lines, reporter constructs containing the RAR beta RARE linked to the HSV-tk promoter were transactivated in the presence of RA by endogenous RARs and co-transfected RAR alpha 1 and RAR beta 2 isoforms, but not by RAR gamam 1. On the contrary, this latter isoform behaved towards the RAR beta RARE as an inhibitor of the transactivation produced by endogenous RARs and by cotransfected RAR alpha 1 and RAR beta 2. RAR gamma 1 also behaved as an antagonist of the transactivation produced by cotransfected RXR alpha. The natural RAR beta gene promoter or RAR beta RARE tk constructs were not activated by the endogenous receptors of normal human keratinocytes (NHK), which are known to contain predominantly RAR gamma 1. It was, however, possible to activate to a certain extent RAR beta RARE-reporter constructs in NHK by co-transfecting RAR alpha 1, RAR beta 2 or RXR alpha. The antagonist behavior of RAR gamma 1 towards the RAR beta RARE may explain why in certain cell types such as keratinocytes, RAR beta is neither expressed nor induced by RA.