F9 embryocarcinoma cells: a cell autonomous model to study the functional selectivity of RARs and RXRs in retinoid signaling

Mouse F9 embryocarcinoma (EC) cells constitute a well established cell-autonomous model system for investigating retinoid signaling in vitro as, depending on culture conditions, retinoic acid (RA) can induce their differentiation into either primitive, parietal or visceral extraembryonic endoderm-like cells. These RA-induced differentiations are accompanied by decreases in proliferation rates, modifications of expression of subsets of RA-target genes, and induction of apoptosis. To elucidate the roles played by the multiple retinoid receptors (RARs and RXRs) in response to RA treatments, F9 EC cells lacking one or several RARs or RXRs were engineered through homologous recombination. Mutated RARs and/or RXRs were then reexpressed in given RAR or RXR null backgrounds. WT and mutant cells were also treated with different combinations of ligands selective for RXRs and/or for each of the three RAR isotypes. These studies lead to the conclusion that most RA-induced events (e.g. primitive and visceral differentiation, growth arrest, apoptosis and activation of expression of a number of genes) are transduced by RARgamma/RXRalpha heterodimers, whereas some other events (e.g. parietal differentiation) are mediated by RARalpha/RXRalpha. heterodimers. They also demonstrate that both AF-1 and AF-2 activation functions of RARs and RXRs, as well as their phosphorylation, are differentially required in these RA-induced events. In RARgamma/RXRalpha heterodimers, the phosphorylation of RARgamma is necessary for triggering primitive differentiation, while that of RXRalpha is required for growth arrest. On the other hand, phosphorylation of RARalpha is necessary for parietal differentiation. Thus, retinoid receptors are sophisticated signal integrators that transduce not only the effects of their cognate ligands, but also those of ligands that bind to membrane receptors.     

Retinoic acid-induced changes in differentiation-defective embryonal carcinoma RAC65 cells.

RAC65 is a mutant clone of mouse embryonal carcinoma cells, P19, which does not undergo terminal differentiation upon treatment with retinoic acid (RA). RAC65 cells express a truncated RA receptor alpha (RAR alpha) which, however, does not fully explain their defect. Here we show that RAC65 cells exhibit an additional defect in RAR alpha mRNA which may reflect a defect in RNA splicing. The parental and mutant cells also differ in their capacities to bind [3H]RA into nuclear fractions and in expression of cellular RA binding protein (CRABP) mRNA after treatment with RA. The combined data suggest that the defect in RAC65 RAR alpha results in reduced expression of the CRABP gene after RA treatment and, therefore, increased flow of RA into the nucleus.     

Retinoic acid stimulates the differentiation of PC12 cells that are deficient in cAMP-dependent protein kinase

Retinoic acid (RA) induced neuronal differentiation in A126-1B2 cells and 123.7 cells, two mutant lines of PC12 that are deficient in cAMP-dependent protein kinase, but not in the parental PC12 cell line. A single exposure to RA was sufficient to cause neurite formation and inhibit cell division for a period of greater than 3 wk, suggesting that RA may cause a long-term, stable change in the state of these cells. In A126-1B2 cells, RA also induced the expression of other markers of differentiation including acetylcholinesterase and the mRNAs for neurofilament (NF-M) and GAP-43 as effectively as nerve growth factor (NGF). Neither NGF nor RA stimulated an increase in the expression of smg-25A in A126-1B2 cells, suggesting that the cAMP-dependent protein kinases may be required for an increase in the expression of this marker. RA also caused a rapid increase in the expression of the early response gene, c-fos, but did not effect the expression of egr-1. RA equivalently inhibited the division of A126-1B2 cells, 123.7 cells and parental PC12 cells, so RA induced differentiation is not an indirect response to growth arrest. In contrast, the levels of retinoic acid receptors (RAR alpha and RAR beta), and retinoic acid binding protein (CRABP) mRNA were strikingly higher in both A126-1B2 cells and 123.7 cells than in the parental PC12 cells. The deficiencies in cAMP-dependent protein kinase may increase the expression of CRABP and the RARs; and, thus, cAMP may indirectly regulate the ability of RA to control neurite formation and neural differentiation. Thus, RA appears to regulate division and differentiation of PC12 cells by a biochemical mechanism that is quite distinct from those used by peptide growth factors.     

TGF-β Modulates the Expression of Retinoic Acid-Induced RAR-β in Primary Cultures of Embryonic Palate Cells

We have previously shown that both transforming growth factor-β (TGF-β) and retinoic acid (HA) regulate the expression of cellular retinoic acid binding proteins (CRABP) I and II and TGF-β3 mRNAs in primary cultures of murine embryonic palate mesenchyreal (MEPM) cells. We now describe additional crosstalk between the RA and TGF-β signal transduction pathways—the ability of TGF-β, including the endogenous form(s), to modulate the expression of the nuclear retinoic acid receptor-β (RAR-β). Northern blot hybridization revealed that RA induced the expression of RAR-β mRNA, there being little or no detectable expression in untreated MEPM cells. Induction by 3.3 μM RA was abrogated by simultaneous treatment with TGF-β1 (5 ng/ml). TGF-β1 alone had no effect on RAR. mRNA expression. Determination of RAR-β mRNA half-life by treatment with actinomycin D indicated that TGF-β1 did not alter the stability of RAR-β mRNA. Conditioned medium (CM) from MEPM cells contained little active TGF-β protein; heat treatment of the CM dramatically increased the amount of active TGF-β as assessed by the mink lung epithelial cell bioassay. Furthermore, heat- or acid-activated CM also inhibited CRABP-I and RA-induced RAR-β expression. The effect of heat-activated conditioned medium could be abrogated with panspecific neutralizing antibodies to TGF-β, confirming that endogenous TGF-β is the biologically active factor in heat-activated CM. These results provide evidence for complex interactions between TGF-β and RA in the regulation of gene expression in embryonic palatal cells and suggest a role for endogenous TGF-β in the regulation of expression of genes encoding elements of the RA signal transduction pathway.     

Retinoic acid resistance of the variant embryonal carcinoma cell line RAC65 is caused by expression of a truncated RARα

P19 embryonal carcinoma (EC) cells differentiate when treated with retinoic acid (RA). The P19 EC-derived mutant cell line RAC65 is resistant to the differentiation-inducing activity of RA. We show that these cells express a truncated retinoic acid receptor alpha(mRAR alpha-RAC65), probably due to the integration of a transposon-like element in the RAR alpha gene. This receptor lacks 71 C-terminal amino acids and terminates in the ligand-binding domain. In CAT assays in RAC65 cells, mRAR alpha-RAC65 fails to trans-activate the RAR beta promoter, which contains a RA-response element. In wild-type P19 EC cells mRAR alpha-RAC65 functions as a dominant-negative repressor of RA-induced RAR beta activation. Gel retardation assays demonstrate that mRAR alpha-RAC65 is still able to bind to the RA-response element of the RAR beta promoter, indicating that competition with functional RARs for the same binding site leads to the observed dominant-negative effect. In addition, in two RAC65 clones in which wild-type hRAR alpha was stably transfected RA-sensitivity was restored and in one RAR beta expression could be induced by RA. Taken together, these data show that the primary cause of RA-resistance of RAC65 cells is the expression of a defective RAR alpha, which prevents the trans-activation of RA-responsive genes and results in a loss of the ability to differentiate.     

Retinoid receptors involved in the effects of retinoic acid on rat testis development

We have previously shown that retinoic acid (RA) is able to act on the development of Leydig, Sertoli, and germ cells in the testis in culture (Livera et al., Biol Reprod 2000; 62:1303-1314). To identify which receptors mediate these effects, we have now added selective agonists and antagonists of retinoic acid receptors (RARs) or retinoid X receptors (RXRs) in the same organotypic culture system. The RAR alpha agonist mimicked most of the effects of RA on the cultured fetal or neonatal testis, whereas the RAR beta, gamma, and pan RXR agonists did not. The RAR alpha agonist decreased the testosterone production, the number of gonocytes, and the cAMP response to FSH of fetal testis explanted at 14.5 days postconception (dpc). The RAR alpha agonist disorganized the cords of the 14.5-dpc cultured testis and increased the cord diameter in cultured 3-days-postpartum (dpp) testis in the same way as RA. All these RA effects could be reversed by an RAR alpha antagonist and were unchanged by an RAR beta/gamma antagonist. The RAR beta agonist, however, increased Sertoli cell proliferation in the 3-dpp testis in the same way as RA, and this effect was blocked by an RAR beta antagonist. The RAR gamma and the pan RXR agonists had no selective effect. These results suggest that all the effects of RA on development of the fetal and neonatal testis are mediated via RAR alpha, except for its effect on Sertoli cell proliferation, which involves RAR beta.     

Combinatorial roles for zebrafish retinoic acid receptors in the hindbrain, limbs and pharyngeal arches

Retinoic acid (RA) signaling regulates multiple aspects of vertebrate embryonic development and tissue patterning, in part through the local availability of nuclear hormone receptors called retinoic acid receptors (RARs) and retinoid receptors (RXRs). RAR/RXR heterodimers transduce the RA signal, and loss-of-function studies in mice have demonstrated requirements for distinct receptor combinations at different stages of embryogenesis. However, the tissue-specific functions of each receptor and their individual contributions to RA signaling in vivo are only partially understood. Here we use morpholino oligonucleotides to deplete the four known zebrafish RARs (raraa, rarab, rarga, and rargb). We show that while all four are required for anterior-posterior patterning of rhombomeres in the hindbrain, there are unique requirements for rarga in the cranial mesoderm for hindbrain patterning, and rarab in lateral plate mesoderm for specification of the pectoral fins. In addition, the alpha subclass (raraa, rarab) is RA inducible, and of these only raraa expression is RA-dependent, suggesting that these receptors establish a region of particularly high RA signaling through positive-feedback. These studies reveal novel tissue-specific roles for RARs in controlling the competence and sensitivity of cells to respond to RA.