Distinct retinoid X receptor-retinoic acid receptor heterodimers are differentially involved in the control of expression of retinoid target genes in F9 embryonal carcinoma cells.

The F9 murine embryonal carcinoma cell line represents a well-established system for the study of retinoid signaling in vivo. We have investigated the functional specificity of different retinoid X receptor (RXR)-retinoic acid (RA) receptor (RAR) isotype pairs for the control of expression of endogenous RA-responsive genes, by using wild-type (WT), RXR alpha(-/-), RAR alpha(-/-), RAR gamma(-/-), RXR alpha(-/-)-RAR alpha(-/-), and RXR alpha(-/-)-RAR gamma(-/-) F9 cells, as well as panRXR and RAR isotype (alpha, beta, and gamma)-selective retinoids. We show that in these cells the control of expression of different sets of RA-responsive genes is preferentially mediated by distinct RXR-RAR isotype combinations. Our data support the conclusion that RXR-RAR heterodimers are the functional units transducing the retinoid signal and indicate in addition that these heterodimers exert both specific and redundant functions on the expression of particular sets of RA-responsive genes. We also show that the presence of a given receptor isotype can hinder the activity of another isotype and therefore that functional redundancy between retinoid receptor isotypes can be artifactually generated by gene knockouts.     

Retinoid X receptor alpha and retinoic acid receptor gamma mediate expression of genes encoding tight-junction proteins and barrier function in F9 cells during visceral endodermal differentiation

Retinoids are critical for differentiation of columnar epithelial cells and for preventing metaplasia of these cells into stratified squamous epithelial cells, in which tight junctions (TJs) are essentially absent. This implies that retinoids might play important roles in regulating the structures and functions of TJs of columnar epithelium. F9 murine embryonal carcinoma cells differentiate into epithelial cells resembling visceral endoderm bearing TJs, when grown in suspension as aggregates in the presence of retinoic acid (RA). We show that RA induces the TJ structure and expression of several TJ-associated molecules, such as ZO-1, occludin, claudin-6, and claudin-7, as well as a barrier function in the genetically engineered cell line F9:rtTA:Cre-ER(T) L32T2, which allows sophisticated genetic manipulations simply by addition of ligands (H. Chiba et al., 2000, Exp. Cell Res. 260, 334-339). Interestingly, our data indicate that a barrier for small substances is generated after that for large ones during de novo formation of TJs. We also compared the RA-induced expression of TJ components and barrier function in RXRalpha(-/-)-RARgamma(-/-) F9 cells with those in wild-type cells and show that the retinoid signals for transduction of these events are mediated by specific RXR-RAR pairs.     

Phosphorylation of activation functions AF-1 and AF-2 of RAR alpha and RAR gamma is indispensable for differentiation of F9 cells upon retinoic acid and cAMP treatment.

The role of RAR alpha 1 and RAR gamma 2 AF-1 and AF-2 activation functions and of their phosphorylation was investigated during RA-induced primitive and parietal differentiation of F9 cells. We found that: (i) primitive endodermal differentiation requires RAR gamma 2, whereas parietal endodermal differentiation requires both RAR gamma 2 and RAR alpha 1, and in all cases AF-1 and AF-2 must synergize; (ii) primitive endodermal differentiation requires the proline-directed kinase site of RAR gamma 2-AF-1, whereas parietal endodermal differentiation additionally requires that of RAR alpha 1-AF-1; (iii) the cAMP-induced parietal endodermal differentiation also requires the protein kinase A site of RAR alpha-AF-2, but not that of RAR gamma; and (iv) the AF-1-AF-2 synergism and AF-1 phosphorylation site requirements for RA-responsive gene induction are promoter context-dependent. Thus, AF-1 and AF-2 of distinct RARs exert specific cellular and molecular functions in a cell-autonomous system mimicking physiological situations, and their phosphorylation by kinases belonging to two main signalling pathways is required to enable RARs to transduce the RA signal during F9 cell differentiation.     

The location and expression of fibroblast growth factor (FGF) in F9 visceral and parietal embryonic cells after retinoic acid-induced differentiation

It is well-established that fibroblast growth factors (FGFs) participate in mesoderm formation and patterning in the developing embryo. To identify cells in mammalian embryos that produce and/or respond to FGFs, we utilized the F9 teratocarcinoma cell system. Undifferentiated F9 cells resemble inner cell mass (ICM) cells of the mouse blastocyst by several criteria including having a characteristic high nuclear to cytoplasmic ratio and by their expression of stage-specific embryonic antigens. F9 stem cells differ from ICM cells by their low spontaneous rate of differentiation and their differentiation potential. ICM cells are heterogeneous with a proportion of the cells maintaining totipotency. In contrast, F9 stem cells appear capable of forming only endodermal derivatives. Retinoic acid (RA) treatment of F9 stem cells is required for them to differentiate, and under different culturing conditions the F9 cells will form either extraembryonic parietal or visceral endoderm. We have previously shown that FGF is synthesized by F9 parietal endoderm, but not by F9 stem cells. Our present study demonstrates that F9 aggregate cultures that contain visceral endoderm cells produce cell-associated-heparin-binding mitogens for 3T3 and endothelial cells, factors with characteristics of FGFs. Furthermore, our studies detect endothelial cell-mitogens within the extracellular matrix (ECM) of F9 parietal endoderm cells, not detected within F9 stem cell 'matrices'. Parietal endoderm cell matrix mitogens could be removed by prior treatment of the ECM with buffers containing heparin or 2 M NaCl, and could be neutralized by basic FGF antibodies.     

Biphasic response to retinoic acid dose in differentiation of F9 cells into primitive endoderm-like cells

The differentiation of F9 cells, as monitored by the secretion of basement membrane proteins, was biphasic depending on the dose of retinoic acid (RA) used. Secretion of laminin A reached a plateau at about 10 nM and increased again at more than 100 mM RA. A similar biphasic response was observed in the secretion of tissue-type plasminogen activator as well. In the presence of RA alone, this biphasic pattern of differentiation was stable for a long period of time, but the addition of 1 mM dibutyryl cAMP changed it to monophasic after 12 days of exposure.     

Effects of tunicamycin on the differentiation of F9 cells induced by either retinoic acid or retinoic acid and dibutyryl cyclic AMP

Tunicamycin (0.5 micrograms/ml) inhibited differentiation of F9 cells treated either with retinoic acid or with retinoic acid and dibutyryl cyclic AMP, as monitored by the activity of alkaline phosphatase and expression of cytokeratins. On the other hand, the pattern of the polysaccharide chain synthesis changed drastically with the treatment irrespective of the presence of tunicamycin. Therefore, phenotypes induced with retinoic acid are dissociated into two categories, one that is directly induced by the drug and the other that is induced indirectly by a mechanism in which glycoproteins play a role.     

Comparative proteomic analysis of differentiation of mouse F9 embryonic carcinoma cells induced by retinoic acid

The multipotent mouse F9 embryonic carcinoma cell is an ideal model system to investigate the mechanism of retinoic acid (RA) in cell differentiation and cell growth control and the biochemical basis of early embryonic development. We reported here a proteomics approach to study protein expression changes during the differentiation of F9 cells into the visceral endoderm. F9 cells were incubated with or without RA at 0, 24, 48, and 72 h. Total proteins extracted were separated by two‐dimensional electrophoresis (2‐DE) and the protein patterns on the gels were comparatively analyzed by computer. Approximately 1,100 protein spots were detected in the F9 proteome, within the pH 3–10 range. Fourteen protein spots which the levels of expression were found to be altered dramatically during the F9 cells differentiating, and were identified by MALDI‐TOF MS or ESI‐MS/MS. These proteins included metabolism enzymes, HSP60s, RAN, hnRNP K, FUBP1, VDAC1, STI1, and prohibitin. These proteins are involved in cellar metabolism, gene expression regulation, stress response, and apoptosis, respectively. The data from proteomic analyze are consistent with the result obtained from Western blot analysis. This study increases our understanding of the proteomics changes during F9 cells differentiation induced by RA. J. Cell. Biochem. 113: 1811–1819, 2012. © 2012 Wiley Periodicals, Inc.     

The phosphorylation site located in the A region of retinoic X receptor alpha is required for the antiproliferative effect of retinoic acid (RA) and the activation of RA target genes in F9 cells

Mouse F9 embryocarcinoma cells constitute a well established cell autonomous model system for investigating retinoic acid (RA) signaling in vitro. RA induces the differentiation of F9 cells grown as monolayers into endodermal-like cells and decreases their rate of proliferation. Knock-out of the retinoic X receptor alpha (RXRalpha) gene abolishes endodermal differentiation and the induction of several endogenous RA-responsive genes. RXRalpha null cells are also drastically impaired in their antiproliferative response to RA. The role of the RXRalpha phosphorylation site located in the N-terminal A region (Ser(22)) has been investigated here by establishing cell lines re-expressing RXRalpha either wild type or mutated at the phosphorylation site (RXRalphaS22A) in a RXRalpha-null background. We show that Ser(22) is dispensable for RA-induced endodermal differentiation but is crucial for the expression of several RA-responsive genes. Ser(22) is also indispensable for the antiproliferative effect of RA and necessary for the RA-induced down-regulation of p21(CIP) and p27(KIP) CKIs proteins that are known to be involved in the control of cell cycle progression.     

Modulation of protein biosynthesis during early stages of differentiation in retinoic acid treated F9 teratocarcinoma cells

Modulation of protein biosynthesis by retinoic acid during induction of differentiation of F9 teratocarcinoma stem cells was investigated by using computerized analysis of double label autoradiography of two-dimensional polyacrylamide gels. As early as 6 h after induction increased synthesis of 5 and decreased synthesis of 2 proteins occur. By 12 h after induction, synthesis of 13 proteins is elevated and by 24 h that of 17. At 24 h the range of stimulation is from two- to fourfold, as demonstrated by a 3H:14C ratio divided by the mode ratio. Examination of the Gaussian distributions of frequency of ratio indicates that many subtle changes in protein synthesis accompany the development of the new phenotype.     

Differential expression of fetomodulin and tissue plasminogen activator to characterize parietal endoderm differentiation of F9 embryonal carcinoma cells.

Fetomodulin is a surface marker protein of differentiated F9 embryonal carcinoma cells. Gene cloning has recently identified it as thrombomodulin which binds thrombin and proteolytically activates protein C. Activity assays and RNA blotting were adopted to analyze F9 cell differentiation with specific reference to another well-characterized marker, tissue plasminogen activator. Retinoic acid induced primitive endoderm differentiation of F9 cells and simultaneously activated tissue plasminogen activator synthesis. This differentiation, however, did not result in fetomodulin expression. When primitive endoderm cells were exposed to 1 mM dibutyryl cyclic AMP, the tissue plasminogen activator level rose further within 6 hr. In contrast, the cofactor activity of fetomodulin stayed below a detectable level for as long as 15 hr and then increased with time. Expression of the two marker proteins appeared to be regulated differently.     

Cyclic AMP analogs and retinoic acid influence the expression of retinoic acid receptor alpha, beta, and gamma mRNAs in F9 teratocarcinoma cells.

Retinoic acid (RA) receptor alpha (RAR alpha) and RAR gamma steady-state mRNA levels remained relatively constant over time after the addition of RA to F9 teratocarcinoma stem cells. In contrast, the steady-state RAR beta mRNA level started to increase within 12 h after the addition of RA and reached a 20-fold-higher level by 48 h. This RA-associated RAR beta mRNA increase was not prevented by protein synthesis inhibitors but was prevented by the addition of cyclic AMP analogs. In the presence of RA, cyclic AMP analogs also greatly reduced the RAR alpha and RAR gamma mRNA levels, even though cyclic AMP analogs alone did not alter these mRNA levels. The addition of either RA or RA plus cyclic AMP analogs did not result in changes in the three RAR mRNA half-lives. These results suggest that agents which elevate the internal cyclic AMP concentration may also affect the cellular response to RA by altering the expression of the RARs.