Retinoic acid modulation of transmembrane signaling. Analysis in F9 teratocarcinoma cells

F9 embryonal mouse teratocarcinoma cells were differentiated to a primitive endoderm-like phenotype by retinoic acid and to a parietal endoderm-like phenotype by retinoic acid in combination with dibutyryl cyclic AMP. The secretion of tissue plasminogen activator (tPA) is a characteristic of the cells displaying the differentiated phenotypes. The fundamental question of whether tPA secretion is regulated acutely by G-protein-mediated transmembrane signaling was explored. Cells differentiated to primitive and parietal endoderm demonstrated a rapid tPA response to stimulation by beta-adrenergic agonist (isoproterenol). Adenylyl cyclase activity in response to isoproterenol and GTP, but not forskolin, was greater in primitive and parietal endoderm than F9 stem cells. Both primitive and parietal endoderm cells, but not F9 stem cells, displayed beta-adrenergic stimulation of cyclic AMP accumulation. Retinoic acid induced F9 stem cells to the primitive endoderm phenotype and increased beta-adrenergic receptor levels 3-fold. Gi alpha 2 levels declined, G beta-subunits increased, and Gs alpha levels were unchanged following differentiation to primitive endoderm. In parietal endoderm cells beta-adrenergic receptors increased 2-fold over F9 stem cells, Gi alpha 2 levels declined even further than in primitive endoderm, G beta-subunits increased compared to F9 stem cells, and Gs alpha levels again were unchanged. The marked potentiation of short-term stimulation of tPA secretion in the differentiated state may be best explained by the retinoic acid-induced increase in expression of beta-adrenergic receptors coupled with a decline in Gi alpha 2 levels. Short-term regulation by G-protein-linked receptors represents a novel mode for the control of tPA secretion.     

Cellular HSP90 (HSP86) mRNA level and in vitro differentiation of mouse embryonal carcinoma (F9) cells

Treatment of mouse embryonal carcinoma (F9) cells with retinoic acid, an inducer of F9 cell differentiation, greatly increased the level of mRNA specific to one of the heat-shock proteins (HSP86). Experiments including the one employing differentiation-resistant mutant F9 cells suggested that the increase represents early molecular events associated with the embryonal differentiation. The increased HSP86 mRNA declined to the original level during further incubation. The presence of cyclic AMP, which stimulates conversion of the retinoic acid-induced primitive endoderm cells to parietal endoderm cells, prevented the decline. These results suggest that not only the elevation of HSP86 mRNA level represents early molecular events in F9 cell differentiation but also that sustaining the elevated level (by cyclic AMP) is associated with further differentiation of the embryonal cells.     

A fucosyltransferase in teratocarcinoma stem cells. Decreased activity accompanying differentiation to parietal endoderm cells

Teratocarcinoma stem cell F9 expressed a potent fucosyltransferase activity acting on asialofetuin. A majority of the product was susceptible to alpha-L-fucosidase I from almond emulsin, indicating that the linkage formed was mainly Fuc alpha 1 leads to 3GlcNAc. The specific activity of the transferase decreased when the stem cells were induced to differentiate into parietal endoderm cells by retinoic acid and dibutyryl cyclic AMP. Furthermore, PYS-2 cell, a parietal endoderm cell line virtually lacked the transferase. The change in the fucosyltransferase activity could be correlated with cell surface changes occurring during differentiation.     

Down-regulation of phospholipase D during differentiation of mouse F9 teratocarcinoma cells.

Phospholipase D has been recognized as playing an important role in signal transduction in many types of cells. We investigated the expression of phospholipase D during the differentiation of F9 embryonal teratocarcinoma cells. The ADP ribosylation factor-dependent phospholipase D activity, as measured by an in vitro assay, and H2O2-induced phospholipase D activity and phospholipase D protein content in whole cells were decreased during the differentiation of F9 cells induced by a combination of dibutyryl cyclic AMP and all-trans retinoic acid. In contrast, these changes were not observed when cells were induced by retinoic acid. These results suggest that down-regulation of phospholipase D protein is associated with differentiation of F9 cells to a parietal endoderm lineage.     

Expression of fibroblast growth factor by F9 teratocarcinoma cells as a function of differentiation

F9 teratocarcinoma stem cells treated with retinoic acid (RA) and dibutyryl cAMP (but2 cAMP) differentiate into embryonic parietal endoderm. Using heparin-affinity chromatography, endothelial cell proliferation assays, immunoprecipitation, and Western analysis with antibodies specific for acidic and basic fibroblast growth factors (FGFs), we detected biologically active FGF in F9 cells only after differentiation. A bovine basic FGF cDNA probe hybridized to 2.2-kb mRNAs in both F9 stem and parietal endoderm cells and to a 3.8-kb mRNA in F9 stem cells. A genomic DNA probe for acidic FGF hybridized to a 5.8-6.0-kb mRNA in both F9 stem and parietal endoderm cells, and to a 6.0-6.3-kb mRNA only in parietal endoderm cells. Although these FGF mRNAs were present in the stem cells, we could find no evidence that F9 stem cells synthesized FGFs, whereas differentiated F9 cells synthesized both acidic and basic FGF-like proteins. We conclude that biologically active factors with properties characteristic of acidic and basic FGF are expressed by F9 parietal endoderm cells after differentiation. Differentiating embryonic parietal endoderm thus may serve as a source of FGF molecules in the developing blastocyst, where these factors appear to play a central role in subsequent embryogenesis.     

Butyrate inhibits the retinoic acid-induced differentiation of F9 teratocarcinoma stem cells

F9 mouse teratocarcinoma stem cells differentiate into parietal endoderm cells in the presence of retinoic acid, dibutyryl cyclic AMP, and theophylline (RACT). When F9 cells are exposed to 2-5 mM sodium butyrate plus RACT, they fail to differentiate. Differentiation is assessed by induction of laminin and collagen IV mRNA, the synthesis of laminin, collagen IV and plasminogen activator proteins, and alterations in cell morphology. Butyrate inhibits differentiation only when added within 8 hr after retinoic acid addition. Thus an early event in retinoid action on F9 cells is butyrate-sensitive. The population doubling time and cell cycle distribution of F9 cells are not altered within the first 24 hr after butyrate addition, suggesting that butyrate does not inhibit differentiation by inhibition of growth or normal cycling. However, butyrate does inhibit histone deacetylation in F9 cells, and this could be the mechanism by which butyrate inhibits differentiation.     

Evidence for the existence of an early common biochemical pathway in the differentiation of F9 cells into visceral or parietal endoderm: Modulation by cyclic AMP

The addition of dibutyryl cyclic AMP (dbcAMP) to aggregate cultures of F9 cells in medium containing retinoic acid (RA) directs the pathway of differentiation into parietal endoderm instead of visceral endoderm. We examined the levels of some of the markers that characterize the two pathways and studied the time of commitment of cells to either direction of differentiation by using immunoprecipitation and enzyme-linked immunosorbent assays (ELISA). For either pathway, the levels and patterns of laminin, type IV collagen, and fibronectin are the same on the first day of differentiation, characterized by slightly decreased levels of laminin and type IV collagen synthesis and an increased level of fibronectin synthesis. These levels reverse on the second day of culture when the pathways diverge markedly. The differentiation pathway, however, can be redirected into the alternate one; parietal endoderm cells become committed after 3 days, whereas visceral endoderm cells are able to change into parietal endoderm cells at any time. Thus, α-fetoprotein (AFP)-producing F9 embryoid bodies switched to dbcAMP-containing medium lose the capacity to synthesize AFP and start to express genes characteristic of parietal endoderm. Our results indicate that at least some visceral endoderm cells may redifferentiate into parietal endoderm cells. These phenomena thus mimic features of endoderm differentiation in the mouse embryo.     

The amount of a specific cellular protein (p53) is a correlate of differentiation in embryonal carcinoma cells

A specific cellular protein of molecular weight of 53–55,000 (p53) has been shown to be induced in all SV40 transformed cells. A similar protein has also been shown to be present in embryonal carcinoma cells and in midgestation murine embryo primary cells, which are not infected by SV40. In embryo cell primaries the amount of the protein was shown to decrease with the increase in the stage of embryo development. As differentiation or decrease in cell growth rate can account for this, and since the growth rate of embryo primary cells cannot be measured, we chose to investigate various embryonal carcinoma cells. We report that the p53 is present in a pluripotent embryonal carcinoma cell OTT6050, and in its differentiated parietal endoderm derivative, PYS-2 cells. The amount of p53 is higher in the undifferentiated EC stem cells than in the differentiated PYS-2 (parietal endoderm) cells. The amount of the protein decreases in F9 embryonal carcinoma cells induced to differentiate to a parietal endoderm cell type by treatment with retinoic acid, as it does following spontaneous differentiation of OTT6050 EC cells. To determine if a change in growth rate, rather than differentiation, might acount for the diminished levels of this protein, the amount ofp53 was measured in growing and in growth arrested cell populations. When the growth rate of F9 cells was reduced by treatment with 8-bromocyclic AMP there was no change in the amount of p53. The half life of the p53 was compared in the undifferentiated and the differentiated cell types to determine if a change in stability might account, in part, for the altered levels of this protein. The p53 is found to be most stable in the SV40 transformed established clonal cells. It is less stable in the fibroblast clonal cells which were not transformed by SV40. The results of these experiments indicate that a decrease in the amount of p53 primarily correlates with differentiation in the embryonal carcinoma cell lines studied and not with cell growth rate. Furthermore, the decrease appears to be related (in part) to the decreased stability of the p53.     

RXRalpha-null F9 embryonal carcinoma cells are resistant to the differentiation, anti-proliferative and apoptotic effects of retinoids.

The F9 murine embryonal carcinoma (EC) cell line, a well established model system for the study of retinoic acid (RA)-induced differentiation, differentiates into cells resembling three types of extra-embryonic endoderm (primitive, parietal and visceral), depending on the culture conditions and RA concentration used. A number of previously identified genes are differentially expressed during this process and serve as markers for the different endodermal cell types. Differentiation is also accompanied by a decreased rate of proliferation and an apoptotic response. Using homologous recombination, we have disrupted both alleles of the retinoid X receptor (RXR) alpha gene in F9 cells to investigate its role in mediating these responses. The loss of RXRalpha expression impaired the morphological differentiation of F9 EC cells into primitive and parietal endoderm, but has little effect on visceral endodermal differentiation. Concomitantly the inducibility of most primitive and parietal endoderm differentiation-specific genes was impaired, while several genes upregulated during visceral endodermal differentiation were induced normally. We also demonstrate that RXRalpha is required for both the anti-proliferative and apoptotic responses in RA-treated F9 cells. Additionally, we provide further evidence that retinoic acid receptor (RAR)-RXR heterodimers are the functional units transducing the effects of retinoids in F9 cells.     

A new differentiated cell line (Dif 5) derived by retinoic acid treatment of F9 teratocarcinoma cells capable of extracellular matrix production and growth in the absence of serum

Treatment of F9 teratocarcinoma cells with all trans retinoic acid (RA) causes them to differentiate into two or three morphologically distinct cell types. Whereas the majority of these retinoid-derived cells exhibit properties resembling parietal endoderm, a small percentage of this differentiated cell population manifests properties distinct from the parietal endoderm cell type. The isolation and partial characterization of such a non-parietal endoderm cell line (Dif 5) derived from F9 cells following prolonged (44 days) exposure to 1 μM retinoic acid are described.Unlike the retinoid-induced parietal endoderm-like cell population, which exhibits a dramatic, characteristic morphological change upon treatment with 8-bromo cAMP, Dif 5 cells do not show any morphological change with exposure to this cAMP analog. Dif 5 cells synthesize and deposit an extracellular matrix consisting of several components of Reichert's membrane (fibronectin, laminin, and type IV collagen). This new cell line does not synthesize α-fetoprotein but does secrete plasminogen activator.An interesting property of these cells is their ability to grow in the absence of serum or other hormonal supplements. Yet the Dif 5 cells do exhibit density-dependent inhibition of growth. Unlike the parent F9 cells or parietal yolk sac (PYS-2) cells, these cells do possess specific cell surface receptors for epidermal growth factor (EGF). The growth-arrested Dif 5 cells can be reinitiated to proliferate by the addition of fetal calf serum (FCS) or EGF.The properties of Dif 5 cells determined fail to fulfill all the characteristics described for either parietal or visceral endodermal cells. This raises the possibility that Dif 5 cells might represent an endodermal cell type which is intermediate in differentiation to either parietal or visceral endoderm but which lacks the biochemical signal to complete this stage of differentiation. This new Dif 5 cell line should be of considerable value in studying the modulation of growth requirements and extracellular matrix formation during early embryonic development.