F9 teratocarcinoma aggregates express villin upon differentiation into visceral endoderm-like cells

The visceral endoderm of the mouse embryo is a polarized epithelium which has recently been shown to express villin, a major actin binding component of absorptive epitheliums. We report here that villin is induced during differentiation of aggregates of the mouse embryonal carcinoma F9, an in vitro system widely used to study extraembryonic endoderm differentiation. Identical results were obtained with a variant of F9 which carries an immortalizing vector. Villin is coexpressed with F-actin and with alpha-foetoprotein, in most of the visceral endoderm-like cells lining the aggregates. This system is potentially useful to study (i) the induction of villin expression and (ii) the establishment of polarity in the visceral endoderm epithelium.     

Effects of growth medium and cyclic AMP analogues on the cAMP-induced differentiation of F9 teratocarcinoma cells

Summary F9 teratocarcinoma cells differentiate into parietal endodermlike cells when treated with retinoic acid (RA) and cyclic AMP (cAMP). We have previously found that F9 cells can be induced to differentiate by treatment with cAMP in the absence of RA. In the course of determining why other investigators had failed to observe cAMP-induced differentiation, we found that the growth medium played an important role in determining the response of F9 cells to differentiating agents. When F9 cells were grown in minimal essential medium (MEM) and treated with either 8-bromo-cAMP (8BrcA) + 1-methyl, 3-isobutylxanthine (MIX), or dibutyryl cAMP (DBcA) + theophylline (T), they differentiated to parietal endodermlike cells without any requirement for exogenous RA. However, when F9 cells were grown in Dulbecco’s modified Eagle’s medium (DME), DBcA/T failed to induce differentiation alone and required exogenous RA to induce formation of parietal endoderm-like cells. 8BrcA/MIX alone was still able to induce some differentiation, although the extent was not as great as those cells grown in MEM. These results could not be explained by the different growth-promoting properties of the two culture media because there was no difference in the doubling time of F9 cells grown in either medium. Likewise, RA and cAMP both inhibited growth to the same extent in either medium. Inasmuch as almost all published reports on F9 cell differentiation have used DME as a growth medium, and RA with or without DBcA/T as the differentiating agents, these studies would not have had the appropriate conditions to detect the cAMP-induced differentiation of F9 cells.     

Dynamic connexin43 expression and gap junctional communication during endoderm differentiation of F9 embryonal carcinoma cells

Gap junctional communication permits the direct intercellular exchange of small molecules and ions. In vertebrates, gap junctions are formed by the conjunction of two connexons, each consisting of a hexamer of connexin proteins, and are either established or degraded depending on the nature of the tissue formed. Gap junction function has been implicated in both directing developmental cell fate decisions and in tissue homeostasis/metabolite exchange. In mouse development, formation of the extra embryonal parietal endoderm from visceral endoderm is the first epithelial-mesenchyme transition to occur. This transition can be mimicked in vitro, by F9 embryonal carcinoma (EC) cells treated with retinoic acid, to form (epithelial) primitive or visceral endoderm, and then with parathyroid hormone-related peptide (PTHrP) to induce the transition to (mesenchymal) parietal endoderm. Here, we demonstrate that connexin43 mRNA and protein expression levels, protein phosphorylation and subcellular localization are dynamically regulated during F9 EC cell differentiation. Dye injection showed that this complex regulation of connexin43 is correlated with functional gap junctional communication. Similar patterns of connexin43 expression, localization and communication were found in visceral and parietal endoderm isolated ex vivo from mouse embryos at day 8.5 of gestation. However, in F9 cells this tightly regulated gap junctional communication does not appear to be required for the differentiation process as such.     

Retinoic acid induces discrete Wnt-signaling-dependent differentiation in F9 cells

Retinoic acid (RA) induces F9 cells, the mouse teratocarcinoma cells, to differentiate into primitive endoderm and further into visceral and parietal endoderm depending on the culture conditions. To elucidate the instructive mechanisms involved in the differentiation steps we investigated the effects of Wnt-signaling members, Wnt3a and β-catenin, on the differentiation of F9 cells and β-catenin-deficient F9 cells (βT cells). RA up-regulated the expression of differentiation markers for primitive, visceral and parietal endoderm in F9 cells but not for visceral endoderm in βT cells. Wnt3a or leukemia inhibitory factor (LIF) inhibited the RA-induced differentiation in F9 cells. LIF but not Wnt3a could inhibit differentiation in βT cells. RA evoked ZO-1α+ signals at cell-to-cell contacts in F9 cells in a Wnt3a sensitive manner. The results suggest that Wnt3a inhibits differentiation into endoderm through a pathway involving β-catenin, and β-catenin might be necessary in the process leading from primitive to visceral endoderm in F9 cells.     

Galpha13 activation rescues moesin-depletion induced apoptosis in F9 teratocarcinoma cells

Mouse F9 cells differentiate into primitive endoderm when treated with retinoic acid (RA) and into parietal endoderm in response to RA and dibutyryl (db-) cAMP. G protein signaling either blocks or mimics RA-induced differentiation, the latter signaling through the Wnt-beta-catenin pathway. In our study, we found that a constitutively active Galpha13 mutant induces F9 cells to differentiate into parietal endoderm in the absence of exogenous agents. Galpha13 expression and subsequent differentiation are accompanied by beta-catenin translocation to the nucleus. Differentiation and changes in cell morphology are supported by rearrangements to the F-actin cytoskeleton. ERM (ezrin-radixin-moesin) proteins, known to link F-actin to transmembrane receptors, are also redistributed during differentiation. Furthermore, morpholino antisense and shRNA approaches show that moesin expression is essential since its knockdown leads to altered F-actin distribution and subsequent apoptosis. Moesin-depleted cells, however, remain attached to the substrate when Galpha13 is constitutively expressed, but they do not differentiate into extraembryonic endoderm. Our study demonstrates a link between Galpha13 signaling that regulates differentiation of F9 cells through primitive to parietal endoderm and a moesin requirement for cell survival.     

A blockade in Wnt signaling is activated following the differentiation of F9 teratocarcinoma cells

Aberrant activation of the Wnt signaling pathway is a common event in human tumor progression. Wnt signaling has also been implicated in maintaining a variety of adult and embryonic stem cells by imposing a restraint to differentiation. To understand the effect of Wnt signaling on the differentiation of epithelial cells, we used mouse teratocarcinoma F9 cells as a model. The F9 cells can be differentiated into visceral endoderm (VE) resembling absorptive columnar epithelial cells. We performed comparative gene expression analysis on retinoic acid-differentiated and undifferentiated F9 cells and confirmed that markers of VE and intestinal epithelium were induced upon differentiation. The induction of these markers by retinoic acid was reduced in the presence of Wnt, although Wnt alone did not change their expression. This suggests that Wnt signaling inhibited the differentiation of F9 cells by altering gene expression. This inhibition was also reflected in the morphology of the F9 cells as their apical-basal polarity was disrupted by inclusion of Wnt during differentiation. These results support a model in which Wnt modulates the expression of genes required for normal terminal differentiation of the stem cells. However, it follows that progenitor cells must escape from Wnt signaling to attain the differentiated state. Accordingly, we found that differentiated F9 cells no longer responded to Wnt and that a blockade in Wnt signaling occurred upstream of Axin. Consistent with this, Wnt negative regulators, such as Dickkopf-1 and Disabled-2, were induced upon the differentiation of F9 cells. We propose that a similar system to produce Wnt inhibitors regulates homeostasis of certain stem cell compartments in vivo.     

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.     

Retinoic acid abolishes the calcitonin gene-related peptide autocrine system in F9 teratocarcinoma cells

Calcitonin gene-related peptide (CGRP), expressed predominantly in F9 embryonal carcinoma cells, is both a potent chemotactic agent and an autocrine growth factor for these cells. We analyzed the effect of retinoic acid (RA)-induced differentiation of F9 cells into primitive parietal endoderm-like cells, on CGRP production and the CGRP responsiveness of these cells. Poly(A) RNA extracted from F9 cells and analysed by Northern blotting and hybridization with a CGRP probe showed a specific band of about 1200 bases corresponding to mature CGRP mRNA. This band was not detected in F9 cells treated for 6 days with RA (differentiated primitive parietal endoderm-like cells) or in PYS cells (established parietal endoderm-like cell line). During RA-induced differentiation of F9 cells, CGRP mRNA levels fell within 24 h after treatment and were almost undetectable after 2 days. RA treatment also reduced CGRP secretion by F9 cells; the effect was maximal at 3 days and remained stable thereafter. Similarly, RA rapidly reduced adenylate cyclase responsiveness to chicken CGRP (cCGRP) and human CGRP (hCGRP). An 80% fall in cAMP release into the culture medium in the presence of CGRP was observed after 24 h of RA treatment. These results demonstrate that RA rapidly abolishes the CGRP autocrine system involved in the proliferation of F9 cells, at the same time inducing their differentiation into primitive parietal endoderm. They point to the interaction between retinoic acid and growth factors in the regulation of cell proliferation and differentiation. J. Cell. Biochem. 64:447–457. © 1997 Wiley-Liss, Inc.     

Cloning and characterization of keratin D,a murine endodermal cytoskeletal protein induced during in vitro differentiation of F9 teratocarcinoma cells

Summary We have identified a cDNA coding for the murine keratin D from a collection of clones representing F9 teratocarcinoma stem cell mRNA sequences. These sequences are synthesized specifically after the addition of retinoic acid and cAMP to the culture medium. The clone is 1,382 nucleotides long and contains the entire information for the active polypeptide, the complete 3 end and most, if not all, of the 5 non-coding region. The mRNA is found in hepatocytes, in PYS-2 cells (an endodermal cell line) and in differentiated (retinoic-acid-treated) F9 cells, but not in untreated F9 cells. The length of the mRNA is 1.4 kb, as estimated by Northern blot hybridization. Southern hybridization performed under very stringent conditions detects a single fragment hybridizing strongly with the cloned cDNA, suggesting that the mouse genome contains only one or very few copies of this gene. We present the first complete sequence of a keratin expressed in simple epithelia, i.e. keratin D, and discuss its structural features.     

Phosphorylation of the β1 Integrin Cytoplasmic Domain: Toward an Understanding of Function and Mechanism

As F9 stem cells differentiate into parietal endoderm they form focal adhesion sites. There is a concomitant decrease in the level of phosphorylation of S785 in the cytoplasmic domain of the β1 integrin subunit. Previous transfection studies demonstrate that site-specific mutations at this residue, mimicking different phosphorylation states, can alter the subcellular localization of the subunit in differentiating F9 cells. We now extend these observations in an attempt to substantiate the function of β1 phosphorylation and determine how the phosphorylation levels are regulated. We show that treatment of parietal endoderm with okadaic acid induces an increase in β1 phosphorylation and selective loss of β1 from focal adhesion sites. Using a PCR approach, we identify two phosphatases expressed in parietal endoderm, including PP2A. Using a crosslinking approach, where antibodies are added to live cells, we show that the catalytic subunit of PP2A co-immunoprecipitates with β1. Immunocytochemistry shows PP2A colocalizing to focal adhesion sites with β1. In addition integrin-linked kinase (ILK) co-immunoprecipitates with β1 in parietal endoderm and localizes to focal adhesion sites. Okadaic acid treatment significantly decreases the level of ILK associated with β1. A possible role for regulated β1 phosphorylation in cell migration is discussed.     

A role for p21 (WAF1) in the cAMP-dependent differentiation of F9 teratocarcinoma cells into parietal endoderm

Combined treatment of teratocarcinoma F9 cells with retinoic acid and dibutyryl-cAMP induces the differentiation into cells with a phenotype resembling parietal endoderm. We show that the levels of cyclin-dependent kinase inhibitor p21/WAF1/Cip1 (p21) protein and mRNA are dramatically elevated at the end of this differentiation, concomitantly with the appearance of p21 in the immunoprecipitated CDK2-cyclin E complex. The induction of differentiation markers could not be achieved by expression of ectopic p21 alone and still required treatment with differentiation agents. Clones of F9 cells transfected with sense or antisense p21 cDNA constructs revealed, upon differentiation, upregulated levels of mRNA for thrombomodulin, a parietal endoderm-specific marker, or increased fraction of cells in sub-G1 phase of the cell cycle, respectively. Consistent with this observation, whereas p21 was strictly nuclear in undifferentiated cells, a large proportion of differentiated cells had p21 localized also in the cytoplasm, a site associated with the antiapoptotic function of p21. Furthermore, p21 activated the thrombomodulin promoter in transient reporter assays and the p21 mutant defective in binding to cyclin E was equally efficient in activation. The promoter activity in differentiated cells was reduced by cotransfection of p21-specific siRNA or antisense cDNA. Coexpression of p21 increased the activity of the GAL-p300(1-1303) fusion protein on the GAL sites-containing TM promoter. This implies that p21 might act through a derepression of the p300 N-terminal-residing repression domain, thereby enhancing the p300 coactivator function. As differentiation of F9 cells into parietal endoderm-like cells requires the cAMP signaling, the results together suggest that the cyclin-dependent kinase inhibitor p21 may promote specifically this pathway in F9 cells.     

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.     

Modification of response of mouse neuroblastoma cells in culture by serum type

Summary The effect of various types of serum on morphological and biochemical changes in mouse neuroblastoma cells (clone NBP₂) in culture was studied. The extent of spontaneous morphological differentiation varied markedly depending upon the type of serum and was maximal in agammaglobulin calf serum (CS). The extent of morphological differentiation after treatment of cells with cyclic AMP-stimulating agents was also dependent upon serum type and was least pronounced in fetal calf serum. The doubling time and extent of clumping varied with the type, of serum. The activity of tyrosine hydroxylase (TH) in NB cells was dependent upon serum type and it was highest in newborn CS and agammaglobulin CS. Although elevation of intracellular levels of cyclic AMP in NBP₂ clone invariably stimulates neurite formation and TH activity, these functions were increased in certain sera without a significant increase in the cellular cyclic AMP levels. The present study shows that neurite formation, growth rate and TH activity are regulated by more than one mode, one of which is mediated by cyclic AMP. The above changes are independently regulated in the sense that the expression of one can be increased in the absence of others. Preliminary reports of this work were presented at the Symposium on Cell Differentiation and Neoplasia, March 1976; American Society for Neurochemistry, March 1978; and the FASEB meetings, April 1978. This work was supported in part by NIH Grant ROESNS 01576, NIH Training Grant 4007072 and Research Scientist Career Development Award MH 42479.     

A further study on the regulation of cyclic nucleotide phosphodiesterase activity in neuroblastoma cells: Effect of growth

Summary Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) phosphodiesterase activity in mouse neuroblastoma cells in culture markedly increased during exponential growth and reached a maximal level at confluency; whereas guanosine 3′, 5′-cyclic monophosphate (cyclic GMP) phosphodiesterase activity only slightly but significantly increased under a similar experimental condition. The increase in cyclic AMP phosphodiesterase activity was blocked by both cycloheximide and dactinomycin, whereas the increase in cyclic GMP phosphodiesterase was blocked by only cycloheximide. When the confluent cells were replated at low density, the cyclic nucleotide phosphodiesterase activity decreased; however, when they were plated at high cell density which equaled confluency, the enzyme activity did not decrease. Unlike cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity did not change significantly in prostaglandin E₁-treated cells, but decreased in cells treated with the inhibitor of phosphodiesterase. Like cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity also did not change in cells treated with serum-free medium, X-irradiation, sodium butyrate and 6-thioguanine. This work was supported by USPHS NS-09230, and DRG-1273 from Damon Runyon-Walter Winchell Cancer Fund.     

H1 Histone and nucleosome repeat length alterations associated with the in vitro differentiation of murine embryonal carcinoma cells to extra-embryonic endoderm

The histone compositions and average distance between nucleosomes have been determined for F9.22 and PSA1 murine embryonal carcinoma cell lines, for primary extra-embryonic endoderm derived from the in vitro differentiation of PSA1 embryonal carcinoma cells, and for two long-term extra-embryonic endodermal cell lines. A change in the relative proportions of two forms of the H1 histones (H1A and H1B) was found to correlate with the extra-embryonic endodermal differentiated phenotype. The embryonal carcinoma cells had a ratio of H1A/H1B of 1.49 or greater. In contrast, extra-embryonic endoderm from either cell lines or freshly isolated from differentiating embryonal carcinoma cell cultures had a ratio of H1A/H1B of less than 0.9. Partial peptide mapping of gel purified H1A and H1B suggest the two proteins differ in primary structure. The nucleosome repeat length of the embryonal carcinoma cell lines was 196 bp of DNA. Primary extra-embryonic endoderm was found to have a value of 205 bp, but the long-term extra-embryonic endodermal cell lines had an average nucleosome repeat length of 187 bp. Since both freshly isolated primary endoderm and the long-term endodermal cell lines express differentiated functions (basement membrane glycoproteins and plasminogen activator activity), there appears to be no simple correlation between the nucleosome repeat length and the expression of these differentiated functions.     

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.