Coordinate Gα13 and Wnt6-β-catenin signaling in F9 embryonal carcinoma cells is required for primitive endoderm differentiation

2009 ◽  
Vol 87 (4) ◽  
pp. 567-580 ◽  
Author(s):  
Roman Krawetz ◽  
Gregory M. Kelly
Keyword(s):  
Retinoic Acid ◽  
Epithelial To Mesenchymal Transition ◽  
Embryonal Carcinoma ◽  
Embryonal Carcinoma Cell ◽  
Primitive Endoderm ◽  
F9 Cells ◽  
Mesenchymal Transition ◽  
Signaling Axis ◽  
Parietal Endoderm ◽  
Constitutively Active

The mouse F9 embryonal carcinoma cell line is ideally suited to study the epithelial-to-mesenchymal transition accompanying the differentiation of primitive to parietal extraembryonic endoderm. In F9 cells, the application of exogenous agents including retinoic acid or activation of signal transduction cascades downstream of G-proteins triggers widespread changes in gene expression and leads to the formation of primitive endoderm. The epithelial-to-mesenchymal transition is completed and parietal endoderm develops as of result of increasing PKA activity in primitive endoderm cells. Expression of a constitutively active form of Gα13(Q226L) is sufficient to induce F9 cells into parietal endoderm and a model is emerging that a signaling axis linking G-protein signaling to RhoA and the ERM protein moesin is required for differentiation. In this study, we found that expression of either p115RhoGEF or a constitutively active, GTPase-deficient form of RhoA(L63) promoted primitive, but not parietal, endoderm formation. The overexpression of Gα13(Q226L) or p115RhoGEF, but not Rho(L63), caused β-catenin to translocate to the nucleus. Surprisingly, the stimulation of the Wnt-β-catenin pathway was accompanied by nuclear β-catenin and primitive endoderm formation, even when a dominant negative was used to block the signaling axis at the level of p115RhoGEF or when ROCK activity was inhibited using the pharmacological agent Y-27632. Together, results indicate that the coordinate signaling by two independent pathways, one involving canonical Wnt-β-catenin activation of target genes and the other with Gα13 signaling to ERM proteins to modulate cytoarchitectural changes, is required during the retinoic acid induced differentiation of F9 cells to primitive endoderm.

Expression of the intracisternal A-particle is elevated during differentiation of embryonal carcinoma cells

1986 ◽  
Vol 6 (1) ◽  
pp. 150-157 ◽  
Author(s):  
C C Howe ◽  
G C Overton
Keyword(s):  
Retinoic Acid ◽  
Embryonal Carcinoma ◽  
Cell Types ◽  
Endogenous Retrovirus ◽  
Cdna Clones ◽  
Embryonal Carcinoma Cell ◽  
Proviral Sequence ◽  
F9 Cells ◽  
Embryonal Carcinoma Cell Line ◽  
Intracisternal A Particle

Three cDNA clones coding for the 3' region of the intracisternal A-particle (IAP), a mouse endogenous retrovirus, were isolated during screening of a library for genes whose expression was modulated during the retinoic acid-induced differentiation of the embryonal carcinoma cell line F9 into parietal endoderm-like (PE-like) cells. In contrast to previously reported results, no IAP transcripts were detected in either F9 cells or two pluripotent cell lines tested. Instead, IAP transcripts as well as IAPs were abundant in the PE-like cells PYS-2 and F9AcCl 9 and in retinoic acid-induced F9 cells but not in the other differentiated cell types of teratocarcinoma origin which were examined. A comparison of the nucleotide sequences of the three IAP cDNA clones with a genomically integrated proviral sequence (MIA14) demonstrated heterogeneity in both length and sequence among the clones. The position of the poly(A) addition site was determined to be 15 nucleotides from the proposed poly(A) addition signal and to occur after the sequence CAGA, not CA, as previously proposed. Length heterogeneity was greatest in a region of TC repeats 80 base pairs 5' to the poly(A) addition site. Additionally, the putative TATAA box found in MIA14 was deleted in the cDNA clones and in the long terminal repeat regions from two other genomic clones examined. The heterogeneity evident among the cDNA clones further demonstrated that at least two distinct IAP genes are activated during differentiation. An analysis of the rate of transcription in isolated nuclei indicated that the activation of expression of IAP genes in PE-like cells is the result of transcriptional regulation. Together, these observations suggest that the modulation of IAP transcription is regulated autonomously rather than by the fortuitous integration of an IAP sequence adjacent to a developmentally regulated cellular gene.

scholarly journals Expression of the intracisternal A-particle is elevated during differentiation of embryonal carcinoma cells.

1986 ◽  
Vol 6 (1) ◽  
pp. 150-157 ◽  
Author(s):  
C C Howe ◽  
G C Overton
Keyword(s):  
Retinoic Acid ◽  
Embryonal Carcinoma ◽  
Cell Types ◽  
Endogenous Retrovirus ◽  
Cdna Clones ◽  
Embryonal Carcinoma Cell ◽  
Proviral Sequence ◽  
F9 Cells ◽  
Embryonal Carcinoma Cell Line ◽  
Intracisternal A Particle

Three cDNA clones coding for the 3' region of the intracisternal A-particle (IAP), a mouse endogenous retrovirus, were isolated during screening of a library for genes whose expression was modulated during the retinoic acid-induced differentiation of the embryonal carcinoma cell line F9 into parietal endoderm-like (PE-like) cells. In contrast to previously reported results, no IAP transcripts were detected in either F9 cells or two pluripotent cell lines tested. Instead, IAP transcripts as well as IAPs were abundant in the PE-like cells PYS-2 and F9AcCl 9 and in retinoic acid-induced F9 cells but not in the other differentiated cell types of teratocarcinoma origin which were examined. A comparison of the nucleotide sequences of the three IAP cDNA clones with a genomically integrated proviral sequence (MIA14) demonstrated heterogeneity in both length and sequence among the clones. The position of the poly(A) addition site was determined to be 15 nucleotides from the proposed poly(A) addition signal and to occur after the sequence CAGA, not CA, as previously proposed. Length heterogeneity was greatest in a region of TC repeats 80 base pairs 5' to the poly(A) addition site. Additionally, the putative TATAA box found in MIA14 was deleted in the cDNA clones and in the long terminal repeat regions from two other genomic clones examined. The heterogeneity evident among the cDNA clones further demonstrated that at least two distinct IAP genes are activated during differentiation. An analysis of the rate of transcription in isolated nuclei indicated that the activation of expression of IAP genes in PE-like cells is the result of transcriptional regulation. Together, these observations suggest that the modulation of IAP transcription is regulated autonomously rather than by the fortuitous integration of an IAP sequence adjacent to a developmentally regulated cellular gene.

scholarly journals Sox7 Plays Crucial Roles in Parietal Endoderm Differentiation in F9 Embryonal Carcinoma Cells through Regulating Gata-4 and Gata-6 Expression

2004 ◽  
Vol 24 (23) ◽  
pp. 10492-10503 ◽  
Author(s):  
Sugiko Futaki ◽  
Yoshitaka Hayashi ◽  
Tomomi Emoto ◽  
Charles N. Weber ◽  
Kiyotoshi Sekiguchi
Keyword(s):  
Transcription Factors ◽  
Retinoic Acid ◽  
Embryonal Carcinoma ◽  
Carcinoma Cells ◽  
Embryonal Carcinoma Cells ◽  
F9 Cells ◽  
Endoderm Differentiation ◽  
Parietal Endoderm ◽  
F9 Embryonal Carcinoma Cells ◽  
Laminin 1

ABSTRACT During early rodent development, the parietal endoderm appears from an inner cell mass and produces large amounts of basement membrane components, such as laminin-1 and collagen IV. To elucidate the regulatory network for gene expression during these procedures, we constructed a series of short interfering RNA expression vectors targeted to various transcription factors, transfected them into F9 embryonal carcinoma cells, and evaluated the effects of the gene silencing on the induction of parietal endoderm differentiation and basement membrane component production by treating F9 cells with all trans-retinoic acid and dibutyryl cyclic AMP. Among the transcription factors tested, silencing of Sox7 or combined silencing of Gata-4 and Gata-6 resulted in suppression of cell shape changes and laminin-1 production, which are the hallmarks of parietal endoderm differentiation. In cells silenced for Sox7, induction of Gata-4 and Gata-6 by retinoic acid and cyclic AMP treatment was inhibited, while induction of Sox7 was not affected in cells silenced for Gata-4 and Gata-6, indicating that Sox7 is an upstream regulatory factor for these Gata factors. Nevertheless, silencing of Sox7 did not totally cancel the action of retinoic acid, since upregulation of coup-tf2, keratin 19, and retinoic acid receptor β2 was not abolished in Sox7-silenced F9 cells. Although overexpression of Sox7 alone was insufficient to induce parietal endoderm differentiation, overexpression of Gata-4 or Gata-6 in Sox7-silenced F9 cells restored the differentiation into parietal endoderm. Sox7 is therefore required for the induction of Gata-4 and Gata-6, and the interplay among these transcription factors plays a crucial role in parietal endoderm differentiation.

Interaction of several related GC-box- and GT-box-binding proteins with the intronic enhancer is required for differential expression of the gb110 gene in embryonal carcinoma cells

1994 ◽  
Vol 14 (9) ◽  
pp. 5786-5793
Author(s):  
L Hamann ◽  
K U Bayer ◽  
K Jensen ◽  
K Harbers
Keyword(s):  
Transcription Factor ◽  
Binding Proteins ◽  
Embryonal Carcinoma ◽  
Regulatory Elements ◽  
Carcinoma Cells ◽  
Minor Importance ◽  
Embryonal Carcinoma Cells ◽  
Enhancer Element ◽  
F9 Cells ◽  
Parietal Endoderm

The molecular mechanisms by which expression of a gene is down-regulated after differentiation of F9 embryonal carcinoma cells into parietal endoderm-like cells was studied by characterizing the cis- and trans-regulatory elements of the gb110 gene. This gene encodes a putative RNA helicase, and its expression is down-regulated when F9 cells are differentiated with retinoic acid and cyclic AMP. The 5'-flanking region of the gene has all of the features of a GC-rich island promoter and seems to play only a minor role, if any, in the regulated expression. A 133-bp enhancer in the first intron was identified by transient chloramphenicol acetyltransferase assays that activated expression in undifferentiated F9 cells about 50- to 100-fold. As this enhancer was not active in differentiated F9 cells, it seems to be the prime mediator of the differentiation-specific down-regulation of the gb110 gene. Four different protein-binding sites, three of which contain GC- and GT-box motifs, were identified in the enhancer element. The fourth site, interacting with previously described transcription factor FTZ-F1/ELP, seems to be of minor importance for the activity of the enhancer. Mutational analysis showed that the cooperative interaction of several most likely related proteins with the three GC- and GT-box motifs was required for full enhancer activity. On the basis of their binding properties, at least two of these proteins seem to be identical or closely related to ubiquitous transcription factor Sp1. One of the GT-box-binding proteins was present in undifferentiated F9 cells but not, however, in its differentiated derivatives. The cell specificity of this transcription factor explains why the gb110 gene is not expressed or expressed only at low levels in parietal endoderm-like cells.

Control of beta-interferon expression in murine embryonal carcinoma F9 cells

1989 ◽  
Vol 9 (8) ◽  
pp. 3553-3556
Author(s):  
M K Francis ◽  
J M Lehman
Keyword(s):  
Retinoic Acid ◽  
Embryonal Carcinoma ◽  
Biologically Active ◽  
Rnase Protection ◽  
F9 Cells ◽  
Differentiated Cells ◽  
Transcriptional Regulatory Mechanism ◽  
Transcriptional Regulatory ◽  
Tissue Culture Model

Murine embryonal carcinoma F9 cells, a tissue culture model for early embryonic development, do not produce interferon (IFN) in response to poly(I-C), as determined by an antiviral assay. RNase protection analyses were used to examine total RNA extracted from the cells for the presence of beta-IFN RNA. Whereas F9 cells differentiated in vitro with retinoic acid produced a biologically active protein as well as beta-IFN RNA in response to poly(I-C), undifferentiated F9 cells produced no detectable beta-IFN RNA even in the presence of cycloheximide, an IFN-superinducing agent. These results show that undifferentiated embryonal carcinoma cells do not accumulate beta-IFN RNA in response to an IFN-inducing agent, suggesting a transcriptional regulatory mechanism. However, this control mechanism is altered upon differentiation, since the gene can be transcriptionally activated in retinoic acid-differentiated cells.

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