12 research outputs found
Regulation of Ras–MAPK pathway mitogenic activity by restricting nuclear entry of activated MAPK in endoderm differentiation of embryonic carcinoma and stem cells
In response to retinoic acid, embryonic stem and carcinoma cells undergo differentiation to embryonic primitive endoderm cells, accompanied by a reduction in cell proliferation. Differentiation does not reduce the activation of cellular MAPK/Erk, but does uncouple mitogen-activated protein kinase (MAPK) activation from phosphorylation/activation of Elk-1 and results in inhibition of c-Fos expression, whereas phosphorylation of the cytoplasmic substrate p90RSK remains unaltered. Cell fractionation and confocal immunofluorescence microscopy demonstrated that activated MAPK is restricted to the cytoplasmic compartment after differentiation. An intact actin and microtubule cytoskeleton appears to be required for the restriction of MAPK nuclear entry induced by retinoic acid treatment because the cytoskeletal disrupting agents nocodazole, colchicine, and cytochalasin D are able to revert the suppression of c-Fos expression. Thus, suppression of cell proliferation after retinoic acid–induced endoderm differentiation of embryonic stem and carcinoma cells is achieved by restricting nuclear entry of activated MAPK, and an intact cytoskeleton is required for the restraint
Perception of differentiation cues by GATA factors in primitive endoderm lineage determination of mouse embryonic stem cells
AbstractThe formation of the primitive endoderm covering the inner cell mass of early mouse embryos can be simulated in vitro by the differentiation of mouse embryonic stem (ES) cells in culture following either aggregation of suspended cells or stimulation of cell monolayers with retinoic acid. The developmentally regulated transcription factors GATA-4 and GATA-6 have determining role in mouse extraembryonic endoderm development. We analyzed the in vitro differentiation of mouse embryonic stem cells deficient of GATA factors and conclude that GATA-4 is required for ES cells to perceive a cell positioning (cell aggregation) signal and GATA-6 is required to sense morphogenic (retinoic acid) signal. The collaboration between GATA-6 and GATA-4, or GATA-6 and GATA-5 which can substitute for GATA-4, is involved in the perception of differentiation cues by embryonic stem cells in their determination of endoderm lineage. This study indicates that the lineage differentiation of ES cells can be manipulated by the expression of GATA factors
Alteration of Differentiation Potentials by Modulating GATA Transcription Factors in Murine Embryonic Stem Cells
Background. Mouse embryonic stem (ES) cells can be differentiated in vitro by aggregation and/or retinoic acid (RA) treatment. The principal differentiation lineage in vitro is extraembryonic primitive endoderm. Dab2, Laminin, GATA4, GATA5, and GATA6 are expressed in embryonic primitive endoderm and play critical roles in its lineage commitment. Results. We found that in the absence of GATA4 or GATA5, RA-induced primitive endoderm differentiation of ES cells was reduced. GATA4 (−/−) ES cells express higher level of GATA5, GATA6, and hepatocyte nuclear factor 4 alpha marker of visceral endoderm lineage. GATA5 (−/−) ES cells express higher level of alpha fetoprotein marker of early liver development. GATA6 (−/−) ES cells express higher level of GATA5 as well as mesoderm and cardiomyocyte markers which are collagen III alpha-1 and tropomyosin1 alpha. Thus, deletion of GATA6 precluded endoderm differentiation but promoted mesoderm lineages. Conclusions. GATA4, GATA5, and GATA6 each convey a unique gene expression pattern and influences ES cell differentiation. We showed that ES cells can be directed to avoid differentiating into primitive endoderm and to adopt unique lineages in vitro by modulating GATA factors. The finding offers a potential approach to produce desirable cell types from ES cells, useful for regenerative cell therapy
Nuclear Entry of Activated MAPK Is Restricted in Primary Ovarian and Mammary Epithelial Cells
The MAPK/ERK1/2 serine kinases are primary mediators of the Ras mitogenic signaling pathway. Phosphorylation by MEK activates MAPK/ERK in the cytoplasm, and phospho-ERK is thought to enter the nucleus readily to modulate transcription.Here, however, we observe that in primary cultures of breast and ovarian epithelial cells, phosphorylation and activation of ERK1/2 are disassociated from nuclear translocalization and transcription of downstream targets, such as c-Fos, suggesting that nuclear translocation is limited in primary cells. Accordingly, in import assays in vitro, primary cells showed a lower import activity for ERK1/2 than cancer cells, in which activated MAPK readily translocated into the nucleus and activated c-Fos expression. Primary cells express lower levels of nuclear pore complex proteins and the nuclear transport factors, importin B1 and importin 7, which may explain the limiting ERK1/2 import found in primary cells. Additionally, reduction in expression of nucleoporin 153 by siRNA targeting reduced ERK1/2 nuclear activity in cancer cells.ERK1/2 activation is dissociated from nuclear entry, which is a rate limiting step in primary cells and in vivo, and the restriction of nuclear entry is disrupted in transformed cells by the increased expression of nuclear pores and/or nuclear transport factors
Recommended from our members
Disassociation of MAPK Activation and c-Fos Expression in F9 Embryonic Carcinoma Cells following Retinoic Acid-induced Endoderm Differentiation
Recommended from our members