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

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

Cell autonomous sorting and surface positioning in the formation of primitive endoderm in embryoid bodies

The differentiation and formation of the primitive endoderm in early embryos can be mimicked in vitro by the aggregation of embryonic stem cells to form embryoid bodies. We present morphological evidence that primitive endoderm cells often first locate in the interior of embryoid bodies and subsequently migrate to the surface. Cell mixing experiments indicate that surface positioning is an intrinsic property of endoderm epithelial cells. Moreover, Disabled-2 (Dab2) is required for surface sorting and positioning of the endoderm cells: when Dab2 expression was eliminated, the differentiated endoderm epithelial cells distributed throughout the interior of the embryoid bodies. Surprisingly, E-cadherin is dispensable for primitive endoderm differentiation and surface sorting in embryoid bodies. These results support the model that primitive endoderm cells first emerge in the interior of the inner cell mass and are subsequently sorted to the surface to form the primitive endoderm