Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture

Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation

Marsupial milk : identifying signals for regulating mammary function and development of the young

The role of milk in providing nutrition for the young is well established. However, it is becoming apparent that milk has a more comprehensive role in programming and regulating growth and development of the suckled young, and an autocrine impact on the mammary gland so that it functions appropriately during the lactation cycle. This central role of milk is best studied in animal models, such as marsupials that have evolved a different lactation strategy to eutherians and allow researchers to more easily identify regulatory mechanisms that are not as readily apparent in eutherian species. For example, the tammar wallaby (Macropus eugenii) has evolved with a unique reproductive strategy of a short gestation, birth of an altricial young and a relatively long lactation during which the mother progressively changes the composition of the major, and many of the minor components of milk. Thus, in contrast to eutherians, there is a far greater investment in development of the young during lactation and it is likely that many of the signals that regulate development of eutherian embryos in utero are delivered by the milk. This requires the co-ordinated development and function of the mammary gland. Inappropriate timing of these signalling events in mammals may result in either limited or abnormal development of the young, and potentially a higher incidence of mature onset disease. The tammar is emerging as an attractive model to better understand the role of milk factors in these processes

The role of ERK1/2, Src Family Kinases and p38 MAPK in the maintenance of EPL cells.

<p><b>A-D.</b> Photomicrographs of mES cells cultured in MEDII for 2 days and subsequently in MEDII containing medium supplemented with DMSO (A), 1 μM PD0325901 (B) 10 μM PP2 (C) and 10 μM SB203580 (D). Cells were stained for alkaline phosphatase activity (red/purple stain). Scale bar = 200 μm. <b>E, F.</b> mES cells were cultured in MEDII for 2 days and subsequently in MEDII containing medium supplemented with DMSO (■), DMSO with 1 μM PD0325901 (E, □), DMSO with 10 μM PP2 (F, □) or DMSO with 10 μM SB203580 (G, □). RNA from these cells was analyzed for expression of <i>Oct4</i>, <i>Sox2</i>, <i>Nanog</i>, <i>Rex1</i>, <i>Spp1</i>, <i>Gbx2</i>, <i>Dnmt3b</i> and <i>Otx2</i> by real-time PCR. Expression was normalized to <i>β-actin</i>. Error bars represent SEM; n = 3. EPL cells cultured in MEDII with inhibitor were compared to EPL cells in MEDII with DMSO, ** <i>p</i> ≤ 0.05, or mES cells, # <i>p</i>≤ 0.05.</p

Inhibition of MEK1 prevents the formation of EPL cells in response to MEDII.

<p>A. mES cells were pre-treated with 1 μM PD0325901 for 60 minutes. 200 μM l-proline was added, as denoted, and the cells incubated for a further 60 minutes. Cells were collected and analysed by western blot for the presence of phosphorylated ERK1 or ERK2. Total ERK1/2 was used as a loading control. The intensity of the pERK2 band was measured using Quantity One software (BioRad) and represented as a proportion of total ERK1/2. Error bars represent SEM; n = 4; * <i>p</i> ≤ 0.05 when compared to mES cells. B-C. mES cells were cultured in MEDII- and DMSO-containing medium (B) and MEDII- and 1μM PD0325901-contianing medium (C) for 3 days. Scale bar = 200 μm. D. MEDII- and DMSO-containing medium (■) and MEDII- and 1μM PD0325901-contianing medium (□) for 3 days. RNA from these cells was analyzed for expression of <i>Oct4</i>, <i>Sox2</i>, <i>Nanog</i>, <i>Rex1</i>, <i>Spp1</i>, <i>Gbx2</i>, <i>Dnmt3b</i>, <i>Otx2</i> and <i>Fgf5</i> by real-time PCR. Expression was normalized to <i>β-actin</i> and expressed relative to mES cells (<i>Fgf5</i> has been expressed relative to MEDII + DMSO). Error bars represent SEM; n = 3. mES cells + MEDII + PD0325901 were compared to mES cells + MEDII + DMSO (** p ≤ 0.05) or mES cells (# p ≤ 0.05).</p

The regulation of progression of the pluripotent lineage in culture.

<p>The cell states represented in vitro, naïve mES cells, primed mES cells and EPL cells have been aligned with the expression of Nanog and Otx2 and with their deduced intracellular signaling activity. Inducers of lineage progression are shown in orange; Calcineurin exerts its effects through dephosphorylation of NFAT and promotes NFAT translocation to the nucleus.</p

Summary of inhibitors used in this study.

<p>Summary of inhibitors used in this study.</p

The role of p38 MAPK in the formation of EPL cells in response to MEDII.

<p><b>A.</b> mES cells were pre-treated with 10 μM SB203580 for 60 minutes. 200 μM l-proline was added, as denoted and the cells incubated for a further 60 minutes. Cells were collected and analysed by western blot for the presence of phosphorylated pHspb2. Total Hspb2 was used as a loading control. <b>B, C.</b> mES cells were cultured medium supplemented with MEDII and DMSO (A) or MEDII and 10 μM SB203580 for 3 days. Scale bar = 200 μm. <b>D.</b> mES cells were cultured in medium supplemented with MEDII and DMSO (■) or MEDII and 10 μM SB203580 (□) for 3 days. RNA from these cells was analyzed for expression of <i>Oct4</i>, <i>Sox2</i>, <i>Nanog Rex1</i>, <i>Spp1</i>, <i>Gbx2</i>, <i>Dnmt3b</i>, <i>Otx2</i> and <i>Fgf5</i> by real-time PCR. Expression was normalized to <i>β-actin</i> and expressed relative to mES cells (<i>Fgf5</i> has been expressed relative to MEDII + DMSO). Error bars represent SEM; n = 4. mES cells + MEDII + SB203580 were compared to mES cells + MEDII + DMSO (** p ≤ 0.05) or mES cells (# p ≤ 0.05). <b>E.</b> mES cells were cultured in ES cell medium supplemented with MEDII, DMSO or MEDII and 10 μM 10 μM SB203580 for 3 days and formed into EBs. EBs were collected on days 2, 3 and 4, RNA was isolated and analyzed for expression of <i>T</i> and <i>Gapdh</i> by RT-PCR. n = 3.</p