It is well known that FGF4 and TGFβ/activin along with other undefined factors maintain TS cells in a self-renewing state that includes repression of differentiation (18
). Even though FGF4 activation of ERK1/2 has defined antiapoptotic functions in TS cell renewal (26
), signals promoting repression of differentiation, including trophoblast EMT, have not been characterized. MEKK4 is highly expressed in TS cells and MEKK4 expression is significantly reduced during differentiation, positioning MEKK4 as an important MAP3K in controlling TS cell function. MEKK4 is a 180-kDa serine/threonine protein kinase that interacts with several proteins known to regulate EMT, including GSK3β (1
), Cdc42 and Rac (11
), axin, the negative regulator of β-catenin (17
), and NIK, the NCK-interacting kinase (A. N. Abell and G. L. Johnson, unpublished data). MEKK4 signals to activate both JNK and p38 (1
). Our results clearly show that MEKK4 kinase activity is required for FGF4-stimulated JNK and p38 activities in TS cells. Kinase-inactive MEKK4 TS cells selectively differentiate to spongiotrophoblasts and syncytiotrophoblasts even in the presence of FGF4 and fibroblast-conditioned medium, indicating that MEKK4 signaling is essential for repression of differentiation. A major phenotype of the differentiation response observed with expression of kinase-inactive MEKK4 was EMT characterized by increased Slug, Twist, MMP2, and cathepsin S, loss of E-cadherin expression, and increased invasiveness.
The p38 MAPK has been shown to regulate EMT during gastrulation and is downstream of a signaling pathway involving NIK (28
), which is a MEKK4 binding partner. A p38-interacting protein (p38IP) required for p38 activation in vivo is required for downregulation of E-cadherin during gastrulation. The p38IP was also found to be involved in neural tube closure, with p38IP mutants having exencephaly and spina bifida (28
). Interestingly, MEKK4 kinase-inactive and knockout mice have defects in p38 activation and display both exencephaly and spina bifida (3
). The p38 MAPK is also implicated in tumor cell EMT, particularly in response to transforming growth factor β (TGF-β) and in some cases tumor necrosis factor alpha.
JNK does not appear to be involved in EMT during gastrulation but is implicated as a positive regulator of TGF-β-induced EMT in several cell types (15
). Genetically, it was recently shown that TGF-β-stimulated EMT of primary murine tracheal epithelial cells was markedly blunted in a JNK1−/−
background but not a JNK2−/−
). The role of JNK in positively regulating EMT in different cell types seems to involve AP-1 and the regulation of cadherins and specific Smads (4
). Little is known about the role of JNK in controlling the EMT response in tissue stem cells.
Our results show that JNK is strongly activated by FGF4. TS cell differentiation induced by withdrawal of FGF4 results in a loss of JNK activity. Inhibition of JNK in TS cells by kinase-inactive MEKK4 or by chemical inhibition promotes the selective differentiation of cells to the spongiotrophoblast and syncytiotrophoblast lineages. These data define a clear role for FGF4 activation of JNK in repression of EMT, a JNK function that is clearly different from the positive regulation of TGF-β-initiated EMT responses in cultures of primary tissue epithelial cells and many transformed cell lines. Primary factors in fibroblast-conditioned medium for maintenance of TS cell renewal include TGF-β/activin (10
), which in combination with FGF4 maintain TS cells in an undifferentiated, self-renewing state. This is an unusual function for TGF-β in primary epithelial cells, where TGF-β often inhibits proliferation of primary epithelial cells via a G1
cell cycle arrest program (23
). Transformation can alter this control and bypass the G1
cell cycle arrest in response to TGF-β (23
). Constitutive FGF4 signaling in TS cells has been shown to suppress the growth inhibitory effects of TGF-β/activin, and removal of FGF4 initiates a trophoblast differentiation program involving TGF-β (10
). Mechanistically, loss of MEKK4 kinase activity removes a critical signaling response that is needed for FGF4 to control the response of other cytokines, such as TGF-β/activin. Our findings are consistent with the work of Erlebacher et al., who proposed that FGF4 signaling in TS cells inhibited specific TGF-β responses (10
). Our findings demonstrate that FGF4 inhibition of TS cell differentiation requires MEKK4 activation of JNK. The increased expression of Gcm1 and MMP2 in MEKK4 kinase-inactive TS cells, whose expression is also induced by JNK inhibition, is consistent with a transcriptional repressor function for MEKK4-JNK signaling in FGF4 control of TS cell differentiation.
The function of MEKK4 control of JNK activity in TS cells in vivo is also consistent with our findings that MEKK4 kinase-inactive trophoblasts have an abnormal differentiation response during placentation. Normally, a gradient of FGF4 is generated by the developing embryo that controls trophoblast differentiation. However, in MEKK4 kinase-inactive trophoblasts the TS cells do not signal properly and have a dysregulated differentiation response. MEKK4 expression is highest in the developing embryo and particularly in TS cells, and expression is dramatically diminished during differentiation. High MEKK4 expression in renewing TS cells and loss of expression during differentiation is consistent with the necessary function of MEKK4 as a critical hub kinase in the maintenance of TS cell renewal.
Finally, our results have significant implications in understanding the integrated signaling responses for control of tissue stem cell maintenance, repression of differentiation, and changes in signaling for commitment of stem cells to differentiate. Kinases such as MEKK4 function as hubs that respond to different stimuli for the selective activation of pathways within a signaling network. MEKK4 coordinates the activity of the JNK and p38 modules in TS cells required for stem cell maintenance. It is likely that kinases other than MEKK4 will have similar functions in different tissue stem cells, and such hub kinases will control signal networks that integrate the complex cytokine profiles present in different stem cell niches. Identifying the kinases with hub-like functions such as MEKK4 will allow a rational chemical biology approach for selectively controlling renewal and differentiation of tissue stem cells from different tissues.