Different properties of SEK1 and MKK7 in dual phosphorylation of stress-induced activated protein kinase SAPK/JNK in embryonic stem cells

Stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK), belonging to the mitogen-activated protein kinase family, plays an important role in stress signaling. SAPK/JNK activation requires the phosphorylation of both Thr and Tyr residues in its Thr-Pro-Tyr motif, and SEK1 and MKK7 have been identified as the dual specificity kinases. In this study, we generated mkk7(-/-) mouse embryonic stem (ES) cells in addition to sek1(-/-) cells and compared the two kinases in terms of the activation and phosphorylation of JNK. Although SAPK/JNK activation by various stress signals was markedly impaired in both sek1(-/-) and mkk7(-/-) ES cells, there were striking differences in the dual phosphorylation profile. The severe impairment observed in mkk7(-/-) cells was accompanied by a loss of the Thr phosphorylation of JNK without marked reduction in its Tyr-phosphorylated level. On the other hand, Thr phosphorylation of JNK in sek1(-/-) cells was also attenuated in addition to a decreased level of its Tyr phosphorylation. Analysis in human embryonic kidney 293T cells transfected with a kinase-dead SEK1 or a Thr-Pro-Phe mutant of JNK1 revealed that SEK1-induced Tyr phosphorylation of JNK1 was followed by additional Thr phosphorylation by MKK7. Furthermore, SEK1 but not MKK7 was capable of binding to JNK1 in 293T cells. These results indicate that the Tyr and Thr residues of SAPK/JNK are sequentially phosphorylated by SEK1 and MKK7, respectively, in the stress-stimulated ES cells.     

Essential function of PTP-PEST during mouse embryonic vascularization, mesenchyme formation, neurogenesis and early liver development

PTP (protein-tyrosine phosphatase)-PEST is a ubiquitously expressed cellular regulator of integrin signalling. It has been shown to bind several molecules such as Shc, paxillin and Grb2, that are involved downstream of FAK (focal adhesion kinase) pathway. Through its specific association to p130cas and further dephosphorylation, PTP-PEST plays a critical role in cell-matrix interactions, which are essential during embryogenesis. We report here that ablation of the gene leads to early embryonic lethality, correlating well with the high expression of the protein during embryonic development. We observed an increased level of tyrosine phosphorylation of p130cas protein in E9.5 PTP-PEST(-/-) embryos, a first evidence of biochemical defect leading to abnormal growth and development. Analysis of null mutant embryos revealed that they reach gastrulation, initiate yolk sac formation, but fail to progress through normal subsequent developmental events. E9.5-10.5 PTP-PEST(-/-) embryos had morphological abnormalities such as defective embryo turning, improper somitogenesis and vasculogenesis, impaired liver development, accompanied by degeneration in both neuroepithelium and somatic epithelia. Moreover, in embryos surviving until E10.5, the caudal region was truncated, with severe mesenchyme deficiency and no successful liver formation. Defects in embryonic mesenchyme as well as subsequent failure of proper vascularization, liver development and somatogenesis, seemed likely to induce lethality at this stage of development, and these results confirm that PTP-PEST plays an essential function in early embryogenesis.     

Perturbation of the hematopoietic system during embryonic liver development due to disruption of polyubiquitin gene Ubc in mice

Disruption of the polyubiquitin gene Ubc leads to a defect in fetal liver development, which can be partially rescued by increasing the amount of ubiquitin. However, it is still not known why Ubc is required for fetal liver development and the nature of the defective cell types responsible for embryonic lethality have not been characterized. In this study, we assessed the cause of embryonic lethality with respect to the fetal liver hematopoietic system. We found that Ubc was highly expressed in the embryonic liver, and the proliferation capacity of fetal liver cells was reduced in Ubc(-/-) embryos. Specifically, Ubc was most highly expressed in hematopoietic cells, and the proliferation capacity of hematopoietic cells was significantly impaired in Ubc(-/-) embryos. While hematopoietic cell and hematopoietic stem cell (HSC) frequency was maintained in Ubc(-/-) embryos, the absolute number of these cells was diminished because of reduced total liver cell number in Ubc(-/-) embryos. Transplantations of fetal liver cells into lethally irradiated recipient mice by non-competitive and competitive reconstitution methods indicated that disruption of Ubc does not significantly impair the intrinsic function of fetal liver HSCs. These findings suggest that disruption of Ubc reduces the absolute number of HSCs in embryonic livers, but has no significant effect on the autonomous function of HSCs. Thus, the lethality of Ubc(-/-) embryos is not the result of intrinsic HSC failure.     

A unique death pathway keeps RIPK1 D325A mutant mice in check at embryonic day 10.5

Tumor necrosis factor receptor-1 (TNFR1) signaling, apart from its pleiotropic functions in inflammation, plays a role in embryogenesis as deficiency of varieties of its downstream molecules leads to embryonic lethality in mice. Caspase-8 noncleavable receptor interacting serine/threonine kinase 1 (RIPK1) mutations occur naturally in humans, and the corresponding D325A mutation in murine RIPK1 leads to death at early midgestation. It is known that both the demise of Ripk1^D325A/D325A embryos and the death of Casp8^−/− mice are initiated by TNFR1, but they are mediated by apoptosis and necroptosis, respectively. Here, we show that the defects in Ripk1^D325A/D325A embryos occur at embryonic day 10.5 (E10.5), earlier than that caused by Casp8 knockout. By analyzing a series of genetically mutated mice, we elucidated a mechanism that leads to the lethality of Ripk1^D325A/D325A embryos and compared it with that underlies Casp8 deletion-mediated lethality. We revealed that the apoptosis in Ripk1^D325A/D325A embryos requires a scaffold function of RIPK3 and enzymatically active caspase-8. Unexpectedly, caspase-1 and caspase-11 are downstream of activated caspase-8, and concurrent depletion of Casp1 and Casp11 postpones the E10.5 lethality to embryonic day 13.5 (E13.5). Moreover, caspase-3 is an executioner of apoptosis at E10.5 in Ripk1^D325A/D325A mice as its deletion extends life of Ripk1^D325A/D325A mice to embryonic day 11.5 (E11.5). Hence, an unexpected death pathway of TNFR1 controls RIPK1 D325A mutation-induced lethality at E10.5.

A study of mice expressing a caspase-8 non-cleavable RIPK1 mutant during embryonic development reveals an unexpected TNFR1-triggered death pathway involving RIPK3, caspase-8, and caspases -1, -11 and -3.     

Embryonic lethality and fetal liver apoptosis in mice lacking all three small Maf proteins

Embryogenesis is a period during which cells are exposed to dynamic changes of various intracellular and extracellular stresses. Oxidative stress response genes are regulated by heterodimers composed of Cap'n'Collar (CNC) and small Maf proteins (small Mafs) that bind to antioxidant response elements (ARE). Whereas CNC factors have been shown to contribute to the expression of ARE-dependent cytoprotective genes during embryogenesis, the specific contribution of small Maf proteins to such gene regulation remains to be fully examined. To delineate the small Maf function in vivo, in this study we examined mice lacking all three small Mafs (MafF, MafG, and MafK). The small Maf triple-knockout mice developed normally until embryonic day 9.5 (E9.5). Thereafter, however, the triple-knockout embryos showed severe growth retardation and liver hypoplasia, and the embryos died around E13.5. ARE-dependent cytoprotective genes were expressed normally in E10.5 triple-knockout embryos, but the expression was significantly reduced in the livers of E13.5 mutant embryos. Importantly, the embryonic lethality could be completely rescued by transgenic expression of exogenous MafG under MafG gene regulatory control. These results thus demonstrate that small Maf proteins are indispensable for embryonic development after E9.5, especially for liver development, but early embryonic development does not require small Mafs.     

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is required for branching morphogenesis in the chorioallantoic placenta

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-associated Kunitz-type serine proteinase inhibitor that was initially identified as a potent inhibitor of hepatocyte growth factor activator. HAI-1 is also a cognate inhibitor of matriptase, a membrane-associated serine proteinase. HAI-1 is expressed predominantly in epithelial cells in the human body. Its mRNA is also abundant in human placenta, with HAI-1 specifically expressed by villous cytotrophoblasts. In order to address the precise roles of HAI-1 in vivo, we generated HAI-1 mutant mice by homozygous recombination. Heterozygous HAI-1+/- mice underwent normal organ development. However, homozygous HAI-1-/- mice experienced embryonic lethality which became evident at embryonic day 10.5 postcoitum (E10.5). As early as E9.5, HAI-1-/- embryos showed growth retardation that did not reflect impaired cell proliferation but resulted instead from failed placental development and function. Histological analysis revealed severely impaired formation of the labyrinth layer, in contrast all other placental layers, such as the spongiotrophoblast layer and giant cell layer, which were formed. Our results indicate that mouse HAI-1 is essential for branching morphogenesis in the chorioallantoic placenta and lack of HAI-1 function may result in placental failure.     

Differential regulation of HSP70 expression by the JNK kinases SEK1 and MKK7 in mouse embryonic stem cells treated with cadmium

JNK, a member of the mitogen-activated protein kinases (MAPKs), is activated by the MAPK kinases SEK1 and MKK7 in response to environmental stresses. In the present study, the effects of CdCl2 treatment on MAPK phosphorylation and HSP70 expression were examined in mouse embryonic stem (ES) cells lacking the sek1 gene, the mkk7 gene, or both. Following CdCl2 exposure, the phosphorylation of JNK, p38, and ERK was suppressed in sek1-/- mkk7-/- cells. When sek1-/- or mkk7-/- cells were treated with CdCl2, JNK phosphorylation, but not the phosphorylation of either p38 or ERK, was markedly reduced, while a weak reduction in p38 phosphorylation was observed in sek1-/- cells. Thus, both SEK1 and MKK7 are required for JNK phosphorylation, whereas their role in p38 and ERK phosphorylation could overlap with that of another kinase. We also observed that CdCl2-induced HSP70 expression was abolished in sek1-/- mkk7-/- cells, was reduced in sek1-/- cells, and was enhanced in mkk7-/- cells. Similarly, the phosphorylation of heat shock factor 1 (HSF1) was decreased in sek1-/- mkk7-/- and sek1-/- cells, but was increased in mkk7-/- cells. Transfection with siRNA specific for JNK1, JNK2, p38, ERK1, or ERK2 suppressed CdCl2-induced HSP70 expression. In contrast, silencing of p38 or p38 resulted in further accumulation of HSP70 protein. These results suggest that HSP70 expression is up-regulated by SEK1 and down-regulated by MKK7 through distinct MAPK isoforms in mouse ES cells treated with CdCl2.     

Physiological roles of SAPK/JNK signaling pathway

Stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK) is activated by many types of cellular stresses and extracellular signals. Recent studies, including the analysis with knockout mice, have led to progress towards understanding the physiological roles of SAPK/JNK activation in embryonic development in addition to immune responses. SAPK/JNK activation plays essential roles in organogenesis during mouse development by regulating cell survival, apoptosis, and proliferation. Two SAPK/JNK activators, SEK1 and MKK7, are required for fetal liver formation and full activation of SAPK/JNK, which responds to various stimuli in an all-or-none manner. This article focuses on physiological roles of SAPK/JNK activation in fetal liver formation and in apoptosis regulation.     

Stress induces mitochondria-mediated apoptosis independent of SAPK/JNK activation in embryonic stem cells

SAPK/JNK, which belongs to the family of mitogen-activated protein kinase (MAPK), is activated by many types of cellular stresses or extracellular signals and is involved in embryonic development, immune responses, and cell survival or apoptosis. However, the physiological roles of SAPK/JNK in the signaling of stress-induced apoptosis are still controversial. To evaluate the precise function, SAPK/JNK-inactivated mouse embryonic stem (ES) cells were generated by disrupting genes of the MAPK activators, SEK1 and MKK7. Although SAPK/JNK activation by various stresses was completely abolished in sek1(-/-) mkk7(-/-) ES cells, apoptotic responses including DNA fragmentation and caspase 3 activation still occurred normally, which displays a sharp contrast to apaf1(-/-) ES cells exhibiting profound defects in the mitochondria-dependent apoptosis. These normal apoptotic responses without SAPK/JNK activation were also observed in fibroblasts derived from sek1(-/-) mkk7(-/-) ES cells. Instead, interleukin-1 beta (IL-1 beta)-induced IL-6 gene expression was greatly suppressed in sek1(-/-) mkk7(-/-) fibroblasts. These results clearly show that SAPK/JNK activation is responsible for the inflammatory cytokine-induced gene expression but not essentially required for the mitochondria-dependent apoptosis at least in ES or fibroblast-like cells, which are prototypes of all cell lineages.     

MKK7 couples stress signalling to G2/M cell-cycle progression and cellular senescence

During the development of multicellular organisms, concerted actions of molecular signalling networks determine whether cells undergo proliferation, differentiation, death or ageing. Here we show that genetic inactivation of the stress signalling kinase, MKK7, a direct activator of JNKs in mice, results in embryonic lethality and impaired proliferation of hepatocytes. Beginning at passage 4-5, mkk7(-/-) mouse embryonic fibroblasts (MEFs) display impaired proliferation, premature senescence and G2/M cell cycle arrest. Similarly, loss of c-Jun or expression of a c-JunAA mutant in which the JNK phosphorylation sites were replaced with alanine results in a G2/M cell-cycle block. The G2/M cell-cycle kinase CDC2 was identified as a target for the MKK7-JNK-c-Jun pathway. These data show that the MKK7-JNK-c-Jun signalling pathway couples developmental and environmental cues to CDC2 expression, G2/M cell cycle progression and cellular senescence in fibroblasts.     

Impaired synergistic activation of stress-activated protein kinase SAPK/JNK in mouse embryonic stem cells lacking SEK1/MKK4: different contribution of SEK2/MKK7 isoforms to the synergistic activation.

Stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK), which is a member of the mitogen-activated protein kinase (MAPK) family, plays an important role in a stress-induced signaling cascade. SAPK/JNK activation requires the phosphorylation of Thr and Tyr residues in its Thr-Pro-Tyr motif, and SEK1 (MKK4) and MKK7 (SEK2) have been identified as the upstream MAPK kinases. Here we examined the activation and phosphorylation sites of SAPK/JNK and differentiated the contribution of SEK1 and MKK7alpha1, -gamma1, and -gamma2 isoforms to the MAPK activation. In SEK1-deficient mouse embryonic stem cells, stress-induced SAPK/JNK activation was markedly impaired, and this defect was accompanied with a decreased level of the Tyr phosphorylation. Analysis in HeLa cells co-transfected with the two MAPK kinases revealed that the Thr and Tyr of SAPK/JNK were independently phosphorylated in response to heat shock by MKK7gamma1 and SEK1, respectively. However, MKK7alpha1 failed to phosphorylate the Thr of SAPK/JNK unless its Tyr residue was phosphorylated by SEK1. In contrast, MKK7gamma2 had the ability to phosphorylate both Thr and Tyr residues. In all cases, the dual phosphorylation of the Thr and Tyr residues was essentially required for the full activation of SAPK/JNK. These data provide the first evidence that synergistic activation of SAPK/JNK requires both phosphorylation at the Thr and Tyr residues in living cells and that the preference for the Thr and Tyr phosphorylation was different among the members of MAPK kinases.     

Somatic excision demonstrates that c-Jun induces cellular migration and invasion through induction of stem cell factor

Cancer cells arise through sequential acquisition of mutations in tumor suppressors and oncogenes. c-Jun, a critical component of the AP-1 complex, is frequently overexpressed in diverse tumor types and has been implicated in promoting cellular proliferation, migration, and angiogenesis. Functional analysis of candidate genetic targets using germ line deletion in murine models can be compromised through compensatory mechanisms. As germ line deletion of c-jun induces embryonic lethality, somatic deletion of the c-jun gene was conducted using floxed c-jun (c-jun(f/f)) conditional knockout mice. c-jun-deleted cells showed increased cellular adhesion, stress fiber formation, and reduced cellular migration. The reduced migratory velocity and migratory directionality was rescued by either c-Jun reintroduction or addition of secreted factors from wild-type cells. An unbiased analysis of cytokines and growth factors, differentially expressed and showing loss of secretion upon c-jun deletion, identified stem cell factor (SCF) as a c-Jun target gene. Immunoneutralizing antibody to SCF reduced migration of wild-type cells. SCF addition rescued the defect in cellular adhesion, cellular velocity, directional migration, transwell migration, and cellular invasion of c-jun(-/-) cells. c-Jun induced SCF protein, mRNA, and promoter activity. Induction of the SCF promoter required the c-Jun DNA-binding domain. c-Jun bound to the SCF promoter in chromatin immunoprecipitation assays. Mutation of the c-Jun binding site abolished c-Jun-mediated induction of the SCF promoter. These studies demonstrate an essential role of c-Jun in cellular migration through induction of SCF.