Ablation of MEKK4 Kinase Activity Causes Neurulation and Skeletal Patterning Defects in the Mouse Embryo

Abstract

Skeletal disorders and neural tube closure defects represent clinically significant human malformations. The signaling networks regulating normal skeletal patterning and neurulation are largely unknown. Targeted mutation of the active site lysine of MEK kinase 4 (MEKK4) produces a kinase-inactive MEKK4 protein (MEKK4 K1361R ). Embryos homozygous for this mutation die at birth as a result of skeletal malformations and neural tube defects. Hindbrains of exencephalic MEKK4 K1361R embryos show a striking increase in neuroepithelial cell apoptosis and a dramatic loss of phosphorylation of MKK3 and -6, mitogen-activated protein kinase kinases (MKKs) regulated by MEKK4 in the p38 pathway. Phosphorylation of MAPK-activated protein kinase 2, a p38 substrate, is also inhibited, demonstrating a loss of p38 activity in MEKK4 K1361R embryos. In contrast, the MEK1/2-extracellular signal-regulated kinase 1 (ERK1)/ERK2 and MKK4-Jun N-terminal protein kinase pathways were unaffected. The p38 pathway has been shown to regulate the phosphorylation and expression of the small heat shock protein HSP27. Compared to the wild type, MEKK4 K1361R fibroblasts showed significantly reduced phosphorylation of p38 and HSP27, with a corresponding heat shock-induced instability of the actin cytoskeleton. Together, these data demonstrate MEKK4 regulation of p38 and that substrates downstream of p38 control cellular homeostasis. The findings are the first demonstration that MEKK4-regulated p38 activity is critical for neurulation.Mitogen-activated protein kinases (MAPKs) can be divided into five distinct groups that include extracellular signal-regulated kinase 1 (ERK1) and ERK2, c-Jun N-terminal kinase (JNK), p38, ERK3 and ERK4, and ERK5 (10, 34). Each MAPK is activated by MAPK kinases (MAP2Ks). ERK1 and ERK2 are phosphorylated by MEK1 and MEK2 and are primarily activated by growth-promoting stimuli. JNK is activated by its MAP2Ks, MAPK kinase 4 (MKK4) and MKK7, whereas p38 is primarily activated by MKK3 and MKK6 (MKK3/6) but can be phosphorylated by MKK4 under some circumstances (4). JNK and p38 are mainly activated by stress stimuli and cytokines but can also be stimulated by growth factors. MAP2Ks are in turn regulated by MAPK kinase kinases (MAP3Ks). MEK kinase 4 (MEKK4) is a MAP3K that regulates p38 and JNK through the phosphorylation of the MAP2Ks MKK3 and MKK6 and MKK4 and MKK7, respectively (11, 31). The physiological role of MEKK4 is poorly defined, and the pathways that promote MEKK4 signaling to MAPKs are largely unknown.Vertebrate development is orchestrated by cell-cell interactions and cytokine gradients that regulate intracellular signaling networks. We have discovered that MEKK4 expression is regulated during development and is a critical node for the control of neural tube closure and skeletal patterning in the developing mouse embryo. In this report, we investigated the function of MEKK4 in development by the targeted inhibition of MEKK4 kinase activity. The MEKK4 gene was altered by the targeted mutation of the active site lys...

MAP3Ks as central regulators of cell fate during development

“…Both MEKK4 knockout and MEKK4 kinase inactive mutant mice are born, but most animals die shortly after birth. These mice exhibit neural tube and skeletal malformations (Abell et al, 2005;Chi et al, 2005). Neural tube defects in MEKK4-deficient or kinase inactive embryos include spina bifida and exencephaly and appear due to increases in apoptosis during neural development.…”

“…The neural tube defect is highly penetrant in the MEKK4 Ϫ/Ϫ mice suggestive of a role in developmental epithelial biology. There is enhanced apoptosis in MEKK4 mutant mice at the time coincident with closure of the neural tube, but no differences in cell proliferation (Abell et al, 2005). Because cardiac chamber partitioning and valve formation also require appropriate timing of cellular apoptosis, alterations in programmed cell death may also lead to heart defects.…”

“…For example, MAP3Ks that are dominant-negative or constitutively active for kinase activity will reveal downstream targets in vivo and phenotypic consequences for loss-of-function or gain-of-function, respectively. In this respect, knock-in mice for a kinase inactive version of MEKK4 exhibit neural tube and skeletal defects (Abell et al, 2005).…”

GADD45β Enhances Col10a1 Transcription via the MTK1/MKK3/6/p38 Axis and Activation of C/EBPβ-TAD4 in Terminally Differentiating Chondrocytes

“…21 and 22). Ablation of the MAPK kinase kinase (MAP3K), MEKK4/MTK1, causes skeletal patterning defects in the mouse embryo (23). Moreover, Col2a1 promoterdriven MKK6 transgene overexpression leads to decreased chondrocyte proliferation and delayed terminal differentiation to hypertrophy (24).…”

Phylogeny of the TRAF/MATH Domain

“…Indeed, TRAF4 seems to regulate JNK activation by binding to MEKK4 and promoting MEKK4 oligomerization 66 . Most interestingly, mice deficient in MEKK4 develop strikingly similar neurulation and skeletal patterning defects to those observed in TRAF4 deficient mice 67,68 .…”