MEKK3 coordinates with FBW7 to regulate WDR62 stability and neurogenesis.

Mutations of WD repeat domain 62 (WDR62) lead to autosomal recessive primary microcephaly (MCPH), and down-regulation of WDR62 expression causes the loss of neural progenitor cells (NPCs). However, how WDR62 is regulated and hence controls neurogenesis and brain size remains elusive. Here, we demonstrate that mitogen-activated protein kinase kinase kinase 3 (MEKK3) forms a complex with WDR62 to promote c-Jun N-terminal kinase (JNK) signaling synergistically in the control of neurogenesis. The deletion of Mekk3, Wdr62, or Jnk1 resulted in phenocopied defects, including premature NPC differentiation. We further showed that WDR62 protein is positively regulated by MEKK3 and JNK1 in the developing brain and that the defects of wdr62 deficiency can be rescued by the transgenic expression of JNK1. Meanwhile, WDR62 is also negatively regulated by T1053 phosphorylation, leading to the recruitment of F-box and WD repeat domain-containing protein 7 (FBW7) and proteasomal degradation. Our findings demonstrate that the coordinated reciprocal and bidirectional regulation among MEKK3, FBW7, WDR62, and JNK1, is required for fine-tuned JNK signaling for the control of balanced NPC self-renewal and differentiation during cortical development.

Platelet MEKK3 regulates arterial thrombosis and myocardial infarct expansion in mice.

MAPKs play important roles in platelet activation. However, the molecular mechanisms by which MAPKs are regulated in platelets remain largely unknown. Real-time polymerase chain reaction and western blot data showed that MEKK3, a key MAP3K family member, was expressed in human and mouse platelets. Then, megakaryocyte/platelet-specific MEKK3-deletion (MEKK3-/- ) mice were developed to elucidate the platelet-related function(s) of MEKK3. We found that agonist-induced aggregation and degranulation were reduced in MEKK3-/- platelets in vitro. MEKK3 deficiency significantly impaired integrin αIIbß3-mediated inside-out signaling but did not affect the outside-in signaling. At the molecular level, MEKK3 deficiency led to severely impaired activation of extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun NH2-terminal kinase 2 but not p38 or ERK5. In vivo, MEKK3-/- mice showed delayed thrombus formation following FeCl3-induced carotid artery injury. Interestingly, the tail bleeding time was normal in MEKK3-/- mice. Moreover, MEKK3-/- mice had fewer microthrombi, reduced myocardial infarction (MI) size, and improved post-MI heart function in a mouse model of MI. These results suggest that MEKK3 plays important roles in platelet MAPK activation and may be used as a new effective target for antithrombosis and prevention of MI expansion.

Micro-CT Imaging Reveals Mekk3 Heterozygosity Prevents Cerebral Cavernous Malformations in Ccm2-Deficient Mice.

Mutations in CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation in humans. Mouse models of CCM disease have been established by deleting Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. However, the full value of these animal models is limited by the lack of an accurate and quantitative method to assess lesion burden and progression. In the present study we have established a refined and detailed contrast enhanced X-ray micro-CT method to measure CCM lesion burden in mouse brains. As this study utilized a voxel dimension of 9.5µm (leading to a minimum feature size of approximately 25µm), it is therefore sufficient to measure CCM lesion volume and number globally and accurately, and provide high-resolution 3-D mapping of CCM lesions in mouse brains. Using this method, we found loss of Ccm1 or Ccm2 in neonatal endothelium confers CCM lesions in the mouse hindbrain with similar total volume and number. This quantitative approach also demonstrated a rescue of CCM lesions with simultaneous deletion of one allele of Mekk3. This method would enhance the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases.

Loss of Arp2/3 induces an NF-κB-dependent, nonautonomous effect on chemotactic signaling.

Arp2/3-branched actin is critical for cytoskeletal dynamics and cell migration. However, perturbations and diseases affecting this network have phenotypes that cannot be fully explained by cell-autonomous effects. In this paper, we report nonautonomous effects of Arp2/3 depletion. We show that, upon Arp2/3 depletion, the expression of numerous genes encoding secreted factors, including chemokines, growth factors, and matrix metalloproteases, was increased, a signature resembling the senescence-associated secretory phenotype. These factors affected epidermal growth factor chemotaxis in a nonautonomous way, resolving the recent contradictions about the role of Arp2/3 in chemotaxis. We demonstrate that these genes were activated by nuclear factor κB via a CCM2­MEKK3 pathway that has been implicated in hyperosmotic stress signaling. Consistent with this, Arp2/3-depleted cells showed misregulation of volume control and reduced actin in the submembranous cortex. The defects in osmotic signaling in the Arp2/3-depleted cells can be rescued by hypoosmotic treatment. Thus, perturbations of Arp2/3 have nonautonomous effects that should be considered when evaluating experimental manipulations and diseases affecting the Arp2/3-actin cytoskeleton.

The kinases MEKK2 and MEKK3 regulate transforming growth factor-ß-mediated helper T cell differentiation.

Mitogen-activated protein kinases (MAPKs) are key mediators of the T cell receptor (TCR) signals but their roles in T helper (Th) cell differentiation are unclear. Here we showed that the MAPK kinase kinases MEKK2 (encoded by Map3k2) and MEKK3 (encoded by Map3k3) negatively regulated transforming growth factor-ß (TGF-ß)-mediated Th cell differentiation. Map3k2(-/-)Map3k3(Lck-Cre/-) mice showed an abnormal accumulation of regulatory T (Treg) and Th17 cells in the periphery, consistent with Map3k2(-/-)Map3k3(Lck-Cre/-) naive CD4(+) T cells' differentiation into Treg and Th17 cells with a higher frequency than wild-type (WT) cells after TGF-ß stimulation in vitro. In addition, Map3k2(-/-)Map3k3(Lck-Cre/-) mice developed more severe experimental autoimmune encephalomyelitis. Map3k2(-/-)Map3k3(Lck-Cre/-) T cells exhibited impaired phosphorylation of SMAD2 and SMAD3 proteins at their linker regions, which negatively regulated the TGF-ß responses in T cells. Thus, the crosstalk between TCR-induced MAPK and the TGF-ß signaling pathways is important in regulating Th cell differentiation.

MEKK3 overexpression contributes to the hyperresponsiveness of IL-12-overproducing cells and CD4+ T conventional cells in nonobese diabetic mice.

Elevated IL-12 production and higher rate of CD4(+) T conventional (Tconv) cell proliferation in NOD mice have been implicated in the progression of type 1 diabetes. However, the underlying mechanisms remain largely unknown, even though enhanced activation of the IkappaB kinase (IKK)/NF-kappaB pathway has been revealed to mediate IL-12 overproduction. In this study, we report that deviated p38 MAPK activation also contributes to elevated IL-12 production with a mechanism involving MAPK-activated protein kinase-2-mediated stabilization of IL-12p40 mRNA. Aberrant p38 activation induced by various inflammatory stimuli in IL-12-overproducing cells is not due to defective MAPK phosphatase-1 induction in NOD mice. Deviated IKK and MAPKs activation also occurs in NOD CD4(+) Tconv cells, which is associated with higher rates of proliferation. All of the above evidence suggests that the signaling defects occur at the level of MAPK kinase kinase (MAK3K or MEKK). Further exploration shows that MEKK3, but not other MAP3Ks, is overexpressed in NOD IL-12-overproducing cells and CD4(+) Tconv cells independent of autoimmune inflammation. MEKK3 knockdown leads to reversal of the deviated IKK and MAPKs activation, resulting in reduced IL-12 production and decreased CD4(+) Tconv cell proliferation. Thus, this study provides a molecular mechanism of the hyperresponsiveness of IL-12-overproducing cells and CD4(+) Tconv cells in NOD mice.

Vasculogenesis driven by bone marrow-derived cells is essential for growth of Ewing's sarcomas.

The role of vasculogenesis as opposed to angiogenesis in tumor formation has been little explored genetically. Endothelial cells that lack the MEK kinase MEKK3 cannot form vessels. In this study, we employed mice with hematopoietic deletions of the Mekk3 gene to evaluate the importance of vasculogenesis in the formation of Ewing's sarcoma tumors. Bone marrow cells (BM) from LacZ(+) Mekk3-deficient conditional knockout mice (Mekk3(Deltaflox/-) mice) were transplanted into irradiated nude mice before injection of Ewing's sarcoma cells. Because the grafted Mekk3(Deltaflox/-) BM cells cannot contribute to vessel development in the same way as the host Mekk3(+/+) endothelial cells, angiogenesis is normal in the model whereas vasculogenesis is impaired. Four weeks after BM transplant, Ewing's sarcoma TC71 or A4573 cells were injected, and tumor growth and vessel density were compared. Strikingly, chimeric mice transplanted with Mekk3(Deltaflox/-) BM exhibited a reduction in tumor growth and vessel density compared with mice transplanted with Mekk3(Deltaflox/+) BM cells. Mekk3(Deltaflox/-) cells that were LacZ positive were visualized within the tumor; however, few of the LacZ(+) cells colocalized with either CD31(+) endothelial cells or desmin(+) pericytes. Quantification of double-positive LacZ(+) and CD31(+) endothelial cells or LacZ(+) and desmin(+) pericytes confirmed that chimeric mice transplanted with Mekk3(Deltaflox/-) BM were impaired for tumor vessel formation. In contrast, siRNA-mediated knockdown of Mekk3 in TC71 Ewing's sarcoma cells had no effect on tumor growth or vessel density. Our findings indicate that vasculogenesis is critical in the expansion of the tumor vascular network.

MEKK3 is essential for lymphopenia-induced T cell proliferation and survival.

T cell homeostasis is crucial for maintaining an efficient and balanced T cell immunity. The interaction between TCR and self peptide (sp) MHC ligands is known to be the key driving force in this process, and it is believed to be functionally and mechanistically different from that initiated by the antigenic TCR stimulation. Yet, very little is known about the downstream signaling events triggered by this TCR-spMHC interaction and how they differ from those triggered by antigenic TCR stimulation. In this study, we show that T cell conditional ablation of MEKK3, a Ser/Thr kinase in the MAPK cascade, causes a significant reduction in peripheral T cell numbers in the conditional knockout mice, but does not perturb thymic T cell development and maturation. Using an adoptive mixed transfer method, we show that MEKK3-deficient T cells are severely impaired in lymphopenia-induced cell proliferation and survival. Interestingly, the Ag-induced T cell proliferation proceeds normally in the absence of MEKK3. Finally, we found that the activity of ERK1/2, but not p38 MAPK, was attenuated during the lymphopenia-driven response in MEKK3-deficient T cells. Together, these data suggest that MEKK3 may play a crucial selective role for spMHC-mediated T cell homeostasis.

MEKK3 initiates transforming growth factor beta 2-dependent epithelial-to-mesenchymal transition during endocardial cushion morphogenesis.

Congenital heart defects occur at a rate of 5% and are the most prevalent birth defects. A better understanding of the complex signaling networks regulating heart development is necessary to improve repair strategies for congenital heart defects. The mitogen-activated protein 3 kinase (MEKK3) is important to early embryogenesis, but developmental processes affected by MEKK3 during heart morphogenesis have not been fully examined. We identify MEKK3 as a critical signaling molecule during endocardial cushion development. We report the detection of MEKK3 transcripts to embryonic hearts before, during, and after cardiac cushion cells have executed epithelial-to-mesenchymal transition (EMT). MEKK3 is observed to endocardial cells of the cardiac cushions with a diminishing gradient of expression into the cushions. These observations suggest that MEKK3 may function during production of cushion mesenchyme as required for valvular development and septation of the heart. We used a kinase inactive form of MEKK3 (MEKK3(KI)) in an in vitro assay that recapitulates in vivo EMT and show that MEKK3(KI) attenuates mesenchyme formation. Conversely, constitutively active MEKK3 (ca-MEKK3) triggers mesenchyme production in ventricular endocardium, a tissue that does not normally undergo EMT. MEKK3-driven mesenchyme production is further substantiated by increased expression of EMT-relevant genes, including TGFbeta(2), Has2, and periostin. Furthermore, we show that MEKK3 stimulates EMT via a TGFbeta(2)-dependent mechanism. Thus, the activity of MEKK3 is sufficient for developmental EMT in the heart. This knowledge provides a basis to understand how MEKK3 integrates signaling cascades activating endocardial cushion EMT.

Tumour necrosis factor-alpha-induced glucose-stimulated insulin secretion inhibition in INS-1 cells is ascribed to a reduction of the glucose-stimulated Ca2+ influx.

The present study was undertaken to determine how tumour necrosis factor-alpha (TNF-alpha) elicits the inhibition of glucose-stimulated insulin secretion (GSIS) in rat insulinoma cells (INS)-1 beta-cells. TNF-alpha pretreatment did not change the expression levels of insulin, PDX-1, glucose transporter 2, glucokinase, K(ATP) channels, Ca(2)(+) channels, and exocytotic molecules and, furthermore, did not reduce the glucose-stimulated ATP level. On the other hand, TNF-alpha reduced the glucose-stimulated influx of Ca(2)(+). The TNF-alpha treatment was thought to activate c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and NF-kappaB inflammatory signals, since TNF-alpha increased phospho-JNK and phospho-p38 and reduced I kappaB levels. Inhibitors of these signaling pathways prevented the TNF-alpha-induced reduction of the Ca(2)(+) influx and GSIS. Overexpression of MEKK3, a possible mediator from the TNF-alpha receptor to the JNK/p38 and NK-kappaB signaling cascade, increased the levels of phospho-JNK, phospho-p38, and NF-kappaB, and reduced the glucose-stimulated Ca(2)(+) influx and GSIS. The reduction of the Ca(2)(+) influx and GSIS in MEKK3-overexpressing INS-1 cells was also prevented by inhibitors of JNK, p38, and NF-kappaB. These data demonstrate that TNF-alpha inhibits GSIS by reducing the glucose-stimulated Ca(2)(+) influx, possibly through the activation of JNK and p38 MAPK and NF-kappaB inflammatory signals. Thus, our findings suggest that the activation of stress and inflammatory signals can contribute to the inhibition of GSIS in the development of diabetes.

MEKK3 is required for endothelium function but is not essential for tumor growth and angiogenesis.

Mitogen-activated protein kinase kinase kinase 3 (MEKK3) plays an essential role in embryonic angiogenesis, but its role in tumor growth and angiogenesis is unknown. In this study, we further investigated the role of MEKK3 in embryonic angiogenesis, tumor angiogenesis, and angiogenic factor production. We found that endothelial cells from Mekk3-deficient embryos showed defects in cell proliferation, apoptosis, and interactions with myocardium in the heart. We also found that MEKK3 is required for angiopoietin-1 (Ang1)-induced p38 and ERK5 activation. To study the role of MEKK3 in tumor growth and angiogenesis, we established both wild-type and Mekk3-deficient tumor-like embryonic stem cell lines and transplanted them subcutaneously into nude mice to assess their ability to grow and induce tumor angiogenesis. Mekk3-deficient tumors developed and grew similarly as control Mekk3 wild-type tumors and were also capable of inducing tumor angiogenesis. In addition, we found no differences in the production of VEGF in Mekk3-deficient tumors or embryos. Taken together, our results suggest that MEKK3 plays a critical role in Ang1/Tie2 signaling to control endothelial cell proliferation and survival and is required for endothelial cells to interact with the myocardium during early embryonic development. However, MEKK3 is not essential for tumor growth and angiogenesis.

Interleukin-1 (IL-1)-induced TAK1-dependent Versus MEKK3-dependent NFkappaB activation pathways bifurcate at IL-1 receptor-associated kinase modification.

Interleukin-1 (IL-1) receptor-associated kinase (IRAK) is phosphorylated after it is recruited to the receptor, subsequently ubiquitinated, and eventually degraded upon IL-1 stimulation. Although a point mutation changing lysine 134 to arginine (K134R) in IRAK abolished IL-1-induced IRAK ubiquitination and degradation, mutations of serines and threonines adjacent to lysine 134 to alanines ((S/T)A (131-144)) reduced IL-1-induced IRAK phosphorylation and abolished IRAK ubiquitination. Through the study of these IRAK modification mutants, we uncovered two parallel IL-1-mediated signaling pathways for NFkappaB activation, TAK1-dependent and MEKK3-dependent, respectively. These two pathways bifurcate at the level of IRAK modification. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classical NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through IkappaBalpha phosphorylation and subsequent dissociation from NFkappaB but without IkappaBalpha degradation. These results provide significant insight to our further understanding of NFkappaB activation pathways.