MEKK2 And MEKK3:From Inflammation To Vascular Development

Cardiovascular and cerebrovascular diseases have become the major causes of death,mainly including stroke and coronary heart disease.Cerebral Cavernous Malformation（CCM）is a cerebrovascular dysplasia and its clinical manifestations are intracranial hemorrhage,stroke,epilepsy and even sudden death.CCM is caused by mutations of three autosomal genes encoding CCM1/KRIT1,CCM2/OSM and CCM3/PDCD10 respectively.CCM2 loss is associated with the CCM disease,manifesting as cerebrovascular lesions that can lead to focal neurological defects and stroke.MEKK3,a highly conserved Serine/Threonine protein kinase belonging to MAP3K superfamily,is essential for embryonic cardiovascular development in mice.MEKK3 can interact with CCM2,but how this interaction is mediated and its relevance to cerebral vasculature are still unknown.Our laboratory found that Mekk3 deficiency in endothelial cells could lead to embryonic lethality because of impaired embryonic vascular development.Inducible endothelial Mekk3 knockout in neonatal mice is lethal due to multiple intracranial haemorrhages and brain blood vessels leakage,as well as other organs haemorrhages.We discover the interaction of MEKK3 N-terminal helix and PB1 domain with the CCM2 harmonin homology domain（HHD）by Co-IP and GST pull down assay,determine a 2.35?co-crystal structure,and find out critical interaction amino acid residues.Structure-directed disruption of the MEKK3:CCM2 interaction with a competitive cell-permeable peptide leads to brain vascular leakage,which resembles that of endothelial Mekk3 deficient mice.Mekk3 deficient endothelial cells show increased Rho-ROCK signaling.Moreover,ROCK inhibitor can rescue the survival of inducible endothelial Mekk3 deficient mice and restore brain vascular permeability.Using CRISPR-Cas9 system,we generate MEKK3-CCM2 interaction sites mutation(Mekk3^mutN)mice to provide a new mouse model for further validating the role of this complex.We conclude that regulation of Rho-ROCK signaling by CCM2:MEKK3complex is required for maintenance of neurovascular integrity,unraveling a mechanism by which CCM2 loss leads to CCM disease.Besides,another Serine/Threonine protein kinase MEKK2,a closely related homolog with MEKK3,has no effect on embryonic cardiovascular development.However,we find that Mekk2 deficient mice are hyposensitive to LPS-induced septic shock,and produce less IL-6,TNFαin vivo.MEKK2 deficient BMDMs also secrete less IL-6,TNFαsignificantly in vitro,and show impaired MAPK and NF-κB pathway activation.Our studies indicate MEKK2 kinase and its mediated signaling may be a new target for septic shock treatment.