The cerebral cavernous malformation proteins CCM2L and CCM2 prevent the activation of the MAP kinase MEKK3

Abstract

Three genes, CCM1, CCM2, and CCM3, interact genetically and biochemically and are mutated in cerebral cavernous malformations (CCM). A recently described member of this CCM family of proteins, CCM2-like (CCM2L), has high homology to CCM2. Here we show that its relative expression in different tissues differs from that of CCM2 and, unlike CCM2, the expression of CCM2L in endothelial cells is regulated by density, flow, and statins. In vitro, both CCM2L and CCM2 bind MEKK3 in a complex with CCM1. Both CCM2L and CCM2 interfere with MEKK3 activation and its ability to phosphorylate MEK5, a downstream target. The in vivo relevance of this regulation was investigated in zebrafish. A knockdown of ccm2l and ccm2 in zebrafish leads to a more severe "big heart" and circulation defects compared with loss of function of ccm2 alone, and also leads to substantial body axis abnormalities. Silencing of mekk3 rescues the big heart and body axis phenotype, suggesting cross-talk between the CCM proteins and MEKK3 in vivo. In endothelial cells, CCM2 deletion leads to activation of ERK5 and a transcriptional program that are downstream of MEKK3. These findings suggest that CCM2L and CCM2 cooperate to regulate the activity of MEKK3.cerebral cavernous malformation | signaling | MAP kinase | expression | endothelium C erebral cavernous malformations (CCMs) are characterized by endothelial cell channels in low-blood flow venous capillaries with poor coverage of pericytes and smooth cells and poorly developed tight and adherens junctions, resulting in increased permeability, hemorrhage, and subsequent neurologic deficits. Germline loss-of-function mutations in any one of three CCM genes-CCM1 (Krit1), CCM2 (OSM), or CCM3 (PDCD10)-cause the familial and sporadic forms of CCMs. The proteins encoded by these genes interact in a cytosolic complex. Critical insights into their in vivo functions have been obtained from genetic manipulation in fish and mice.In zebrafish, null mutations in either ccm1 (santa, san) or ccm2 (valentine, vtn) result in an enlarged heart ("big heart") and dilation of the subintestinal vessels and posterior cardinal vein, and morpholino (MO) studies indicate that ccm1 and ccm2 are in the same pathway during zebrafish cardiovascular development (1, 2). The contribution of ccm3 or its downstream effectors in the ccm1/2 pathway in zebrafish is less clear (3, 4). CCM proteins have been shown to regulate the signal strength of several pathways acting both as down-regulators and as activators. Reduced expression of CCM1, CCM2, or CCM3 increases RhoA activation (5). CCM2 and CCM3 siRNAs increase phosphorylation of AKT and the MAP kinases p38 and ERK1/2 (6). CCM1 promotes NOTCH activation (7) and deficiency in CCM proteins leads to endothelial-mesenchymal transition (EMT) by up-regulating TGF-β/BMP signaling (8). ccm1 and ccm2 deletion in zebrafish leads to the up-regulation of β1-integrin signaling and klf2 expression that results in a proangiogenic program (9). Ccm2-like (Ccm2l), a recently described paralog of Ccm2, was sho...

HIF-1α and HIF-2α induced angiogenesis in gastrointestinal vascular malformation and reversed by thalidomide

“…cerebral cavernous malformation)1314 and cancer angiogenesis151617. HUVECs purchased from the Institute of Cell Biology of the Chinese Academy of Sciences (Shanghai, China) were also used in our previous in vitro studies1218.…”

Oxidative stress and inflammation in cerebral cavernous malformation disease pathogenesis: Two sides of the same coin

“…In addition, there is evidence that CCM proteins ensure the quiescence of endothelial cells and inhibit angiogenic responses by either activating the Delta-Notch signaling axis (Schulz et al, 2015, Wustehube et al, 2010, You et al, 2013), inhibiting vascular endothelial growth factor (VEGF) and MAPK/ERK signaling (Cullere et al, 2015, DiStefano et al, 2014, Zhu et al, 2010), or preventing the β1 integrin-dependent activation of a mechanotransduction pathway mediated by the blood flow-sensitive transcription factor Kruppel-like factor 2 (KLF2) (Renz et al, 2015). …”

“…Loss-of-function of CCM proteins has also been shown to trigger β-catenin and transforming growth factor beta/bone morphogenetic protein (TGFβ/BMP) signaling-driven endothelial-to-mesenchymal transition (EndMT) (Bravi et al, 2016, Bravi et al, 2015, Guan and Couldwell, 2013, Maddaluno et al, 2013), as well as activation of MEKK3 signaling and increased expression of the mechanosensitive transcription factors KLF2 and KLF4 (Cullere et al, 2015, Cuttano et al, 2016, Fisher et al, 2015; Zhou et al, 2015; Zhou et al, 2016). Both of these mechanisms have been suggested to be key determinants of CCM disease pathogenesis (Maddaluno et al, 2013, Bravi et al, 2016, Cuttano et al, 2016, Zhou et al, 2016).…”

Cerebral Cavernous Malformations: Review of the Genetic and Protein–Protein Interactions Resulting in Disease Pathogenesis

“…Work by Cullere et al in 2015 further elucidated the relationship between CCM2 and CCM2L and their mechanisms of action (15). These authors demonstrated that both CCM2 and CCM2L could bind to and inhibit MEKK3 in a complex with KRIT1.…”