Rho Kinase Inhibition Rescues the Endothelial Cell Cerebral Cavernous Malformation Phenotype

Abstract

Cerebral cavernous malformations (CCM)3 are clusters of leaky, dilated capillaries in the central nervous system that occur in ϳ0.5% of the general population and up to 1.5% of the Hispanic population (1). CCM frequently lead to clinical sequelae such as hemorrhage, epilepsy, and neurological deficits (1). The disease is associated with both germline and somatic mutations in one of three genes, ccm1, -2, or -3 (2). The three CCM proteins form a common complex, indicating that they function coordinately (3, 4); each lacks defined catalytic domains, indicating that they are scaffold-or adaptor-like proteins for organization of protein complexes (3-5). The identical disease phenotype produced upon loss of any one of the three CCM proteins suggests that they coordinately regulate a common mechanism required for vascular integrity (3,4,6,7).It was recently shown that loss of endothelial cell expression of CCM2 resulted in activation of the GTPase RhoA (7,8). Crose et al. (8) demonstrated that CCM2 knockdown in brain microvascular endothelial cells resulted in defective RhoA degradation because of the dysregulation of Smurf1, a CCM2 binding partner, and an ubiquitin-protein isopeptide ligase (E3) that controls RhoA degradation. RhoA overabundance induced by loss of CCM2 was shown to increase cytoskeletal stability, inhibit vessel-like tube formation, and increase endothelial cell monolayer permeability (7,8). Herein, we show that loss of CCM1, -2, or -3 expression results in a common phenotype associated with RhoA overexpression and activation. We define ROCK as a critical RhoA effector whose increased activation dysregulates endothelial cell function. ROCK is activated by RhoA and phosphorylates several substrates, including myosin light chain, myosin light chain phosphatase, and LIM kinase for the regulation of actin cytoskeletal dynamics (9). ROCK has also been shown to regulate vascular permeability and has been a drug discovery target for regulation of vascular bed diseases (10). Our findings show that ROCK inhibition rescues extracellular matrix invasion and vessel-like tube formation, two endothelial cell functions disrupted by loss of CCM protein expression. EXPERIMENTAL PROCEDURESEstablishment of Knockdown Cell Lines-Lentiviral genespecific shRNAs in pLKO.1 were obtained from the University of North Carolina -Chapel Hill Lenti-shRNA Core Facility.RhoA Biosensor-Imaging and image processing were performed as described (11).Tube Formation Assay and Live Cell Imaging-7.0 ϫ 10 Ϫ4 cells were incubated for 15 h on Matrigel (BD Biosciences) and stained with rhodamine phalloidin as described previously (8).Imaging was performed on either a Pathway (BD Biosciences) or a Cellomics ArrayScan (Thermo Scientific). For live cell imaging, six fields of cells were imaged via transmitted light every 10 min for 15 h. Cellomics ArrayScan software was used to quantitate mean tube area. Statistical Significance-Where indicated, statistical significance was calculated using the two-tailed Student's t test. See supplemental mater...

Structure-Function Analysis of Core STRIPAK Proteins

“…The polarized localization of the Golgi has also been intimately linked to the small proteins of the Rho-GTPase family (26,37). In light of the defects in Rho signaling following modulation of CCM1, CCM2, or CCM3 expression (12)(13)(14), it is tempting to postulate that CCM3, MST4, and perhaps STRIPAK may regulate Golgi polarization via regulation of Rho-GTPases. Whether striatins, CCM3, and MST4 play a role in all aspects of Golgi polarization, including cell migration, remains to be answered.…”

“…Interestingly, TORC2 controls actin cytoskeleton assembly across multiple species, in part via regulation of the Rho1 GTPases (43)(44)(45). CCM disease, CCM3, and MST4 are intimately linked to Rho signaling in human cells (12)(13)(14)46), suggesting that this function of STRIPAK has been evolutionarily conserved. In addition to these roles in cytoskeleton and membrane dynamics, a surprising recent report implicated the Drosophila STRIPAK complex (including CCM3) in Hippo signaling (47), indicating that STRIPAK may control multiple signaling pathways.…”

“…11). Recent studies have implicated defective Rho signaling as one of the consequences of depletion (or overexpression) of the CCM1, CCM2, and CCM3 proteins (12)(13)(14). Further links between CCM3 and its kinase partners and cytoskeletal dynamics via the Golgi were also uncovered.…”

CCM1 and the second life of proteins in adhesion complexes

“…85 Also, for both CCM2 and CCM3, an inhibitory role toward RHO has been described. 82,83,86 How inhibition of RHO/ROCK by the CCM proteins is achieved is not known and it will be of interest to further investigate whether CCM proteins might influence the balance between RHO and RAC signaling.…”

“…Recently, several groups have reported an inhibitory role of CCM1 toward RHO signaling, which would improve the endothelial barrier function. 82,83 Activation of RHO results in ROCK-mediated phosphorylation of several substrates involved in regulation of actin cytoskeletal dynamics, like myosin light chain and LIM kinase. Furthermore, ROCK is described to phosphorylate Occludin and Claudin-5 in brain endothelium, and thereby, enhances leakiness.…”