Rho kinase inhibition rescues the endothelial cell cerebral cavernous malformation phenotype.
Cerebral cavernous malformations (CCM) are vascular lesions causing seizures and stroke. Mutations causing inactivation of one of three genes, ccm1, -2, or -3, are sufficient to induce vascular endothelial cell defects resulting in CCM. Herein, we show that loss of expression of the CCM1, -2, or -3 proteins causes a marked increase in expression of the GTPase RhoA. Live cell imaging with a RhoA-specific biosensor demonstrates increased RhoA activity with loss of CCM1, -2, or -3, with an especially pronounced RhoA activation in both the cytosol and the nucleus with loss of CCM1 expression. Increased RhoA activation was associated with Rho kinase-dependent phosphorylation of myosin light chain 2. Functionally, loss of CCM1, -2, or -3 inhibited endothelial cell vessel-like tube formation and extracellular matrix invasion, each of which is rescued by chemical inhibition or short hairpin RNA knockdown of Rho kinase. The findings, for the first time, define a signaling network for CCM1, -2, and -3 in CCM pathology, whereby loss of CCM1, -2, or -3 protein expression results in increased RhoA activity, with the activation of Rho kinase responsible for endothelial cell dysregulation. The results define Rho kinase as a therapeutic target to rescue endothelial cells from loss of CCM protein function.
Borikova
Asya L
AL
Department of Pharmacology and Lineberger Comprehensive Cancer Center, School of Dentistry, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Dibble
Christopher F
CF
Sciaky
Noah
N
Welch
Christopher M
CM
Abell
Amy N
AN
Bencharit
Sompop
S
Johnson
Gary L
GL
D000577:Amides / Q000494:pharmacology; D051017:Apoptosis Regulatory Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D015374:Biosensing Techniques; D024722:Cardiac Myosins / Q000378:metabolism; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D002460:Cell Line; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D020786:Hemangioma, Cavernous, Central Nervous System / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D000075927:KRIT1 Protein; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D018994:Myosin Light Chains / Q000378:metabolism; D010641:Phenotype; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D011725:Pyridines / Q000494:pharmacology; D034622:RNA Interference; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D000577:Amides; D051017:Apoptosis Regulatory Proteins; C502544:CCM2 protein, human; D002352:Carrier Proteins; D000075927:KRIT1 Protein; C107928:KRIT1 protein, human; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D018994:Myosin Light Chains; C495390:PDCD10 protein, human; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D011725:Pyridines; C107184:myosin light chain 2; C066737:RHOA protein, human; C108830:Y 27632; C515852:ROCK2 protein, human; D054460:rho-Associated Kinases; D024722:Cardiac Myosins; D020742:rhoA GTP-Binding Protein