Cerebral cavernous malformation is a vascular disease associated with activated RhoA signaling

Abstract

Cerebral cavernous malformation (CCM) involves the homozygous inactivating mutations of one of three genes, ccm1, -2, or -3 resulting in hyperpermeable blood vessels in the brain. The CCM1, -2, and -3 proteins form a complex to organize the signaling networks controlling endothelial cell physiology including actin dynamics, tube formation, and adherens junctions. The common biochemical defect with the loss of CCM1, -2, or -3 is increased RhoA activity leading to the activation of Rho-associated coiled coil-forming kinase (ROCK). Inhibition of the ROCK rescues CCM endothelial cell dysfunction, suggesting that the inhibition of RhoA-ROCK signaling may be a therapeutic strategy to prevent or arrest the progression of the CCM lesions.

Figure 1

Defined structural domains and interacting proteins for CCM1, -2, and -3. Interactions were defined using multiple techniques, including coimmunoprecipitation coupled with mass spectroscopy and/or immunoblotting and yeast 2-hybrid screens. The CCM1 four-point-one, ezrin, radixin, moesin (FERM) domain binds multiple proteins. The NPAY, NPLF, and NPYF sequences in CCM1 bind to the PTB domains. CCM1 also encodes a nuclear localization (NLS) sequence. CCM2 encodes a PTB domain and Karet domain. CCM3 has a FRAP-ATM-TRAP (FAT) domain that binds MST4, STK24, and STK25 serine/threonine protein kinases.

Figure 2

CCM proteins regulate RhoA activation and signaling. (A) The CCM proteins form a ternary complex and negatively regulate RhoA activity and protein levels. CCM2 associates with the E3 ubiquitin ligase Smurf1, which targets active RhoA for degradation. RhoA is activated by GEF-mediated GDP to GTP exchange after ligand binding to a GPCR or RTK. Activated RhoA activates both mDia and ROCK. mDia stimulates action polymerization. The ROCK phosphorylates and inhibits the MLC phosphatase (MLCPase) and directly phosphorylates the MLC and LIMK. LIMK phosphorylates the actin severing protein cofilin, leading to a decreased activity. This process leads to the stabilization and cross-linking of actin and myosin. (B) The RhoA-bound ROCK phosphorylates multiple substrates that affect global actin cytoskeletal dynamics, which in the context of CCM leads to stress fiber formation and junctional protein destabilization. Current pharmacological approaches to treat CCM based on this model are through the inhibition of RhoA geranylgeranylation with statins or through ROCK inhibition to restore proper endothelial cytoskeletal dynamics.