14-325 Sigma-AldrichRac/cdc42 Assay Reagent (PAK1 PBD, agarose), 300 µg

Cerebral cavernous malformations (CCMs) are characterized by abnormally dilated intracranial capillaries that have a propensity to bleed. The development of some CCMs in humans has been attributed to mutations in CCM1 and CCM2 genes. In animal models, major cardiovascular defects caused by both gene mutations have been observed. However, the effects of the loss of Ccm function on the microvasculature in animal models are less defined. Using high-resolution imaging in vivo, we demonstrated that the loss of Ccm1 in zebrafish embryos leads to failed microvascular lumenization during angiogenesis due to impaired intraendothelial vacuole formation and fusion. No developmental changes during vasculogenesis and the initial stage of angiogenesis were observed, being in contrast to prior reports. In vivo zebrafish studies were further substantiated by in vitro findings in human endothelial cells that elucidated the biochemical pathways of CCM1 deficiency. We found that CCM1 regulates angiogenic microvascular lumen formation through Rac1 small GTPase. In summary, Ccm1 has been identified as a key angiogenic modulator in microvascular tubulogenesis. Additionally, the microvascular pathology observed in developing Ccm1 mutant zebrafish embryos mirrors that seen in human CCM lesions, suggesting that zebrafish might provide a superior animal model to study the pathogenesis of human CCM.

Rac1-GTPase activation plays a key role in the development and progression of cardiac remodeling. Therefore, we engineered a transgenic mouse model by over-expressing the cDNA of a constitutively active form of Zea maize Rac gene (ZmRacD) specifically in the hearts of FVB/N mice. Echocardiography and MRI analyses showed cardiac hypertrophy in old transgenic mice as evidenced by increased left ventricular (LV) mass and LV mass/body weight ratio which are associated with relative ventricular chamber dilation and systolic dysfunction. LV hypertrophy in the hearts of old transgenic mice was further confirmed by increased heart weight/body weight ratio and by histopathology analysis. The cardiac remolding in the old transgenic mice was coupled with increased myocardial Rac-GTPase activity (372%) and ROS production (462%). There were also increases in the α(1) (224%) β(1) (240%) integrins expression. This led to the activation of hypertrophic signaling pathways e.g. ERK1/2 (295%) and JNK (223%). Pravastatin treatment led to inhibition of Rac-GTPase activity and integrins signaling. Interestingly, activation of ZmRacD expression with thyroxin (T4) led to cardiac dilation and systolic dysfunction in adult transgenic mice within two weeks. In conclusion, this is the first study to show the conservation of Rho/Rac proteins between plant and animal kingdoms in-vivo. Additionally, ZmRacD is a novel transgenic model that gradually develops cardiac phenotype with aging. Furthermore, the shift from cardiac hypertrophy to dilated hearts via T4 treatment will provide us with an excellent system to study the temporal changes in cardiac signaling from adaptive to maladaptive hypertrophy and heart failure.

Although stroke is a common cause of death and a major cause of disability all over the world, genetic components of common forms of ischemic stroke are largely unknown. To identify susceptibility genes of atherothrombotic stroke, we performed a large case-control association study and a replication study in a total of 2775 cases with atherothrombotic stroke and 2839 controls. Through the analysis in 860 cases and 860 age- and sex-matched controls, we found that a single-nucleotide polymorphism (SNP), rs2280887, in the ARHGEF10 gene was significantly associated with atherothrombotic stroke even after the adjustment of multiple testing by a permutation test [unadjusted P = 1.2 x 10(-6), odds ratio = 1.80, 95% confidence interval (CI) = 1.42-2.28]. This association was replicated in independent 1915 cases and 1979 controls. Subsequent fine mapping found another three SNPs which showed similar association due to strong linkage disequilibrium to rs2280887 (r(2) > 0.95). In the functional analyses of these four highly associated SNPs, using luciferase assay and electrophoretic mobility shift assay we found that rs4376531 affected ARHGEF10 transcriptional activity due to the different Sp1-binding affinity. In small GTPase activity assay, we found that a gene product of ARHGEF10 specifically activated RhoA. A population-based cohort study revealed the subjects with rs4376531 CC or CG to increase the incidence of ischemic stroke (P = 0.033, hazard ratio = 1.79, 95% CI = 1.05-3.04). Our data suggest that the functional SNP of ARHGEF10 confers the susceptibility to atherothrombotic stroke.

The overall goal of this study was to determine the molecular basis by which mixed-lineage kinase 3 (MLK3) kinase and its signaling pathways are negatively regulated by the pro-survival Akt pathway in cerebral ischemia. We demonstrated that tyrosine phosphorylation of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) underlies the increased Akt-Ser473 phosphorylation by orthovanadate. Co-immunoprecipitation analysis revealed that endogenous Akt physically interacts with Rac1 in the hippocampal CA1 region, and this interaction is promoted on tyrosine phosphatase inhibition. The elevated Akt activation can deactivate MLK3 by phosphorylation at the Ser71 residue of Rac1, a small Rho family of guanidine triphosphatases required for MLK3 autophosphorylation. Subsequently, inhibition of c-Jun N-terminal kinase 3 (JNK3) results in decreased serine phosphorylation of 14-3-3, a cytoplasmic anchor of Bax, and prevents ischemia-induced mitochondrial translocation of Bax, release of cytochrome c and activation of caspase 3. At the same time, the expression of Fas-ligand decreases in the CA1 region after inhibition of c-Jun activation. The neuroprotective effect of Akt activation is significant in the CA1 region after global cerebral ischemia. Our results suggest that the activation of the pro-apoptotic MLK3/JNK3 cascade induced by ischemic stress can be suppressed through activation of the anti-apoptotic phosphatidylinositol 3-kinase/Akt pathway, which provides a direct link between Akt and the family of stress-activated kinases.

BACKGROUND: Ras proteins play an essential role in the transduction of signals from a wide range of cell-surface receptors to the nucleus. These signals may promote cellular proliferation or differentiation, depending on the cell background. It is well established that Ras plays an important role in the transduction of mitogenic signals from activated growth-factor receptors, leading to cell-cycle entry. However, important questions remain as to whether Ras controls signalling events during cell-cycle progression and, if so, at which point in the cell-cycle it is activated. RESULTS: To address these questions we have developed a novel, functional assay for the detection of cellular activated Ras. Using this assay, we found that Ras was activated in HeLa cells, following release from mitosis, and in NIH 3T3 fibroblasts, following serum-stimulated cell-cycle entry. In each case, peak Ras activation occurred in mid-G1 phase. Ras activation in HeLa cells at mid-G1 phase was dependent on RNA and protein synthesis and was not associated with tyrosine phosphorylation of Shc proteins and their binding to Grb2. Significantly, activation of Ras and the extracellular-signal regulated (ERK) sub-group of mitogen-activated protein kinases were not temporally correlated during G1-phase progression. CONCLUSIONS: Activation of Ras during mid-G1 phase appears to differ in many respects from its rapid activation by growth factors, suggesting a novel mechanism of regulation that may be intrinsic to cell-cycle progression. Furthermore, the temporal dissociation between Ras and ERK activation suggests that Ras targets alternate effector pathways during G1-phase progression.