KRIT-1/CCM1 is a Rap1 effector that regulates endothelial cell–cell junctions

Cerebral cavernous malformation (CCM), a disease associated with defective endothelial junctions, result from autosomal dominant CCM1 mutations that cause loss of KRIT-1 protein function, though how the loss of KRIT-1 leads to CCM is obscure. KRIT-1 binds to Rap1, a guanosine triphosphatase that maintains the integrity of endothelial junctions. Here, we report that KRIT-1 protein is expressed in cultured arterial and venous endothelial cells and is present in cell–cell junctions. KRIT-1 colocalized and was physically associated with junctional proteins via its band 4.1/ezrin/radixin/moesin (FERM) domain. Rap1 activity regulated the junctional localization of KRIT-1 and its physical association with junction proteins. However, the association of the isolated KRIT-1 FERM domain was independent of Rap1. Small interfering RNA–mediated depletion of KRIT-1 blocked the ability of Rap1 to stabilize endothelial junctions associated with increased actin stress fibers. Thus, Rap1 increases KRIT-1 targeting to endothelial cell–cell junctions where it suppresses stress fibers and stabilizes junctional integrity

Regulation of Endothelial Cell Behavior and Vascular Function by Krev Interaction Trapped Protein 1 (KRIT1)

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2016.Disruption of endothelial cell-cell contact is a key event in many cardiovascular diseases and is a characteristic of pathologically activated vascular endothelium. Krev Interaction Trapped Protein 1 (KRIT1) is a scaffold protein that co-localizes with β- catenin at endothelial adherens junctions and is necessary for maintaining a stable endothelial monolayer. In endothelial cells, depletion of KRIT1 leads to monolayer leak, dissociation of β-catenin from VE cadherin, stress fiber formation, and cell migration. Heterozygous loss of KRIT1 in humans is linked to the neurovascular disease Cerebral Cavernous Malformation (CCM). CCM presents itself as areas of focal hemorrhage in the central nervous system and is characterized by the loss of endothelial cell-cell contact. However, the role of KRIT in CCM pathogenesis is poorly understood and its regulation and role in endothelial cell biology needs to be studied. In this thesis project, we demonstrate that Phospholipase C epsilon (PLCε), a lipase and Rap1 GEF, regulates endothelial barrier stability, potentially upstream of KRIT. Additionally, we describe a novel role of KRIT1 in regulating vascular endothelial growth factor (VEGF). Knockdown of KRIT1 expression in endothelial cells increases nuclear β-catenin, which in turn up-regulates VEGF protein levels. This subsequently activates vascular endothelial growth factor receptor 2 (VEGFR2) on endothelial cells, leading to monolayer leak, cell migration, stress fiber formation, and increased phosphorylation of β-catenin. However, the initial disruption of β-catenin from VE-cadherin and its nuclear translocation is only partially dependent on this VEGF mediated pathway. To determine whether this pathway was relevant to CCM pathogenesis, we utilized an endothelial null KRIT1 mouse model. Endothelial specific KRIT1 knockout mice develop areas of focal hemorrhage in the cerebellum, mirroring human CCM, as well as exhibiting hyperpermeability and increased vascular density in their cremaster muscle. Using SU5416, a VEGFR2 inhibitor, we were able to reduce the lesion number and lesion hemorrhage, reverse the cremaster permeability, and reduce the vascular density. In summary, we have uncovered a novel regulatory pathway of KRIT1 and identified a potential therapeutic avenue for CCM pathogenesis and potentially other vascular disorders

Oxidative Stress and Inflammation in Cerebral Cavernous Malformation Disease Pathogenesis: Two Sides of the Same Coin

AbstractCerebral Cavernous Malformation (CCM) is a vascular disease of proven genetic origin, which may arise sporadically or is inherited as an autosomal dominant condition with incomplete penetrance and highly variable expressivity. CCM lesions exhibit a range of different phenotypes, including wide inter-individual differences in lesion number, size, and susceptibility to intracerebral hemorrhage (ICH). Lesions may remain asymptomatic or result in pathological conditions of various type and severity at any age, with symptoms ranging from recurrent headaches to severe neurological deficits, seizures, and stroke. To date there are no direct therapeutic approaches for CCM disease besides the surgical removal of accessible lesions. Novel pharmacological strategies are particularly needed to limit disease progression and severity and prevent de novo formation of CCM lesions in susceptible individuals.Useful insights into innovative approaches for CCM disease prevention and treatment are emerging from a growing understanding of the biological functions of the three known CCM proteins, CCM1/KRIT1, CCM2 and CCM3/PDCD10. In particular, accumulating evidence indicates that these proteins play major roles in distinct signaling pathways, including those involved in cellular responses to oxidative stress, inflammation and angiogenesis, pointing to pathophysiological mechanisms whereby the function of CCM proteins may be relevant in preventing vascular dysfunctions triggered by these events. Indeed, emerging findings demonstrate that the pleiotropic roles of CCM proteins reflect their critical capacity to modulate the fine-tuned crosstalk between redox signaling and autophagy that govern cell homeostasis and stress responses, providing a novel mechanistic scenario that reconciles both the multiple signaling pathways linked to CCM proteins and the distinct therapeutic approaches proposed so far. In addition, recent studies in CCM patient cohorts suggest that genetic susceptibility factors related to differences in vascular sensitivity to oxidative stress and inflammation contribute to inter-individual differences in CCM disease susceptibility and severity.This review discusses recent progress into the understanding of the molecular basis and mechanisms of CCM disease pathogenesis, with specific emphasis on the potential contribution of altered cell responses to oxidative stress and inflammatory events occurring locally in the microvascular environment, and consequent implications for the development of novel, safe, and effective preventive and therapeutic strategies

Activation of m-Calpain (Calpain II) by Epidermal Growth Factor Is Limited by Protein Kinase A Phosphorylation of m-Calpain

We have shown previously that the ELR-negative CXC chemokines interferon-inducible protein 10, monokine induced by gamma interferon, and platelet factor 4 inhibit epidermal growth factor (EGF)-induced m-calpain activation and thereby EGF-induced fibroblast cell motility (H. Shiraha, A. Glading, K. Gupta, and A. Wells, J. Cell Biol. 146:243-253, 1999). However, how this cross attenuation could be accomplished remained unknown since the molecular basis of physiological m-calpain regulation is unknown. As the initial operative attenuation signal from the CXCR3 receptor was cyclic AMP (cAMP), we verified that this second messenger blocked EGF-induced motility of fibroblasts (55% ± 4.5% inhibition) by preventing rear release during active locomotion. EGF-induced calpain activation was inhibited by cAMP activation of protein kinase A (PKA), as the PKA inhibitors H-89 and Rp-8Br-cAMPS abrogated cAMP inhibition of both motility and calpain activation. We hypothesized that PKA might negatively modulate m-calpain in an unexpected manner by directly phosphorylating m-calpain. A mutant human large subunit of m-calpain was genetically engineered to negate a putative PKA consensus sequence in the regulatory domain III (ST369/370AA) and was expressed in NR6WT mouse fibroblasts to represent about 30% of total m-calpain in these cells. This construct was not phosphorylated by PKA in vitro while a wild-type construct was, providing proof of the principle that m-calpain can be directly phosphorylated by PKA at this site. cAMP suppressed EGF-induced calpain activity of cells overexpressing a control wild-type human m-calpain (83% ± 3.7% inhibition) but only marginally suppressed that of cells expressing the PKA-resistant mutant human m-calpain (25% ± 5.5% inhibition). The EGF-induced motility of the cells expressing the PKA-resistant mutant also was not inhibited by cAMP. Structural modeling revealed that new constraints resulting from phosphorylation at serine 369 would restrict domain movement and help “freeze” m-calpain in an inactive state. These data point to a novel mechanism of negative control of calpain activation, direct phosphorylation by PKA

Phospholipase Cε Modulates Rap1 Activity and the Endothelial Barrier

<div><p>The phosphoinositide-specific phospholipase C, PLCε, is a unique signaling protein with known roles in regulating cardiac myocyte growth, astrocyte inflammatory signaling, and tumor formation. PLCε is also expressed in endothelial cells, however its role in endothelial regulation is not fully established. We show that endothelial cells of multiple origins, including human pulmonary artery (HPAEC), human umbilical vein (HUVEC), and immortalized brain microvascular (hCMEC/D3) endothelial cells, express PLCε. Knockdown of PLCε in arterial endothelial monolayers decreased the effectiveness of the endothelial barrier. Concomitantly, RhoA activity and stress fiber formation were increased. PLCε-deficient arterial endothelial cells also exhibited decreased Rap1-GTP levels, which could be restored by activation of the Rap1 GEF, Epac, to rescue the increase in monolayer leak. Reintroduction of PLCε rescued monolayer leak with both the CDC25 GEF domain and the lipase domain of PLCε required to fully activate Rap1 and to rescue endothelial barrier function. Finally, we demonstrate that the barrier promoting effects PLCε are dependent on Rap1 signaling through the Rap1 effector, KRIT1, which we have previously shown is vital for maintaining endothelial barrier stability. Thus we have described a novel role for PLCε PIP₂ hydrolytic and Rap GEF activities in arterial endothelial cells, where PLCε-dependent activation of Rap1/KRIT1 signaling promotes endothelial barrier stability.</p></div