The G-protein-coupled receptor (GPCR)-kinase-interacting proteins 1 and 2 (GIT1 and GIT2) are ubiquitous multidomain proteins involved in diverse cellular processes. They traffic between three distinct cellular compartments (cytoplasmic complexes, focal adhesions and the cell periphery) through interactions with proteins including ARF, Rac1 and Cdc42 GTPases, p21-activated kinase (PAK), PAK-interacting exchange factor (PIX), the kinase MEK1, phospholipase Cγ (PLCγ) and paxillin. GITs and PIX cooperate to form large oligomeric complexes to which other proteins are transiently recruited. Activation of Rac1 and Cdc42 drives association of PAK with these oligomers, which unmasks the paxillin-binding site in GITs that recruits them to focal complexes. There, they regulate cytoskeletal dynamics by feedback inhibition of Rac1. GITs also participate in receptor internalization by regulating membrane trafficking between the plasma membrane and endosomes, targeting ARF GTPases through their ARF GTPase-activating protein (ARF-GAP) activity. Furthermore, GITs act as scaffolds to control spatial activation of several signaling molecules. Finally, recent results suggest pathogenic roles for GIT proteins in Huntington's disease and HIV infection.

Many findings suggest that steady laminar flow in blood vessels activates signal transduction events that lead to expression of atheroprotective genes (1, 2). The nature and magnitude of shear stress plays an important role in long-term maintenance of the structure and function of the blood vessel. In ''linear'' areas of the vasculature blood flows in ordered laminar patterns in a pulsatile fashion and endothelial cells (ECs) experience pulsatile shear stress with fluctuations in magnitude that yield a mean positive shear stress. At areas of abrupt curvatures in the vasculature, as in the carotid bifurcation, the laminar flow of blood is disrupted and separate flow patterns result (3-8). The significance of these flow patterns is demonstrated by studies that correlate development of atherosclerotic lesions (fatty streaks and small plaques) with areas of the carotid that experience these flow reversals with low time-averaged shear stress (3, 4). Regions of the carotid bifurcation that experience steady nonoscillatory shear stress as the result of laminar blood flow patterns are relatively protected from atherosclerosis. Examples of the atheroprotective nature of steady laminar flow are inhibition of E-selectin expression and suppression of vascular cell adhesion molecule 1 (VCAM-1) induction by cytokines such as IL-1 and tumor necrosis factor ␣

Background The G-protein-coupled receptor (GPCR)-kinase interacting protein-1 (GIT1) is a multi-domain scaffold protein that participates in many cellular functions including receptor internalization, focal adhesion remodeling, and signaling by both GPCRs and tyrosine kinase receptors. However, there have been no in vivo studies of GIT1 function to date. Methods and Results To determine essential functions of GIT1 in vivo, we generated a traditional GIT1 knockout (KO) mouse. GIT1 KO mice exhibited ∼60% perinatal mortality. Pathologic examination showed that the major abnormality in GIT1-KO mice was impaired lung development characterized by markedly reduced numbers of pulmonary blood vessels and increased alveolar spaces. Since vascular endothelial growth factor (VEGF) is essential for pulmonary vascular development, we investigated the role of GIT1 in VEGF signaling in the lung and cultured endothelial cells (EC). Because activation of phospholipase-Cγ (PLCγ and ERK1/2 by angiotensin II requires GIT1, we hypothesized that GIT1 mediates VEGF-dependent pulmonary angiogenesis by modulating PLCγ and ERK1/2 activity in EC. In cultured EC, knockdown of GIT1 decreased VEGF-mediated phosphorylation of PLCγ and ERK1/2. PLCγ and ERK1/2 activity in lungs from GIT1 KO mice was reduced postnatally. Conclusions Our data support a critical role for GIT1 in pulmonary vascular development by regulating VEGF-induced PLCγ and ERK1/2 activation.

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Abstract-c-Jun NH 2 -terminal kinase (JNK) is activated by a number of cellular stimuli including reactive oxygen species (ROS). Previous studies have demonstrated that fluid shear stress (flow) inhibits cytokine-induced JNK activation in endothelial cells (ECs). In the present study, we show JNK activation by ROS in ECs and hypothesized that flow inhibits ROS-induced JNK activation in ECs via modulation of cellular protection systems against ROS. JNK was activated by 300 mol/L hydrogen peroxide (H 2 O 2 ) in bovine lung microvascular ECs (BLMVECs) with a peak at 60 minutes after stimulation (6.3Ϯ1.2-fold increase). Preexposure of BLMVECs to physiological steady laminar flow (shear stressϭ12 dyne/cm 2 ) for 10 minutes significantly decreased H 2 O 2 -induced JNK activation. Thioredoxin and glutathione are cellular antioxidants that protect cells against ROS. Flow induced a significant increase in the ratio of reduced glutathione to oxidized glutathione consistent with a 1.6-fold increase in glutathione reductase (GR) activity. Preincubation of BLMVECs with the GR inhibitor, 1,3 bis-(2 chloroethyl)-1-nitrosourea, abolished the inhibitory effect of flow. In contrast, preincubation of BLMVECs with azelaic acid, a specific inhibitor for thioredoxin reductase, did not alter the effect of flow on H 2 O 2 -induced JNK activation. Overexpression of GR mimicked the effect of flow to inhibit JNK activation. These results suggest that flow activates GR, an important regulator of the intracellular redox state of glutathione, and exerts a protective mechanism against oxidative stress in endothelial cells. Key Words: oxidative stress Ⅲ endothelial cell Ⅲ signal transduction A growing body of evidence indicates that reactive oxygen species (ROS) are involved in cardiovascular diseases such as atherosclerosis, 1 reperfusion injury, 2 heart failure, 3-5 and hypertension. 6,7 Previous studies have clarified that ROS are important regulators of signaling events in pathological cardiovascular condition. 8,9 Among cellular signals, members of mitogen-activated protein (MAP) kinases play critical roles in cellular proliferation, inflammation, and apoptosis. 10 -12 We have demonstrated that hydrogen peroxide (H 2 O 2 ) activates MAP kinases in cultured cells derived from vascular tissue. [13][14][15] c-Jun NH 2 -terminal kinase (JNK) is activated by a number of cellular stimuli including proinflammatory cytokines and ROS. 16,17 It is speculated that JNK plays an important role in proatherogenic signal events through phosphorylation of c-Jun, activation of AP-1, and stimulation of proinflammatory gene expression such as ICAM-1. Our previous studies have demonstrated that laminar fluid shear stress (flow) inhibits cytokine-induced JNK activation in vascular endothelial cells. 18,19 These results are consistent with the concept that flow exerts an atheroprotective effect against inflammatory cytokines. 20 -23 Cardiovascular risk factors such as smoking, hypercholesterolemia, diabetes, and hypertension have been shown to share a common...