G-Protein-Coupled Receptor Kinase Interacting Protein-1 Is Required for Pulmonary Vascular Development

Aab Cardiovascular Research Institute and the Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Circulation (Impact Factor: 14.43). 04/2009; 119(11):1524-32. DOI: 10.1161/CIRCULATIONAHA.108.823997
Source: PubMed


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

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    • "Expression analyses suggest widespread expression of GIT2, whereas GIT1 is localised to endothelial cells, cells lining the bronchi and the bile duct (Schmalzigaug et al., 2007). Although GIT1-knockout mice have impaired vascularisation of the developing lung (Pang et al., 2009), no vascular phenotype has been reported for the GIT2 knockout (Schmalzigaug et al., 2009). This might be due to functional redundancy between these two GIT proteins. "
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    ABSTRACT: RhoJ is a RhoGTPase expressed in endothelial cells and tumour cells which regulates cell motility, invasion, endothelial tube formation and focal adhesion numbers. This study aimed to further delineate the molecular function of RhoJ. Using timelapse microscopy RhoJ was found to regulate focal adhesion disassembly; siRNA-mediated knockdown of RhoJ increased focal adhesion disassembly time, while expression of an active mutant (daRhoJ) decreased it. Further, daRhoJ co-precipitated with the GIT-PIX complex, a regulator of focal adhesion disassembly. An interaction between daRhoJ and GIT1 was confirmed using yeast-2-hybrid, which depended on the Spa homology domain of GIT1. GIT1, GIT2, β-PIX and RhoJ all co-localised in focal adhesions and depended on each other for their recruitment to focal adhesions. Functionally, the GIT-PIX complex regulated endothelial tube formation, with knockdown of GIT1/2 or β-PIX phenocopying RhoJ knockdown. RhoJ knockout mice showed reduced tumour growth and diminished tumour vessel density, identifying a role for RhoJ in mediating tumour angiogenesis. These studies give novel insight into the molecular function of RhoJ in regulating cell motility and tumour vessel formation.
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    • "Through these domains, GIT1 interacts with diverse proteins including ARF6, MEK, phospholipase C-γ (PLCγ), p21-activated kinase (PAK)-interacting exchange factor (PIX) and paxillin [20], [21]. GIT1 has diverse biological functions, which we have shown to include a critical role in pulmonary vascular development by regulating VEGF induced PLCγ and ERK1/2 activation [22]. GIT1 is also upregulated in atherosclerotic plaques and regulates endothelial cell and vascular smooth muscle cell migration [23]. "
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    ABSTRACT: G protein coupled receptor kinase 2 (GRK2) interacting protein-1 (GIT1), is a scaffold protein that plays an important role in angiogenesis and osteoclast activity. We have previously demonstrated that GIT1 knockout (GIT1 KO) mice have impaired angiogenesis and dysregulated osteoclast podosome formation leading to a reduction in the bone resorbing ability of these cells. Since both angiogenesis and osteoclast-mediated bone remodeling are involved in the fracture healing process, we hypothesized that GIT1 participates in the normal progression of repair following bone injury. In the present study, comparison of fracture healing in wild type (WT) and GIT1 KO mice revealed altered healing in mice with loss of GIT1 function. Alcian blue staining of fracture callus indicated a persistence of cartilagenous matrix in day 21 callus samples from GIT1 KO mice which was temporally correlated with increased type 2 collagen immunostaining. GIT1 KO mice also showed a decrease in chondrocyte proliferation and apoptosis at days 7 and 14, as determined by PCNA and TUNEL staining. Vascular microcomputed tomography analysis of callus samples at days 7, 14 and 21 revealed decreased blood vessel volume, number, and connection density in GIT1 KO mice compared to WT controls. Correlating with this, VEGF-A, phospho-VEGFR2 and PECAM1 (CD31) were decreased in GIT1 KO mice, indicating reduced angiogenesis with loss of GIT1. Finally, calluses from GIT1 KO mice displayed a reduced number of tartrate resistant acid phosphatase-positive osteoclasts at days 14 and 21. Collectively, these results indicate that GIT1 is an important signaling participant in fracture healing, with gene ablation leading to reduced callus vascularity and reduced osteoclast number in the healing callus.
    PLoS ONE 02/2014; 9(2):e89127. DOI:10.1371/journal.pone.0089127 · 3.23 Impact Factor
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    • "In addition to their roles as regulators of GPCR internalization and resensitization [1] [4], in vitro, GIT proteins have been investigated for their participation in focal adhesion dynamics, cell migration [5] [6], and as scaffolding proteins directing the spatial localization of signaling molecules such as MEK1 and ERK1/2 [7]. In vivo, GIT expression has been shown to regulate emotional function [8] [9] vascular development [10] and mitochondrial biogenesis [11]. Little, however, is known about the role of GIT proteins in bone physiology. "
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    ABSTRACT: G protein-coupled receptor kinase interacting protein 2 (GIT2) is a signaling scaffold protein involved in the regulation of cytoskeletal structure, membrane trafficking, and G protein-coupled receptor internalization. Since dynamic cytoskeletal reorganization plays key roles both in osteoblast differentiation and in the maintenance of osteoclast polarity during bone resorption, we hypothesized that skeletal physiology would be altered in GIT2(-/-) mice. We found that adult GIT2(-/-) mice have decreased bone mineral density and bone volume in both the trabecular and cortical compartments. This osteopenia was associated with decreased numbers of mature osteoblasts, diminished osteoblastic activity, and increased marrow adiposity, suggesting a defect in osteoblast maturation. In vitro, mesenchymal stem cells derived from GIT2(-/-) mice exhibited impaired differentiation into osteoblasts and increased adipocyte differentiation, consistent with a role for GIT2 in mesenchymal stem cell fate determination. Despite elevated osteoclast inducing cytokines and osteoclast numbers, GIT2(-/-) mice also exhibit impaired bone resorption, consistent with a further role for GIT2 in regulating osteoclast function. Collectively, these findings underscore the importance of the cytoskeleton in both osteoblast and osteoclast function and demonstrate that GIT2 plays essential roles in skeletal metabolism, affecting both bone formation and bone resorption in vivo.
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