Jinjiang Pang

Phospholipase Cε hydrolyzes perinuclear phosphatidylinositol 4-phosphate to regulate cardiac hypertrophy.

Phospholipase Cε (PLCε) is a multifunctional enzyme implicated in cardiovascular, pancreatic, and inflammatory functions. Here we show that conditional deletion of PLCε in mouse cardiac myocytes protects from stress-induced pathological hypertrophy. PLCε small interfering RNA (siRNA) in ventricular myocytes decreases endothelin-1 (ET-1)-dependent elevation of nuclear calcium and activation of nuclear protein kinase D (PKD). PLCε scaffolded to muscle-specific A kinase-anchoring protein (mAKAP), along with PKCε and PKD, localizes these components at or near the nuclear envelope, and this complex is required for nuclear PKD activation. Phosphatidylinositol 4-phosphate (PI4P) is identified as a perinuclear substrate in the Golgi apparatus for mAKAP-scaffolded PLCε. We conclude that perinuclear PLCε, scaffolded to mAKAP in cardiac myocytes, responds to hypertrophic stimuli to generate diacylglycerol (DAG) from PI4P in the Golgi apparatus, in close proximity to the nuclear envelope, to regulate activation of nuclear PKD and hypertrophic signaling pathways.

GIT1 Mediates VEGF-Induced Podosome Formation in Endothelial Cells Critical Role for PLCγ

Objective—We and others showed that tyrosine kinase receptors (TKRs) such as the epidermal growth factor receptor stimulate G protein–coupled receptor (GPCR) kinase-interacting protein 1 (GIT1) phosphorylation via c-Src, which is required for phospholipase C-&ggr; (PLC&ggr;) activation, indicating that GIT1 participates in TKR signaling. VEGF is the most important TKR in endothelial cells (ECs); essential for cell survival, migration, and angiogenesis. Podosomes, actin-rich structures, were found to contribute to EC migration, tissue invasion, and matrix remodeling, suggesting a role for podosomes in angiogenesis. Because GIT1 is a substrate of c-Src, and podosome formation is c-Src dependent, we hypothesized that GIT1 plays an important role in VEGF-induced EC podosome formation and cell migration. Methods and Results—Exposure of ECs to VEGF for 30 minutes stimulated GIT1 colocalization with podosomes. Depletion of GIT1 by siRNA significantly decreased VEGF-induced podosome formation. A key role for PLC&ggr; was suggested by several experiments. Double staining PLC&ggr; and actin showed colocalization of PLC&ggr; with podosomes. Podosome formation was dramatically reduced by PLC&ggr; inhibitor U73122, Src inhibitor PP2, or expression of dominant negative small GTPases. Therefore, VEGF-induced EC podosome formation is dependent on Src, GIT1, PLC&ggr;, and small GTPases. In addition, matrix metalloprotease 2 (MMP2) and MT-MMP1 were detected at sites of VEGF-induced podosomes. Depletion of GIT1 by siRNA also significantly inhibited VEGF-induced MMP2 activation and extracellular matrix (ECM) degradation. Therefore, GIT1 mediates VEGF-induced matrix metalloproteinase (MMP) activation and ECM degradation by regulating podosome formation. Finally, depletion of GIT1 by siRNA significantly decreased VEGF-induced cell migration. Conclusions—These data indicate that GIT1 is an essential mediator for VEGF-induced EC podosome formation and cell migration via PLC&ggr;.

Hexarelin suppresses high lipid diet and vitamin D3-induced atherosclerosis in the rat

Growth hormone-releasing peptides (GHRP) and ghrelin are synthetic and natural ligands of growth hormone secretagogue receptor (GHSR) respectively and are shown to exert protective actions on cardiac dysfunction. Because ghrelin has been reported to inhibit proinflammatory responses in human endothelium and GHSR has been identified in blood vessels, we hypothesized that GHRP could alleviate the development of atherosclerosis (As). Atherosclearosis was induced by a short period (4 days) of vitamin D(3) and chronic (three months) intragastric feeding of high fat emulsion (containing 0.5% propylthiouracil) in adult SD rats. Some As rats received chronic hexarelin (a variant of GHRP) injection (SC BID, 30 days) and normal rats received placebo as control. Significant atherosclerosis developed in animals fed with the emulsion. Serum total cholesterol and LDL-c increased, and HDL-c and aortic nitric oxide (NO) decreased significantly in As group. Hexarelin suppressed the formation of atherosclerotic plaques and neointima, partially reversed serum HDL-c/LDL-c ratio and increased the levels of serum NO and aortic mRNAs of eNOS, GHSR and CD36 in As rats. Hexarelin also decreased [(3)H]-TdR incorporation in cultured vascular smooth muscle cell (VSMC) and calcium sedimentation in aortic wall. Furthermore, foam cell formation induced by ox-LDL was decreased by hexarelin. In conclusion, hexarelin suppresses high lipid diet and vitamin D3-induced atherosclerosis in rats, possibly through upregulating HDL-c/LDL-c ratio, vascular NO production and downregulating the VSMC proliferation, aortic calcium sedimentation and foam cell formation. These novel anti-atherosclerotic actions of hexarelin suggest that the peptide might have a clinical potential in treating atherosclerosis.

Gas6–Axl Receptor Signaling Is Regulated by Glucose in Vascular Smooth Muscle Cells

Objective—The receptor tyrosine kinase Axl and its ligand Gas6 are involved in the development of renal diabetic disease. In vascular smooth muscle cells (VSMCs) Axl is activated by reactive oxygen species and stimulates migration and cell survival, suggesting a role for Axl in the vascular complications of diabetes. Methods and Results—We investigated the effect of varying glucose concentration on Axl signaling in VSMCs. Glucose exerted powerful effects on Gas6-Axl signaling with greater activation of Akt and mTOR in low glucose, and greater activation of ERK1/2 in high glucose. Plasma membrane distribution and tyrosine phosphorylation of Axl were not affected by glucose. However, coimmunoprecipitation studies demonstrated that glucose changed the interaction of Axl with its binding partners. Specifically, binding of Axl to the p85 subunit of PI3-kinase was increased in low glucose, whereas binding to SHP-2 was increased in high glucose. Furthermore, Gas6-Axl induced migration was increased in high glucose, whereas Gas6-Axl mediated inhibition of apoptosis was greater in low glucose. Conclusion—This study demonstrates a role for glucose in altering Axl signaling through coupling to binding partners and suggests a mechanism by which Axl contributes to VSMC dysfunction in diabetes.

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

Background— 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. Methods and Results— To determine essential functions of GIT1 in vivo, we generated a traditional GIT1 knockout mouse. GIT1 knockout mice exhibited ≈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-Cγ (PLCγ) 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 PLCγ and ERK1/2 activity in endothelial cells. In cultured endothelial cells, knockdown of GIT1 decreased VEGF-mediated phosphorylation of PLCγ and ERK1/2. PLCγ and ERK1/2 activity in lungs from GIT1 knockout 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.

GIT1 Mediates HDAC5 Activation by Angiotensin II in Vascular Smooth Muscle Cells

Objective—The G protein–coupled receptor (GPCR)-kinase2 interacting protein1 (GIT1) is a scaffold protein involved in angiotensin II (Ang II) signaling. Histone deacetylase-5 (HDAC5) has emerged as an important substrate of calcium/calmodulin-dependent protein kinase II (CamK II) in GPCR signaling. Here we investigated the hypothesis that Ang II–mediated vascular smooth muscle cell (VSMC) gene transcription involves GIT1-CamK II–dependent phosphorylation of HDAC5. Methods and Results—Ang II rapidly stimulated phosphorylation of HDAC5 at Ser498 in VSMCs. Knockdown of GIT1 significantly decreased HDAC5 phosphorylation induced by Ang II. The involvement of Src, phospholipase &ggr; (PLC&ggr;), and CamK II in GIT1-mediated HDAC5 phosphorylation was demonstrated. The association of GIT1 and CamK II was constitutive but increased after stimulation with Ang II. Moreover, the interaction of GIT1 and CamK II through the ARF GTPase-activating protein (ARF-GAP) and coiled-coil domains of GIT1 was essential for the phosphorylation of HDAC5. Finally, knockdown of GIT1 decreased myocyte enhancer factor 2 transcriptional activity induced by Ang II. Conclusions—This study identifies a novel function for GIT1 as a mediator of Ang II–induced VSMC gene transcription via a Src-PLC&ggr;-CamK II-HDAC5 signaling pathway.

Thioredoxin-interacting protein mediates sustained VEGFR2 signaling in endothelial cells required for angiogenesis.

Objective—Thioredoxin-interacting protein (TXNIP) is an &agr;-arrestin protein whose function is important for the regulation of vascular endothelial growth factor receptor 2 (VEGFR2) signaling and endothelial cell survival. Because VEGFR2 is critical for angiogenesis, we explored the role of TXNIP in VEGF-induced angiogenesis. Approach and Results—TXNIP knockdown inhibited VEGF-induced endothelial cell tube formation and proliferation in cultured human umbilical vein endothelial cell. To elucidate the mechanism by which TXNIP altered VEGFR2 signaling in human umbilical vein endothelial cell, we studied phosphorylation of VEGFR2, phospholipase C gamma-1 (PLC&ggr;1), endothelial NO synthase, and Akt (known as protein kinase B). TXNIP knockdown significantly decreased phosphorylation of VEGFR2 and PLC&ggr;1 at times >5 minutes, but phosphorylation was unchanged at 2 minutes, as was Akt and endothelial NO synthase phosphorylation. Cell-surface biotinylation assay showed that TXNIP knockdown significantly attenuated VEGFR2 internalization. These results suggested that TXNIP was required for sustained VEGFR2 signaling, which is mediated largely by internalized VEGFR2. Rab5 knockdown to inhibit the trafficking and fusion of early endosomes significantly blocked VEGF-induced VEGFR2 internalization and phosphorylation of VEGFR2 and PLC&ggr;1. Immunofluorescence and coimmunoprecipitation showed that TXNIP was part of a complex that included Rab5 and VEGFR2. Finally, TXNIP knockdown prevented the association of VEGFR2 and Rab5. Conclusions—Our results show that TXNIP is essential for VEGFR2 internalization in Rab5 positive endosomes, which is required for endothelial cell growth and angiogenesis.

An epidermal growth factor (EGF) -dependent interaction between GIT1 and sorting nexin 6 promotes degradation of the EGF receptor

G‐protein coupled receptor (GPCR) ki‐nase‐2 interacting protein 1 (GIT1) is a multifunctional scaffolding protein that regulates epidermal growth factor receptor (EGFR) signaling pathways. We demonstrate that GIT1 interacts with sorting nexin 6 (SNX6), a member of the SNX family that increases EGFR trafficking between endosomes and lysosomes, thereby enhancing EGFR degradation. The GIT1‐SNX6 interaction is increased 3‐fold after treatment with EGF for 60 min. The second coiled‐coil domain (CC2;aa 424–474) of GIT1 mediates binding to SNX6. Subcellular fractionation and confocal microscopy data indicate that GIT1 and SNX6 interact in endosomes. Knockdown of GIT1 expression by small interfering RNA decreased the rate of EGF‐induced EGFR degradation. Expression of exogenous GIT1 or SNX6 alone did not alter EGFR degradation;however, coexpression of GIT1 and SNX6 decreased EGFR levels both basally and in response to EGF. In contrast, expression of GIT1(CC2 deleted) and SNX6 did not reduce EGFR levels, demonstrating that the interaction between GIT1 and SNX6 was required to regulate EGFR trafficking. Phosphorylation of the EGFR substrate phospholipase C‐γ was decreased by coexpression of GIT1 and SNX6. These data demonstrate an endosomal, EGF‐regulated interaction between SNX6 and GIT1 that enhances degradation of the EGFR, and thereby alters EGFR signaling. Our findings suggest a new role for GIT1 in tyrosine kinase receptor trafficking.—Cavet, M. E., Pang, J., Yin, G., Berk, B. C. An epidermal growth factor (EGF) ‐dependent interaction between GIT1 and sorting nexin 6 promotes degradation of the EGF receptor. FASEB J. 22, 3607–3616 (2008)

Chronic administration of hexarelin attenuates cardiac fibrosis in the spontaneously hypertensive rat

Cardiac fibrosis is a hallmark of heart disease and plays a vital role in cardiac remodeling during heart diseases, including hypertensive heart disease. Hexarelin is one of a series of synthetic growth hormone secretagogues (GHSs) possessing a variety of cardiovascular effects via action on GHS receptors (GHS-Rs). However, the role of hexarelin in cardiac fibrosis in vivo has not yet been investigated. In the present study, spontaneously hypertensive rats (SHRs) were treated with hexarelin alone or in combination with a GHS-R antagonist for 5 wk from an age of 16 wk. Hexarelin treatment significantly reduced cardiac fibrosis in SHRs by decreasing interstitial and perivascular myocardial collagen deposition and myocardial hydroxyproline content and reducing mRNA and protein expression of collagen I and III in SHR hearts. Hexarelin treatment also increased matrix metalloproteinase (MMP)-2 and MMP-9 activities and decreased myocardial mRNA expression of tissue inhibitor of metalloproteinase (TIMP)-1 in SHRs. In addition, hexarelin treatment significantly attenuated left ventricular (LV) hypertrophy, LV diastolic dysfunction, and high blood pressure in SHRs. The effect of hexarelin on cardiac fibrosis, blood pressure, and cardiac function was mediated by its receptor, GHS-R, since a selective GHS-R antagonist abolished these effects and expression of GHS-Rs was upregulated by hexarelin treatment. In summary, our data demonstrate that hexarelin reduces cardiac fibrosis in SHRs, perhaps by decreasing collagen synthesis and accelerating collagen degradation via regulation of MMPs/TIMP. Hexarelin-reduced systolic blood pressure may also contribute to this reduced cardiac fibrosis in SHRs. The present findings provided novel insights and underscore the therapeutic potential of hexarelin as an antifibrotic agent for the treatment of cardiac fibrosis.

G-Protein–Coupled Receptor-2–Interacting Protein-1 Is Required for Endothelial Cell Directional Migration and Tumor Angiogenesis via Cortactin-Dependent Lamellipodia Formation

Objective—Recent evidence suggests G-protein–coupled receptor-2–interacting protein-1 (GIT1) overexpression in several human metastatic tumors, including breast, lung, and prostate. Tumor metastasis is associated with an increase in angiogenesis. We have showed previously that GIT1 is required for postnatal angiogenesis during lung development. However, the functional role of GIT1 in pathological angiogenesis during tumor growth is unknown. Approach and Results—In the present study, we show inhibition of angiogenesis in matrigel implants as well as reduced tumor angiogenesis and melanoma tumor growth in GIT1-knockout mice. We demonstrate that this is a result of impaired directional migration of GIT1-depleted endothelial cells toward a vascular endothelial growth factor gradient. Cortactin-mediated lamellipodia formation in the leading edge is critical for directional migration. We observed a significant reduction in cortactin localization and lamellipodia formation in the leading edge of GIT1-depleted endothelial cells. We specifically identified that the Spa homology domain (aa 250–420) of GIT1 is required for GIT1–cortactin complex localization to the leading edge. The mechanisms involved extracellular signal-regulated kinases 1 and 2–mediated Cortactin-S405 phosphorylation and activation of Rac1/Cdc42. Finally, using gain of function studies, we show that a constitutively active mutant of cortactin restored directional migration of GIT1-depleted cells. Conclusion—Our data demonstrated that a GIT1–cortactin association through GIT1-Spa homology domain is required for cortactin localization to the leading edge and is essential for endothelial cell directional migration and tumor angiogenesis.