De Novo Elastin Assembly Alleviates Development of Supravalvular Aortic Stenosis-Brief Report.

BACKGROUND: A series of incurable cardiovascular disorders arise due to improper formation of elastin during development. Supravalvular aortic stenosis (SVAS), resulting from a haploinsufficiency of ELN, is caused by improper stress sensing by medial vascular smooth muscle cells, leading to progressive luminal occlusion and heart failure. SVAS remains incurable, as current therapies do not address the root issue of defective elastin. METHODS: We use SVAS here as a model of vascular proliferative disease using both human induced pluripotent stem cell-derived vascular smooth muscle cells and developmental Eln+/- mouse models to establish de novo elastin assembly as a new therapeutic intervention. RESULTS: We demonstrate mitigation of vascular proliferative abnormalities following de novo extracellular elastin assembly through the addition of the polyphenol epigallocatechin gallate to SVAS human induced pluripotent stem cell-derived vascular smooth muscle cells and in utero to Eln+/- mice. CONCLUSIONS: We demonstrate de novo elastin deposition normalizes SVAS human induced pluripotent stem cell-derived vascular smooth muscle cell hyperproliferation and rescues hypertension and aortic mechanics in Eln+/- mice, providing critical preclinical findings for the future application of epigallocatechin gallate treatment in humans.

Thioredoxin-2 inhibits mitochondrial reactive oxygen species generation and apoptosis stress kinase-1 activity to maintain cardiac function.

BACKGROUND: Thioredoxin 2 (Trx2) is a key mitochondrial protein that regulates cellular redox and survival by suppressing mitochondrial reactive oxygen species generation and by inhibiting apoptosis stress kinase-1 (ASK1)-dependent apoptotic signaling. To date, the role of the mitochondrial Trx2 system in heart failure pathogenesis has not been investigated. METHODS AND RESULTS: Western blot and histological analysis revealed that Trx2 protein expression levels were reduced in hearts from patients with dilated cardiomyopathy, with a concomitant increase in ASK1 phosphorylation/activity. Cardiac-specific Trx2 knockout mice develop spontaneous dilated cardiomyopathy at 1 month of age with increased heart size, reduced ventricular wall thickness, and a progressive decline in left ventricular contractile function, resulting in mortality due to heart failure by ≈4 months of age. The progressive decline in cardiac function observed in cardiac-specific Trx2 knockout mice was accompanied by the disruption of mitochondrial ultrastructure, mitochondrial membrane depolarization, increased mitochondrial reactive oxygen species generation, and reduced ATP production, correlating with increased ASK1 signaling and increased cardiomyocyte apoptosis. Chronic administration of a highly selective ASK1 inhibitor improved cardiac phenotype and reduced maladaptive left ventricular remodeling with significant reductions in oxidative stress, apoptosis, fibrosis, and cardiac failure. Cellular data from Trx2-deficient cardiomyocytes demonstrated that ASK1 inhibition reduced apoptosis and reduced mitochondrial reactive oxygen species generation. CONCLUSIONS: Our data support an essential role for mitochondrial Trx2 in preserving cardiac function by suppressing mitochondrial reactive oxygen species production and ASK1-dependent apoptosis. Inhibition of ASK1 represents a promising therapeutic strategy for the treatment of dilated cardiomyopathy and heart failure.

AIP1 mediates vascular endothelial cell growth factor receptor-3-dependent angiogenic and lymphangiogenic responses.

OBJECTIVE: To investigate the novel function of ASK1-interacting protein-1 (AIP1) in vascular endothelial cell growth factor receptor (VEGFR)-3 signaling, and VEGFR-3-dependent angiogenesis and lymphangiogenesis. APPROACH AND RESULTS: AIP1, a signaling scaffold protein, is highly expressed in the vascular endothelium. We have previously reported that AIP1 functions as an endogenous inhibitor in pathological angiogenesis by blocking VEGFR-2 activity. Surprisingly, here we observe that mice with a global deletion of AIP1-knockout mice (AIP1-KO) exhibit reduced retinal angiogenesis with less sprouting and fewer branches. Vascular endothelial cell (but not neuronal)-specific deletion of AIP1 causes similar defects in retinal angiogenesis. The reduced retinal angiogenesis correlates with reduced expression in VEGFR-3 despite increased VEGFR-2 levels in AIP1-KO retinas. Consistent with the reduced expression of VEGFR-3, AIP1-KO show delayed developmental lymphangiogenesis in neonatal skin and mesentery, and mount weaker VEGF-C-induced cornea lymphangiogenesis. In vitro, human lymphatic endothelial cells with AIP1 small interfering RNA knockdown, retinal endothelial cells, and lymphatic endothelial cells isolated from AIP1-KO all show attenuated VEGF-C-induced VEGFR-3 signaling. Mechanistically, we demonstrate that AIP1 via vegfr-3-specific miR-1236 increases VEGFR-3 protein expression and that, by directly binding to VEGFR-3, it enhances VEGFR-3 endocytosis and stability. CONCLUSION: Our in vivo and in vitro results provide the first insight into the mechanism by which AIP1 mediates VEGFR-3-dependent angiogenic and lymphangiogenic signaling.

TNF, acting through inducibly expressed TNFR2, drives activation and cell cycle entry of c-Kit+ cardiac stem cells in ischemic heart disease.

TNF, signaling through TNFR2, has been implicated in tissue repair, a process that in the heart may be mediated by activated resident cardiac stem cells (CSCs). The objective of our study is to determine whether ligation of TNFR2 can induce activation of resident CSCs in the setting of ischemic cardiac injury. We show that in human cardiac tissue affected by ischemia heart disease (IHD), TNFR2 is expressed on intrinsic CSCs, identified as c-kit(+)/CD45(-)/VEGFR2(-) interstitial round cells, which are activated as determined by entry to cell cycle and expression of Lin-28. Wild-type mouse heart organ cultures subjected to hypoxic conditions both increase cardiac TNF expression and show induced TNFR2 and Lin-28 expression in c-kit(+) CSCs that have entered cell cycle. These CSC responses are enhanced by exogenous TNF. TNFR2(-/-) mouse heart organ cultures subjected to hypoxia increase cardiac TNF but fail to induce CSC activation. Similarly, c-kit(+) CSCs isolated from mouse hearts exposed to hypoxia or TNF show induction of Lin-28, TNFR2, cell cycle entry, and cardiogenic marker, α-sarcomeric actin (α-SA), responses more pronounced by hypoxia in combination with TNF. Knockdown of Lin-28 by siRNA results in reduced levels of TNFR2 expression, cell cycle entry, and diminished expression of α-SA. We conclude that hypoxia-induced c-kit(+) CSC activation is mediated by TNF/TNFR2/Lin-28 signaling. These observations suggest that TNFR2 signaling in resident c-kit(+) CSCs induces cardiac repair, findings which provide further understanding of the unanticipated harmful effects of TNF blockade in human IHD.

AIP1 suppresses atherosclerosis by limiting hyperlipidemia-induced inflammation and vascular endothelial dysfunction.

OBJECTIVE: Apoptosis signal-regulating kinase 1-interacting protein-1 (AIP1) is a signaling adaptor molecule implicated in stress and apoptotic signaling induced by proinflammatory mediators. However, its function in atherosclerosis has not been established. In the present study, we use AIP1-null (AIP1(-/-)) mice to examine its effect on atherosclerotic lesions in an apolipoprotein E-null (ApoE(-/-)) mouse model of atherosclerosis. APPROACH AND RESULTS: ApoE(-/-) control mice developed atherosclerosis in the aortic roots and descending aortas on Western-type diet for 10 weeks, whereas the atherosclerotic lesions are significantly augmented in ApoE(-/-)AIP1(-/-) double knockout (DKO) mice. DKO mice show increases in plasma inflammatory cytokines with no significant alterations in body weight, total cholesterol levels, or lipoprotein profiles. Aortas in DKO mice show increased inflammation and endothelial cell (EC) dysfunction with nuclear factor-κB activity, correlating with increased accumulation of macrophages in the lesion area. Importantly, macrophages from DKO donors are not sufficient to augment inflammatory responses and atherogenesis when transferred to ApoE-KO recipients. Mechanistic studies suggest that AIP1 is highly expressed in aortic EC, but not in macrophages, and AIP1 deletion in EC significantly enhance oxidized low-density lipoprotein-induced nuclear factor-κB signaling, gene expression of inflammatory molecules, and monocyte adhesion, suggesting that vascular EC are responsible for the increased inflammatory responses observed in DKO mice. CONCLUSIONS: Our data demonstrate that loss of AIP1 in aortic EC primarily contributes to the exacerbated lesion expansion in the ApoE(-/-)AIP1(-/-) mice, revealing an important role of AIP1 in limiting inflammation, EC dysfunction, and atherosclerosis.

Endothelium-derived neuregulin protects the heart against ischemic injury.

BACKGROUND: Removal of cardiac endothelial cells (EC) has been shown to produce significant detrimental effects on the function of adjacent cardiac myocytes, suggesting that EC play a critical role in autocrine/paracrine regulation of the heart. Despite this important observation, the mediators of the protective function of EC remain obscure. Neuregulin (NRG, a member of the epidermal growth factor family) is produced by EC and cardiac myocytes contain receptors (erbB) for this ligand. We hypothesized that NRG is an essential factor produced by EC, which promotes cardioprotection against ischemic injury. METHODS AND RESULTS: We demonstrate that human cardiac EC express and release NRG in response to hypoxia-reoxygenation. Under conditions where hypoxia--reoxygenation causes significant cardiac myocyte cell death, NRG can significantly decrease apoptosis of isolated adult ventricular myocytes. Coculturing adult murine myocytes with human umbilical vein, murine lung microvascular, or human coronary artery EC can also protect myocytes against hypoxia--reoxygenation--induced apoptosis. These protective effects are abolished by NRG gene deletion or silencing of NRG expression in EC. Finally, endothelium-selective deletion of NRG in vivo leads to significantly decreased tolerance to ischemic insult, as demonstrated by impaired postischemic contractile recovery in a perfused whole-organ preparation and larger infarct sizes after coronary artery ligation. CONCLUSION: Together, these data demonstrate that EC-derived NRG plays an important role in cardiac myocyte protection against ischemic injury in the heart and supports the idea that manipulation of this signaling pathway may be an important clinical target in this setting.

Mitochondrial dysfunction induces ALK5-SMAD2-mediated hypovascularization and arteriovenous malformations in mouse retinas.

Although mitochondrial activity is critical for angiogenesis, its mechanism is not entirely clear. Here we show that mice with endothelial deficiency of any one of the three nuclear genes encoding for mitochondrial proteins, transcriptional factor (TFAM), respiratory complex IV component (COX10), or redox protein thioredoxin 2 (TRX2), exhibit retarded retinal vessel growth and arteriovenous malformations (AVM). Single-cell RNA-seq analyses indicate that retinal ECs from the three mutant mice have increased TGFß signaling and altered gene expressions associated with vascular maturation and extracellular matrix, correlating with vascular malformation and increased basement membrane thickening in microvesels of mutant retinas. Mechanistic studies suggest that mitochondrial dysfunction from Tfam, Cox10, or Trx2 depletion induces a mitochondrial localization and MAPKs-mediated phosphorylation of SMAD2, leading to enhanced ALK5-SMAD2 signaling. Importantly, pharmacological blockade of ALK5 signaling or genetic deficiency of SMAD2 prevented retinal vessel growth retardation and AVM in all three mutant mice. Our studies uncover a novel mechanism whereby mitochondrial dysfunction via the ALK5-SMAD2 signaling induces retinal vascular malformations, and have therapeutic values for the alleviation of angiogenesis-associated human retinal diseases.

Metalloproteinase-dependent cleavage of neuregulin and autocrine stimulation of vascular endothelial cells.

Inflammation is often accompanied by robust angiogenesis. Vascular endothelial cells (ECs) express erbB receptors and their ligand, neuregulin-1, and can respond to neuregulin by proliferation and angiogenesis. We hypothesized that some growth factor-like responses of ECs to inflammatory cytokines can be explained by cleavage of transmembrane neuregulin with subsequent release of its extracellular epidermal growth factor-like-containing domain and autocrine activation. Using a model of cultured human ECs, we found that interleukin-6 or interferon-gamma causes rapid cleavage and release of transmembrane neuregulin. Inhibitors of metalloproteinases abolish this effect. The addition of an inhibitor of tumor necrosis factor-alpha converting enzyme (TACE) blocks cytokine-induced neuregulin release. Silencing of TACE expression increases the amount of basal proneuregulin present in ECs but does not block neuregulin release in response to phorbol myristate acetate (PMA), suggesting that other proteinases are responsible for mediating protein kinase C-dependent cleavage. Cytokines capable of inducing neuregulin cleavage stimulated ERK activation and in vitro angiogenesis (Matrigel cord formation). This effect is blocked by inhibitors that block neuregulin cleavage, erbB protein tyrosine kinase inhibitors, or antineuregulin-neutralizing antibodies. Cytokine-activated metalloproteinase cleavage of neuregulin may play an important role in autocrine activation of EC signaling pathways, contributing to key biological effects, perhaps including inflammation-associated angiogenesis.

Functional role of phosphodiesterase 3 in cardiomyocyte apoptosis: implication in heart failure.

BACKGROUND: Myocyte apoptosis plays an important role in pathological cardiac remodeling and the progression of heart failure. cAMP signaling is crucial in the regulation of myocyte apoptosis and cardiac remodeling. Multiple cAMP-hydrolyzing phosphodiesterases (PDEs), such as PDE3 and PDE4, coexist in cardiomyocytes and elicit differential temporal/spatial regulation of cAMP signaling. However, the role of PDE3 and PDE4 in the regulation of cardiomyocyte apoptosis remains unclear. Although chronic treatment with PDE3 inhibitors increases mortality in patients with heart failure, the contribution of PDE3 expression/activity in heart failure is not well known. METHODS AND RESULTS: In this study we report that PDE3A expression and activity were significantly reduced in human failing hearts as well as mouse hearts with chronic pressure overload. In primary cultured cardiomyocytes, chronic inhibition of PDE3 but not PDE4 activity by pharmacological agents or adenovirus-delivered antisense PDE3A promoted cardiomyocyte apoptosis. Both angiotensin II (Ang II) and the beta-adrenergic receptor agonist isoproterenol selectively induced a sustained downregulation of PDE3A expression and induced cardiomyocyte apoptosis. Restoring PDE3A via adenovirus-delivered expression of wild-type PDE3A1 completely blocked Ang II- and isoproterenol-induced cardiomyocyte apoptosis, suggesting the critical role of PDE3A reduction in cardiomyocyte apoptosis. Moreover, we defined a crucial role for inducible cAMP early repressor expression in PDE3A reduction-mediated cardiomyocyte apoptosis. CONCLUSIONS: Our results suggest that PDE3A reduction and consequent inducible cAMP early repressor induction are critical events in Ang II- and isoproterenol-induced cardiomyocyte apoptosis and may contribute to the development of heart failure. Drugs that maintain PDE3A function may represent an attractive therapeutic approach to treat heart failure.

Insulin-like growth factor-1 enhances inflammatory responses in endothelial cells: role of Gab1 and MEKK3 in TNF-alpha-induced c-Jun and NF-kappaB activation and adhesion molecule expression.

Insulin-like growth factor (IGF)-1 and the type I IGF-1 receptor are important regulators of vascular function that may contribute to cardiovascular disease. We hypothesized that IGF-1 causes endothelial cell dysfunction and expression of neutrophil and monocyte adhesion molecules by enhancing pro-inflammatory cytokine signal transduction. Long-term IGF-1 treatment of endothelial cells potentiated c-Jun and nuclear factor NF-kappaB activation by tumor necrosis factor (TNF)-alpha and enhanced TNF-alpha-mediated adhesion molecule expression. In response to IGF-1 treatment, the expression of kinases in the c-Jun/c-Jun NH(2)-terminal kinase signaling pathway (MEKK1, MEK4, and JNK1/2) was unchanged, but expressions of insulin receptor substrate-1 and Grb2-associated binder-1 (Gab1) were significantly decreased. Because Gab1 is involved in both c-Jun and NF-kappaB activation by TNF-alpha, we focused on Gab1-dependent signaling. Gab1 inhibited c-Jun and NF-kappaB transcriptional activation by TNF-alpha. Interestingly, Gab1 inhibited c-Jun transcriptional activity induced by MEKK3 but not MEKK1 and MEK4. Gab1 associated with MEKK3, and a catalytically inactive form of MEKK3 inhibited TNF-alpha-induced c-Jun and NF-kappaB transcriptional activation, suggesting a critical role for Gab1 and MEKK3 in TNF-alpha signaling. These data demonstrate that Gab1 and MEKK3 play important roles in endothelial cell inflammation via regulating the activation of c-Jun and NF-kappaB. Furthermore, the IGF-1-mediated downregulation of Gab1 expression represents a novel mechanism to promote vascular inflammation and atherosclerosis.

Inhibition of tumor necrosis factor-[alpha]-induced SHP-2 phosphatase activity by shear stress: a mechanism to reduce endothelial inflammation.

OBJECTIVE: Atherosclerosis preferentially occurs in areas of turbulent flow, whereas laminar flow is atheroprotective. Inflammatory cytokines have been shown to stimulate adhesion molecule expression in endothelial cells that may promote atherosclerosis, in part, by stimulating c-Jun N-terminal kinase (JNK) and nuclear factor (NF)-kappaB transcriptional activity. METHODS AND RESULTS: Because Src kinase family and Src homology region 2-domain phosphatase-2 (SHP-2) may regulate JNK activation, we studied the effect of shear stress on endothelial inflammation and JNK. Human umbilical vein endothelial cells preexposed to flow showed decreased tumor necrosis factor (TNF)-alpha-induced c-Jun and NF-kappaB transcriptional activation. TNF-alpha-mediated JNK, c-Jun, and NF-kappaB activation required Src and SHP-2 activity. Shear stress significantly inhibited SHP-2 phosphatase activity without affecting TNF-alpha-induced Src family kinase activation. Because MEKK3 and Gab1 are critical for TNF-alpha-induced c-Jun and NF-kappaB activation, we determined the role of SHP-2 phosphatase activity in MEKK3 signaling. A catalytically inactive form of SHP-2 increased MEKK3/Gab1 interaction and inhibited MEKK3 (but not MEKK1)-mediated c-Jun and NF-kappaB activation. CONCLUSIONS: These results suggest that SHP-2 is a key mediator for the inhibitory effects of shear stress on TNF-alpha signaling in part via regulating MEKK3/Gab1 interaction, MEKK3 signaling, and subsequent adhesion molecule expression.

Activation of mitogen-activated protein kinases and p90 ribosomal S6 kinase in failing human hearts with dilated cardiomyopathy.

OBJECTIVE: A new member of the MAP kinase family, big MAP kinase-1 (BMK1), has been recently identified to promote cell growth and attenuate apoptosis. P90 ribosomal S6 kinase (p90RSK), one of the potentially important substrates of extracellular signal regulated kinase (ERK), regulates gene expression in part via phosphorylation of CREB and the Na(+)/H(+) exchanger. Recently, we have demonstrated that the activity of BMK1, Src (the upstream regulator of BMK1) and p90RSK was increased in hypertrophied myocardium induced by pressure-overload in the guinea pig. However, the abundance and activity of these kinases in human hearts are unknown. METHODS: In addition to the three classical MAP kinases (ERK, p38 kinase, and c-Jun NH(2)-terminal kinase (JNK)), we examined the protein expression and activity of Src, BMK1, and p90RSK in explanted hearts from patients with dilated cardiomyopathy (n=9). Normal donor hearts, which were not suitable for transplant for technical reasons, were used as controls (n=5). RESULTS: There were no significant differences in the levels of protein expression of these kinases between normal and failing hearts. ERK1/2 and p90RSK were activated in heart failure compared to control (P<0.01 and P<0.03, respectively), while the activity of p38 kinase was decreased (P<0.05) and the activity of JNK was unchanged in heart failure. By contrast, the activities of Src and BMK1 were significantly reduced in end-stage heart failure compared to normal donor hearts (P<0.05). CONCLUSION: These data suggest that multiple MAP kinases, p90RSK, and Src are differentially regulated in human failing myocardium of patients with idiopathic dilated cardiomyopathy and may be involved in the pathogenesis of this complex disease.

Novel action and mechanism of auranofin in inhibition of vascular endothelial growth factor receptor-3-dependent lymphangiogenesis.

Auranofin is a gold compound initially developed for the treatment of rheumatoid arthritis. Recent data suggest that auranofin has promise in the treatment of other inflammatory and proliferative diseases. However, the mechanisms of action of auranofin have not been well defined. In the present study, we identify vascular endothelial growth factor receptor-3 (VEGFR3), an endothelial cell (EC) surface receptor essential for angiogiogenesis and lymphangiogenesis, as a novel target of auranofin. In both primary EC and EC cell lines, auranofin induces downregulation of VEGFR3 in a dose-dependent manner. Auranofin at high doses (≥1 µM) decreases cellular survival protein thioredoxin reductase (TrxR2), TrxR2-dependent Trx2 and transcription factor NF-κB whereas increases stress signaling p38MAPK, leading to EC apoptosis. However, auranofin at low doses (≤0.5 µM) specifically induces downregulation of VEGFR3 and VEGFR3-mediated EC proliferation and migration, two critical steps required for in vivo lymphangiogenesis. Mechanistically, we show that auranofin-induced VEGFR3 downregulation is blocked by antioxidant N-acetyl-L-cysteine (NAC) and lysosome inhibitor chloroquine, but is promoted by proteasomal inhibitor MG132. These results suggest that auranofin induces VEGFR3 degradation through a lysosome-dependent pathway. Auranofin may be a potent therapeutic agent for the treatment of lymphangiogenesis-dependent diseases such as lymphedema and cancer metastasis.

SOCS1 prevents graft arteriosclerosis by preserving endothelial cell function.

OBJECTIVES: The aim of this study was to determine the role of suppressor of cytokine signaling 1 (SOCS1) in graft arteriosclerosis (GA). BACKGROUND: GA, the major cause of late cardiac allograft failure, is initiated by immune-mediated endothelial activation resulting in vascular inflammation and consequent neointima formation. SOCS1, a negative regulator of cytokine signaling, is highly expressed in endothelial cells (ECs) and may prevent endothelial inflammatory responses and phenotypic activation. METHODS: Clinical specimens of coronary arteries with GA, with atherosclerosis, or without disease were collected for histological analysis. SOCS1 knockout or vascular endothelial SOCS1 (VESOCS1) transgenic mice were used in an aorta transplant model of GA. Mouse aortic ECs were isolated for in vitro assays. RESULTS: Dramatic but specific reduction of endothelial SOCS1 was observed in human GA and atherosclerosis specimens, which suggested the importance of SOCS1 in maintaining normal endothelial function. SOCS1 deletion in mice resulted in basal EC dysfunction. After transplantation, SOCS1-deficient aortic grafts augmented leukocyte recruitment and neointima formation, whereas endothelial overexpression of SOCS1 diminished arterial rejection. Induction of endothelial adhesion molecules in early stages of GA was suppressed by the VESOCS1 transgene, and this effect was confirmed in cultured aortic ECs. Moreover, VESOCS1 maintained better vascular function during GA progression. Mechanistically, endothelial SOCS1, by modulating both basal and cytokine-induced expression of the adhesion molecules platelet/endothelial cell adhesion molecule-1, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, restrained leukocyte adhesion and transendothelial migration during inflammatory cell infiltration. CONCLUSIONS: SOCS1 prevents GA progression by preserving endothelial function and attenuating cytokine-induced adhesion molecule expression in vascular endothelium.

Thioredoxin and redox signaling in vasculature-studies using Trx2 endothelium-specific transgenic mice.

Increasing evidence supports that reactive oxygen species (ROS) generated from mitochondria in vasculature significantly contribute to human disease. The mitochondrial antioxidant systems, particularly the redox protein thioredoxin-2 (Trx2), provide a primary line of defense against cellular ROS. Using endothelial cell culture and endothelial cell-specific transgenesis of Trx2 gene in mice, we demonstrate the critical roles of Trx2 in regulating endothelium functions. Here, we describe the methods related to generation and characterization of the Trx2 transgenic mice, and the in vivo functional assays associated with Trx2 activities. These methods could be applied to functional analyses for other redox genes.

The novel role of the C-terminal region of SHP-2. Involvement of Gab1 and SHP-2 phosphatase activity in Elk-1 activation.

SHP-2, a nontransmembrane-type protein-tyrosine phosphatase that contains two Src homology 2 (SH2) domains, is thought to participate in growth factor signal transduction pathways via SH2 domain interactions. To determine the role of each region of SHP-2 in platelet-derived growth factor signaling assayed by Elk-1 activation, we generated six deletion mutants of SHP-2. The large SH2 domain deletion SHP-2 mutant composed of amino acids 198-593 (SHP-2-(198-593)), but not the smaller SHP-2-(399-593), showed significantly higher SHP-2 phosphatase activity in vitro. In contrast, SHP-2-(198-593) mutant inhibited wild type SHP-2 phosphatase activity, whereas SHP-2-(399-593) mutant increased activity. To understand these functional changes, we focused on the docking protein Gab1 that assembles signaling complexes. Pull-down experiments with Gab1 suggested that the C-terminal region of SHP-2 as well as the SH2 domains (N-terminal region) associated with Gab1, but the SHP-2-(198-593) mutant did not associate with Gab1. SHP-2-(1-202) or SHP-2-(198-593) inhibited platelet-derived growth factorinduced Elk-1 activation, but SHP-2-(399-593) increased Elk-1 activation. Co-expression of SHP-2-(1-202) with SHP-2-(399-593) inhibited SHP-2-(399-593)/Gab1 interaction, and the SHP-2-(399-593) mutant induced SHP-2 phosphatase and Elk-1 activation, supporting the autoinhibitory effect of SH2 domains on the C-terminal region of SHP-2. These data suggest that both SHP-2/Gab1 interaction in the C-terminal region of SHP-2 and increased SHP-2 phosphatase activity are important for Elk-1 activation. Furthermore, we identified a novel sequence for SHP-2/Gab1 interactions in the C-terminal region of SHP-2.

ERK1/2 associates with the c-Met-binding domain of growth factor receptor-bound protein 2 (Grb2)-associated binder-1 (Gab1): role in ERK1/2 and early growth response factor-1 (Egr-1) nuclear accumulation.

Endothelial cell (EC) migration contributes to reendothelialization after angioplasty or rupture of atherosclerotic plaques. Extracellular signal-regulated kinase (ERK)1/2 translocates to the nucleus and activates transcription factors such as Ets-like transcription factor-1 and early growth response factor-1 (Egr-1) during reendothelialization. Because ERK1/2 does not possess a nuclear localization signal (NLS), its mechanism of translocation and accumulation in the nucleus remains unclear. Because Gab1 has a putative NLS in its N-terminal region, and Gab1 associates with phosphorylated ERK1/2, we hypothesized that Gab1 participates in ERK1/2 and Egr-1 nuclear accumulation. Using regenerating EC as a model system, we found that endogenous growth factor receptor-bound protein 2-associated binder-1 (Gab1) translocates into the nucleus in migrating EC. Wild-type red fluorescent protein-tagged Gab1 could be observed in both nucleus and cytoplasm, whereas the putative NLS deletion mutant (deltaNLS-Gab1) specifically localized in the cytoplasm. In addition, reduction of Gab1 expression by antisense Gab1 oligos or overexpression of deltaNLS-Gab1 inhibited serum-induced ERK1/2 and Egr-1 nuclear accumulation, suggesting a functional role for the NLS of Gab1 and a role for Gab1-ERK1/2 interactions in ERK1/2-Egr-1 nuclear accumulation. To investigate whether Gab1-ERK1/2 interaction is critical for ERK1/2 and Egr-1 nuclear accumulation, we created a dominant-negative Gab1 construct that consisted of the c-Met binding domain (amino acids 442-536) of Gab1. We found that overexpression of the c-Met binding domain of Gab1 disrupted serum-induced Gab1-ERK1 interaction and inhibited ERK1 and Egr-1 nuclear accumulation. These data suggest that Gab1-ERK1/2 binding and their nuclear translocation play a crucial role in Egr-1 nuclear accumulation.