SHP2 mediates gp130-dependent cardiomyocyte hypertrophy via negative regulation of skeletal alpha-actin gene

Morphological and biochemical phenotypes of cardiomyocyte hypertrophy are determined by neurohumoral factors. Stimulation of G protein-coupled receptor (GPCR) results in uniform cell enlargement in all directions with an increase in skeletal α-actin (α-SKA) gene expression, while stimulation of gp130 receptor by interleukin-6 (IL-6)-related cytokines induces longitudinal elongation with no increase in α-SKA gene expression. Thus, α-SKA is a discriminating marker for hypertrophic phenotypes; however, regulatory mechanisms of α-SKA gene expression remain unknown. Here, we clarified the role of SH2-containing protein tyrosine phosphatase 2 (SHP2) in α-SKA gene expression. In neonatal rat cardiomyocytes, endothelin-1 (ET-1), a GPCR agonist, but not leukemia inhibitory factor (LIF), an IL-6-related cytokine, induced RhoA activation and promotes α-SKA gene expression via RhoA. In contrast, LIF, but not ET-1, induced activation of SHP2 in cardiomyocytes, suggesting that SHP2 might negatively regulate α-SKA gene expression downstream of gp130. Therefore, we examined the effect of adenovirus-mediated overexpression of wild-type SHP2 (SHP2^WT), dominant-negative SHP2 (SHP2^C/S), or β-galactosidase (β-gal), on α-SKA gene expression. LIF did not upregulate α-SKA mRNA in cardiomyocytes overexpressing either β-gal or SHP2^WT. In cardiomyocytes overexpressing SHP2^C/S, LIF induced upregulation of α-SKA mRNA, which was abrogated by concomitant overexpression of either C3-toxin or dominant-negative RhoA. RhoA was activated after LIF stimulation in the cardiomyocytes overexpressing SHP2^C/S, but not in myocytes overexpressing β-gal. Furthermore, SHP2 mediates LIF-induced longitudinal elongation of cardiomyocytes via ERK5 activation. Collectively, these findings indicate that SHP2 negatively regulates α-SKA expression via RhoA inactivation and suggest that SHP2 implicates ERK5 in cardiomyocyte elongation downstream of gp130.     

SHP2-mediated signaling cascade through gp130 is essential for LIF-dependent I CaL, [Ca2+]i transient, and APD increase in cardiomyocytes

Leukemia inhibitory factor (LIF), a cardiac hypertrophic cytokine, increases L-type Ca(2+) current (I(CaL)) via ERK-dependent and PKA-independent phosphorylation of serine 1829 in the Cav(1.2) subunit. The signaling cascade through gp130 is involved in this augmentation. However, there are two major cascades downstream of gp130, i.e. JAK/STAT3 and SHP2/ERK. In this study, we attempted to clarify which of these two cascades plays a more important role. Knock-in mouse line, in which the SHP2 signal was disrupted (gp130(F759/F759) group), and wild-type mice (WT group) were used. A whole-cell patch clamp experiment was performed, and intracellular Ca(2+) concentration ([Ca(2+)](i) transient) was monitored. The I(CaL) density and [Ca(2+)](i) transient were measured from the untreated cells and the cells treated with LIF or IL-6 and soluble IL-6 receptor (IL-6+sIL-6r). Action potential duration (APD) was also recorded from the ventricle of each mouse, with or without LIF. Both LIF and IL-6+sIL-6r increased I(CaL) density significantly in WT (+27.0%, n=16 p<0.05, and +32.2%, n=15, p<0.05, respectively), but not in gp130(F759/F759) (+9.4%, n=16, NS, and -6.1%, n=13, NS, respectively). Administration of LIF and IL-6+sIL-6r increased [Ca(2+)](i) transient significantly in WT (+18.8%, n=13, p<0.05, and +32.0%, n=21, p<0.05, respectively), but not in gp130(F759/F759) (-3.8%, n=7, NS, and -6.4%, n=10, NS, respectively). LIF prolonged APD(80) significantly in WT (10.5+/-4.3%, n=12, p<0.05), but not in gp130(F759/F759) (-2.1+/-11.2%, n=7, NS). SHP2-mediated signaling cascade is essential for the LIF and IL-6+sIL-6r-dependent increase in I(CaL), [Ca(2+)](i) transient and APD.     

Grb2-associated binder-1 plays a central role in the hepatocyte growth factor enhancement of hepatoma growth inhibition by K vitamin analog compound 5

Compound 5 (Cpd 5), a K vitamin analog, has been shown to inhibit Hep3B human hepatoma cell growth in cultures and rat hepatoma growth in vivo through prolonged epidermal growth factor receptor (EGFR)-extracellular response kinase (ERK) phosphorylation, and hepatocyte growth factor (HGF) synergizes with Cpd 5 to enhance the inhibition of Hep3B cell and rat hepatoma growth. To explore the mechanisms mediating the HGF/Cpd 5 synergy, we examined the possible involvement of the Grb2-associated binder-1 (Gab1) docking protein because it interacts with both EGFR and HGF receptor c-Met pathways. We found that HGF enhanced Cpd 5-induced c-Met phosphorylation at Tyr-1349, a binding site for Gab1, resulting in increased c-Met binding to Gab1, and induced strong and prolonged Gab1 tyrosine phosphorylation. Prolonged Gab1 phosphorylation by HGF/Cpd 5 in turn enhanced the ability of Gab1 to bind to protein tyrosine phosphatase SHP2 and enhanced the activation of its downstream mitogen-activated protein kinase pathway. In contrast, this same HGF/Cpd 5 treatment inhibited Gab1 binding to phosphatidylinositol 3-kinase (PI3K), leading to the inactivation of the PI3K-Akt pathway. The inhibition of Akt phosphorylation by HGF/Cpd 5 further activated the Raf-MEK-ERK signaling cascade via an Akt-Raf1 interaction, leading to strong and prolonged ERK phosphorylation. The transfection of Hep3B cells with mutated Gab1 (Gab1 Y627F), which had lost its ability to bind SHP2, antagonized HGF/Cpd 5-induced ERK phosphorylation, whereas the transfection of Hep3B cells with mutated Gab1 3YF, which lost its ability to bind PI3K, further enhanced HGF/Cpd 5-induced ERK phosphorylation and cell growth inhibition. CONCLUSION: Gab1 plays a central role in regulating HGF/Cpd 5 synergy in their actions on Hep3B cell growth inhibition.     

Gab-family adapter proteins act downstream of cytokine and growth factor receptors and T- and B-cell antigen receptors

We previously found that the adapter protein Gab1 (110 kD) is tyrosine-phosphorylated and forms a complex with SHP-2 and PI-3 kinase upon stimulation through either the interleukin-3 receptor (IL-3R) or gp130, the common receptor subunit of IL-6-family cytokines. In this report, we identified another adapter molecule (100 kD) interacting with SHP-2 and PI-3 kinase in response to various stimuli. The molecule displays striking homology to Gab1 at the amino acid level; thus, we named it Gab2. It contains a PH domain, proline-rich sequences, and tyrosine residues that bind to SH2 domains when they are phosphorylated. Gab1 is phosphorylated on tyrosine upon stimulation through the thrombopoietin receptor (TPOR), stem cell factor receptor (SCFR), and T-cell and B-cell antigen receptors (TCR and BCR, respectively), in addition to IL-3R and gp130. Tyrosine phosphorylation of Gab2 was induced by stimulation through gp130, IL-2R, IL-3R, TPOR, SCFR, and TCR. Gab1 and Gab2 were shown to be substrates for SHP-2 in vitro. Overexpression of Gab2 enhanced the gp130 or Src-related kinases-mediated ERK2 activation as that of Gab1 did. These data indicate that Gab-family molecules act as adapters for transmitting various signals.     

Gab1, SHP2, and protein kinase A are crucial for the activation of the endothelial NO synthase by fluid shear stress

Fluid shear stress enhances NO production in endothelial cells by a mechanism involving the activation of the phosphatidylinositol 3-kinase and the phosphorylation of the endothelial NO synthase (eNOS). We investigated the role of the scaffolding protein Gab1 and the tyrosine phosphatase SHP2 in this signal transduction cascade in cultured and native endothelial cells. Fluid shear stress elicited the phosphorylation and activation of Akt and eNOS as well as the tyrosine phosphorylation of Gab1 and its association with the p85 subunit of phosphatidylinositol 3-kinase and SHP2. Overexpression of a Gab1 mutant lacking the pleckstrin homology domain abrogated the shear stress-induced phosphorylation of Akt but failed to affect the phosphorylation or activity of eNOS. The latter response, however, was sensitive to a protein kinase A (PKA) inhibitor. Mutation of Gab1 Tyr627 to phenylalanine (YF-Gab1) to prevent the binding of SHP2 completely prevented the shear stress-induced phosphorylation of eNOS, leaving the Akt response intact. A dominant-negative SHP2 mutant prevented the activation of PKA and phosphorylation of eNOS without affecting that of Akt. Moreover, shear stress elicited the formation of a signalosome complex including eNOS, Gab1, SHP2 and the catalytic subunit of PKA. In isolated murine carotid arteries, flow-induced vasodilatation was prevented by a PKA inhibitor as well as by overexpression of either the YF-Gab1 or the dominant-negative SHP2 mutant. Thus, the shear stress-induced activation of eNOS depends on Gab1 and SHP2, which, in turn, regulate the phosphorylation and activity of eNOS by a PKA-dependent but Akt-independent mechanism.     

Role of the docking protein Gab2 in beta(1)-integrin signaling pathway-mediated hematopoietic cell adhesion and migration

Gab2, a newly identified pleckstrin homology domain-containing docking protein, is a major binding protein of SHP-2 tyrosine phosphatase in interleukin (IL)-3-stimulated hematopoietic cells. Its signaling mechanism remains largely unknown. We report here an important regulatory role for Gab2 in beta(1) integrin signaling pathway that mediates hematopoietic cell adhesion and migration. Cross-linking of the beta(1) integrin on Ba/F3 cells induced rapid tyrosine phosphorylation of Gab2 and its association with Syk kinase, SHP-2 phosphatase, and the p85 subunit of phosphatidylinositol (PI)-3 kinase. In addition, Gab2 was also constitutively associated with SHP-1 phosphatase via its C-terminal Src homology 2 domain. Overexpression of the pleckstrin homology domain or a mutant Gab2 molecule lacking SHP-2 binding sites resulted in significant reductions in Ba/F3 cell adhesion and migration. Biochemical analyses revealed that enforced expression of Gab2 mutant molecules dramatically reduced beta(1)-integrin ligation-triggered PI3 kinase activation, whereas Erk kinase activation remained unaltered. Furthermore, transduction of primary hematopoietic progenitor cells from viable motheaten mice with these mutant Gab2 molecules also significantly ameliorated their enhanced migration capacity associated with the SHP1 gene mutation. Taken together, these results suggest an important signaling role for Gab2 in regulating hematopoietic cell adhesion and migration.     

IL-6 trans-signaling via STAT3 directs T cell infiltration in acute inflammation

Interleukin (IL)-6 signaling through its soluble receptor (IL-6 transsignaling) directs transition between innate and acquired immune responses by orchestrating the chemokine-directed attraction and apoptotic clearance of leukocytes. Through analysis of mononuclear cell infiltration in WT and IL-6-deficient mice during peritoneal inflammation, we now report that IL-6 selectively governs T cell infiltration by regulating chemokine secretion (CXCL10, CCL4, CCL5, CCL11, and CCL17) and chemokine receptor (CCR3, CCR4, CCR5, and CXCR3) expression on the CD3+ infiltrate. Although blockade of IL-6 trans-signaling prevented chemokine release, chemokine receptor expression remained unaltered suggesting that this response is regulated by IL-6 itself. To dissect the signaling events promoting T cell migration, inflammation was established in knock-in mice expressing mutated forms of the universal signal-transducing element for IL-6-related cytokines gp130. In mice (gp130Y757F/Y757F) deficient in SHP2 and SOCS3 binding, but presenting hyperactivation of STAT1/3, T cell recruitment and CCL5 expression was enhanced. Conversely, both of these parameters were suppressed in mice with ablated gp130-mediated STAT1/3 activation (gp130DeltaSTAT/DeltaSTAT). T cell migration was related to STAT3 activity, because monoallelic deletion of Stat3 in gp130(Y757F/Y757F) mice (gp130Y757F/Y757F:Stat3+/-) corrected the exaggerated responses observed in gp130Y757F/Y757F mice. Consequently, STAT3 plays a defining role in IL-6-mediated T cell migration.     

G-CSF-induced tyrosine phosphorylation of Gab2 is Lyn kinase dependent and associated with enhanced Akt and differentiative, not proliferative, responses

The granulocyte colony-stimulating factor receptor (G-CSFR) transduces intracellular signals for myeloid cell proliferation, survival, and differentiation through the recruitment of nonreceptor protein tyrosine kinases Lyn and janus kinase 2 (Jak2). This results in the tyrosine phosphorylation of a small set of positive and negative adapters and effectors. Grb2-associated binder-2 (Gab2) is a newly described adapter molecule, preferentially expressed in hematopoietic cells and associated with phosphatidylinositol 3 (PI3) kinase. Studies suggest that Gab2 plays both positive and negative roles in cytokine receptor signaling. To investigate the role Gab2 plays in G-CSF receptor-mediated signaling, we have analyzed its activation state and correlated that with wild-type and mutant G-CSF receptors stably expressed in the murine factor-dependent Ba/F3 cell lines. G-CSF-induced tyrosine phosphorylation of Gab2 occurred in the wild-type and single Y-to-F mutants (Y704F, Y729F, and Y744F), but not in the ADA and W650R loss-of-function mutants. Cells expressing truncated proximal G-CSFR, the tyrosine-null (Y4F) G-CSFR, or Y764F mutant receptors had decreased phosphorylation of Gab2. Specific inhibitors of Src kinase (PD173 and PP1) but not Jak2 kinase (AG490) blocked Gab2 phosphorylation. Phosphorylation of Gab2 occurred in wild-type, but not Lyn-deficient, G-CSFR-transfected DT40 B cells. These data propose that Lyn, not Jak2, phosphorylates Gab2 and that maximal phosphorylation of Gab2 requires Y764, a Grb2-binding site. Serine phosphorylation of Akt, a marker of PI3-kinase activity, was detected in both wild-type and truncated proximal domain receptors, but not in the ADA and W650R mutants. Levels of phospho-Akt and phospho-extracellular signal-regulated kinase (phospho-ERK) were greater in proximal truncated than in wild-type G-CSFR cells, suggesting that Gab2 is dissociated from PI3 kinase or ERK activities. Overexpression of Gab2 enhanced the phosphorylation state of Akt, but not of ERK. This inhibited the proliferation of wild-type and truncated G-CSFR-transfected Ba/F3 cells and enhanced their myeloid differentiation. All together, these data indicate that G-CSF treatment leads to Lyn-mediated tyrosine phosphorylation of Gab2, which may serve as an important intermediate of enhanced Akt activity and myeloid differentiation, not growth/survival response.     

Cardiotrophin-1 stimulation of cardiac fibroblast growth: roles for glycoprotein 130/leukemia inhibitory factor receptor and the endothelin type A receptor

Cardiotrophin-1 (CT-1), a member of the interleukin-6 superfamily, and endothelin-1 (ET-1) are potent hypertrophic factors in cardiomyocytes. Although CT-1 and ET-1 gene expression in the heart is upregulated in experimental heart failure, their role in the activation of the cardiac fibroblast is unknown. This study was designed to identify the presence and action of CT-1 and its receptor complex, glycoprotein130 (gp130) and leukemia inhibitory factor (LIF) receptor, on cardiac fibroblast growth in cultured adult canine cardiac fibroblasts. In addition, we investigated the interaction between CT-1/gp130/LIF receptor and ET-1/endothelin type A (ET(A)) receptor axis. Immunohistochemistry was performed using the indirect immunoperoxidase method, while we assessed the cell cycle of cardiac fibroblasts by flow cytometry, DNA synthesis by [(3)H]thymidine incorporation, and collagen synthesis by [(3)H]proline incorporation, respectively. CT-1 and gp130/LIF receptor were widely present in the cytoplasm of the cardiac fibroblasts. Exogenous CT-1 markedly stimulated [(3)H]thymidine and [(3)H]proline incorporations (P<0.01), with accumulation of cells in the S phase. Blockade of gp130 or LIF receptor inhibited basal growth as well as CT-1- or ET-1-stimulated cardiac fibroblast growth. The specific ET(A) receptor antagonist, BQ123, significantly inhibited CT-1-stimulated DNA synthesis. This study demonstrates that CT-1 and its receptors are present in cardiac fibroblasts. In addition, growth of these cells stimulated by endogenous and exogenous CT-1 requires gp130/LIF receptor as well as ET(A) receptor activation. We conclude that gp130/LIF receptor and ET(A) receptor activation are essential for cardiac fibroblast growth by CT-1 and that there is synergism with ET-1/ET(A) receptor axis.     

SHP2-mediated signaling cascade through gp130 is essential for LIF-dependent ICaL, [Ca2+]i transient,and APD increase in cardiomyocytes

Leukemia inhibitory factor (LIF), a cardiac hypertrophic cytokine, increases L-type Ca²⁺ current (I_CaL) via ERK-dependent and PKA-independent phosphorylation of serine 1829 in the Cav_1.2 subunit. The signaling cascade through gp130 is involved in this augmentation. However, there are two major cascades downstream of gp130, i.e. JAK/STAT3 and SHP2/ERK. In this study, we attempted to clarify which of these two cascades plays a more important role. Knock-in mouse line, in which the SHP2 signal was disrupted (gp130^F759/F759 group), and wild-type mice (WT group) were used. A whole-cell patch clamp experiment was performed, and intracellular Ca²⁺ concentration ([Ca²⁺]_i transient) was monitored. The I_CaL density and [Ca²⁺]_i transient were measured from the untreated cells and the cells treated with LIF or IL-6 and soluble IL-6 receptor (IL-6 + sIL-6r). Action potential duration (APD) was also recorded from the ventricle of each mouse, with or without LIF. Both LIF and IL-6 + sIL-6r increased I_CaL density significantly in WT (+ 27.0%, n = 16 p < 0.05, and + 32.2%, n = 15, p < 0.05, respectively), but not in gp130^F759/F759 (+ 9.4%, n = 16, NS, and − 6.1%, n = 13, NS, respectively). Administration of LIF and IL-6 + sIL-6r increased [Ca²⁺]_i transient significantly in WT (+ 18.8%, n = 13, p < 0.05, and + 32.0%, n = 21, p < 0.05, respectively), but not in gp130^F759/F759 (− 3.8%, n = 7, NS, and − 6.4%, n = 10, NS, respectively). LIF prolonged APD₈₀ significantly in WT (10.5 ± 4.3%, n = 12, p < 0.05), but not in gp130^F759/F759 (− 2.1 ± 11.2%, n = 7, NS). SHP2-mediated signaling cascade is essential for the LIF and IL-6 + sIL-6r-dependent increase in I_CaL, [Ca²⁺]_i transient and APD.     

Treatment with insulin uncovers the motogenic capacity of nitric oxide in aortic smooth muscle cells: dependence on Gab1 and Gab1-SHP2 association

Contrary to the antimotogenic effect of NO in dedifferentiated vascular smooth muscle cells (VSMCs), we have reported that NO stimulates the motility of differentiated cultured VSMC isolated from adult rats. This process involves upregulation of protein tyrosine phosphatase SHP2, followed by downregulation of RhoA activity. In the present study, we tested the hypothesis that insulin alters the motogenic phenotype of cultured rat aortic smooth muscle cells exposed to NO from inhibition to stimulation of cell motility. We demonstrate for the first time that NO stimulates the motility of VSMCs cultured for several days in the presence but not the absence of insulin. Moreover, we show that NO blocks PDGF-induced cell motility in insulin-naive but not in insulin-treated cells. We also demonstrate that the scaffold adapter protein Gab1, considered a physiological activator of protein tyrosine phosphatase SHP2, increases cell motility in the presence but not the absence of insulin. In cells cultured in the presence of insulin, overexpression of Gab1 mimics, whereas a dominant-negative allele of Gab1 (Gab1YF) blocks, the motility-stimulatory effect of NO. Cotransfection experiments with dominant-negative Gab1 and wild-type SHP2 or wild-type Gab1 and dominant-negative SHP2 indicate that the two proteins work together as a functional unit to induce motility. Because chronic insulin can increase the levels of phosphatidylinositol 3 (PI3) kinase in several models of hyperinsulinemia, we also tested the potential involvement of this enzyme in mechanisms leading to increased cell motility. We found that the motogenic effect of NO, Gab1, and SHP2 was blocked by the selective PI3 kinase inhibitor LY294002, suggesting a requirement of PI3 kinase in mediating motogenesis. These observations may be relevant to molecular mechanisms related to the pathogenesis of vascular disease in hyperinsulinemic diabetes. The full text of this article is available online at http://www.circresaha.org.     

gp130 signaling pathways: Recent advances and implications for cardiovascular disease

gp130 was initially identified as a signal-transducing receptor component that associates with the interleukin 6 receptor (IL-6R) when the receptor is occupied by interleukin 6 (IL-6). It has been revealed that the receptor complexes for IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OM), and ciliary neurotrophic factor (CNTF) utilize this gp130 protein as a common signal-transducing component, explaining how these cytokines mediate overlapping biological function. Recent observations with mice lacking gp130 or having continuously activated gp130 protein have disclosed an important biological function of gp130 in cardiovascular system: the former mice show extremely hypoplastic development of the ventricular myocardium at 16.5 days postcoitum (dpc), and the latter exhibit hypertrophy of myocardium. These cardiovascular abnormalities are considered to be the results of the perturbation of gp130, which also transduces the signal of cardiotrophin-1 (CT-1), a recently isolated factor causing hypertrophy in cultured cardiomyocytes and having sequence similarity with IL-6, IL-11, LIF, CNTF, and OM. In fact, CT-1 shares gp130 with these cytokines as a critical signaling component. Besides various well-established mechanisms by which cardiac growth and development are regulated, a gp130 signaling may be a newly discovered mechanism that regulates these events.     

Suppression of leukemia inhibitor factor in lymphoid tissue in primary HIV infection: absence of HIV replication in gp130-positive cells

BACKGROUND: Leukemia inhibitor factor (LIF), a member of the interleukin-6 cytokine family, has recently been shown to inhibit HIV-1 replication both in vivo and in vitro. OBJECTIVE: LIF and its corresponding receptors gp130 and LIF receptor-alpha (LIFR-alpha) were studied in lymphoid tissue (LT) to reveal potential systemic immunoregulatory effects during the course of HIV-1 infection. METHODS: LIF, gp130, LIFR-alpha and HIV-1 replicating cells were identified at the single cell level by immunohistochemistry and quantified by computerized in situ imaging in tonsil and lymph nodes biopsies (LT) from patients with primary HIV-1 infection (PHI), chronic HIV-1 infection (cHI), long-term non-progressors (LTNP) and HIV-1 seronegative controls. RESULTS: LIF and its receptors, gp130 and LIFR-alpha were significantly (P < 0.005) upregulated in LT from PHI patients as compared with HIV-1 seronegative controls. Expression of LIF in cHI was comparable to LIF levels in HIV-1 seronegative controls whereas LTNP showed significantly reduced LIF expression (P < 0.05). LIF receptors, gp130 and LIFR-alpha were significantly upregulated in cHI (P < 0.005) but downregulated in LTNP (P < 0.05 and P < 0.005, respectively). LIF expressing cells could be demonstrated in LT 2 days after onset of acute retroviral syndrome. LIF expression was evident in CD3, CD4 and CD8 cells. Furthermore, high plasma viral load was associated with high expression of LIF in LT. Finally, no HIV-1 replication could be found in CD4 gp130-positive cells in PHI. CONCLUSIONS: LIF, gp130 and LIFR-alpha showed increased expression in LT from patients with PHI. Furthermore, HIV-1 replication did not occur in cells expressing the LIF signaling receptor, gp130, indicating that LIF may be associated with control of HIV-1 replicating cells in vivo.