PDK1 Recruitment to the SHPS-1 Signaling Complex Enhances Insulin-like Growth Factor-I-stimulated AKT Activation and Vascular Smooth Muscle Cell Survival

In vascular smooth muscle cells, exposed to hyperglycemia and insulin-like growth factor-I (IGF-I), SHPS-1 functions as a scaffold protein, and a signaling complex is assembled that leads to AKT activation. However, the underlying mechanism by which formation of this complex activates the kinase that phosphorylates AKT (Thr³⁰⁸) is unknown. Therefore, we investigated the mechanism of PDK1 recruitment to the SHPS-1 signaling complex and the consequences of disrupting PDK1 recruitment for downstream signaling. Our results show that following IGF-I stimulation, PDK1 is recruited to SHPS-1, and its recruitment is mediated by Grb2, which associates with SHPS-1 via its interaction with Pyk2, a component of the SHPS-1-associated complex. A proline-rich sequence in PDK1 bound to an Src homology 3 domain in Grb2 in response to IGF-I. Disruption of Grb2-PDK1 by expression of either a Grb2 Src homology 3 domain or a PDK1 proline to alanine mutant inhibited PDK1 recruitment to SHPS-1, leading to impaired IGF-I-stimulated AKT Thr³⁰⁸ phosphorylation. Following its recruitment to SHPS-1, PDK1 was further activated via Tyr^373/376 phosphorylation, and this was required for a maximal increase in PDK1 kinase activity and AKT-mediated FOXO3a Thr³² phosphorylation. PDK1 recruitment was also required for IGF-I to prevent apoptosis that occurred in response to hyperglycemia. Assembly of the Grb2-PDK1 complex on SHPS-1 was specific for IGF-I signaling because inhibiting PDK1 recruitment to SHPS-1 had no effect on EGF-stimulated AKT Thr³⁰⁸ phosphorylation. These findings reveal a novel mechanism for recruitment of PDK1 to the SHPS-1 signaling complex, which is required for IGF-I-stimulated AKT Thr³⁰⁸ phosphorylation and inhibition of apoptosis.     

Role of SHPS-1 in the regulation of insulin-like growth factor I-stimulated Shc and mitogen-activated protein kinase activation in vascular smooth muscle cells

Insulin-like growth factor I (IGF-I) stimulates smooth muscle cell (SMC) proliferation, and the mitogen-activated protein kinase (MAPK) pathway plays an important role in mediating IGF-I-induced mitogenic signaling. Our prior studies have shown that recruitment of Src homology 2 domain tyrosine phosphatase (SHP-2) to the membrane scaffolding protein Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) is required for IGF-I-dependent MAPK activation. The current studies were undertaken to define the upstream signaling components that are required for IGF-I-stimulated MAPK activation and the role of SHPS-1 in regulating this process. The results show that IGF-I-induced Shc phosphorylation and its subsequent binding to Grb2 is required for sustained phosphorylation of MAPK and increased cell proliferation in SMCs. Furthermore, for Shc to be phosphorylated in response to IGF-I requires that Shc must associate with SHPS-1 and this association is mediated in part by SHP-2. Preincubation of cells with a peptide that contains a phospho-tyrosine binding motif sequence derived from SHPS-1 inhibited IGF-I-stimulated SHP-2 transfer to SHPS-1, the association of Shc with SHPS-1, and IGF-I-dependent Shc phosphorylation. Expression of an SHPS-1 mutant that did not bind to Shc or SHP-2 resulted in decreased Shc and MAPK phosphorylation in response to IGF-I. In addition, SMCs expressing a mutant form of the beta3 subunit of the alphaVbeta3, which results in impairment of SHP-2 transfer to SHPS-1, also showed attenuated IGF-I-dependent Shc and MAPK phosphorylation. Further analysis showed that Shc and SHP-2 can be coimmunoprecipitated after IGF-I stimulation. A cell-permeable peptide that contained a polyproline sequence from Shc selectively inhibited Shc/SHP-2 association and impaired Shc but not SHP-2 binding to SHPS-1. Exposure to this peptide also inhibited IGF-I-stimulated Shc and MAPK phosphorylation. Cells expressing a mutant form of Shc with the four prolines substituted with alanines showed no Shc/SHPS-1 association in response to IGF-I. We conclude that SHPS-1 functions as an anchor protein that recruits both Shc and SHP-2 and that their recruitment is necessary for IGF-I-dependent Shc phosphorylation, which is required for an optimal mitogenic response in SMCs.     

p66shc negatively regulates insulin-like growth factor I signal transduction via inhibition of p52shc binding to Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 leading to impaired growth factor receptor-bound protein-2 membrane recruitment

Our previous studies have indicated an essential role of p52shc in mediating IGF-I activation of MAPK in smooth muscle cells (SMC). However, the role of the p66 isoform of shc in IGF-I signal transduction is unclear. In the current study, two approaches were employed to investigate the role of p66shc in mediating IGF-I signaling. Knockdown p66shc by small interfering RNA enhanced IGF-I-stimulated p52shc tyrosine phosphorylation and growth factor receptor-bound protein-2 (Grb2) association, resulting in increased IGF-I-dependent MAPK activation. This was associated with enhanced IGF-I-stimulated cell proliferation. In contrast, knockdown of p66shc did not affect IGF-I-stimulated IGF-I receptor tyrosine phosphorylation. Overexpression of p66shc impaired IGF-I-stimulated p52shc tyrosine phosphorylation and p52shc-Grb2 association. In addition, IGF-I-dependent MAPK activation was also impaired, and SMC proliferation in response to IGF-I was inhibited. IGF-I-dependent cell migration was enhanced by p66shc knockdown and attenuated by p66shc overexpression. Mechanistic studies indicated that p66shc inhibited IGF-I signal transduction via competitively inhibiting the binding of Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to SHP substrate-1 (SHPS-1), leading to the disruption of SHPS-1/SHP-2/Src/p52shc complex formation, an event that has been shown previously to be essential for p52shc phosphorylation and Grb2 recruitment. These findings indicate that p66shc functions to negatively regulate the formation of a signaling complex that is required for p52shc activation in response to IGF-I, thus leading to attenuation of IGF-I-stimulated cell proliferation and migration.     

The association between integrin-associated protein and SHPS-1 regulates insulin-like growth factor-I receptor signaling in vascular smooth muscle cells

Growth factor signaling is usually analyzed in isolation without considering the effect of ligand occupancy of transmembrane proteins other than the growth factor receptors themselves. In smooth muscle cells, the transmembrane protein Src homology 2 domain containing protein tyrosine phosphatase substrate-1 (SHPS-1) has been shown to be an important regulator of insulin-like growth factor-I (IGF-I) signaling. SHPS-1 is phosphorylated in response to IGF-I, leading to recruitment of Src homology 2 domain tyrosine phosphatase (SHP-2). Subsequently, SHP-2 is transferred to IGF-I receptor and regulates the duration of IGF-I receptor phosphorylation. Whether ligand occupancy of SHPS-1 influences SHPS-1 phosphorylation or SHP-2 recruitment, thereby altering growth factor signaling, is unknown. Previous studies have shown that integrin associated protein (IAP) associates with SHPS-1. We undertook these studies to determine whether this interaction controlled SHPS-1 phosphorylation and/or SHP-2 recruitment and thereby regulated IGF-I signaling. Disruption of IAP-SHPS-1 binding, by using an IAP monoclonal antibody or cells expressing mutant forms of IAP that did not bind to SHPS-1, inhibited IGF-I-stimulated SHPS-1 phosphorylation and SHP-2 recruitment. This was associated with a lack of SHP-2 transfer to IGF-I receptor and sustained receptor phosphorylation. This resulted in an inability of IGF-I to stimulate sustained mitogen-activated protein kinase activation, cell proliferation, and cell migration. The effect was specific for IGF-I because disruption of the IAP-SHPS-1 interaction had no effect on platelet-derived growth factor-stimulated SHPS-1 phosphorylation or cell migration. In summary, our results show that 1) ligand occupancy of SHPS-1 is a key determinant of its ability to be phosphorylated after IGF-I stimulation, and 2) the interaction between IAP and SHPS-1 is an important regulator of IGF-I signaling because disruption of the results in impaired SHP-2 recruitment and subsequent inhibition of IGF-I-stimulated cell proliferation and migration.     

The role of Src kinase in insulin-like growth factor-dependent mitogenic signaling in vascular smooth muscle cells

Activation of the MAPK pathway mediates insulin-like growth factor-I (IGF-I)-dependent proliferation in vascular smooth muscle cells (SMC). Our previous studies have shown that IGF-I-induced Shc phosphorylation is necessary for sustained activation of MAPK and increased cell proliferation of SMCs, and both Shc and the tyrosine phosphatase SHP-2 must be recruited to the membrane protein SHPS-1 in order for Shc to be phosphorylated. These studies were undertaken to determine whether Src kinase activity is required to phosphorylate Shc in response to IGF-I in SMC and because SHP-2 binds to Src whether their interaction was also required for IGF-I-stimulated mitogenesis. Our results show that IGF-I induces activation of Src kinase and that is required for Shc phosphorylation and for optimal MAPK activation. We tested whether Shc is a substrate of c-Src in SMC by disrupting Src/Shc association using a peptide containing a YXXL (Tyr328) motif sequence derived from Src. The peptide blocked the binding of Src and Shc in vitro and in vivo. Cells expressing a mutant Src (Src-FF) that had Tyr328/Tyr358 substituted with phenylalanines (Src-FF) showed defective Src/Shc binding, impaired IGF-I-dependent Shc phorylation, and impaired mitogenesis. This supports the conclusion that Src phosphorylates Shc. IGF-I induced both Src/SHP-2 and Src/SHPS-1 association. SMCs expressing an SHP-2 mutant that had the polyproline-rich region of SH2 deleted (SHP-2Delta10) had disrupted SHP-2/Src association, impaired IGF-I-dependent Shc phosphorylation, and an attenuated mitogenic response. IGF-I-induced association of Src and SHPS-1 was also impaired in SHP-2Delata10-expressing cells, although SHP-2/SHPS-1 association was unaffected. Upon IGF-I stimulation, a complex assembles on SHPS-1 that contains SHP-2, c-Src, and Shc wherein Src phosphorylates Shc, a signaling step that is necessary for an optimal mitogenic response.     

IGF-I stimulates cooperative interaction between the IGF-I receptor and CSK homologous kinase that regulates SHPS-1 phosphorylation in vascular smooth muscle cells

IGF-I plays an important role in smooth muscle cell proliferation and migration. In vascular smooth muscle cells cultured in 25 mm glucose, IGF-I stimulated a significant increase in Src homology 2 domain containing protein tyrosine phosphatase substrate-1 (SHPS-1) phosphorylation compared with 5 mm glucose and this increase was required for smooth muscle cell proliferation. A proteome-wide screen revealed that carboxyl-terminal SRC kinase homologous kinase (CTK) bound directly to phosphotyrosines in the SHPS-1 cytoplasmic domain. Because the kinase(s) that phosphorylates these tyrosines in response to IGF-I is unknown, we determined the roles of IGF-I receptor (IGF-IR) and CTK in mediating SHPS-1 phosphorylation. After IGF-I stimulation, CTK was recruited to IGF-IR and subsequently to phospho-SHPS-1. Expression of an IGF-IR mutant that eliminated CTK binding reduced CTK transfer to SHPS-1, SHPS-1 phosphorylation, and cell proliferation. IGF-IR phosphorylated SHPS-1, which provided a binding site for CTK. CTK recruitment to SHPS-1 resulted in a further enhancement of SHPS-1 phosphorylation. CTK knockdown also impaired IGF-I-stimulated SHPS-1 phosphorylation and downstream signaling. Analysis of specific tyrosines showed that mutation of tyrosines 428/452 in SHPS-1 to phenylalanine reduced SHPS-1 phosphorylation but allowed CTK binding. In contrast, the mutation of tyrosines 469/495 inhibited IGF-IR-mediated the phosphorylation of SHPS-1 and CTK binding, suggesting that IGF-IR phosphorylated Y469/495, allowing CTK binding, and that CTK subsequently phosphorylated Y428/452. Based on the above findings, we conclude that after IGF-I stimulation, CTK is recruited to IGF-IR and its recruitment facilitates CTK's subsequent association with phospho-SHPS-1. This results in the enhanced CTK transfer to SHPS-1, and the two kinases then fully phosphorylate SHPS-1, which is necessary for IGF-I stimulated cellular proliferation.     

Insulin-like growth factor-I stimulates Shc-dependent phosphatidylinositol 3-kinase activation via Grb2-associated p85 in vascular smooth muscle cells

Insulin-like growth factor-I (IGF-I) stimulates vascular smooth muscle cell proliferation and migration by activating both MAPK and phosphatidylinositol 3-kinase (PI3K). Vascular smooth muscle cells (VSMCs) maintained in 25 mm glucose sustain MAPK activation via increased Shc phosphorylation and Grb2 association resulting in an enhanced mitogenic response compared with cells grown in 5 mm glucose. PI3K plays a major role in IGF-I-stimulated VSMC migration, and hyperglycemia augments this response. In contrast to MAPK activation the role of Shc in modulating PI3K in response to IGF-I has not been determined. In this study we show that impaired Shc association with Grb2 results in decreased Grb2-p85 association, SHPS-1-p85 recruitment, and PI3K activation in response to IGF-I. Exposure of VSMCs to cell-permeable peptides, which contained polyproline sequences from p85 proposed to mediate Grb2 association, resulted in inhibition of Grb2-p85 binding and AKT phosphorylation. Transfected cells that expressed p85 mutant that had specific prolines mutated to alanines resulted in less Grb2-p85 association, and a Grb2 mutant (W36A/W193A) that attenuated p85 binding showed decreased association of p85 with SHPS-1, PI3K activation, AKT phosphorylation, cell proliferation, and migration in response to IGF-I. Cellular exposure to 25 mm glucose, which is required for Shc phosphorylation in response to IGF-I, resulted in enhanced Grb2 binding to p85, activation of PI3K activity, and increased AKT phosphorylation as compared with cells exposed to 5 mm glucose. We conclude that in VSMCs exposed to hyperglycemia, IGF-I stimulation of Shc facilitates the transfer of Grb2 to p85 resulting in enhanced PI3K activation and AKT phosphorylation leading to enhanced cell proliferation and migration.     

Interaction between insulin-like growth factor-I receptor and alphaVbeta3 integrin linked signaling pathways: cellular responses to changes in multiple signaling inputs

Integrins are heterodimeric transmembrane proteins that mediate cell attachment to extracellular matrix, migration, division, and inhibition of apoptosis. Because growth factors are also important for these processes, there has been interest in cooperative signaling between growth factor receptors and integrins. IGF-I is an important growth factor for vascular cells. One integrin, alphaVbeta3, that is expressed in smooth muscle cells modulates IGF-I actions. Ligand occupancy of alphaVbeta3 is required for IGF-I to stimulate cell migration and division. Src homology 2 containing tyrosine phosphatase (SHP-2) is a tyrosine phosphatase whose recruitment to signaling molecules is stimulated by growth factors including IGF-I. If alphaVbeta3 ligand occupancy is inhibited, there is no recruitment of SHP-2 to alphaVbeta3 and its transfer to downstream signaling molecules is blocked. Ligand occupancy of alphaVbeta3 stimulates tyrosine phosphorylation of the beta3-subunit, resulting in recruitment of SHP-2. This transfer is mediated by an insulin receptor substrate-1-related protein termed DOK-1. Subsequently, SHP-2 is transferred to another transmembrane protein, SHPS-1. This transfer requires IGF-I receptor-mediated tyrosine phosphorylation of SHPS-1, which contains two YXXL motifs that mediate SHP-2 binding. The transfer of SHP-2 to SHPS-1 is also required for recruitment of Shc to SHPS-1. Ligand occupancy of alphaVbeta3 results in sustained Shc phosphorylation and enhanced Shc recruitment. Shc activation results in induction of MAPK. Inhibition of the Shc/SHPS-1 complex formation results in failure to achieve sustained MAPK activation and an attenuated mitogenic response. Thus, within the vessel wall, a mechanism exists whereby ligand occupancy of the alphaVbeta3 integrin is required for assembly of a multicomponent membrane signaling complex that is necessary for cells to respond optimally to IGF-I.     

Insulin-like growth factor-I (IGF-I) induces epidermal growth factor receptor transactivation and cell proliferation through reactive oxygen species

Insulin-like growth factor-I (IGF-I) plays an important role in proliferation of vascular smooth muscle cells (VSMCs). However, the mechanism that IGF-I induces VSMCs proliferation is not completely understood. In this study, we determined (a) whether and how IGF-I induces transactivation of epidermal growth factor receptor (EGFR) in primary rat aortic VSMCs, (b) the contribution of EGFR to IGF-I-stimulated activation of extracellular signal-regulated kinase (ERK) and cell proliferation, and (c) the role of reactive oxygen species (ROS) in the cellular function. We showed that IGF-I induced phosphorylation of EGFR and ERK1/2 in VSMCs. AG1478, an EGFR inhibitor, inhibited IGF-I-induced phoshorylation of EGFR and ERK1/2. IGF-I stimulated ROS production and Src activation. Antioxidants inhibited IGF-I-induced ROS generation and activation of EGFR, ERK, and Src. Src kinase inhibitor PP1 and Src siRNA blocked IGF-I-induced activation of EGFR and ERK1/2. Inhibition of IGF-I-stimulated EGFR activation inhibited IGF-I-induced VSMC proliferation. These results suggest that (1) IGF-I induces EGFR activation through production of ROS and ROS-mediated Src activation in VSMCs, and (2) EGFR transactivation is required for IGF-I-induced VSMC proliferation.     

Expression of the human beta3 integrin subunit in mouse smooth muscle cells enhances IGF-I-stimulated signaling and proliferation

Optimal stimulation of signal transduction and biological functions by IGF-I in porcine smooth muscle cells (pSMC) requires ligand occupancy of the alphaVbeta3 integrin. Binding of heparin-binding domain (HBD) of vitronectin (VN) to the cysteine loop (C-loop) region of beta3 is required for pSMC to respond optimally to IGF-I stimulation. Mouse smooth muscle cells (mSMC), which express a form of beta3 whose sequence within the C-loop region is different than porcine or human beta3, do not respond optimally to IGF-I, and IGF-I stimulated beta3 and SHPS-1 phosphorylation which are necessary for optimal IGF-I signaling were undetectable. VN also had no effect on IGF-I stimulated the cell proliferation. In contrast, when human beta3 (hbeta3) was introduced into mSMC, there was an enhanced VN binding in spite of an equivalent amount of total beta3 expression, and IGF-I-dependent beta3, and SHPS-1 phosphorylation were detected. In addition, there was enhanced IGF-I-stimulated Shc association with SHPS-1, Shc tyrosine phosphorylation, Shc and Grb2 association, and MAP kinase activation leading to increased cell proliferation. These enhancements could be further augmented by adding a peptide containing the HBD of VN. To determine if these changes were mediated by the C-loop region of beta3, an antibody that reacts with that region of beta3 was utilized. The addition of the hbeta3 C-loop antibody abolished VN-induced enhancement of IGF-I signaling and IGF-I-stimulated cell proliferation. These results strongly support the conclusion that optimal SMC responsiveness to IGF-I requires ligand interaction with the C-loop domain of hbeta3.     

Nox4 NAD(P)H oxidase mediates Src-dependent tyrosine phosphorylation of PDK-1 in response to angiotensin II: role in mesangial cell hypertrophy and fibronectin expression

Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to hypertrophy and extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces an increase in PDK-1 (3-phosphoinositide-dependent protein kinase-1) kinase activity that required its phosphorylation on tyrosine 9 and 373/376. Introduction into the cells of PDK-1, mutated on these tyrosine residues or kinase-inactive, attenuates Ang II-induced hypertrophy and fibronectin accumulation. Ang II-mediated PDK-1 activation and tyrosine phosphorylation (total and on residues 9 and 373/376) are inhibited in cells transfected with small interfering RNA for Src, indicating that Src is upstream of PDK-1. In cells expressing oxidation-resistant Src mutant C487A, Ang II-induced hypertrophy and fibronectin expression are prevented, suggesting that the pathway is redox-sensitive. Ang II also up-regulates Nox4 protein, and siNox4 abrogates the Ang II-induced increase in intracellular reactive oxygen species (ROS) generation. Small interfering RNA for Nox4 also inhibits Ang II-induced activation of Src and PDK-1 tyrosine phosphorylation (total and on residues 9 and 373/376), demonstrating that Nox4 functions upstream of Src and PDK-1. Importantly, inhibition of Nox4, Src, or PDK-1 prevents the stimulatory effect of Ang II on fibronectin accumulation and cell hypertrophy. This work provides the first evidence that Nox4-derived ROS are responsible for Ang II-induced PDK-1 tyrosine phosphorylation and activation through stimulation of Src. Importantly, this pathway contributes to Ang II-induced MC hypertrophy and fibronectin accumulation. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal hypertrophy and fibrosis.     

The molecular functions of Shp2 in the Ras/Mitogen-activated protein kinase (ERK1/2) pathway

Shp2 is a ubiquitous tyrosine phosphatase containing Src Homology 2 domains which plays major biological functions in response to various growth factors, hormones or cytokines. This is essentially due to its particularity of promoting the activation of the Ras/Mitogen-activated protein kinase pathway. Recent progresses have been made in the understanding of the molecular mechanisms involved in this regulation. We review here, and discuss the physiological relevance, of the following molecular functions of Shp2 that have been proposed to couple the phosphatase to Ras activation: promoter of Grb2/Sos recruitment through direct binding to Grb2, binding partner and regulator of SHPS-1, negative regulator of Sprouty, negative regulator of RasGAP recruitment, and activator of Src through dephosphorylation of Src-regulatory proteins.     

Impaired Shc, Ras, and MAPK activation but normal Akt activation in FL5.12 cells expressing an insulin-like growth factor I receptor mutated at tyrosines 1250 and 1251

The Y1250F/Y1251F mutant of the insulin-like growth factor I receptor (IGF-IR) has tyrosines 1250 and 1251 mutated to phenylalanines and is deficient in IGF-I-mediated suppression of apoptosis in FL5.12 lymphocytic cells. To address the mechanism of loss of function in this mutant we investigated signaling responses in FL5.12 cells overexpressing either a wild-type (WT) or Y1250F/Y1251F (mutant) IGF-IR. Cells expressing the mutant receptor were deficient in IGF-I-induced phosphorylation of the JNK pathway and had decreased ERK and p38 phosphorylation. IGF-I induced phosphorylation of Akt was comparable in WT and mutant expressing cells. The decreased activation of the mitogen-activated protein kinase (MAPK) pathways was accompanied by greatly decreased Ras activation in response to IGF-I. Although phosphorylation of Gab2 was similar in WT and mutant cell lines, phosphorylation of Shc on Tyr(313) in response to IGF-I was decreased in cells expressing the mutant receptor, as was recruitment of Grb2 and Ship to Shc. However, phosphorylation of Shc on Tyr(239), the Src phosphorylation site, was normal. A role for JNK in the survival of FL5.12 cells was supported by the observation that the JNK inhibitor SP600125 suppressed IGF-I-mediated protection from apoptosis. Altogether these data demonstrate that phosphorylation of Shc, and assembly of the Shc complex necessary for activation of Ras and the MAPK pathways are deficient in cells expressing the Y1250F/Y1251F mutant IGF-IR. This would explain the loss of IGF-I-mediated survival in FL5.12 cells expressing this mutant and may also explain why this mutant IGF-IR is deficient in functions associated with cellular transformation and cell migration in fibroblasts and epithelial tumor cells.     

Phosphatidylinositol 3-kinase-dependent membrane recruitment of Rac-1 and p47phox is critical for alpha-platelet-derived growth factor receptor-induced production of reactive oxygen species

Platelet-derived growth factor (PDGF) plays a critical role in the pathogenesis of proliferative diseases. NAD(P)H oxidase (Nox)-derived reactive oxygen species (ROS) are essential for signal transduction by growth factor receptors. Here we investigated the dependence of PDGF-AA-induced ROS production on the cytosolic Nox subunits Rac-1 and p47(phox), and we systematically evaluated the signal relay mechanisms by which the alphaPDGF receptor (alphaPDGFR) induces ROS liberation. Stimulation of the alphaPDGFR led to a time-dependent increase of intracellular ROS levels in fibroblasts. Pharmacological inhibitor experiments and enzyme activity assays disclosed Nox as the source of ROS. alphaPDGFR activation is rapidly followed by the translocation of p47(phox) and Rac-1 from the cytosol to the cell membrane. Experiments performed in p47(phox)(-/-) cells and inhibition of Rac-1 or overexpression of dominant-negative Rac revealed that these Nox subunits are required for PDGF-dependent Nox activation and ROS liberation. To evaluate the signaling pathway mediating PDGF-AA-dependent ROS production, we investigated Ph cells expressing mutant alphaPDGFRs that lack specific binding sites for alphaPDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase (PI3K), phospholipase Cgamma, and SHP-2). Lack of PI3K signaling (but not Src, phospholipase Cgamma, or SHP-2) completely abolished PDGF-dependent p47(phox) and Rac-1 translocation, increase of Nox activity, and ROS production. Conversely, a mutant alphaPDGFR able to activate only PI3K was sufficient to mediate these subcellular events. Furthermore, the catalytic PI3K subunit p110alpha (but not p110beta) was identified as the crucial isoform that elicits alphaPDGFR-mediated production of ROS. Finally, bromodeoxyuridine incorporation and chemotaxis assays revealed that the lack of ROS liberation blunted PDGF-AA-dependent chemotaxis but not cell cycle progression. We conclude that PI3K/p110alpha mediates growth factor-dependent ROS production by recruiting p47(phox) and Rac-1 to the cell membrane, thereby assembling the active Nox complex. ROS are required for PDGF-AA-dependent chemotaxis but not proliferation.     

TGF-beta 1 modulation of IGF-I signaling pathway in rat thyroid epithelial cells

Transforming growth factor beta 1 (TGF-beta 1) and insulin-like growth factor I (IGF-I) have contrasting effects on cell cycle regulation in thyroid cells and TGF-beta 1 induces a dramatic decrease in IGF-I-induced cell proliferation. The aim of the present study was to investigate the molecular mechanism of cross-talk between TGF-beta 1 and IGF-I in FRTL-5 cells. TGF-beta 1 affected IGF-I-stimulated insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation and its association with Grb2 protein. Moreover, TGF-beta 1 decreased the IGF-I-induced tyrosine phosphorylation of the adaptor protein CrkII and its association with the IGF-I receptor. These results were accompanied by TGF-beta 1 inhibition of IGF-I-stimulated mitogen-activated protein kinase phosphorylation and activation. Conversely, TGF-beta 1 did not alter IGF-I-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity, IGF-I-induced tyrosine phosphorylation of Shc, and its binding to Grb2. Taken together, these findings provide a molecular basis for the growth-inhibitory action of TGF-beta 1 on the IGF-I-induced mitogenic effect.     

Glucose regulation of integrin-associated protein cleavage controls the response of vascular smooth muscle cells to insulin-like growth factor-I

Vascular smooth muscle cells (SMC) maintained in high glucose are more responsive to IGF-I than SMC maintained in normal glucose due to a difference in the Shc phosphorylation response. In this study we aimed to determine the mechanism by which glucose regulates the sensitivity of SMC to IGF-I. For Shc to be phosphorylated in response to IGF-I it must be recruited to tyrosine-phosphorylated sites on Src homology 2 domain-containing phosphatase (SHP) substrate-1 (SHPS-1). The association of integrin-associated protein (IAP) with SHPS-1 is required for SHPS-1 tyrosine phosphorylation. When SMC were grown in 5 mm glucose, the amount of intact IAP was reduced, compared with SMC grown in 25 mm glucose. This reduction was due to proteolytic cleavage of IAP. Proteolysis of IAP resulted in loss of its SHPS-1 binding site, which led to loss of SHPS-1 phosphorylation. Analysis of the conditioned medium showed that there was more protease activity in the medium from SMC cultured in 5 mm glucose as compared with 25 mm. Inhibition of matrix metalloprotease-2 synthesis using RNA interference or its activity using a specific protease inhibitor protected IAP from cleavage. This protection was associated with an increase in IAP-SHPS-1 association, increased recruitment and phosphorylation of Shc, and increased cell growth in response to IGF-I. Our results show that the enhanced response of SMC in 25 mm glucose to IGF-I is due to the protection of IAP from proteolytic degradation, thereby increasing its association with SHPS-1 and allowing the formation of the SHPS-1-Shc signaling complex.     

Two novel proteins that are linked to insulin-like growth factor (IGF-I) receptors by the Grb10 adapter and modulate IGF-I signaling

Grb10 is a protein that binds to the intracellular domains of activated tyrosine kinase receptors, including insulin-like growth factor (IGF-I) and insulin receptors. This occurs through the interaction of two C-terminal Grb10 motifs (BPS and Src homology domains) with receptor phosphotyrosine residues. Published data from transfection/overexpression studies support both positive and negative regulatory effects of Grb10, thus leaving its physiological role unclear. Because Grb10 has the structure of an adapter protein, the objective of this study was to determine whether Grb10 links other proteins to IGF-I receptors and thus modulates IGF-I signaling. Using yeast two-hybrid screening, the N terminus of Grb10 was shown to interact with two novel proteins, designated GIGYF1 (Grb10 interacting GYF protein 1) and GIGYF2. Mutation analysis indicates that a 17-amino acid sequence in GIGYF1 and GIGYF2, homologous to the GYF domain described previously, binds to tandem proline-rich regions in the N terminus of Grb10. In IGF-I receptor-expressing R+ fibroblasts, there is detectable binding of a Myc-tagged fragment of GIGYF1 to Grb10 in the basal state. Stimulation with IGF-I results in increased binding of GIGYF1 to Grb10 and transient binding of both Grb10 and GIGYF1 to IGF-I receptors, presumably via the adapter function of Grb10. At later time points, GIGYF1 dissociates, but Grb10 remains linked to IGF-I receptors. Overexpression of the Grb10 binding fragment of GIGYF1 in R+ cells results in a significant increase in IGF-I-stimulated receptor tyrosine phosphorylation. In conclusion, we have identified two members of a novel protein family, which become transiently linked to IGF-I receptors by the Grb10 adapter protein following IGF-I stimulation. Grb10 and GIGYFs may act cooperatively to regulate receptor signaling.     

Occupation of alphavbeta3-integrin by endogenous ligands modulates IGF-I receptor activation and proliferation of human intestinal smooth muscle

We have previously shown that endogenous IGF-I regulates growth of human intestinal smooth muscle cells by stimulating proliferation and inhibiting apoptosis. In active Crohn's disease, expression of IGF-I and the alpha(v)beta(3)-integrin receptor ligands fibronectin and vitronectin is increased. The aim of the present study was to determine whether occupation of the alpha(v)beta(3)-receptor influences IGF-I receptor tyrosine kinase activation and function in human intestinal smooth muscle cells. In untreated cells, IGF-I elicited time-dependent tyrosine phosphorylation of its cognate receptor that was maximal within 2 min and sustained for 30 min. In the presence of the alpha(v)beta(3)-ligand fibronectin, IGF-I-stimulated IGF-I receptor activation was augmented. Conversely, in the presence of the alpha(v)beta(3)-specific disintegrin echistatin, IGF-I-stimulated IGF-I receptor tyrosine kinase phosphorylation was inhibited. IGF-I-stimulated IGF-I receptor activation was accompanied by recruitment of the adapter protein IRS-1, activation of Erk1/2, p70S6 kinase, and proliferation. These effects were augmented by fibronectin and attenuated by echistatin. IGF-I also elicited time-dependent recruitment of protein tyrosine phosphatase SHP-2 that coincided with dephosphorylation of the tyrosine phosphorylated IGF-I receptor tyrosine kinase. The alpha(v)beta(3)-disintegrin echistatin accelerated the rate of SHP-2 recruitment and deactivation of the IGF-I receptor tyrosine kinase. The results show that occupancy of the alpha(v)beta(3)-integrin receptor modulates IGF-I-induced IGF-I receptor activation and function in human intestinal muscle cells. We hypothesize that the concomitant increases in the expression of alpha(v)beta(3)-ligands and of IGF-I in active Crohn's disease may contribute to muscle hyperplasia and stricture formation by acting in concert to augment IGF-I-stimulated IGF-I receptor tyrosine kinase activity and IGF-I-mediated muscle cell growth.     

Regulation of Monocyte Adhesion and Migration by Nox4

We showed that metabolic disorders promote thiol oxidative stress in monocytes, priming monocytes for accelerated chemokine-induced recruitment, and accumulation at sites of vascular injury and the progression of atherosclerosis. The aim of this study was to identify both the source of reactive oxygen species (ROS) responsible for thiol oxidation in primed and dysfunctional monocytes and the molecular mechanisms through which ROS accelerate the migration and recruitment of monocyte-derived macrophages. We found that Nox4, a recently identified NADPH oxidase in monocytes and macrophages, localized to focal adhesions and the actin cytoskeleton, and associated with phospho-FAK, paxillin, and actin, implicating Nox4 in the regulation of monocyte adhesion and migration. We also identified Nox4 as a new, metabolic stress-inducible source of ROS that controls actin S-glutathionylation and turnover in monocytes and macrophages, providing a novel mechanistic link between Nox4-derived H₂O₂ and monocyte adhesion and migration. Actin associated with Nox4 was S-glutathionylated, and Nox4 association with actin was enhanced in metabolically-stressed monocytes. Metabolic stress induced Nox4 and accelerated monocyte adhesion and chemotaxis in a Nox4-dependent mechanism. In conclusion, our data suggest that monocytic Nox4 is a central regulator of actin dynamics, and induction of Nox4 is the rate-limiting step in metabolic stress-induced monocyte priming and dysfunction associated with accelerated atherosclerosis and the progression of atherosclerotic plaques.