RESUMEN
Pain of unknown etiology is frequent in individuals with the tumor predisposition syndrome neurofibromatosis 1 (NF1), even when tumors are absent. Nerve Schwann cells (SCs) were recently shown to play roles in nociceptive processing, and we find that chemogenetic activation of SCs is sufficient to induce afferent and behavioral mechanical hypersensitivity in wild-type mice. In mouse models, animals showed afferent and behavioral hypersensitivity when SCs, but not neurons, lacked Nf1. Importantly, hypersensitivity corresponded with SC-specific upregulation of mRNA encoding glial cell line-derived neurotrophic factor (GDNF), independently of the presence of tumors. Neuropathic pain-like behaviors in the NF1 mice were inhibited by either chemogenetic silencing of SC calcium or by systemic delivery of GDNF-targeting antibodies. Together, these findings suggest that alterations in SCs directly modulate mechanical pain and suggest cell-specific treatment strategies to ameliorate pain in individuals with NF1.
Asunto(s)
Hipersensibilidad , Neuralgia , Neurofibromatosis 1 , Animales , Ratones , Neurofibromatosis 1/genética , Nocicepción , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Células de SchwannRESUMEN
Vascular smooth muscle cells maintained in normal (5.6 mm) glucose respond to insulin-like growth factor-I (IGF-I) with increased protein synthesis but do not proliferate. In contrast, hyperglycemia alters responsiveness to IGF-I, resulting in increased SHPS-1 phosphorylation and assembly of a signaling complex that enhances MAPK and phosphatidylinositol 3-kinase pathways. Hyperglycemia also reduces the basal IRS-1 concentration and IGF-I-stimulated IRS-1-linked signaling. To determine if failure to down-regulate IRS-1 alters vascular smooth muscle cell (VSMC) responses to IGF-I, we overexpressed IRS-1 in VSMCs maintained in high glucose. These cultures showed reduced SHPS-1 phosphorylation, transfer of SHP-2 to SHPS-1, and impaired Shc and MAPK phosphorylation and cell proliferation in response to IGF-I. In vitro studies demonstrated that SHPS-1 was a substrate for type I IGF receptor (IGF-IR) and that IRS-1 competitively inhibited SHPS-1 phosphorylation. Exposure of VSMC cultures to a peptide that inhibited IRS-1/IGF-IR interaction showed that IRS-1 binding to IGF-IR impairs SHPS-1 phosphorylation in vivo. IRS-1 also sequestered SHP-2. Expression of an IRS-1 mutant (Y1179F/Y1229F) reduced IRS-1/SHP-2 association, and exposure of cells expressing the mutant to the inhibitory peptide enhanced SHPS-1 phosphorylation and SHP-2 transfer. This result was confirmed by expressing an IRS-1 mutant that had both impaired binding to IGF-IR and to SHP-2 IGF-I increased SHPS-1 phosphorylation, SHP-2 association with SHPS-1, Shc MAPK phosphorylation, and proliferation in cells expressing the mutant. We conclude that IRS-1 is an important factor for maintaining VSMCs in the non-proliferative state and that its down-regulation is a component of the VSMC response to hyperglycemic stress that results in an enhanced response to IGF-I.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Antígenos de Diferenciación/genética , Línea Celular , Proliferación Celular/efectos de los fármacos , Glucosa/farmacología , Humanos , Proteínas Sustrato del Receptor de Insulina/genética , Factor I del Crecimiento Similar a la Insulina/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Mutación , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Ratas , Receptor IGF Tipo 1/genética , Receptores Inmunológicos/genética , Proteínas Adaptadoras de la Señalización Shc , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/fisiología , Edulcorantes/farmacología , PorcinosRESUMEN
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.
Asunto(s)
Antígeno CD47/metabolismo , Glucosa/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Humanos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Unión Proteica/efectos de los fármacos , Interferencia de ARN , Receptores Inmunológicos/metabolismo , Transducción de Señal/efectos de los fármacos , PorcinosRESUMEN
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.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Integrina beta3/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Aorta/citología , Células Cultivadas , Medio de Cultivo Libre de Suero , Proteína Adaptadora GRB2/metabolismo , Humanos , Integrina beta3/química , Ligandos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Datos de Secuencia Molecular , Miocitos del Músculo Liso/metabolismo , Fosforilación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores Inmunológicos/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Tirosina/metabolismo , Vitronectina/química , Vitronectina/metabolismoRESUMEN
The interaction between the arginine glycine and aspartic acid motif (RGD) of integrin ligands such as vitronectin and the integrin receptor alphaVbeta3 in mediating cell attachment has been well described. Similarly, the ability of disintegrins, small RGD containing peptides, to inhibit cell attachment and other cellular processes has also been studied extensively. Recently, we characterized a second site of interaction between vitronectin and its integrin partner. We determined that amino acids within the heparin-binding domain of vitronectin bind to a cysteine loop (C-loop) region of beta3 and that this interaction is required for the positive effects of alphaVbeta3 ligand occupancy on IGF-I signaling in smooth muscle cells. In this study we examine the signaling events activated following ligand binding of disintegrins to the alphaVbeta3 and the ability of these signals to be regulated by binding of the heparin-binding domain of vitronectin. We demonstrate that disintegrin ligand binding activates a series of events including the sequential activation of the tyrosine kinases c-Src and Syk. This leads to the activation of calpain and the cleavage of the beta3 cytoplasmic tail. Addition of vitronectin or a peptide homologous to the heparin-binding domain inhibited activation of this pathway. Our results suggest that the signaling events that occur following ligand binding to the alphaVbeta3 integrin reflects a balance between the effects mediated through the RGD binding site interaction and the effects mediated by the heparin binding site interaction and that for intact vitronectin the effect of the heparin-binding domain predominates.
Asunto(s)
Integrina alfaVbeta3/metabolismo , Transducción de Señal , Vitronectina/metabolismo , Animales , Sitios de Unión , Proteína Tirosina Quinasa CSK , Desintegrinas/metabolismo , Heparina , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ligandos , Miocitos del Músculo Liso/metabolismo , Unión Proteica , Proteínas Tirosina Quinasas/metabolismo , Porcinos , Quinasa Syk , Familia-src QuinasasRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica , Factor I del Crecimiento Similar a la Insulina/metabolismo , Sistema de Señalización de MAP Quinasas , Receptores Inmunológicos/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Membrana Celular/metabolismo , Proliferación Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Proteína Adaptadora GRB2/metabolismo , Vectores Genéticos , Humanos , Immunoblotting , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Datos de Secuencia Molecular , Músculo Liso Vascular/citología , Mutación , Péptidos/química , Fosforilación , Estructura Terciaria de Proteína , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteínas Tirosina Fosfatasas/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Porcinos , Factores de TiempoRESUMEN
IGF-I stimulation of smooth muscle cell (SMC) migration and proliferation requires alphaVbeta3 ligand occupancy. We hypothesized that changes in the levels of extracellular matrix proteins induced by alterations in glucose concentrations may regulate the ability of SMCs to respond to IGF-I. IGF-I stimulated migration and proliferation of SMCs that had been maintained in 25 mM glucose containing media, but it had no stimulatory effect when tested using SMCs that had been grown in 5 mM glucose. IGF-I stimulated an increase in Shc phosphorylation and enhanced activation of the MAPK pathway in SMCs grown in 25 mM glucose, whereas in cells maintained in 5 mM glucose, IGF-I had no effect on Shc phosphorylation, and the MAPK response to IGF-I was markedly reduced. In cells grown in 25 mM glucose, the levels of alphaVbeta3 ligands, e.g. osteopontin, vitronectin, and thrombospondin, were all significantly increased, compared with cells grown in 5 mM glucose. The addition of these alphaVbeta3 ligands to SMCs grown in 5 mM glucose was sufficient to permit IGF-I-stimulated Shc phosphorylation and downstream signaling. Because we have shown previously that alphaVbeta3 ligand occupancy is required for IGF-I-stimulated Shc phosphorylation and stimulation of SMC growth, our data are consistent with a model in which 25 mM glucose stimulates increases in the concentrations of these extracellular matrix proteins, thus enhancing alphaVbeta3 ligand occupancy, which leads to increased Shc phosphorylation and enhanced cell migration and proliferation in response to IGF-I.
Asunto(s)
Glucosa/farmacología , Hiperglucemia/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Músculo Liso Vascular/citología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Aorta/citología , Células CHO , División Celular/efectos de los fármacos , División Celular/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Cricetinae , Cricetulus , Matriz Extracelular/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Integrina alfaVbeta3/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Osteopontina/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Porcinos , Trombospondinas/metabolismo , Vitronectina/metabolismoRESUMEN
PURPOSE: To investigate the role of hyperglycemia in regulating the proliferative response of retinal endothelial cells (RECs) to insulin-like growth factor (IGF)-I. METHODS: The regulation of IGF-I signaling by glucose concentration was assessed by biochemical analysis of primary RECs grown in media containing normal (5 mM) and high (25 mM) glucose. Cell counting was used to asses the proliferative response to IGF-I. RESULTS: Glucose (25 mM) enhanced the proliferative response of RECs to IGF-I. Phosphorylation of the adaptor protein Shc (Src homology 2 domain containing) transforming protein 1) was increased in RECs grown in high glucose. For Shc to be phosphorylated, it must be recruited to the cytoplasmic domain of the transmembrane protein SHPS-1 (SHP substrate-1). Shc recruitment to SHPS-1 was increased when RECs were grown in high glucose. The difference in Shc recruitment to SHPS-1 was attributable to a difference in SHPS-1 phosphorylation that is required for Shc recruitment. This, in turn, was attributable to an increase in SHPS-1 association with integrin-associated protein (IAP), which is necessary for SHPS-1 phosphorylation. The difference in response under the two different glucose conditions appeared to be attributable to changes in the activation of the integrin alphaVbeta3, since blocking alphaVbeta3 in high glucose inhibited the response to IGF-I, whereas addition of the active region of vitronectin to RECs grown in normal glucose enhanced their response. CONCLUSIONS: This study demonstrates that hyperglycemic conditions enhance the response of RECs to IGF-I by increasing the association of IAP with SHPS-1 permitting the formation of the SHPS-1-Shc signaling complex, which is required for the proliferative response to IGF-I.
Asunto(s)
Retinopatía Diabética/metabolismo , Hiperglucemia/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Antígenos de Diferenciación/metabolismo , Antígeno CD47/metabolismo , Bovinos , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Retinopatía Diabética/patología , Glucosa/farmacología , Humanos , Hiperglucemia/patología , Factor I del Crecimiento Similar a la Insulina/farmacología , Ligandos , Fosforilación/efectos de los fármacos , Epitelio Pigmentado Ocular/patología , Receptores Inmunológicos/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal/efectos de los fármacos , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
Under usual conditions, the role of IGF-I in vascular cell types is to maintain cellular protein synthesis and cell size, and even excess IGF-I does not stimulate proliferation. In pathophysiologic states, such as hyperglycemia, smooth muscle cells (SMC) dedifferentiate and change their responsiveness to IGF-I. During hyperglycemia IGF-I stimulates both SMC migration and proliferation. Our laboratory has investigated the molecular mechanism by which this change is mediated. During hyperglycemia SMC secrete increased concentrations of thrombospondin, vitronectin and osteopontin, ligands for the integrin alphaVbeta3. Activation of alphaVbeta3 stimulates recruitment of a tyrosine phosphatase, SHP-2. Exposure of SMC to IGF-I results in phosphorylation of the transmembrane protein, SHPS-1, which provides a docking site for alphaVbeta3-associated SHP-2. After IGF-I stimulation SHP-2 associates with Src kinase, which associates with the signaling protein Shc. Src phosphorylates Shc, resulting in activation of MAP kinases, which are necessary both for stimulation of cell proliferation and migration. Blocking activation of alphaVbeta3 results in an inability of IGF-I to stimulate Shc phosphorylation. Under conditions of normoglycemia, there are insufficient alphaVbeta3 ligands to recruit SHP-2, and no increase in Shc phosphorylation can be demonstrated in SMC. In contrast, if alphaVbeta3 ligands are added to cells in normal glucose, the signaling events that are necessary for Shc phosphorylation can be reconstituted. Therefore when SMC are exposed to normal glucose they are protected from excessive stimulation of mitogenesis by IGF-I. With hyperglycemia there is a marked increased in alphaVbeta3 ligands and Shc phosphorylation in response to IGF-I is sustained. These findings indicate that in SMC hyperglycemic stress leads to altered IGF-I signaling, which allows the cells to undergo a mitogenic response, and which may contribute to the development of atherosclerosis.
Asunto(s)
Hiperglucemia/fisiopatología , Factor I del Crecimiento Similar a la Insulina/farmacología , Integrina alfaVbeta3/fisiología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Estrés Fisiológico/fisiopatología , Animales , Glucemia/fisiología , Modelos Biológicos , Miocitos del Músculo Liso/metabolismoRESUMEN
The response of smooth muscle cells to IGF-I requires ligand occupancy of the alphaVbeta3 integrin. We have shown that vitronectin (Vn) is required for IGF-I-stimulated migration or proliferation, whereas the anti-alphaVbeta3 monoclonal antibody, LM609, which inhibits ligand binding, blocks responsiveness of these cells to IGF-I. The amino acids 177-184 ((177)CYDMKTTC(184)) within the extracellular domain of beta3 have been proposed to confer the ligand specificity of alphaVbeta3; therefore, we hypothesized that ligand binding to the 177-184 cysteine loop of beta3 may be an important regulator of the cross talk between alphaVbeta3 and IGF-I in SMCs. Here we demonstrate that blocking ligand binding to a specific amino acid sequence within the beta3 subunit of alphaVbeta3 (i.e. amino acids 177-184) blocked Vn binding to the beta3 subunit of alphaVbeta3 and correspondingly beta3 phosphorylation was decreased. In the presence of this antibody, IGF-I-stimulated Shc phosphorylation and ERK 1/2 activation were impaired, and this was associated with an inhibition in the ability of IGF-I to stimulate an increase in migration or proliferation. Furthermore, in cells expressing a mutated form of beta3 in which three critical residues within the 177-184 sequence were altered beta3 phosphorylation was decreased. This was associated with a loss of IGF-I-stimulated Shc phosphorylation and impaired smooth muscle cell proliferation in response to IGF-I. In conclusion, we have demonstrated that the 177-184 sequence of beta3 is necessary for Vn binding to alphaVbeta3 and that ligand occupancy of this site is necessary for an optimal response of smooth muscle cells to IGF-I.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/farmacología , Integrina alfaVbeta3/metabolismo , Integrina beta3/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Vitronectina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Anticuerpos/farmacología , Movimiento Celular , Proliferación Celular , Células Cultivadas , Humanos , Integrina alfaVbeta3/antagonistas & inhibidores , Ligandos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Datos de Secuencia Molecular , Miocitos del Músculo Liso/metabolismo , Fosforilación , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Vitronectina/antagonistas & inhibidoresRESUMEN
We have shown that vitronectin (Vn) binding to a cysteine loop sequence within the extracellular domain of the beta3-subunit (amino acids 177-184) of alphaVbeta3 is required for the positive effects of Vn on IGF-I signaling. When Vn binding to this sequence is blocked, IGF-I signaling in smooth muscle cells is impaired. Because this binding site is distinct from the site on beta3 to which the Arg-Gly-Asp sequence of extracellular matrix ligands bind (amino acids 107-171), we hypothesized that the region of Vn that binds to the cysteine loop on beta3 is distinct from the region that contains the Arg-Gly-Asp sequence. The results presented in this study demonstrate that this heparin binding domain (HBD) is the region of Vn that binds to the cysteine loop region of beta3 and that this region is sufficient to mediate the positive effects of Vn on IGF-I signaling. We provide evidence that binding of the HBD of Vn to alphaVbeta3 has direct effects on the activation state of beta3 as measured by beta3 phosphorylation. The increase in beta3 phosphorylation associated with exposure of cells to this HBD is associated with enhanced phosphorylation of the adaptor protein Src homology 2 domain-containing transforming protein C and enhanced activation MAPK, a downstream mediator of IGF-I signaling. We conclude that the interaction of the HBD of Vn binding to the cysteine loop sequence of beta3 is necessary and sufficient for the positive effects of Vn on IGF-I-mediated effects in smooth muscle cells.
Asunto(s)
Heparina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Vitronectina/química , Vitronectina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , ADN/genética , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Músculo Liso/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Porcinos , Vitronectina/genéticaRESUMEN
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.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación , Proteínas Portadoras/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Antígenos CD/fisiología , Antígeno CD47 , Proteínas Portadoras/fisiología , Movimiento Celular , Células Cultivadas , Clonación Molecular , Péptidos y Proteínas de Señalización Intracelular , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína/fisiología , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Transducción de Señal , PorcinosRESUMEN
IGF-I stimulates smooth muscle cell (SMC) migration and the phosphatidylinositol-3 (PI-3) kinase pathway plays an important role in mediating the IGF-I-induced migratory response. Prior studies have shown that the tyrosine phosphatase Src homology 2 domain tyrosine phosphatase (SHP)-2 is necessary to activate PI-3 kinase in response to growth factors and expression of a phosphatase inactive form of SHP-2 (SHP-2/C459S) impairs IGF-I-stimulated cell migration. However, the mechanism by which SHP-2 phosphatase activity or the recruitment of SHP-2 to other signaling molecules contributes to IGF-I stimulated PI-3 kinase activation has not been determined. SMCs that had stable expression of SHP-2/C459S had reduced cell migration and Akt activation in response to IGF-I, compared with SMC-expressing native SHP-2. Similarly in cells expressing native SHP-2, IGF-I induced SHP-2 binding to p85, whereas in cells expressing SHP-2/C459S, there was no increase. Because the C459S substitution results in loss of the ability of SHP-2 to disassociate from its substrates, making it inaccessible not only to p85 but also the other proteins, a p85 mutant in which tyrosines 528 and 556 were changed to phenylalanines was prepared to determine whether this would disrupt the p85/SHP-2 interaction and whether the loss of this specific interaction would alter IGF-I stimulated the cell migration. Substitution for these tyrosines in p85 resulted in loss of SHP-2 recruitment and was associated with a reduction in association of the p85/p110 complex with insulin receptor substrate-1. Cells stably expressing this p85 mutant also showed a decrease in IGF-I-stimulated PI-3 kinase activity and cell migration. Preincubation of cells with a cell-permeable peptide that contains the tyrosine556 motif of p85 also disrupted SHP-2 binding to p85 and inhibited the IGF-I-induced increase in cell migration. The findings indicate that tyrosines 528 and 556 in p85 are required for SHP-2 association. SHP-2 recruitment to p85 is required for IGF-I-stimulated association of the p85/p110 complex with insulin receptor substrate-1 and for the subsequent activation of the PI-3 kinase pathway leading to increased cell migration.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/metabolismo , Músculo Liso/citología , Fosfatidilinositol 3-Quinasas/química , Monoéster Fosfórico Hidrolasas/química , Animales , Aorta/metabolismo , Movimiento Celular , Células Cultivadas , Activación Enzimática , Fibroblastos/metabolismo , Humanos , Immunoblotting , Inmunoprecipitación , Modelos Estadísticos , Mutación , Fenilalanina/química , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Porcinos , Tirosina/químicaRESUMEN
Insulin-like growth factor-I (IGF-I) stimulates vascular smooth muscle cell (SMC) proliferation and migration. The response of smooth muscle cells to IGF-I is determined not only by activation of the IGF-I receptor but also by at least three other transmembrane proteins, alphaVbeta3, integrin-associated protein (IAP), and SHPS-1. This regulation seems to be attributable to their ability to regulate the transfer of SHP-2 phosphatase, a key component of IGF-I signaling. Ligand occupancy of SHPS-1 with IAP is required for the recruitment and transfer of SHP-2 and subsequent signaling in response to IGF-I. The extracellular matrix protein thrombospondin-1 stimulates an increase in the cell proliferation response to IGF-I. Because thrombospondin-1 is a ligand for IAP, we wished to determine whether the enhancing effect of thrombospondin-1 was mediated through IAP binding. To examine the effect of thrombospondin-1 binding to IAP, we used a peptide termed 4N1K derived from the IAP binding site of thrombospondin-1. Preincubation with 4N1K increased IGF-I-stimulated mitogen-activated protein kinase activation and DNA synthesis. This enhancement seemed to be attributable to its ability to increase the duration of IGF-I-stimulated receptor and insulin receptor substrate-1 (IRS-1) phosphorylation. Preincubation with 4N1K delayed IGF-I stimulation of SHPS-1 phosphorylation (attributable to an alteration in IAP-SHPS-1 interaction), resulting in a delay in SHP-2 recruitment. This delay in SHP-2 transfer seems to account for the increase in the duration of IGF-I receptor phosphorylation and for enhanced downstream signaling. These observations support the conclusion that thrombospondin-1 and IGF-I seem to function coordinately in stimulating smooth muscle proliferation via the thrombospondin-1 interaction with IAP.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación , Proteínas Portadoras/metabolismo , Músculo Liso Vascular/metabolismo , Fragmentos de Péptidos/farmacología , Receptor IGF Tipo 1/metabolismo , Receptores Inmunológicos , Trombospondina 1/metabolismo , Animales , Sitios de Unión/fisiología , Antígeno CD47 , División Celular/efectos de los fármacos , Células Cultivadas , ADN/biosíntesis , Activación Enzimática/efectos de los fármacos , Ligandos , Glicoproteínas de Membrana/metabolismo , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Fragmentos de Péptidos/química , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Porcinos , Trombospondina 1/químicaRESUMEN
Ligand occupancy of the alphaVbeta3 integrin is required for IGF-I receptor (IGF-IR) phosphorylation of an appropriate duration and for stimulation of IGF-I actions. In vascular smooth muscle cells (SMCs), the tyrosine phosphatase SHP-2 regulates the duration of IGF-IR phosphorylation and biological actions. We determined the role of ligand occupancy of the alphaVbeta3 integrin on beta3 phosphorylation and studied the role of beta3 phosphorylation in regulating both SHP-2 recruitment to the cell membrane and IGF-I-dependent biological responses. Vitronectin binding to alphaVbeta3 induced tyrosine phosphorylation of the beta3-subunit in subconfluent SMCs and was accompanied by increased association of SHP-2 with beta3. In confluent SMCs, the beta3-subunit was constitutively phosphorylated leading to basal binding of SHP-2. The Src kinase inhibitor PP2 caused a concentration-dependent decrease in beta3 phosphorylation and resulted in decreased SHP-2 association with beta3 and with the cell membrane. In contrast to control cells, SMCs expressing a mutant beta3 that had two tyrosines changed to phenylalanines showed a 89.9 +/- 1.2% decrease in beta3 phosphorylation. This decrease was associated with reduced SHP-2 binding to nonphosphorylated beta3 and a corresponding decrease in the membrane association of SHP-2. When IGF-I was added to cells expressing mutant beta3, SHP-2 was not recruited to the Src homology 2 domain-containing tyrosine phosphatase substrate-1 or to IGF-IR. This was associated with prolonged IGF-IR phosphorylation and an impaired cellular DNA synthesis response to IGF-I. These results define a mechanism by which ligand occupancy of alphaVbeta3 regulates the SMC response to IGF-I.
Asunto(s)
Membrana Celular/metabolismo , Integrina alfaVbeta3/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptor IGF Tipo 1/metabolismo , ADN/biosíntesis , Humanos , Integrina alfaVbeta3/genética , Péptidos y Proteínas de Señalización Intracelular , Ligandos , Mutación , Fosforilación , Fosfotransferasas , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 11RESUMEN
The alphaVbeta3 integrin is an important determinant of IGF-I-stimulated receptor phosphorylation and biological actions. Blocking ligand occupancy of alphaVbeta3 with the distintegrin echistatin reduces IGF-I-stimulated receptor phosphorylation, and it inhibits cellular migration and DNA synthesis responses to IGF-I. We have shown that recruitment of the tyrosine phosphatase Src-homology 2-containing phosphotyrosine phosphatase-2 (SHP-2) to the IGF-I receptor (IGF-IR) is an important determinant of the duration of IGF-IR phosphorylation. These studies were undertaken to determine whether an alteration in the recruitment of SHP-2 to the receptor in the presence of echistatin could account for the decrease in receptor phosphorylation. Following an overnight exposure of smooth muscle cell cultures to echistatin, the addition of IGF-I was accompanied by rapid dephosphorylation of IGF-IR compared with cells exposed to media alone. This was associated with an increase in the rate of SHP-2 recruitment to the IGF-IR. In cells expressing a catalytically inactive form of SHP-2, prior exposure to echistatin had no effect on the rate of receptor dephosphorylation. In contrast to the usual physiologic situation in which following IGF-I exposure SHP-2 is recruited to IGF-IR via SHP-2 substrate-1 (SHPS-1) in the presence of echistatin, SHPS-1 was not used for SHP-2 recruitment. Our findings show that IRS-1 may substitute for SHPS-1 under these conditions. These results demonstrate that the activation state of alphaVbeta3 is an important regulator of the duration of IGF-IR phosphorylation and subsequent downstream signaling and that this regulation is mediated through changes in the subcellular localization of SHP-2.
Asunto(s)
Antígenos de Diferenciación , Integrina alfaVbeta3/fisiología , Proteínas Tirosina Fosfatasas/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptores Inmunológicos , Dominios Homologos src , Expresión Génica , Humanos , Técnicas de Inmunoadsorción , Factor I del Crecimiento Similar a la Insulina/farmacología , Péptidos y Proteínas de Señalización Intercelular , Péptidos y Proteínas de Señalización Intracelular , Cinética , Glicoproteínas de Membrana/metabolismo , Mutagénesis , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Péptidos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteína Fosfatasa 2 , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteínas Tirosina Fosfatasas/análisis , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas con Dominio SH2 , TransfecciónRESUMEN
Integral membrane proteins that are present on cell surfaces bind to extracellular ligands, and this binding influences multiple cellular processes. Three cell surface proteins, alpha V beta 3 integrin, integrin associated protein, and SHPS-1, have been shown to modulate both IGF-I receptor-linked signaling and cellular growth and migration responses that are stimulated by IGF-I. Ligand occupancy of these three proteins influences the recruitment of the phosphatase SHP-2 to the IGF-I receptor and thereby modulates the duration of IGF-I receptor tyrosine phosphorylation. In addition, changes in ligand occupancy of these three integral membrane proteins can regulate the transfer of SHP-2 phosphatase to downstream signaling molecules, which is also required for stimulation of cell migration and DNA synthesis by IGF-I. Determination of the spectrum of ligands for these three integral membrane proteins and the mechanisms by which each ligand functions to alter IGF-I signaling are important objectives of future research.
Asunto(s)
Proteínas de la Membrana/fisiología , Receptor IGF Tipo 1/fisiología , Transducción de Señal/fisiología , Animales , Humanos , Integrina alfaVbeta3/metabolismo , Integrinas/fisiología , LigandosRESUMEN
This study determined if blocking ligand occupancy of the αVß3 integrin could inhibit the pathophysiologic changes that occur in the early stages of diabetic nephropathy (DN). Diabetic rats were treated with either vehicle or a monoclonal antibody that binds the ß3 subunit of the αVß3 integrin. After 4 weeks of diabetes the urinary albumin to creatinine ratio (UACR) increased in both diabetic animals that subsequently received vehicle and in the animals that subsequently received the anti-ß3 antibody compared with control nondiabetic rats. After 8 weeks of treatment the UACR continued to rise in the vehicle-treated rats; however it returned to levels comparable to control nondiabetic rats in rats treated with the anti-ß3 antibody. Treatment with the antibody prevented the increase of several profibrotic proteins that have been implicated in the development of DN. Diabetes was associated with an increase in phosphorylation of the ß3 subunit in kidney homogenates from diabetic animals, but this was prevented by the antibody treatment. This study demonstrates that, when administered after establishment of early pathophysiologic changes in renal function, the anti-ß3 antibody reversed the effects of diabetes normalizing albuminuria and profibrotic proteins in the kidney to the levels observed in nondiabetic control animals.
Asunto(s)
Albuminuria/prevención & control , Anticuerpos Monoclonales/farmacología , Diabetes Mellitus Experimental/tratamiento farmacológico , Nefropatías Diabéticas/prevención & control , Integrina alfaVbeta3/antagonistas & inhibidores , Riñón/efectos de los fármacos , Albuminuria/diagnóstico , Albuminuria/etiología , Albuminuria/orina , Animales , Biomarcadores/orina , Colágeno Tipo IV/metabolismo , Creatinina/orina , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/diagnóstico , Diabetes Mellitus Experimental/metabolismo , Nefropatías Diabéticas/diagnóstico , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/orina , Fibrosis , Integrina alfaVbeta3/inmunología , Integrina alfaVbeta3/metabolismo , Riñón/metabolismo , Riñón/patología , Ligandos , Masculino , Proteínas de la Membrana/metabolismo , Fosforilación , Unión Proteica , Ratas Sprague-Dawley , Estreptozocina , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Hyperglycemia stimulates secretion of αVß3 ligands from vascular cells, including endothelial cells, resulting in activation of the αVß3 integrin. This study determined whether blocking ligand occupancy of αVß3 would inhibit the development of diabetic nephropathy. Ten diabetic pigs received an F(ab)2 fragment of an antibody directed against the extracellular domain of the ß3-subunit, and 10 received a control IgG F(ab)2 for 18 weeks. Nondiabetic pigs excreted 115 ± 50 µg of protein/mg creatinine compared with control F(ab)2-treated diabetic animals (218 ± 57 µg/mg), whereas diabetic animals treated with the anti-ß3 F(ab)2 excreted 119 ± 55 µg/mg (P < .05). Mesangial volume/glomerular volume increased to 21 ± 2.4% in control-treated diabetic animals compared with 14 ± 2.8% (P < .01) in animals treated with active antibody. Diabetic animals treated with control F(ab)2 had significantly less glomerular podocin staining compared with nondiabetic animals, and this decrease was attenuated by treatment with anti-ß3 F(ab)2. Glomerular basement membrane thickness was increased in the control, F(ab)2-treated diabetic animals (212 ± 14 nm) compared with nondiabetic animals (170 ± 8.8 nm), but it was unchanged (159.9 ± 16.4 nm) in animals receiving anti-ß3 F(ab)2. Podocyte foot process width was greater in control, F(ab)2-treated, animals (502 ± 34 nm) compared with animals treated with the anti-ß3 F(ab)2 (357 ± 47 nm, P < .05). Renal ß3 tyrosine phosphorylation decreased from 13 934 ± 6437 to 6730 ± 1524 (P < .01) scanning units in the anti-ß3-treated group. We conclude that administration of an antibody that inhibits activation of the ß3-subunit of αVß3 that is induced by hyperglycemia attenuates proteinuria and early histologic changes of diabetic nephropathy, suggesting that it may have utility in preventing the progression of this disease complication.
Asunto(s)
Nefropatías Diabéticas/etiología , Integrina alfaVbeta3/metabolismo , Animales , Anticuerpos Monoclonales , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Membrana Basal Glomerular/patología , Integrina alfaVbeta3/antagonistas & inhibidores , Masculino , Ratones Endogámicos BALB C , Podocitos/patología , Proteinuria/etiología , PorcinosRESUMEN
PURPOSE: Burn injury is associated with early apoptotic death of T cells. Insulin-like growth factor-1 (IGF-I) is able to protect T cells from apoptosis. Association of IGF-I with its IGFBP (Binding Protein)-1 limits its bioavailability and serine phosphorylation of IGFBP-1 lowers this further because of an increased affinity for IGF-I. The level of phosphorylated IGFBP-1 has been shown to increase in pediatric burn patients. Thus we hypothesized that a longitudinal study of burn patients would demonstrate 1) increased IGFBP-1 levels, 2) increased IGFBP-1 phosphorylation and 3) decreased IGF-I levels over time. METHODS: We conducted a prospective observational study in adult burn patients admitted to UNC Jaycee Burn Center. Plasma levels of insulin, insulin-like growth factor 1 (IGF-I) and insulin-like growth factor binding protein 1 (IGGBP-1) were measured on admission up to 10 days post admission. ELISA was used to measure serum levels of insulin, IGF-I and IGFBP-1. Serine phosphorylation of IGFBP-1 was measured by Western blot with and without the incubation of calf intestinal phosphatase (CIP). Significant findings: There was a significant positive correlation of increasing %TBSA burn and increasing levels of serum IGFBP-1 from admittance blood draws. Levels of IGF-I also decreased with increasing Total Body Surface Area (TBSA, p<0.05). In patients studied longitudinally (n=84) we found that IGFBP-1 levels are significantly (p<0.05) increased 1-72 hours post burn (mean±SEM serum concentration; burn=172±23 ng/mL, normal=13±3 ng/mL) and that levels of IGF-I are reduced. IGFBP-1 is serine phosphorylated in burn patients. In patients surviving past 72 hours IGFBP-1 remained phosphorylated over the study period. CONCLUSIONS: IGFBP-1 and its serine phosphorylation regulate and limit IGF-I bioavailability. Our results suggest that increases in IGFBP-1 and persistent serine phosphorylation of IGFBP-1 correlate with the severity of burn injury, and may contribute to burn-associated T cell apoptosis and subsequent immune dysfunction by reducing the bioavailability of this important cell survival factor.