EGFR and FGFR signaling through FRS2 is subject to negative feedback control by ERK1/2

Fibroblast growth factor (FGF) receptor substrate 2 (FRS2) is a membrane-anchored docking protein that has been shown to play an important role in linking FGF, nerve growth factor (NGF) and glial cell-derived neurotrophic factor (GDNF) receptors with the Ras/mitogen-activated protein (MAP) kinase signaling cascade. Here we provide evidence that FRS2 can also play a role in epidermal growth factor (EGF) signaling. Upon EGF stimulation, FRS2 mediates enhanced MAPK activity and undergoes phosphorylation on tyrosine as well as serine/threonine residues. This involves the direct interaction of the FRS2 PTB domain with the EGFR and results in a significantly altered mobility of FRS2 in SDS-PAGE which is also observed in FGF stimulated cells. This migration shift of FRS2 is completely abrogated by U0126, a specific MAPK kinase 1 (MEK1) inhibitor, suggesting that ERK1/2 acts as serine/threonine kinase upstream of FRS2. Indeed, we show that the central portion of FRS2 constitutively associates with ERK1/2, whereas the FRS2 carboxy-terminal region serves as substrate for ERK2 phosphorylation in response to EGF and FGF stimulation. Notably, tyrosine phosphorylation of FRS2 is enhanced when ERK1/2 activation is inhibited after both EGF and FGF stimulation. These results indicate a ligand-stimulated negative regulatory feedback loop in which activated ERK1/2 phosphorylates FRS2 on serine/threonine residues thereby down-regulating its tyrosine phosphorylation. Our findings support a broader role of FRS2 in EGFR-controlled signaling pathways in A-431 cells and provide insight into a molecular mechanism for ligand-stimulated feedback regulation with FRS2 as a central regulatory switch point.     

Fibroblast growth factor 16 and 18 are expressed in human cardiovascular tissues and induce on endothelial cells migration but not proliferation

Endothelial cells line the blood vessel and precursor endothelial cells appear to have a pivotal effect on the organ formation of the heart, the embryonic development of the kidney, and the liver. Several growth factors including the fibroblast growth factors (FGF) seem to be involved in these processes. Ligands such as basic FGF produced and secreted by endothelial cells may also coordinate cellular migration, differentiation, and proliferation under pathological conditions including wound healing, tumorgenesis, and fibrogenesis in the adult. Recently we demonstrated the expression of two secreted FGFs, FGF16, and FGF18, in HUVEC and in rat aortic tissue. In the present report, we confirmed by RT-PCR analysis that FGF18 is wildly expressed in the cardiovascular tissue, while FGF16 showed a more restricted expression pattern. HUVEC clearly demonstrated chemotaxis towards FGF16 and FGF18. Both FGFs also enhanced cell migration in response to mechanical damage. However, recombinant FGF16 and FGF18 failed to induce endothelial cell proliferation or sprouting in a three-dimensional in vitro angiogenesis assay. Fgf18 expression was earlier reported in the liver, and we detected FGF18 expression in liver vascular and liver sinusoidal endothelial cells (LSECs), but not in hepatic parenchymal cells. Recombinant FGF18 stimulated DNA synthesis in primary hepatocytes, suggesting, that endothelial FGF18 might have a paracrine function in promoting growth of the parenchymal tissue. Interestingly, FGF2, which is mitogenic on endothelial cells and hepatocytes stimulates a sustained MAPK activation in both cell types, while FGF18 causes a short transient activation of the MAPK pathway in endothelial cells but a sustained activation in hepatocytes. Therefore, the difference in the time course of MAPK activation by the different FGFs appears to be the cause for the different cellular responses.     

The protein tyrosine phosphatase, Shp2, is required for the complete activation of the RAS/MAPK pathway by brain-derived neurotrophic factor

Brain-derived neurotrophic factor (BDNF) and other neurotrophins induce a unique prolonged activation of mitogen-activated protein kinase (MAPK) compared with growth factors. Characterization and kinetic and spatial modeling of the signaling pathways underlying this prolonged MAPK activation by BDNF will be important in understanding the physiological role of BDNF in many complex systems in the nervous system. In addition to Shc, fibroblast growth factor receptor substrate 2 (FRS2) is required for the BDNF-induced activation of MAPK. BDNF induces phosphorylation of FRS2. However, BDNF does not induce phosphorylation of FRS2 in cells expressing a deletion mutant of TrkB (TrkBDeltaPTB) missing the juxtamembrane NPXY motif. This motif is the binding site for SHC. NPXY is the consensus sequence for phosphotyrosine binding (PTB) domains, and notably, FRS2 and SHC contain PTB domains. This NPXY motif, which contains tyrosine 484 of TrkB, is therefore the binding site for both FRS2 and SHC. Moreover, the proline containing region (VIENP) of the NPXY motif is also required for FRS2 and SHC phosphorylation, which indicates this region is an important component of FRS2 and SHC recognition by TrkB. Previously, we had found that the phosphorylation of FRS2 induces association of FRS2 and growth factor receptor binding protein 2 (Grb2). Now, we have intriguing data that indicates BDNF induces association of the SH2 domain containing protein tyrosine phosphatase, Shp2, with FRS2. Moreover, the PTB association motif of TrkB containing tyrosine 484 is required for the BDNF-induced association of Shp2 with FRS2 and the phosphorylation of Shp2. These results imply that FRS2 and Shp2 are in a BDNF signaling pathway. Shp2 is required for complete MAPK activation by BDNF, as expression of a dominant negative Shp2 in cells attenuates BDNF-induced activation of MAPK. Moreover, expression of a dominant negative Shp2 attenuates Ras activation showing that the protein tyrosine phosphatase is required for complete activation of MAPKs by BDNF. In conclusion, Shp2 regulates BDNF signaling through the MAPK pathway by regulating either Ras directly or alternatively, by signaling components upstream of Ras. Characterization of MAPK signaling controlled by BDNF is likely to be required to understand the complex physiological role of BDNF in neuronal systems ranging from the regulation of neuronal growth and survival to the regulation of synapses.     

The docking protein FRS2alpha controls a MAP kinase-mediated negative feedback mechanism for signaling by FGF receptors

The docking protein FRS2alpha functions as a major mediator of signaling by FGF and NGF receptors. Here we demonstrate that, in addition to tyrosine phosphorylation, FRS2alpha is phosphorylated by MAP kinase on multiple threonine residues in response to FGF stimulation or by insulin, EGF, and PDGF, extracellular stimuli that do not induce tyrosine phosphorylation of FRS2alpha. Prevention of FRS2alpha threonine phosphorylation results in constitutive tyrosine phosphorylation of FRS2alpha in unstimulated cells and enhanced tyrosine phosphorylation of FRS2alpha, MAPK stimulation, cell migration, and proliferation in FGF-stimulated cells. Expression of an FRS2alpha mutant deficient in MAPK phosphorylation sites induces anchorage-independent cell growth and colony formation in soft agar. These experiments reveal a novel MAPK-mediated, negative feedback mechanism for control of signaling pathways that are dependent on FRS2 and a mechanism for heterologous control of signaling via FGF receptors.     

Keratinocyte growth factor receptor ligands target the receptor to different intracellular pathways

The keratinocyte growth factor receptor (KGFR)/fibroblast growth factor receptor 2b is activated by high-affinity-specific interaction with two different ligands, keratinocyte growth factor (KGF)/fibroblast growth factor (FGF)7 and FGF10/KGF2, which are characterized by an opposite requirement of heparan sulfate proteoglycans and heparin for binding to the receptor. We investigated here the possible different endocytic trafficking of KGFR, induced by the two ligands. Immunofluorescence and immunoelectron microscopy analysis showed that KGFR internalization triggered by either KGF or FGF10 occurs through clathrin-coated pits. Immunofluorescence confocal microscopy using endocytic markers as well as tumor susceptibility gene 101 (TSG101) silencing demonstrated that KGF drives KGFR to the degradative pathway, while FGF10 targets the receptor to the recycling endosomes. Biochemical analysis showed that KGFR is ubiquitinated and degraded after KGF treatment but not after FGF10 treatment, and that the alternative fate of KGFR might depend on the different ability of the receptor to phosphorylate the fibroblast growth factor receptor substrate 2 (FRS2) substrate and to recruit the ubiquitin ligase c-Cbl. The recycling endocytic pathway followed by KGFR upon FGF10 stimulation correlates with the higher mitogenic activity exerted by this ligand on epithelial cells compared with KGF, suggesting that the two ligands may play different functional roles through the regulation of the receptor endocytic transport.     

Expression and function of fibroblast growth factor (FGF) 9 in hepatic stellate cells and its role in toxic liver injury

Hepatic injury and regeneration of the liver are associated with activation of hepatic stellate cells (HSC). Fibroblast growth factors (FGFs) and their receptors are important regulators of repair in various tissues. HSC express FGFR3IIIc as well as FGFGR4 and different spliced FGFR1IIIc and FGFR2IIIc isoforms which differ in the presence or absence of the acid box and of the first Ig-like domain. Expression of FGF9, known to be capable to activate the HSC FGFR2/3-isoforms, was increased in HSC in liver slice cultures after exposition to carbon tetrachloride, as an acute liver injury model. FGF9 significantly stimulated 3-H thymidine incorporation of hepatocytes, but failed to induce DNA synthesis in HSC despite the fact that FGF9 induced a sustained activation of extracellular signal-related kinases (ERK) 1/2. FGF9 induced an increased phosphorylation of Tyr436 of the fibroblast growth factor receptor substrate (FRS) 2, while phosphorylation of Tyr196 which is required for efficient Grb2 recruitment remained unchanged. Our findings suggest that HSC FGF9 provide a paracrine mitogenic signal to hepatocytes during acute liver injury, while the autocrine FGF9 signaling appears to be not sufficient to induce cell proliferation.     

Overexpression of Spry1 in chondrocytes causes attenuated FGFR ubiquitination and sustained ERK activation resulting in chondrodysplasia

The FGF signaling pathway plays essential roles in endochondral ossification by regulating osteoblast proliferation and differentiation, chondrocyte proliferation, hypertrophy, and apoptosis. FGF signaling is controlled by the complementary action of both positive and negative regulators of the signal transduction pathway. The Spry proteins are crucial regulators of receptor tyrosine kinase-mediated MAPK signaling activity. Sprys are expressed in close proximity to FGF signaling centers and regulate FGFR-ERK-mediated organogenesis. During endochondral ossification, Spry genes are expressed in prehypertrophic and hypertrophic chondrocytes. Using a conditional transgenic approach in chondrocytes in vivo, the forced expression of Spry1 resulted in neonatal lethality with accompanying skeletal abnormalities resembling thanatophoric dysplasia II, including increased apoptosis and decreased chondrocyte proliferation in the presumptive reserve and proliferating zones. In vitro chondrocyte cultures recapitulated the inhibitory effect of Spry1 on chondrocyte proliferation. In addition, overexpression of Spry1 resulted in sustained ERK activation and increased expression of p21 and STAT1. Immunoprecipitation experiments revealed that Spry1 expression in chondrocyte cultures resulted in decreased FGFR2 ubiquitination and increased FGFR2 stability. These results suggest that constitutive expression of Spry1 in chondrocytes results in attenuated FGFR2 degradation, sustained ERK activation, and up-regulation of p21Cip and STAT1 causing dysregulated chondrocyte proliferation and terminal differentiation.     

Identification of the mechanisms regulating the differential activation of the mapk cascade by epidermal growth factor and nerve growth factor in PC12 cells

In PC12 cells, epidermal growth factor (EGF) transiently stimulates the mitogen-activated protein (MAP) kinases, ERK1 and ERK2, and provokes cellular proliferation. In contrast, nerve growth factor (NGF) stimulation leads to the sustained activation of the MAPKs and subsequently to neuronal differentiation. It has been shown that both the magnitude and longevity of MAPK activation governs the nature of the cellular response. The activations of MAPKs are dependent upon two distinct small G-proteins, Ras and Rap1, that link the growth factor receptors to the MAPK cascade by activating c-Raf and B-Raf, respectively. We found that Ras was transiently stimulated upon both EGF and NGF treatment of PC12 cells. However, EGF transiently activated Rap1, whereas NGF stimulated prolonged Rap1 activation. The activation of the ERKs was due almost exclusively (>90%) to the action of B-Raf. The transient activation of the MAPKs by EGF was a consequence of the formation of a short lived complex assembling on the EGF receptor itself, composed of Crk, C3G, Rap1, and B-Raf. In contrast, NGF stimulation of the cells resulted in the phosphorylation of FRS2. FRS2 scaffolded the assembly of a stable complex of Crk, C3G, Rap1, and B-Raf resulting in the prolonged activation of the MAPKs. Together, these data provide a signaling link between growth factor receptors and MAPK activation and a mechanistic explanation of the differential MAPK kinetics exhibited by these growth factors.     

Cellular signaling by fibroblast growth factor receptors

The 22 members of the fibroblast growth factor (FGF) family of growth factors mediate their cellular responses by binding to and activating the different isoforms encoded by the four receptor tyrosine kinases (RTKs) designated FGFR1, FGFR2, FGFR3 and FGFR4. Unlike other growth factors, FGFs act in concert with heparin or heparan sulfate proteoglycan (HSPG) to activate FGFRs and to induce the pleiotropic responses that lead to the variety of cellular responses induced by this large family of growth factors. A variety of human skeletal dysplasias have been linked to specific point mutations in FGFR1, FGFR2 and FGFR3 leading to severe impairment in cranial, digital and skeletal development. Gain of function mutations in FGFRs were also identified in a variety of human cancers such as myeloproliferative syndromes, lymphomas, prostate and breast cancers as well as other malignant diseases. The binding of FGF and HSPG to the extracellular ligand domain of FGFR induces receptor dimerization, activation and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of the receptor molecule. A variety of signaling proteins are phosphorylated in response to FGF stimulation including Shc, phospholipase-Cgamma, STAT1, Gab1 and FRS2alpha leading to stimulation of intracellular signaling pathways that control cell proliferation, cell differentiation, cell migration, cell survival and cell shape. The docking proteins FRS2alpha and FRS2beta are major mediators of the Ras/MAPK and PI-3 kinase/Akt signaling pathways as well as negative feedback mechanisms that fine-tune the signal that is initiated at the cell surface following FGFR stimulation.     

The docking protein Gab1 is an essential component of an indirect mechanism for fibroblast growth factor stimulation of the phosphatidylinositol 3-kinase/Akt antiapoptotic pathway

The docking protein Gab1 has been implicated as a mediator of multiple signaling pathways that are activated by a variety of receptor tyrosine kinases and cytokines. We have previously proposed that fibroblast growth factor 1 (FGF1) stimulation of tyrosine phosphorylation of Gab1 and recruitment of phosphatidylinositol (PI) 3-kinase are mediated by an indirect mechanism in which the docking protein fibroblast receptor substrate 2alpha (FRS2alpha) plays a critical role. In this report, we explore the role of Gab1 in FGF1 signaling by using mouse embryo fibroblasts (MEFs) derived from Gab1(-/-) or FRS2alpha(-/-) mice. We demonstrate that Gab1 is essential for FGF1 stimulation of both PI 3-kinase and the antiapoptotic protein kinase Akt, while FGF1-induced mitogen-activated protein kinase (MAPK) stimulation is not affected by Gab1 deficiency. To test the indirect mechanism for FGF1 stimulation of PI 3-kinase and Akt, we use a chimeric docking protein composed of the membrane targeting signal and the phosphotyrosine-binding domain of FRS2alpha fused to the C-terminal portion of Gab1, the region including the binding sites for the complement of signaling proteins that are recruited by Gab1. We demonstrate that expression of the chimeric docking protein in Gab1(-/-) MEFs rescues PI 3-kinase and the Akt responses, while expression of the chimeric docking protein in FRS2alpha(-/-) MEFs rescues stimulation of both Akt and MAPK. These experiments underscore the essential role of Gab1 in FGF1 stimulation of the PI 3-kinase/Akt signaling pathway and provide further support for the indirect mechanism for FGF1 stimulation of PI 3-kinase involving regulated assembly of a multiprotein complex.     

The Shb adaptor protein binds to tyrosine 766 in the FGFR-1 and regulates the Ras/MEK/MAPK pathway via FRS2 phosphorylation in endothelial cells

Stimulation of fibroblast growth factor receptor-1 (FGFR-1) is known to result in phosphorylation of tyrosine 766 and the recruitment and subsequent activation of phospholipase C-gamma (PLC-gamma). To assess the role of tyrosine 766 in endothelial cell function, we generated endothelial cells expressing a chimeric receptor, composed of the extracellular domain of the PDGF receptor-alpha and the intracellular domain of FGFR-1. Mutation of tyrosine 766 to phenylalanine prevented PLC-gamma activation and resulted in a reduced phosphorylation of FRS2 and reduced activation of the Ras/MEK/MAPK pathway relative to the wild-type chimeric receptor. However, FGFR-1-mediated MAPK activation was not dependent on PKC activation or intracellular calcium, both downstream mediators of PLC-gamma activation. We report that the adaptor protein Shb is also able to bind tyrosine 766 in the FGFR-1, via its SH2 domain, resulting in its subsequent phosphorylation. Overexpression of an SH2 domain mutant Shb caused a dramatic reduction in FGFR-1-mediated FRS2 phosphorylation with concomitant perturbment of the Ras/MEK/MAPK pathway. Expression of the chimeric receptor mutant and the Shb SH2 domain mutant resulted in a similar reduction in FGFR-1-mediated mitogenicity. We conclude, that Shb binds to tyrosine 766 in the FGFR-1 and regulates FGF-mediated mitogenicity via FRS2 phosphorylation and the subsequent activation of the Ras/MEK/MAPK pathway.     

Phosphorylation of carboxyl-terminal tyrosines modulates the specificity of Sprouty-2 inhibition of different signaling pathways

Sprouty proteins are evolutionarily conserved negative feedback regulators of multiple receptor tyrosine kinases. Mammalian versions of these proteins differentially regulate signaling induced by the fibroblast and the epidermal growth factors (FGF and EGF, respectively). Herein we show that, although both growth factors elevate expression of Sprouty-2, FGF- and not EGF-induced activation of the Erk/MAPK pathway is inhibited by Sprouty-2. Attenuation of FGF-signaling is accompanied by the induction of Sprouty-2 phosphorylation on the amino-terminal as well as carboxyl-terminal tyrosine residues, which are less effectively modified upon EGF treatment. Mutagenesis of carboxyl-terminal tyrosines, especially a newly identified phosphorylation site, tyrosine 227, impaired the inhibitory activity of Sprouty-2. These results attribute a novel role for carboxyl-terminal tyrosine residues and yet unidentified phosphotyrosine-binding proteins in the differential regulation of Sprouty-2 activity.     

Phosphoprotein Enriched in Astrocytes 15 kDa (PEA-15) Reprograms Growth Factor Signaling by Inhibiting Threonine Phosphorylation of Fibroblast Receptor Substrate 2α

Changes in cellular expression of phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) are linked to insulin resistance, tumor cell invasion, and cellular senescence; these changes alter the activation of the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase pathway. Here, we define the mechanism whereby increased PEA-15 expression promotes and sustains ERK1/2 activation. PEA-15 binding prevented ERK1/2 membrane recruitment and threonine phosphorylation of fibroblast receptor substrate 2α (FRS2α), a key link in fibroblast growth factor (FGF) receptor activation of ERK1/2. This reduced threonine phosphorylation led to increased FGF-induced tyrosine phosphorylation of FRS2α, thereby enhancing downstream signaling. Conversely, short hairpin RNA-mediated depletion of endogenous PEA-15 led to reduced FRS2α tyrosine phosphorylation. Thus, PEA-15 interrupts a negative feedback loop that terminates growth factor receptor signaling downstream of FRS2α. This is the dominant mechanism by which PEA-15 activates ERK1/2 because genetic deletion of FRS2α blocked the capacity of PEA-15 to activate the MAP kinase pathway. Thus, PEA-15 prevents ERK1/2 localization to the plasma membrane, thereby inhibiting ERK1/2-dependent threonine phosphorylation of FRS2α to promote activation of the ERK1/2 MAP kinase pathway.     

A permeable FGF-1 nuclear localization sequence peptide induces DNA synthesis independently of Ras activation

A 26-amino-acid peptide (designated PFNP) composed of the nuclear localization signal of fibroblast growth factor (FGF)-1 and a membrane-permeable peptide is known to mimic FGF-1's ability to stimulate DNA synthesis in various cell types at low cell densities. The underlying molecular mechanism is unknown, however. Here we show that PFNP activity is inhibited in murine fibroblasts by a tyrosine kinase inhibitor, that PFNP does not bind to the FGF receptor, and that PFNP does not induce phosphorylation of the FGF receptor substrate. In addition, expression of a dominant-negative form of Ras, which abolished the activities of epidermal growth factor (EGF) and heparin-binding EGF, had no affect on PFNP-induced DNA synthesis. Despite this apparent Ras independence, PFNP activity correlated with phosphorylation of ERK1/2 MAP kinases and was concentration dependently inhibited by inhibitors of ERK1/2 MAP kinase phosphorylation. These results indicate that whereas Ras activation is dispensable for PFNP-induced DNA synthesis, activation of tyrosine kinases and ERK1/2 kinases, albeit independently of the FGF receptor system, is crucial. Interestingly, FGF-1 signaling was predominantly Ras-independent when the cell density was optimum for PFNP, suggesting that PFNP and FGF-1 share the same signaling mechanism.     

Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway

Sprouty (Spry) inhibits signalling by receptor tyrosine kinases; however, the molecular mechanism underlying this function has not been defined. Here we show that after stimulation by growth factors Spry1 and Spry2 translocate to the plasma membrane and become phosphorylated on a conserved tyrosine. Next, they bind to the adaptor protein Grb2 and inhibit the recruitment of the Grb2-Sos complex either to the fibroblast growth factor receptor (FGFR) docking adaptor protein FRS2 or to Shp2. Membrane translocation of Spry is necessary for its phosphorylation, which is essential for its inhibitor activity. A tyrosine-phosphorylated octapeptide derived from mouse Spry2 inhibits Grb2 from binding FRS2, Shp2 or mouse Spry2 in vitro and blocks activation of the extracellular-signal-regulated kinase (ERK) in cells stimulated by growth factor. A non-phosphorylated Spry mutant cannot bind Grb2 and acts as a dominant negative, inducing prolonged activation of ERK in response to FGF and promoting the FGF-induced outgrowth of neurites in PC12 cells. Our findings suggest that Spry functions in a negative feedback mechanism in which its inhibitor activity is controlled rapidly and reversibly by post-translational mechanisms.     

A model of cytokine shedding induced by low doses of gamma radiation

A model for sustained shedding of epidermal growth factor (EGF) in response to low doses of gamma radiation was developed based on a time delay in the feedback from mitogen-activated protein kinase (MAPK) activation to metalloprotease activity in an autocrine signaling process. We determined the kinetic parameters of our model using the data available for MAPK activation by gamma irradiation in the 1-2-Gy dose range and then showed that predictions of the model were consistent with experimental results for the kinetics of EGF shedding into the growth medium after exposure of human mammary epithelial cells to 1-5 cGy of gamma radiation in the presence of antibodies that block ligand binding to EGF receptors. The model allowed us to estimate the rate of radiation-induced cytokine release per cell from measurements of EGF concentration in the growth medium and to assess the effectiveness of EGF shedding and subsequent diffusion through the medium as a mechanism for signal transmission between hit cells and bystanders.     

Activation of the ERK1/2 and p38 mitogen-activated protein kinase pathways mediates fibroblast growth factor-induced growth arrest of chondrocytes

Fibroblast growth factors (FGFs) regulate long bone development by affecting the proliferation and differentiation of chondrocytes. FGF treatment inhibits the proliferation of chondrocytes both in vitro and in vivo, but the signaling pathways involved have not been clearly identified. In this report we show that both the MEK-ERK1/2 and p38 MAPK pathways, but not phospholipase C gamma or phosphatidylinositol 3-kinase, play a role in FGF-mediated growth arrest of chondrocytes. Chemical inhibitors of the MEK1/2 or the p38 MAPK pathways applied to rat chondrosarcoma (RCS) chondrocytes significantly prevented FGF-induced growth arrest. The retinoblastoma family members p107 and p130 were previously shown to be essential effectors of FGF-induced growth arrest in chondrocytes. The dephosphorylation of p107, one of the earliest events in RCS growth arrest, was significantly blocked by MEK1/2 inhibitors but not by the p38 MAPK inhibitors, whereas that of p130, which occurs later, was partially prevented both by the MEK and p38 inhibitors. Furthermore, by expressing the nerve growth factor (NGF) receptor, TrkA, and the epidermal growth factor (EGF) receptor, ErbB1, in RCS cells we show that NGF treatment of the transfected cells caused growth inhibition, whereas EGF did not. FGF- and NGF-induced growth inhibition is accompanied by a strong and sustained activation of ERK1/2 and p38 MAPK and a decrease of AKT phosphorylation, whereas EGF induces a much more transient activation of p38 and ERK1/2 and increases AKT phosphorylation. These results indicate that inhibition of chondrocyte proliferation by FGF requires both ERK1/2 and p38 MAPK signaling and also suggest that sustained activation of these pathways is required to achieve growth inhibition.     

Compartmentalizing Proximal FGFR1 Signaling in Ovine Placental Artery Endothelial Cell Caveolae

Caveolae orchestrate the dominant placental angiogenic growth factor fibroblast growth factor 2 (FGF2) signaling primarily via FGF receptor 1 (FGFR1) in placental artery endothelial cells; however, how the proximal FGF2/FGFR1 signaling is organized in the caveolae is obscure. We have shown in the present study that the FGFR substrate 2alpha (FRS2alpha) is physically associated with FGFR1, and both are targeted to the caveolae via interaction with caveolin-1 in ovine fetoplacental artery endothelial cells. Treatment with FGF2 rapidly stimulated time- and concentration-dependent FRS2alpha tyrosine phosphorylation and recruited the cytosolic growth factor receptor-bound protein 2 (GRB2)-GRB2-associated binding protein 1 (GAB1) complex to the caveolae, where they formed a ternary complex with FRS2alpha. Disruption of caveolae by cholesterol depletion with methyl-beta-cyclodextrin inhibited FGF2-induced FRS2alpha tyrosine phosphorylation, and it blocked the FGF2-induced recruitment of GRB2 and GAB1 to the caveolae and formation of the FRS2alpha-GRB2-GAB1 complex in the caveolae, as well as activation of the PI3K/AKT1 and MAPK1/2 pathways. Thus, these findings have demonstrated that the proximal fibroblast growth factor (FGF2/FGFR1) signaling is compartmentalized in the placental endothelial caveolae via the FGFR substrate 2α that mediates formation of a FRS2α-GRB2-GAB1 complex.     

肽类生长因子对抗癌基因的诱导作用

观察了肽类生长因子对２ＢＳ细胞中抗癌基因表达的影响。结果表明,ＥＧＦ或ＦＧＦ均可明显诱导２ＢＳ细胞中Ｒｂ基因的表达,其幅度分别为３０５％、２４３％;ＥＧＦ也可诱导２ＢＳ细胞中ＴＧＦβ１基因表达增加３０－５０％,但ＦＧＦ对ＴＧＦβ１的表达无明显影响;ＥＧＦ或ＦＧＦ对ｐ５３基因的表达均无明显诱导作用。上述结果为生长因子作用机制研究及生长因子与抗癌基因的关系提供了新的线索。