The conformation-dependent interaction of alpha 2-macroglobulin with vascular endothelial growth factor. A novel mechanism of alpha 2-macroglobulin/growth factor binding

alpha(2)-Macroglobulin (alpha(2)M) is a highly conserved proteinase inhibitor present in human plasma at high concentration (2-4 mg/ml). alpha(2)M exists in two conformations, a native form and an activated, receptor-recognized form. While alpha(2)M binds to numerous cytokines and growth factors, in most cases, the nature of the alpha(2)M interaction with these factors is poorly understood. We examined in detail the interaction between alpha(2)M and vascular endothelial growth factor (VEGF) and found a novel and unexpected mechanism of interaction as demonstrated by the following observations: 1) the binding of VEGF to alpha(2)M occurs at a site distinct from the recently characterized growth factor binding site; 2) VEGF binds different forms of alpha(2)M with distinct spatial arrangement, namely to the interior of methylamine or ammonia-treated alpha(2)M and to the exterior of native and proteinase-converted alpha(2)M; and 3) VEGF (molecular mass approximately 40 kDa) can access the interior of receptor-recognized alpha(2)M in the absence of a proteinase trapped within the molecule. VEGF bound to receptor-recognized forms of alpha(2)M is internalized and degraded by macrophages via the alpha(2)M receptor, the low density lipoprotein receptor-related protein. Oxidation of both native and receptor-recognized alpha(2)M results in significant inhibition of VEGF binding. We also examined the biological significance of this interaction by studying the effect of alpha(2)M on VEGF-induced cell proliferation and VEGF-induced up-regulation of intracellular Ca(2+) levels. We demonstrate that under physiological conditions, alpha(2)M does not impact the ability of VEGF to induce cell proliferation or up-regulate Ca(2+).     

Mitogenic effect of transforming growth factor beta 1 on human fibroblasts involves the induction of platelet-derived growth factor alpha receptors

Platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta), potent modulators of mesenchymal cell growth and differentiation, are often colocalizable in vivo. Previous in vitro studies in fibroblastic cell lines have shown variable, even antagonistic effects of TGF-beta on the mitogenic action of PDGF. This study demonstrates that in diploid human dermal fibroblasts, TGF-beta 1 is weakly mitogenic in the absence of serum or purified growth factors, and that TGF-beta 1 potentiates DNA synthesis in PDGF-stimulated fibroblasts with delayed kinetics when compared to stimulation with PDGF alone. TGF-beta 1 enhances mitogenic potency of all three PDGF isoforms and increases receptor binding of both 125I PDGF-AA and 125I PDGF-BB, consistent with the increased expression of the alpha type PDGF receptor. The induction of PDGF alpha receptor subunits by TGF-beta may play a role in enhancing the proliferative potential of human fibroblasts in certain physiologic and pathologic conditions.     

Retinyl acetate inhibits platelet-derived growth factor-induced Ca2+ signals in C3H 10T1/2 fibroblasts

Mitogenic stimulation of density-arrested C3H 10T1/2 mouse fibroblasts by serum or purified platelet-derived growth factor (PDGF) was potently inhibited by retinyl acetate (RAc; IC50 = 0.1 microgram/ml, 0.3 x 10(-6) M) when administered during the first 2 hours of mitogen exposure. This inhibitory effect of RAc coincided with a period early in the cell growth-division cycle when density-arrested C3H 10T1/2 cells stimulated by PDGF were found to require physiological levels of extracellular Ca2+ for the transition from G0 to G1 of the cell cycle. To determine if the inhibitory effect of RAc was mediated through alterations in the Ca2+ signaling pathway induced by mitogens, we examined Fura-2-loaded fibroblasts for changes in the Ca2+ response elicited by PDGF. Addition of PDGF (5 ng/ml) induced a transient increase in the [Ca2+]i that was not significantly effected by the extracellular Ca2+ concentration. Treatment of cells with RAc caused a concentration- and time-dependent inhibition of this PDGF-stimulated Ca2+ flux (IC50 = 0.45 microgram/ml or 1.5 x 10(-6) M; t1/2 = 15 min), whereas release of intracellularly stored Ca2+ by thrombin was unaffected by RAc (1.2 micrograms/ml, 4 x 10(-6) M). Treatment with RAc did not significantly affect PDGF binding to cell surface receptors or the generation of inositol phosphates. These results suggest that the mechanism by which RAc inhibits PDGF- or serum-induced mitogenesis is through modulation of the Ca2+ signal stimulated by PDGF, and thereby depriving the cell of a rise in intracellular Ca2+ necessary for progression through the cell cycle.     

Platelet-derived growth factor-stimulated migration of murine fibroblasts is associated with epidermal growth factor receptor expression and tyrosine phosphorylation

Previous studies have shown that epidermal growth factor (EGF) synergizes with various extracellular matrix components in promoting the migration of B82L fibroblasts expressing wild-type EGF receptors and that functional EGF receptors are critical for the conversion of B82L fibroblasts to a migratory cell type (). In the present study, we examined the effects of platelet-derived growth factor (PDGF) on the motility of B82L fibroblasts using a microchemotaxis chamber. We found that PDGF can enhance fibronectin-induced migration of B82L fibroblasts expressing wild-type EGF receptors (B82L-clone B3). However, B82L cells that lack the EGF receptor (B82L-parental) or that express an EGF receptor that is kinase-inactive (B82L-K721M) or C-terminally truncated (B82L-c'973) exhibit little PDGF-stimulated migration. In addition, none of these three cell lines exhibit the capacity to migrate to fibronectin alone. These observations indicate that, similar to cell migration toward fibronectin, PDGF-induced cell migration of B82L fibroblasts is augmented by the expression of an intact EGF receptor kinase. The loss of PDGF-stimulated motility in B82L cells that do not express an intact EGF receptor does not appear to result from a gross dysfunction of PDGF receptors, because ligand-stimulated tyrosine phosphorylation of the PDGF-beta receptor and the activation of mitogen-activated protein kinases are readily detectable in these cells. Moreover, an interaction between EGF and PDGF receptor systems is supported by the observation that the EGF receptor exhibits an increase in phosphotyrosine content in a time-dependent fashion upon the addition of PDGF. Altogether, these studies demonstrate that the expression of EGF receptor is critical for PDGF-stimulated migration of murine B82L fibroblasts and suggest a role for the EGF receptor downstream of PDGF receptor activation in the signaling events that lead to PDGF-stimulated cell motility.     

Interferon-γ modulates lung macrophage production of PDGF-BB and fibroblast growth

Platelet-derived growth factor (PDGF) is a potent mediator of fibroblast proliferation and chemotaxis. We have studied here the cytokine interferon-γ (IFN-γ) which is known to prime macrophages for increased PDGF production. Thus, we postulated that IFN-γ would act as a positive regulator of PDGF-BB secretion by rat alveolar macrophages, and in addition we asked whether or not the IFN-γ (a known anti-mitogenic cytokine) would block the growth response of primary lung fibroblasts to the PDGF-BB. Macrophages incubated with IFN-γ or iron spheres alone for 24 h secreted 2.5-fold more PDGF-BB than control macrophages incubated in serum-free medium. Preincubation of macrophages with IFN-γ prior to the addition of iron spheres synergistically increased PDGF-BB production 2–10-fold after 24 h. In contrast, when IFN-γ was added to quiescent rat lung fibroblasts (RLFs) in the presence of PDGF-BB, the cytokine induced a concentration-dependent decrease in cell growth, while IFN-γ alone did not affect proliferation. [¹²⁵I]PDGF-BB receptor assays showed that neither preincubation nor coincubation of RLF with IFN-γ affected PDGF-BB binding to its receptors.     

Differential inhibition of transforming growth factor beta 1 and beta 2 activity by alpha 2-macroglobulin.

Affinity labeling and immunoprecipitation studies demonstrate that alpha 2-macroglobulin (alpha 2M) is the major serum-binding protein for transforming growth factors beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2). Purified alpha 2M inhibits the binding of both 125I-TGF-beta 1 and 125I-TGF-beta 2 to cell surface receptors at I50 values of 200 and 10 micrograms/ml, respectively. alpha 2M (200 micrograms/ml) does not block TGF-beta 1 inhibition of CCL-64 mink lung cell growth but reduces this activity of TGF-beta 2 10-fold. The electrophoretic migration of 125I-TGF-beta.alpha 2M complexes on polyacrylamide gels under nondenaturing conditions demonstrates that alpha 2M has 10-fold greater affinity for TGF-beta 2 than for TGF-beta 1. Each of these complexes comigrates as a single band with the fast form of alpha 2M. We suggest that alpha 2M is an important differential regulator of the biological activities of TGF-beta 1 and TGF-beta 2 in vivo.     

Inhibition of MG-63 cell proliferation and PDGF-stimulated cellular processes by inhibitors of phosphatidylinositol 3-kinase

Studies on a platelet-derived growth factor (PDGF) responsive osteosarcoma cell line, MG-63, were initiated to determine the effects of phosphatidylinositol (Ptdlns) 3-kinase inhibitors on serum-stimulated cell proliferation and PDGF-stimulated DNA replication, actin rearrangements, or Ptdlns 3-kinase activity. In a dose-dependent manner, the fungal metabolite wortmannin and a quercetin derivative, LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), inhibited serum-stimulated MG-63 cell proliferation. The mitogenic effects of PDGF on MG-63 cells, as determined by incorporation of [³H]-thymidine, were also substantially inhibited in the presence of 0.10 μM wortmannin or 10 μM LY294002. Furthermore, MG-63 cells stimulated by PDGF form distinct actin-rich, finger-like membrane projections which are completely inhibited by either 0.10 μM wortmannin or 10 μM LY294002. At these same concentrations, wortmannin and LY294002 were also effective at reducing levels of phosphatidylinositol 3-phosphate in PDGF-stimulated MG-63 cells. Treatment of these cells with increasing concentrations of wortmannin reduced the level of PDGF stimulated tyrosine phosphorylation of the PDGF receptor but did not significantly affect the amount of the Ptdlns 3-kinase regulatory subunit, p85, associated with the receptor. Additionally, pretreatment of cells with 0.250 μM wortmannin followed by stimulation with PDGF resulted in a slightly reduced level of receptor autokinase activity; however, similar treatment with 50 μM LY294002 did not affect the level of autokinase activity. These results demonstrate the effects of two different Ptdlns 3-kinase inhibitors on serum- and PDGF-stimulated MG-63 cell proliferation and PDGF-stimulated morphological changes and suggest a greater role for Ptdlns 3-kinase in these processes. J. Cell. Biochem. 64:182–195. © 1997 Wiley-Liss, Inc.     

Imatinib mesylate inhibits platelet derived growth factor stimulated proliferation of rheumatoid synovial fibroblasts

Synovial fibroblast is the key cell type in the growth of the pathological synovial tissue in arthritis. Here, we show that platelet-derived growth factor (PDGF) is a potent mitogen for synovial fibroblasts isolated from patients with rheumatoid arthritis. Inhibition of PDGF-receptor signalling by imatinib mesylate (1muM) completely abrogated the PDGF-stimulated proliferation and inhibited approximately 70% of serum-stimulated proliferation of synovial fibroblasts. Similar extent of inhibition was observed when PDGF was neutralized with anti-PDGF antibodies, suggesting that imatinib mesylate does not inhibit pathways other than those mediated by PDGF-receptors. No signs of apoptosis were detected in synovial fibroblasts cultured in the presence of imatinib. These results suggest that imatinib mesylate specifically inhibits PDGF-stimulated proliferation of synovial fibroblasts, and that inhibition of PDGF-receptors could represent a feasible target for novel antirheumatic therapies.     

Binding and internalization of 125I-alpha 2-macroglobulin by cultured fibroblasts

The binding and internalization of 125I-labeled alpha 2-macroglobulin (125I-alpha 2M) was studied in cultured fibroblasts. Two classes of binding sites were detected on cell surfaces. One class corresponds to the previously described, high affinity and low capacity sites. The other class of binding sites may mediate uptake of high physiological blood levels of 125I-alpha 2M and has not been described previously. At 0 degrees C, this lower affinity class saturates at approximately 1,000 micrograms/ml and has a capacity of approximately 600,000 sites/cell. The lower affinity class accounts for the vast majority of cellular receptors for alpha 2M. An assay employing pepsin at pH 4 was developed to distinguish between surface-bound and internalized 125I-alpha 2M. Cellular uptake of 125I-alpha 2M at 37 degrees C has a component which saturates between 200 and 1,000 micrograms/ml and the rate of internalization of this component was approximately 1,700,000 molecules/cell/h. One mM Ca2+ was required for cell uptake of 125I-alpha 2M at 37 degrees C. Ca2+ was also required for binding at 0 degrees C to both low and high affinity classes of binding sites for 125I-alpha 2M. The transglutaminase inhibitors bacitracin, monodansylcadaverine, and N-benzyloxycarboxyl-5-diazo-4-oxonorvaline paranitrophenyl ester all inhibited cellular internalization of 125I-alpha 2M at 37 degrees C. Each of these three compounds selectively reduced 125I-alpha 2M binding to the high affinity, low capacity component at 0 degrees C. Based on the current binding studies and previous studies using electron microscopy which showed that bacitracin and other transglutaminase inhibitors block clustering of alpha 2M-receptor complexes in coated pits, we suggest that the inhibitors block the accumulation of occupied lower affinity alpha 2M receptors in coated pits where they acquire a higher apparent affinity.     

Altered regulation of platelet-derived growth factor A-chain and c-fos gene expression in senescent progeria fibroblasts

The study of human genetic disorders known as premature aging syndromes may provide insight into the mechanisms of cellular senescence. These diseases are clinically characterized by the premature onset and accelerated progression of numerous features normally associated with human aging. Previous studies have indicated that fibroblasts derived from premature aging syndrome patients have in vitro growth properties similar to senescent fibroblasts from normal individuals. As an initial approach to determine whether gene expression is altered in premature aging syndrome fibroblasts, RNA was prepared from various cell strains and used for gel blot hybridization experiments. Although normal fibroblasts only express platelet-derived growth factor (PDGF) A-chain mRNA for a brief period following mitogenic stimulation, one strain of Hutchinson-Gilford (progeria) syndrome fibroblasts, AG3513, constitutively expresses PDGF A-chain mRNA and PDGF-AA homodimers. The PDGF A-chain gene does not appear to be amplified or rearranged in these fibroblasts. AG3513 progeria fibroblasts have properties characteristic of senescent cells, including an altered morphology and a diminished mitogenic response to growth promoters. The diminished response of AG3513 progeria fibroblasts to PDGF stimulation was examined in some detail. Studies using 125I-PDGF-BB, which binds with high affinity to both A- and B-type PDGF receptors, indicate that normal and AG3513 progeria fibroblasts have a similar number of PDGF receptors. Although receptor autophosphorylation occurs normally in PDGF-stimulated AG3513 progeria fibroblasts, c-fos mRNA induction does not. The senescent phenotype of AG3513 fibroblasts is probably unrelated to their constitutive PDGF A-chain gene expression; further studies are necessary in order to directly address this issue. Also, additional analysis of this progeria fibroblast strain may provide information on the control of mitogen-inducible gene expression in normal cells.     

Reversible binding of platelet-derived growth factor-AA, -AB, and -BB isoforms to a similar site on the "slow" and "fast" conformations of alpha 2-macroglobulin.

The mechanism by which the platelet-derived growth factor (PDGF)-binding protein, alpha 2-macroglobulin (alpha 2M), modulates PDGF bioactivity is unknown, but could involve reversible PDGF-alpha 2M binding. Herein we report that greater than 70% of 125I-PDGF-BB or -AB complexed to alpha 2M was dissociated by SDS-denaturation followed by SDS-polyacrylamide gel electrophoresis, i.e. most of the binding was noncovalent. Reduction of the PDGF.alpha 2M complex following denaturation dissociated the cytokine from alpha 2M by greater than 90%, suggesting covalent disulfide bond formation. Approximately 50% of the growth factor was dissociated by lowering the pH from 7.5 to 4.0. 125I-PDGF-BB bound alpha 2M in a time-dependent manner (t1/2 = approximately 1 h), reaching equilibrium after 4 h. The 125I-PDGF.BB/alpha 2M complex dissociated more slowly (t1/2 = approximately 2.5 h). "Slow" and "fast" alpha 2M bound nearly equal amounts of PDGF-AB or -BB. Trypsin treatment converted PDGF-BB/alpha 2M complex to the fast conformation but did not release bound 125I-PDGF-BB. All PDGF-isoforms (AA, -AB, and -BB) competed for binding with 125I-PDGF-BB binding to slow alpha 2M and fast alpha 2M-methylamine by 65-80%. Other cytokines that bind alpha 2M (transforming growth factor-beta 1 and -beta 2, tumor necrosis factor-alpha, basic fibroblast growth factor, interleukin -1 beta, and -6) did not compete for 125I-PDGF-BB binding slow alpha 2M, but transforming growth factor-beta 1 and basic fibroblast growth factor inhibited 125I-PDGF-BB binding alpha 2M-methylamine by 30-50%. The reversible nature of the PDGF.alpha 2M complex could allow for targeted PDGF release near mesenchymal cells which possess PDGF receptors.     

Interaction of lecithin:cholesterol acyltransferase (LCAT).alpha 2-macroglobulin complex with low density lipoprotein receptor-related protein (LRP). Evidence for an alpha 2-macroglobulin/LRP receptor-mediated system participating in LCAT clearance.

The reaction of lecithin:cholesterol acyltransferase (LCAT) with high density lipoproteins (HDL) is of critical importance in reverse cholesterol transport, but the structural and functional pathways involved in the regulation of LCAT have not been established. We present evidence for the direct binding of LCAT to alpha(2)-macroglobulin (alpha(2)M) in human plasma to form a complex 18.5 nm in diameter. Forty percent of plasma LCAT-HDL was associated with alpha(2)M; moreover, most of the LCAT in cerebrospinal fluid and in the medium of cultured human hepatoma cell line was associated with alpha(2)M. Purified recombinant human LCAT (rLCAT) labeled with (125)I bound to native and methylamine-activated alpha(2)M (alpha(2)M-MA) in vitro in a time- and concentration-dependent manner, and this binding did not depend on the presence of lipid. rLCAT bound to alpha(2)M-MA with greater affinity than to alpha(2)M. Furthermore, rLCAT did not activate alpha(2)M as phosphatidylcholine-specific phospholipase C does. Reconstituted HDL particles (LpA-I) inhibited the binding of rLCAT to alpha(2)M more efficiently than native HDL(3) did. LCAT associated with alpha(2)M was enzymatically inactive under both endogenous and exogenous assay conditions. Purified rLCAT alone did not bind to low density lipoprotein receptor-related protein (LRP) as lipoprotein lipase (LPL) does; however, when rLCAT was combined with alpha(2)M-MA to form a complex, binding, internalization, and degradation of rLCAT took place in LRP-expressing cells (LRP (+/+)) but not in cells deficient in LRP (LRP (-/-)). It is concluded that the binding of LCAT to alpha(2)M inhibits its enzymatic activity. Furthermore, the finding supports the possibility that the LRP receptor can act in vivo to mediate clearance of the LCAT-alpha(2)M complex and may significantly influence the bioavailability of LCAT.     

A chemically modified preparation of alpha2-macroglobulin binds beta-amyloid peptide with increased affinity and inhibits Abeta cytotoxicity

Macromolecules that bind beta-amyloid peptide (Abeta) and neutralize Abeta cytotoxicity offer a promising new approach for treating Alzheimer's disease. When the plasma protein, alpha2-macroglobulin (alpha2M), is treated with methylamine (alpha2M-MA), it undergoes conformational change and acquires Abeta-binding activity. In this study, we demonstrate that a chemically stabilized preparation of human alpha2M conformational intermediates (alpha2M-cis-Pt/MA) binds Abeta with greatly increased affinity, compared with alpha2M-MA. alpha2M-cis-Pt/MA was generated by reacting alpha2M with the protein cross-linking reagent, cis-Pt, followed by methylamine. Increased Abeta-binding to alpha2M-cis-Pt/MA was demonstrated by co-migration of radio-iodinated proteins in non-denaturing PAGE, chemical cross-linking, and co-immunoprecipitation. The apparent K(D) for Abeta-binding to alpha2M-cis-Pt/MA was decreased 10-fold, compared with alpha2M-MA, to 29 nm. Native alpha2M demonstrated negligible Abeta-binding, as anticipated. alpha2M-cis-Pt/MA markedly counteracted Abeta-induced C6 cell apoptosis. Essentially complete inhibition of apoptosis was observed even when the Abeta was present at fourfold molar excess to alpha2M-cis-Pt/MA. Under equivalent conditions, alpha2M-MA inhibited apoptosis by 25 +/- 6%. When Abeta and alpha2M-cis-Pt/MA were added to human plasma in vitro, significant binding was detected. No binding was observed when an equivalent concentration of native alpha2M or alpha2M-MA was added to plasma. We propose that alpha2M-cis-Pt/MA is a novel alternative to Abeta-specific antibodies, for studying the efficacy of Abeta-binding agents in vitro and in vivo.     

Kinetic analysis of platelet-derived growth factor receptor/phosphoinositide 3-kinase/Akt signaling in fibroblasts

Isoforms of the serine-threonine kinase Akt coordinate multiple cell survival pathways in response to stimuli such as platelet-derived growth factor (PDGF). Activation of Akt is a multistep process, which relies on the production of 3'-phosphorylated phosphoinositide (PI) lipids by PI 3-kinases. To quantitatively assess the kinetics of PDGF receptor/PI 3-kinase/Akt signaling in fibroblasts, a systematic study of this pathway was performed, and a mechanistic mathematical model that describes its operation was formulated. We find that PDGF receptor phosphorylation exhibits positive cooperativity with respect to PDGF concentration, and its kinetics are quantitatively consistent with a mechanism in which receptor dimerization is initially mediated by the association of two 1:1 PDGF/PDGF receptor complexes. Receptor phosphorylation is transient at high concentrations of PDGF, consistent with the loss of activated receptors upon endocytosis. By comparison, Akt activation responds to lower PDGF concentrations and exhibits more sustained kinetics. Further analysis and modeling suggest that the pathway is saturated at the level of PI 3-kinase activation, and that the p110alpha catalytic subunit of PI 3-kinase contributes most to PDGF-stimulated 3'-PI production. Thus, at high concentrations of PDGF the kinetics of 3'-PI production are limited by the turnover rate of these lipids, while the Akt response is additionally influenced by the rate of Akt deactivation.     

Interactions between the receptors for platelet-derived growth factor and epidermal growth factor

Preincubation of Swiss 3T3 cells or human fibroblasts with purified platelet-derived growth factor (PDGF) at 4 degrees C or 37 degrees C rapidly inhibits subsequent binding of 125I-epidermal growth factor (125I-EGF). The effect does not result from competition by PDGF for binding to the EGF receptor since (a) very low concentrations of PDGF are effective, (b) cells with EGF receptors but no PDGF receptors are not affected, and (c) the inhibition persists even if the bound PDGF is eluted before incubating the cells with 125I-EGF. PDGF does not affect 125I-insulin binding nor does EGF affect 125I-PDGF binding under these conditions. Endothelial cell-derived growth factor also competes for binding to PDGF receptors and inhibits 125I-EGF binding. The inhibition demonstrated by PDGF seems to result from an increase in the Kd for 125I-EGF binding with no change in the number of EGF receptors.     

Correlation between trypsin binding to a specific receptor and prostacyclin production in cultured vascular endothelial cells

The correlation between the binding and processing of trypsin and its effect on prostacyclin (PGI2) production in cultured adult bovine aortic endothelial (ABAE) cells was studied. ABAE cells demonstrated an ability to produce PGI2 in a dose-response manner to trypsin at the range of 0.1-2.0 micrograms/ml with a saturation at a concentration of 1 microgram/ml. Likewise, 125I-trypsin binding to the cultured cells increased in a dose-response way and reached saturation at a concentration of about 1 microgram/ml; 125I-trypsin was bound to a specific high-affinity cell-surface receptor with a dissociation constant (Kd) of 1.5 X 10(-8) M and each of the confluent ABAE cells has about 1.2 X 10(5) such receptors sites. The cell-surface receptor for trypsin displayed specific characteristics and an excess amount of unlabeled trypsin successfully abolished 125I-trypsin binding while thrombin in excess failed to compete for 125I-trypsin binding. Only a small fraction of the cell-surface-bound 125I-trypsin was internalized and subsequently degraded by ABAE cells as compared to the process of 125I-trypsin internalization by human skin fibroblasts (HSF). This study demonstrated that the stimulatory effect of trypsin on prostacyclin production and release by ABAE cells might be mediated by a specific cell-surface receptor for trypsin on these cells distinct from the thrombin receptor.     

Tyrosine phosphatase SHP-2 is involved in regulation of platelet-derived growth factor-induced migration.

SHP-2 is a ubiquitously expressed Src homology-2-containing cytosolic tyrosine phosphatase that binds to and becomes tyrosine-phosphorylated by the activated platelet-derived growth factor receptor-beta (PDGFR-beta). Removal of the binding site on the receptor, by mutation of Tyr1009 to Phe1009 (denoted Y1009F), led to loss of PDGF-stimulated phosphatase activity in cells expressing the mutated receptor, and these cells failed to form membrane edge ruffles and to migrate toward PDGF. Furthermore, treatment with phosphatase inhibitors phenylarsine oxide (PAO) and orthovanadate led to loss of PDGF-stimulated phosphatase activity and attenuated PDGF-stimulated migration of wild type PDGFR-beta cells. Treatment of wild type PDGFR-beta cells with combinations of PAO or orthovanadate and phosphatidylinositol 3-kinase inhibitors wortmannin or LY294002 resulted in a synergistic inhibition of PDGFR-beta-mediated cell migration. PDGF stimulation of wild type PDGFR-beta cells led to induction of p125 focal adhesion kinase (FAK) activity at low concentrations of the growth factor and a decrease at higher concentrations. In the mutant Y1009F cells and in wild type PDGFR-beta cells treated with PAO and orthovanadate, FAK activity was not increased in response to PDGF. These results suggest that SHP-2 activity is involved in regulation of FAK activity and thereby of cell migration through PDGFR-beta, independently of phosphatidylinositol 3-kinase.     

Regulation of transferrin receptor expression at the cell surface by insulin-like growth factors, epidermal growth factor and platelet-derived growth factor.

Addition of platelet-derived growth factor (PDGF), recombinant insulin-like growth factor I (rIGF-I) or epidermal growth factor (EGF) to BALB/c 3T3 fibroblasts causes a marked increase in the binding of [125I]diferric transferrin to cell surface receptors. This effect is very rapid and is complete within 5 min. The effect of EGF is transient, with [125I]diferric transferrin binding returning to control values within 25 min. In contrast, PDGF and rIGF-I cause a prolonged stimulation of [125I]diferric transferrin binding that could be observed for up to 2 h. The increase in the binding of [125I]diferric transferrin caused by growth factors was investigated by analysis of the binding isotherm. Epidermal growth factor, PDGF and rIGF-I were found to increase the cell surface expression of transferrin receptors rather than to alter the affinity of the transferrin receptors. This result was confirmed in human fibroblasts by the demonstration that EGF, PDGF and rIGF-I could stimulate the binding of a monoclonal antibody directed against the transferrin receptor (OKT9) to the cell surface. Furthermore, PDGF and rIGF-I stimulated the sustained uptake of [59Fe]diferric transferrin by BALB/c 3T3 fibroblasts, while EGF transiently increased uptake. Thus the effect of these growth factors to increase the cell surface expression of the transferrin receptor appears to have an important physiological consequence.     

Eicosapentaenoic acid inhibits PDGF-induced mitogenesis and cyclin D1 expression via TGF-beta in mesangial cells

Eicosapentaenoic acid (EPA), an omega-3 polyunsaturated fatty acid derived from fish oil, is efficacious in glomerular diseases where mesangial proliferation is a key event. We examined the mechanisms of action of EPA on platelet-derived growth factor (PDGF)-stimulated rat mesangial cell mitogenesis. EPA dose-dependently inhibited PDGF-stimulated [(3)H]-thymidine incorporation. PDGF-induced PDGF receptor autophosphorylation, an initial event for PDGF signaling, was not affected by 2 micro g/ml EPA. Similarly, PDGF-stimulated activation of extracellular signal-regulated kinase (ERK) was not altered. On the other hand, EPA inhibited cyclin-dependent kinase 4 (CDK4) activation and cyclin D1 protein induction, a critical step for G1/S progression. TGF-beta secretion assessed by ELISA and bioassay was increased by EPA at 18 h. Coincubation with anti-TGF-beta antibody inhibited the EPA-induced suppression of [(3)H]-thymidine incorporation and cyclin D1 expression. SB203580, an inhibitor of p38, a downstream kinase of TGF-beta, did not affect EPA's growth inhibitory effect. These results demonstrate that EPA inhibits PDGF-stimulated mesangial cell mitogenesis and cyclin D1 expression via TGF-beta.