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PubMed1996

Ca2+ channel blockers modulate metabolism of collagens within the extracellular matrix.

The extracellular matrix (ECM) is an intricate network composed of an array of macromolecules capable of regulating the functional responsiveness of cells. Its composition greatly varies among different types of tissue, and dysregulation of its metabolism may contribute to vascular remodeling during the pathogenesis of various diseases, including atherosclerosis. In view of their antiatherosclerotic effects, the role of Ca2+ channel blockers in the metabolism of ECM was examined. Nanomolar concentrations of the five Ca2+ channel blockers amlodipine, felodipine, manidipine, verapamil, or diltiazem significantly decreased both the constitutive and platelet-derived growth factor BB-dependent collagen deposition in the ECM formed by human vascular smooth muscle cells and fibroblasts. The drugs inhibited the expression of fibrillar collagens type I and III and of basement membrane type IV collagen. Furthermore, Ca2+ channel blockers specifically increased the proteolytic activity of the 72-kDa type IV collagenase as shown by gelatin zymography and inhibited the transcription of tissue inhibitor of metalloproteinases-2.

Amlodipine↗
PubMed1996

Separation of the arterial wall from blood contact using hydrogel barriers reduces intimal thickening after balloon injury in the rat: the roles of medial and luminal factors in arterial healing.

The objective of this study was to clarify the relative roles of medial versus luminal factors in the induction of thickening of the arterial intima after balloon angioplasty injury. Platelet-derived growth factor (PDGF) and thrombin, both associated with thrombosis, and basic fibroblast growth factor (bFGF), stored in the arterial wall, have been implicated in this process. To unequivocally isolate the media from luminally derived factors, we used a 20-microns thick hydrogel barrier that adhered firmly to the arterial wall to block thrombus deposition after balloon-induced injury of the carotid artery of the rat. Thrombosis, bFGF mobilization, medial repopulation, and intimal thickening were measured. Blockade of postinjury arterial contact with blood prevented thrombosis and dramatically inhibited both intimal thickening and endogenous bFGF mobilization. By blocking blood contact on the two time scales of thrombosis and of intimal thickening, and by using local protein release to probe, by reconstitution, the individual roles of PDGF-BB and thrombin, we were able to conclude that a luminally derived factor other than PDGF or thrombin is required for the initiation of cellular events leading to intimal thickening after balloon injury in the rat. We further conclude that a luminally derived factor is required for mobilization of medial bFGF.

Angioplasty, Balloon↗
PubMed1998

Tumorigenic conversion of immortal human keratinocytes through stromal cell activation.

The stromal microenvironment plays a crucial role in tumor development and progression. One of the most potent activators of stromal cells is the platelet-derived growth factor (PDGF). To investigate the role of PDGF in epithelial tumor development we stably transfected immortal nontumorigenic human keratinocytes with the PDGF-B cDNA. Transfected HaCaT cells overexpressed PDGF-B but remained negative for the PDGF receptors alpha and beta (mRNA). Thus, they did not exhibit autocrine growth stimulation in vitro, but proliferation of cocultured fibroblasts was enhanced and this effect was inhibited by a neutralizing antibody to PDGF-BB. After subcutaneous injection into nude mice the transfected cells maintained high PDGF expression and formed progressively enlarging, rapidly proliferating cysts, classified as benign tumors. During early tumor development (up to 2 months), PDGF-B transfectants induced marked mesenchymal cell proliferation and angiogenesis, yet this effect vanished at later stages (2-6 months) concomitantly with increased epithelial cell proliferation and enhanced tumor growth. These results demonstrate that an activated stromal environment can promote tumorigenic conversion of nontumorigenic keratinocytes by inducing sustained epithelial hyperproliferation. This effect is apparently caused by a dual action of PDGF-BB: (i) PDGF-BB can promote tumor growth by inducing angiogenesis and stroma formation, and (ii) PDGF-activated stromal cells maintain elevated keratinocyte proliferation via a paracrine mechanism. Thus, PDGF, a major factor activated in wound healing, may play an important role as an endogenous promoter in epithelial tumor formation.

Animals↗
PubMed1999

Platelet-derived growth factor beta receptor regulates interstitial fluid homeostasis through phosphatidylinositol-3' kinase signaling.

Platelet-derived growth factor (PDGF) isoforms lead to mitogenic, survival, and chemotactic responses in a variety of mesenchymal cell types during development and in the adult. We have studied the importance of phosphatidylinositol-3' kinase (PI3K) signaling in these responses by mutating the PI3K-binding sites in the PDGF-beta receptor by gene targeting in embryonic stem cells. Homozygous mutant mice developed normally; however, cells derived from the mutants were less chemotactic and had largely lost their ability to contract collagen gels in response to PDGF. Injection of a mast cell degranulating agent in mice led to a decrease in interstitial fluid pressure resulting in edema formation. In contrast to wild-type mice, mutant mice were unable to normalize the pressure after treatment with PDGF. Taken together, these observations suggest a function for PDGF signaling through PI3K in interstitial fluid homeostasis by modulating the tension between cells and extracellular matrix structures.

3T3 Cells↗
PubMed1995

Inhibition of platelet-derived growth factor-BB-induced fibroblast proliferation by plasmin-activated alpha 2-macroglobulin is mediated via an alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein-dependent mechanism.

alpha 2-Macroglobulin (alpha 2M) is a potentially important regulator of platelet-derived growth factor-BB (PDGF-BB)-stimulated cell growth due to our previous observation that PDGF-BB binds to alpha 2M noncovalently (Bonner, J. C., Goodell, A. L., Lasky, J. A., and Hoffman, M. R. (1992) J. Biol. Chem. 267, 12837-12844). We examined the in vitro effect of native and plasmin-activated (receptor-recognized) alpha 2M on the PDGF-BB-induced proliferation of mouse Swiss 3T3 and rat lung fibroblasts. Nondenaturing polyacrylamide gel electrophoresis showed that plasmin converted alpha 2M to its electrophoretically "fast" form at a 2:1 molar ratio and that 125I-PDGF-BB bound both alpha 2M and alpha 2M-plasmin. PDGF-BB-induced growth was not affected by native alpha 2M (0.3 microM) or plasmin (0.6 microM). The combination of plasmin and alpha 2M (2:1 molar ratio) inhibited PDGF-BB-induced cell proliferation 80-90%. Complexes of PDGF-BB.alpha 2M purified by gel filtration chromatography retained growth promoting activity, but the PDGF-BB.alpha 2M-plasmin complex did not. Preincubation of fibroblasts (37 degrees C for 24 h) with alpha 2M-plasmin did not change 125I-PDGF-BB binding or affect gene expression of the 6.5-kilobase PDGF-alpha receptor or 5.2-kilobase PDGF-beta receptor mRNA. However, preincubation with alpha 2M-plasmin (0-4 degrees C for 4 h) increased 125I-PDGF-BB binding 2-fold, and this increase was blocked by a receptor-associated protein antagonist of the alpha 2M-receptor/low density lipoprotein receptor-related protein. The receptor-associated protein antagonist blocked 125I-alpha 2M-methylamine binding, inhibited PDGF-BB-alpha 2M-plasmin uptake from fibroblast-cultured supernatants, and abolished the inhibitory effect of alpha 2M-plasmin on PDGF-stimulated growth. These data suggest that inhibition of PDGF-stimulated proliferation by alpha 2M-plasmin is mediated in part by clearance of PDGF-BB-alpha 2M-plasmin through the lipoprotein receptor-related protein.

Animals↗
PubMed1995

Demonstration of functionally different interactions between phospholipase C-gamma and the two types of platelet-derived growth factor receptors.

Phosphorylated tyrosine residues in receptor tyrosine kinases serve as binding sites for signal transduction molecules. We have identified two autophosphorylation sites, Tyr-988 and Tyr-1018, in the platelet-derived growth factor (PDGF) alpha-receptor carboxyl-terminal tail, which are involved in binding of phospholipase C-gamma (PLC-gamma). The capacities of the Y988F and Y1018F mutant PDGF alpha-receptors, expressed in porcine aortic endothelial cells, to bind PLC-gamma are 60 and 5% of that of the wild-type receptor, respectively. Phosphorylated but not unphosphorylated peptides containing Tyr-1018 are able to compete with the intact receptor for binding to immobilized PLC-gamma SH2 domains; a phosphorylated Tyr-988 peptide competes 10 times less efficiently. The complex between PLC-gamma and the PDGF alpha-receptor is more stable than that of PLC-gamma and the PDGF beta-receptor. However, PDGF stimulation results in a smaller fraction of tyrosine-phosphorylated PLC-gamma and a smaller accumulation of inositol trisphosphate in cells expressing the alpha-receptor as compared with cells expressing the beta-receptor. We conclude that phosphorylated Tyr-988 and Tyr-1018 in the PDGF alpha-receptor carboxyl-terminal tail bind PLC-gamma, but this association leads to only a relatively low level of tyrosine phosphorylation and activation of PLC-gamma.

Amino Acid Sequence↗
PubMed1995

Growth factor-induced phosphorylation and activation of aortic smooth muscle Na+/Ca2+ exchanger.

Although the Na+/Ca2+ exchanger is one of the major Ca2+ extrusion systems in excitable tissues, little is known about its regulation via protein phosphorylation. We now present evidence that the Na+/Ca2+ exchanger is phosphorylated in quiescent and growth factor-stimulated cultured aortic smooth muscle cells. The Na+/Ca2+ exchanger was isolated from 32P-labeled cells by immunoprecipitation with a specific polyclonal antibody. Phosphorylation of the exchanger was increased by up to 1.7-fold in response to platelet-derived growth factor-BB (PDGF-BB), alpha-thrombin, or phorbol 12-myristate 13-acetate (PMA). However, angiotensin II did not enhance the phosphorylation significantly. The extent of phosphorylation appeared to correlate with the growth factor-induced increase in cell 1,2-diacylglycerol. At least four phosphopeptides (P1 to P4) were detected by tryptic phosphopeptide map analysis of the phosphorylated exchanger, suggesting that phosphorylation occurred at multiple sites. PDGF-BB and PMA increased phosphorylation of the same phosphopeptides (in particular P1). Phosphorylated amino acids were exclusively serine residues in both quiescent and stimulated cells. We found that growth factors enhanced Na+/Ca2+ exchange activity and that there was a good correlation between the growth factor-induced stimulations of phosphorylation and exchange activity. PDGF-BB-induced activation of the exchanger was abolished by prior long treatment of cells with PMA. These results suggest that the Na+/Ca2+ exchanger is activated by protein kinase C-dependent phosphorylation in response to growth factors in vascular smooth muscle cells.

Animals↗
PubMed1995

Differential effects of platelet-derived growth factor BB on p125 focal adhesion kinase and paxillin tyrosine phosphorylation and on cell migration in rabbit aortic vascular smooth muscle cells and Swiss 3T3 fibroblasts.

In rabbit aortic vascular smooth muscle cells (VSMC) platelet-derived growth factor BB (PDGF-BB) stimulated the tyrosine phosphorylation of phospholipase C-gamma, p120 GTPase-activating protein, and the p85 alpha subunit of phosphatidylinositol 3'-kinase only at high concentrations (5-25 ng/ml). In contrast, PDGF-BB induced a rapid and concentration-dependent increase in p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation, which was half-maximal and maximum at 1 and 2.5 ng/ml, respectively. Saliently, stimulation of p125FAK tyrosine phosphorylation was sustained at up to 100 ng/ml PDGF-BB and for prolonged times of treatment. With similar concentration dependence, PDGF-BB stimulated the tyrosine phosphorylation of the 68-kDa focal adhesion-associated protein, paxillin. PDGF-BB also induced p125FAK and paxillin tyrosine phosphorylation in human aortic VSMC. PDGF-BB caused no detectable disruption of the actin cytoskeleton in VSMC. PDGF-BB stimulated rabbit VSMC migration with a very similar concentration dependence to that for p125FAK and paxillin tyrosine phosphorylation. PDGF-BB was equally effective in stimulating p125FAK and paxillin tyrosine phosphorylation under conditions similar to those used for cell migration. In Swiss 3T3 fibroblasts, PDGF-BB and -AA stimulated p125FAK tyrosine phosphorylation and cell migration only at low concentrations, and stimulation was abolished at 10-25 ng/ml. PDGF-AA failed to stimulate tyrosine phosphorylation, mitogenesis, and chemotaxis in rabbit VSMC, and immunoblot analysis showed that rabbit VSMC expressed PDGF beta-receptors but no alpha-receptors. These results implicate p125FAK in the chemotactic response to PDGF-BB and suggest that the ability of PDGF-BB to trigger the p125FAK pathway may be dependent both upon cell type and receptor isotype expression.

3T3 Cells↗
PubMed1995

Sphingosine 1-phosphate rapidly activates the mitogen-activated protein kinase pathway by a G protein-dependent mechanism.

Addition of sphingosine 1-phosphate induces proliferation of quiescent Swiss 3T3 fibroblasts by unknown mechanisms. To identify the pathways involved, the ability of sphingosine 1-phosphate to activate mitogen-activated protein (MAP) kinase was studied. Sphingosine 1-phosphate rapidly activated the Raf/MAP kinase kinase (MKK)/MAP kinase pathway, and the concentration dependence for MAP kinase activation correlated with that for induction of DNA synthesis. Both MKK1 and MKK2 were activated by sphingosine 1-phosphate, assessed by specific immune complex kinase assays. Prior treatment of the Swiss 3T3 cells with pertussis toxin inhibited 70-80% of the sphingosine 1-phosphate-stimulated MAP kinase activity. Thus, one of the direct or indirect targets of exogenous sphingosine 1-phosphate appears to be a G(i)/G(o) protein.

3T3 Cells↗
PubMed1995

Differential binding and regulation of platelet-derived growth factor A and B chain isoforms by alpha 2-macroglobulin.

Alpha 2-Macroglobulin (alpha 2M) is a multifunctional secreted glycoprotein that serves as a ubiquitous proteinase inhibitor and as a binding protein for platelet-derived growth factor (PDGF) BB and homologues of PDGF-BB secreted in culture by macrophages. The interaction of alpha 2M with PDGF-A chain molecules has not been addressed. This is a potentially important issue because fibroblasts and smooth muscle cells produce PDGF-AA, whereas macrophages produce mainly PDGF-BB. Recombinant human 125I-PDGF-B chain molecules (AB and BB) bound to plasma-derived, native human, or bovine alpha 2M and trypsin-activated alpha 2M on Superose 6 fast protein liquid chromatography gel filtration and on nondenaturing polyacrylamide gel electrophoresis, whereas 125I-PDGF-AA did not. Similar results were obtained with 125I-PDGF isoforms binding to immobilized bovine alpha 2M and alpha 2M-methylamine. The same differential pattern of unlabeled PDGF isoforms binding to alpha 2M was observed by Western blotting of PDGF. Human lung fibroblasts secreted alpha 2M as measured by Western blotting, and fibroblast-derived alpha 2M possessed the same differential binding pattern for PDGF isoforms as did plasma-derived alpha 2M. The specific binding of PDGF-AB and -BB to these fibroblasts was inhibited by native bovine alpha 2M, although PDGF-AA binding was not affected. Native alpha 2M preferentially blocked fibroblast chemotaxis to the PDGF-B chain dimers. These data suggest that only PDGF-B chain dimers, such as those produced by macrophages or released from platelets, are regulated by alpha 2M and that PDGF-AA produced by fibroblasts and smooth muscle cells is not controlled by this cytokine-binding protein.

Animals↗
PubMed1995

Synergistic effects of growth factors on the regulation of smooth muscle cell scavenger receptor activity.

Rabbit smooth muscle cells (SMC) express types I and II scavenger receptors (ScR) that are up-regulated by platelet secretion products. In the current studies we investigated the effect of growth factors secreted by platelets on ScR activity in rabbit and human SMC. Platelet-derived growth factor (PDGF BB) and transforming growth factor beta 1 (TGF-beta 1) at 10 ng/ml increased ScR activity in rabbit SMC (by approximately 4- and 2-fold, respectively) but not in human SMC. Epidermal growth factor (EGF) or insulin-like growth factor I (IGF-I) alone had little effect on SMC ScR activity. The growth factors had synergistic effects on ScR activity and on types I and II ScR mRNA expression. In rabbit SMC, PDGF BB, EGF, and TGF-beta 1 together stimulated ScR activity 12-fold. In human SMC, EGF and TGF-beta 1, together with either IGF-I or PDGF BB, stimulated receptor activity approximately 7-fold. Growth factor-mediated induction of ScR activity in rabbit and human SMC was blocked by the tyrosine kinase inhibitor tyrphostin 47, whereas the induction of ScR activity in rabbit but not human SMC was blocked by the protein kinase C inhibitor MDL.29,152. Studies using neutralizing antibodies demonstrated that TGF-beta 1 is the predominant factor in in vitro preparations of platelet secretory products which regulates ScR activity. The growth factors that act synergistically in regulating ScR activity in vitro are all present in atherosclerotic lesions, where they are produced by macrophages, endothelial cells, SMC, and platelets. The data suggest that these growth factors may regulate ScR activity in SMC in vivo and contribute to foam cell formation.

Animals↗
PubMed1995

Structural role of extracellular domain 1 of alpha-platelet-derived growth factor (PDGF) receptor for PDGF-AA and PDGF-BB binding.

The purpose of this study was to bacterially express, purify, and refold combinations of the extracellular immunoglobulin (Ig)-like domains (2-3, 1-3, and 1-5) of the human alpha-platelet-derived growth factor receptor (alpha PDGFR) to characterize molecular interactions with its ligand, platelet-derived growth factor (PDGF). The far UV circular dichroism spectroscopy of the alpha-PDGFR extracellular domains (ECDs) revealed a predominantly beta-sheet protein, with a structure consistent with folded Ig-like domains. The addition of PDGF-BB to these ECD types changed the conformation of all three types with a decrease in mean residue ellipticity in the following rank order: 1-5 = 1-3 > 2-3. In striking contrast, addition of PDGF-AA to these ECD types markedly changed the conformation of ECD 2-3, by an increased mean residue ellipticity but no changes were observed for ECDs 1-3 and 1-5. PDGF-AA bound to the immobilized ECD types 2-3, 1-3, and 1-5 at concentrations of 20, 11, and 7.5 nM, respectively. In contrast, PDGF-BB bound the ECD types 2-3, 1-3, and 1-5 at concentrations of 3, 3, and 2.2 nM, respectively. Scatchard analysis of binding studies using labeled ECDs indicated that PDGF-BB bound ECD 1-3 and ECD 2-3 with KD values of 74 and 72 nM, respectively. While, PDGF-AA bound ECD 1-3 and ECD 2-3 with KD values of 33 and 87 nM, respectively. Therefore, our results indicated that the loss of ECD 1 impaired the binding affinity of alpha PDGFR ECD 1-3 toward PDGF-AA without having a similar effect on PDGF-BB binding. Together all of our data suggest that ECD 1 is differentially required for proper orientation of PDGF-AA but not PDGF-BB binding determinant within ECDs 2 and 3.

Becaplermin↗
PubMed1995

Cell type-specific modulation of cell growth by transforming growth factor beta 1 does not correlate with mitogen-activated protein kinase activation.

Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional cytokine that positively or negatively regulates the proliferation of various types of cells. In this study we have examined whether or not the activation of the mitogen-activated protein (MAP) kinases is involved in the transduction of cell growth modulation signals of TGF-beta 1, as MAP kinase activity is known to be closely associated with cell cycle progression. Although TGF-beta 1 stimulated the growth of quiescent Balb 3T3 and Swiss 3T3 cells, it failed to detectably stimulate the tyrosine phosphorylation and activation of the 41- and 43-kDa MAP kinases at any time point up to the reinitiation of DNA replication. TGF-beta 1 also failed to stimulate the expression of the c-fos gene. Furthermore, TGF-beta 1 synergistically enhanced the mitogenic action of epidermal growth factor (EGF) without affecting EGF-induced MAP kinase activation in these fibroblasts, and it inhibited the EGF-stimulated proliferation of mouse keratinocytes (PAM212) without inhibiting EGF-induced MAP kinase activation. Thus, the ability of TGF-beta 1 to modulate cell proliferation is apparently not associated with the activation of MAP kinases. In this respect, TGF-beta 1 is clearly distinct from the majority, if not all, of peptide growth factors, such as platelet-derived growth factor and EGF, whose ability to modulate cell proliferation is closely associated with the activation of MAP kinases. These results also suggest that the activation of MAP kinases is not an absolute requirement for growth factor-stimulated mitogenesis.

3T3 Cells↗
PubMed1996

Sphingosine and sphingosine 1-phosphate differentially modulate platelet-derived growth factor-BB-induced Ca2+ signaling in transformed oligodendrocytes.

The roles of sphingosine and sphingosine 1-phosphate in Ca2+ signaling following platelet-derived growth factor (PDGF) receptor stimulation were investigated in the oligodendrocyte cell line CEINGE cl3, using single-cell fura-2 microfluorimetry and videoimaging. Two different Ca2+ responses were observed, which differed in their delays and kinetics. The first response, which occurred after a shorter delay, exhibited a single Ca2+ peak often followed by a plateau, while the second type of response was characterized by a longer delay and by Ca2+ spikes with different frequencies and amplitudes. The latter phenomenon was never observed after stimulation of G protein-coupled receptors for ATP, ET-1, and BK. The incubation with the inhibitor of sphingosine kinase, DL-threo-dihydrosphingosine, significantly increased the percentage of cells responding to PDGF-BB exposure with Ca2+ spikes (87 versus 47%), while it did not modify the Ca2+ response elicited by exposure to ATP, ET-1, or BK. Exposure to exogenous 10 microM sphingosine or 1 microM sphingosine 1-phosphate produced oscillatory and non-oscillatory Ca2+ responses, respectively, similar to those elicited by PDGF-BB. A second application of PDGF-BB, 30 min after the first, was normally ineffective in producing a Ca2+ response. However, if the second exposure was preceded by the inhibition of sphingosine 1-phosphate formation, an oscillatory Ca2+ response occurred in all cells. We conclude that intracellular levels of sphingosine and sphingosine 1-phosphate may differentially modulate Ca2+ signaling triggered by PDGF receptor stimulation in CEINGE cl3-transformed oligodendrocytes.

Becaplermin↗
PubMed1996

The HIV-1 nef protein interferes with phosphatidylinositol 3-kinase activation 1.

nef is a human immunodeficiency virus (HIV) gene encoding a 27-kDa myristoylated protein with structural features of a signal transducing molecule, but whose functions are largely unknown. We studied the interactions of Nef with the signal transduction pathways triggered by the platelet-derived growth factor (PDGF) receptor. The association of phosphatidylinositol (PI) 3-kinase with the activated receptor was severely impaired by nef expression. Conversely, PDGF-induced receptor tyrosine phosphorylation, binding to phospholipase C-gamma and to Ras-GAP were not modified. Microtubule-associated protein kinase activation and intracellular calcium influx in response to PDGF were either unaffected or only slightly enhanced. Nef significantly reduced the proliferative response to the growth factor, while the chemotactic response was unchanged. These data show that Nef affects selectively the PI 3-kinase signaling pathway and suggest that this interference results in some of the HIV adverse effects on host cell functions.

3T3 Cells↗
PubMed1996

Molecular cloning and characterization of SmLIM, a developmentally regulated LIM protein preferentially expressed in aortic smooth muscle cells.

Differentiated, quiescent vascular smooth muscle cells assume a dedifferentiated, proliferative phenotype in response to injury, one of the hallmarks of arteriosclerosis. Members of the LIM family of zinc-finger proteins are important in the differentiation of various cells including striated muscle. We describe here the molecular cloning and characterization of a developmentally regulated smooth muscle LIM protein, SmLIM, that is expressed preferentially in the rat aorta. This 194-amino acid protein has two LIM domains, and comparisons of rat SmLIM with its mouse and human homologues reveal high levels of amino acid sequence conservation (100 and 99%, respectively). SmLIM is a nuclear protein and maps to human chromosome 3. SmLIM mRNA expression was high in aorta but not in striated muscle and low in other smooth muscle tissues such as intestine and uterus. In contrast with arterial tissue, SmLIM mRNA was barely detectable in venous tissue. The presence of SmLIM expression within aortic smooth muscle cells was confirmed by in situ hybridization. In vitro, SmLIM mRNA levels decreased by 80% in response to platelet-derived growth factor-BB in rat aortic smooth muscle cells. In vivo, SmLIM mRNA decreased by 60% in response to vessel wall injury during periods of maximal smooth muscle cell proliferation. The down-regulation of SmLIM by phenotypic change in vascular smooth muscle cells suggests that it may be involved in their growth and differentiation.

Amino Acid Sequence↗
PubMed1996

Identification of a domain within the carboxyl-terminal region of the beta platelet-derived growth factor (PDGF) receptor that mediates the high transforming activity of PDGF.

We have reported previously that a chimeric platelet-derived growth factor receptor (PDGFR) possessing the ligand binding domain of the alpha PDGFR and the intracellular domain of the beta PDGFR (alpha 340 beta 342 R) was markedly more efficient than the wild type alpha PDGFR (alpha RWT) in its ability to enhance PDGF-A transforming activity in NIH/3T3 fibroblasts. To determine the region within the cytoplasmic domain of beta PDGFR that confers this higher transforming activity, we generated several additional alpha/beta PDGFR chimerae. When a chimeric PDGFR possessing the first 933 amino-terminal amino acids from the alpha PDGFR and the final 165 amino acids from the carboxyl-terminal of the beta PDGFR (alpha 933 beta 942 R) was cotransfected with the PDGF-A gene into NIH/3T3 cells, it showed a similar high efficiency to enhance PDGF-A chain transforming activity as alpha 340 beta 342 R. However, when chimeric PDGFRs in which either the kinase insert domain (alpha beta RKI) or the last 79 amino acids from the carboxyl-terminal end of the beta PDGFR (alpha 1024 beta 1028 R) were substituted into alpha PDGFR sequences were cotransfected with PDGF-A, they showed similar low efficiencies in enhancing transforming activity as the alpha RWT. These results predicted that the 86 amino acids following the tyrosine kinase 2 domain of beta PDGFR (amino acid residues 942-1027) were responsible for the higher transforming activity of beta PDGFR. To confirm this finding, we next constructed a chimera in which amino acid residues 942-1028 of the beta PDGFR (alpha beta 942-1028R) were substituted for those in the alpha PDGFR. Cotransfection experiments indicated that alpha beta 942-1028R increased transforming activity of PDGF-A to similar extent as the alpha 933 beta 942R, or alpha 340 beta 342R. Therefore, our findings define a critical domain within the noncatalytic region of beta PDGFR intracellular domain that confers the higher focus forming activity mediated by the beta PDGFR.

3T3 Cells↗
PubMed1996

Differential modulation of G1 cyclins and the Cdk inhibitor p27kip1 by platelet-derived growth factor and plasma factors in density-arrested fibroblasts.

Stimulation of quiescent Balb/c 3T3 fibroblasts into S phase requires the synergistic action of platelet-derived growth factor (PDGF) and progression factors found in platelet-poor plasma (PPP). Traverse of the G1/S phase boundary and the initiation of DNA replication require functional cyclin E-cyclin-dependent kinase (Cdk) 2 and cyclin A-Cdk2 complexes; however, the mechanisms by which PDGF and PPP regulate Cdk2 activation are not known. Density-arrested fibroblasts contain low levels of cyclins E and A, and high levels of the Cdk inhibitor p27kip1. Exposure of PDGF, which stimulates cell cycle entry but not progression through G1, induces the formation of cyclin D1-Cdk4 complexes that bind p27kip1 and titrate the pool of Kip1 available to inhibit Cdk2. In addition, PDGF stimulates a moderate transient reduction in the abundance of p27kip1 protein. However, limited expression of cyclin E and cyclin A is observed after PDGF treatment, and in the absence of PPP, p27 levels are sufficient to bind and inactivate existing cyclin-Cdk complexes. Although plasma does not significantly increase the proportion of Kip1 bound to cyclin D1-Cdk4, stimulation of PDGF-treated cells with plasma does overcome the threshold inhibition of p27kip1 by further increasing the expression of cyclins E and A and decreasing the amount of Kip1 over a prolonged time period. Our results indicate that the distinct mitogenic activities of PDGF and PPP differentially influence the activation of cyclin E- and cyclin A-associated kinases that ultimately regulate entry into S phase.

1-Methyl-3-isobutylxanthine↗