TEL/PDGFbetaR induces hematologic malignancies in mice that respond to a specific tyrosine kinase inhibitor.
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The TEL/PDGFbetaR fusion protein is expressed as the consequence of a recurring t(5;12) translocation associated with chronic myelomonocytic leukemia (CMML). Unlike other activated protein tyrosine kinases associated with hematopoietic malignancies, TEL/PDGFbetaR is invariably associated with a myeloid leukemia phenotype in humans. To test the transforming properties of TEL/PDGFbetaR in vivo, and to analyze the basis for myeloid lineage specificity in humans, we constructed transgenic mice with TEL/PDGFbetaR expression driven by a lymphoid-specific immunoglobulin enhancer-promoter cassette. These mice developed lymphoblastic lymphomas of both T and B lineage, demonstrating that TEL/PDGFbetaR is a transforming protein in vivo, and that the transforming ability of this fusion is not inherently restricted to the myeloid lineage. Treatment of TEL/PDGFbetaR transgenic animals with a protein tyrosine kinase inhibitor with in vitro activity against PDGFbetaR (CGP57148) resulted in suppression of disease and a prolongation of survival. A therapeutic benefit was apparent both in animals treated before the development of overt clonal disease and in animals transplanted with clonal tumor cells. These results suggest that small-molecule tyrosine kinase inhibitors may be effective treatment for activated tyrosine kinase-mediated malignancies both early in the course of disease and after the development of additional transforming mutations. (+info)
The bovine papillomavirus E5 protein requires a juxtamembrane negative charge for activation of the platelet-derived growth factor beta receptor and transformation of C127 cells.
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The bovine papillomavirus E5 gene encodes a 44-amino-acid, homodimeric transmembrane protein that is the smallest known transforming protein. The E5 protein transforms cultured fibroblasts by forming a stable complex with the endogenous platelet-derived growth factor (PDGF) beta receptor through transmembrane and juxtamembrane interactions, leading to sustained receptor activation. Aspartic acid 33 in the extracellular juxtamembrane region of the E5 protein is important for cell transformation and interaction with the PDGF beta receptor. A. N. Meyer et al. (Proc. Natl. Acad. Sci USA 91:4634-4638, 1994) speculated that this residue interacted with lysine 499 on the receptor. We constructed E5 mutants containing all possible substitutions at position 33, as well as several double mutants containing substitutions at aspartic acid 33 and at glutamic acid 36, and we examined the ability of these mutants to transform C127 mouse fibroblasts and to bind to and induce activation of the PDGF beta receptor. There was an excellent correlation between the transformation activities of the various mutants and their ability to bind to and activate the PDGF beta receptor. Analysis of the mutants demonstrated that a juxtamembrane negative charge on the E5 protein was required for cell transformation and for productive interaction with the PDGF beta receptor and indicated that aspartic acid 33 was more important for these activities than was glutamic acid 36. These results are consistent with the existence of an essential juxtamembrane salt bridge between lysine 499 on the PDGF beta receptor and an acidic residue in the C terminus of the E5 protein and lend support to our proposed model for the complex between the E5 dimer and the PDGF beta receptor. (+info)
Cell surface retention sequence binding protein-1 interacts with the v-sis gene product and platelet-derived growth factor beta-type receptor in simian sarcoma virus-transformed cells.
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The cell surface retention sequence (CRS) binding protein-1 (CRSBP-1) is a newly identified membrane glycoprotein which is hypothesized to be responsible for cell surface retention of the oncogene v-sis and c-sis gene products and other secretory proteins containing CRSs. In simian sarcoma virus-transformed NIH 3T3 cells (SSV-NIH 3T3 cells), a fraction of CRSBP-1 was demonstrated at the cell surface and underwent internalization/recycling as revealed by cell surface 125I labeling and its resistance/sensitivity to trypsin digestion. However, the majority of CRSBP-1 was localized in intracellular compartments as evidenced by the resistance of most of the 35S-metabolically labeled CRSBP-1 to trypsin digestion, and by indirect immunofluorescent staining. CRSBP-1 appeared to form complexes with proteolytically processed forms (generated at and/or after the trans-Golgi network) of the v-sis gene product and with a approximately 140-kDa proteolytically cleaved form of the platelet-derived growth factor (PDGF) beta-type receptor, as demonstrated by metabolic labeling and co-immunoprecipitation. CRSBP-1, like the v-sis gene product and PDGF beta-type receptor, underwent rapid turnover which was blocked in the presence of 100 microM suramin. In normal and other transformed NIH 3T3 cells, CRSBP-1 was relatively stable and did not undergo rapid turnover and internalization/recycling at the cell surface. These results suggest that in SSV-NIH 3T3 cells, CRSBP-1 interacts with and forms ternary and binary complexes with the newly synthesized v-sis gene product and PDGF beta-type receptor at the trans-Golgi network and that the stable binary (CRSBP-1.v-sis gene product) complex is transported to the cell surface where it presents the v-sis gene product to unoccupied PDGF beta-type receptors during internalization/recycling. (+info)
The role of alpha and beta platelet-derived growth factor receptor in the vascular response to injury in nonhuman primates.
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Restenosis remains a significant clinical problem associated with mechanical interventional procedures for arterial revascularization or repair, including coronary angioplasty and stenting. Studies with rodents have established that platelet-derived growth factor (PDGF), a potent chemotactic and mitogenic agent for vascular smooth muscle cells, is a key mediator of lesion formation after vascular injury. To further explore this hypothesis in a more clinically relevant model, neutralizing monoclonal antibodies (mAbs) were used to examine the effect of selective inhibition of alpha or beta PDGF receptor (PDGFR) on neointima formation in nonhuman primates. Carotid arteries were injured by surgical endarterectomy and femoral arteries by balloon catheter dilatation. Immunostaining revealed that both injuries induced cell proliferation and the upregulation of beta PDGFR but not alpha PDGFR. By 7 days after injury, beta PDGFR staining was limited to the luminal region of the media, the small areas of neointima, and the adventitia. Nearly all bromodeoxyuridine-positive cells were found in these regions as well. After 30 days, a concentric neointima that stained strongly for beta PDGFR had formed in the carotid and femoral arteries. Treatment of baboons with anti-beta PDGFR mAb 2A1E2 for 6 days after injury reduced the carotid artery and femoral artery lesion sizes by 37% (P<0.05) and 48% (P<0.005), respectively, when measured at 30 days. Under the same conditions, treatment with anti-alpha PDGFR mAb 2H7C5 had no effect. These findings suggest that PDGF mediates neointima formation through the beta PDGFR, and that antagonism of this pathway may be a promising therapeutic strategy for reducing clinical restenosis. (+info)
Increased mitogenicity of an alphabeta heterodimeric PDGF receptor complex correlates with lack of RasGAP binding.
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The different platelet-derived growth factor (PDGF) isoforms cause activation of their alpha and beta protein tyrosine kinase receptors through dimerization. Homodimerization as well as heterodimerization of receptors occur. It has been shown previously that the heterodimeric receptor complex mediates a stronger mitogenic response than either of the homodimeric complexes. In this report, we show that in cells expressing both PDGF alpha- and beta-receptors, stimulation with PDGF-AB, which leads to preferential heterodimer formation, leads to a very low degree of phosphorylation of Tyr771 in the beta-receptor. In contrast, Tyr771 is phosphorylated in a homodimeric complex of beta-receptors. Phosphorylated Tyr771 is a binding site for RasGAP; an analogous site is not present in the alpha-receptor, which lacks the ability to associate with RasGAP. The lowered phosphorylation of Tyr771 in the heterodimeric receptor complex correlates with lowered association with RasGAP, as well as with a more efficient activation of Ras and MAP kinase, which is consistent with the increased mitogenicity elicited by PDGF-AB, compared to PDGF-AA or PDGF-BB. (+info)
Tyrosine phosphatase SHP-2 is involved in regulation of platelet-derived growth factor-induced migration.
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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. (+info)
Activation of microvascular pericytes in autoimmune Raynaud's phenomenon and systemic sclerosis.
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OBJECTIVE: To determine the temporal and spatial relationship between platelet-derived growth factor beta (PDGFbeta) receptors, PDGF-AB/BB, and activated pericytes across the Raynaud's phenomenon (RP) and systemic sclerosis (SSc; scleroderma) disease spectrum. METHODS: Monoclonal antibodies against PDGFbeta receptors, PDGF-AB/BB, and high molecular weight-melanoma-associated antigen (HMW-MAA), a marker for activated pericytes, were used to immunohistochemically analyze serial sections of skin biopsy tissue from patients with RP and from scleroderma patients. To delineate cell-specific PDGFbeta receptor expression, double immunofluorescence-stained sections were analyzed using computer-aided image analysis and confocal microscopy. RESULTS: PDGFbeta receptor-expressing cells and HMW-MAA-expressing pericytes were found in biopsy samples from autoimmune RP patients and in both early fibrotic and early nonfibrotic scleroderma skin, but not in normal or primary RP or late-stage scleroderma skin. PDGF-AB/BB was expressed within the epidermis, at the epidermal/dermal junction, and by dermal macrophages. Analysis of juxtaposed serial sections revealed an increased frequency of receptor expression in microvessels from autoimmune RP and early scleroderma skin (P < 0.01). Double-labeling studies using confocal microscopy showed that, in vivo, PDGFbeta receptors were predominantly expressed by microvascular pericytes from both autoimmune RP and early scleroderma skin. CONCLUSION: PDGFbeta receptors are expressed by activated microvascular pericytes in patients with autoimmune RP and in early SSc patients, but not in those with primary RP or late-stage scleroderma. These findings suggest that features of autoimmune RP are distinct from those of primary RP, and that microvascular pericytes may be an important link between chronic microvascular damage and fibrosis. (+info)
Chimera analysis reveals that fibroblasts and endothelial cells require platelet-derived growth factor receptorbeta expression for participation in reactive connective tissue formation in adults but not during development.
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The hypothesis that platelet-derived growth factor (PDGF) plays an important role in repair of connective tissue has been difficult to test experimentally, in part because the disruption of any of the PDGF ligand and receptor genes is embryonic lethal. We have developed a method that circumvents the embryonic lethality of the PDGF receptor (R)beta-/- genotype and minimizes the tendency of compensatory processes to mask the phenotype of gene disruption by comparing the behavior of wild-type and PDGFRbeta-/- cells within individual chimeric mice. This quantitative chimera analysis method has revealed that during development PDGFRbeta expression is important for all muscle lineages but not for fibroblast or endothelial lineages. Here we report that fibroblasts and endothelial cells, but not leukocytes, are dependent on PDGFRbeta expression during the formation of new connective tissue in and around sponges implanted under the skin. Even the 50% reduction in PDGFRbeta gene dosage in PDGFRbeta+/- cells reduces fibroblast and endothelial cell participation by 85%. These results demonstrate that the PDGFRbeta/PDGF B-chain system plays an important direct role in driving both fibroblast and endothelial cell participation in connective tissue repair, that cell behavior can be regulated by relatively small changes in PDGFRbeta expression, and that the functions served by PDGF in wound healing are different from the roles served during development. (+info)