PDGF (alpha)-receptor is unresponsive to PDGF-AA in aortic smooth muscle cells from the NG2 knockout mouse.
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A line of null mice has been produced which fails to express the transmembrane chondroitin sulfate proteoglycan NG2. Homozygous NG2 null mice do not exhibit gross phenotypic differences from wild-type mice, suggesting that detailed analyses are required to detect subtle alterations caused by the absence of NG2. Accordingly, dissociated cultures of aortic smooth muscle cells from null mice were compared to parallel cultures from wild-type mice for their ability to proliferate and migrate in response to specific growth factors. Both null and wild-type smooth muscle cells exhibited identical abilities to proliferate and migrate in response to PDGF-BB. In contrast, only the wild-type cells responded to PDGF-AA in both types of assays. NG2 null cells failed to proliferate or migrate in response to PDGF-AA, implying a defect in the signaling cascade normally initiated by activation of the PDGF (alpha)-receptor. In agreement with this idea, activation of the extracellular signal-regulated kinase (ERK) in response to PDGF-AA treatment occured only in wild-type cells. Failure to observe autophosphorylation of the PDGF (alpha)-receptor in PDGF-AA-treated null cells indicates that the absence of NG2 causes a defect in signal transduction at the level of (alpha)-receptor activation. (+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)
Rib truncations and fusions in the Sp2H mouse reveal a role for Pax3 in specification of the ventro-lateral and posterior parts of the somite.
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The splotch (Pax3) mouse mutant serves as a model for developmental defects of several types, including defective migration of dermomyotomal cells to form the limb musculature. Here, we describe abnormalities of the ribs, neural arches, and acromion in Sp2H homozygous embryos, indicating a widespread dependence of lateral somite development on Pax3 function. Moreover, the intercostal and body wall muscles, derivatives of the ventrolateral myotome, are also abnormal in Sp2H homozygotes. Pax3 is expressed in the dermomyotome, but not in either the sclerotome or the myotome, raising the possibility that Pax3-dependent inductive influences from the dermomyotome are necessary for early specification of lateral sclerotome and myotome. Support for this idea comes from analysis of gene expression markers of lateral sclerotome (tenascin-C and scleraxis) and myotome (myogenin, MyoD, and Myf5). All exhibit ventrally truncated domains of expression in Sp2H homozygotes, potentially accounting for the rib and intercostal muscle truncations. In contrast, the medial sclerotomal marker Pax1 is expressed normally in mutant embryos, arguing that Pax3 is not required for development of the medial sclerotome. Most of the somitic markers show ectopic expression in anteroposterior and mediolateral dimensions, suggesting a loss of definition of somite boundaries in splotch and explaining the rib and muscle fusions. An exception is Myf5, which is not ectopically expressed in Sp2H homozygotes, consistent with the previous suggestion that Pax3 and Myf5 function in different pathways of skeletal myogenesis. PDGFalpha and its receptor are candidates for mediating signalling between myotome and sclerotome. We find that both genes are misexpressed in Sp2H embryos, suggesting that PDGFalpha/PDGFRalpha may function downstream of Pax3, accounting for the close similarities between the splotch and Patch mutant phenotypes. Our findings point to additional regulatory functions for the Pax3 transcription factor, apart from those already demonstrated for development of the neural tube, neural crest, and dermomyotome. (+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)
Roles for PDGF-A and sonic hedgehog in development of mesenchymal components of the hair follicle.
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Skin appendages, such as hair, develop as a result of complex reciprocal signaling between epithelial and mesenchymal cells. These interactions are not well understood at the molecular level. Platelet-derived growth factor-A (PDGF-A) is expressed in the developing epidermis and hair follicle epithelium, and its receptor PDGF-Ralpha is expressed in associated mesenchymal structures. Here we have characterized the skin and hair phenotypes of mice carrying a null mutation in the PDGF-A gene. Postnatal PDGF-A-/- mice developed thinner dermis, misshapen hair follicles, smaller dermal papillae, abnormal dermal sheaths and thinner hair, compared with wild-type siblings. BrdU labeling showed reduced cell proliferation in the dermis and in the dermal sheaths of PDGF-A-/- skin. PDGF-A-/- skin transplantation to nude mice led to abnormal hair formation, reproducing some of the features of the skin phenotype of PDGF-A-/- mice. Taken together, expression patterns and mutant phenotypes suggest that epidermal PDGF-A has a role in stimulating the proliferation of dermal mesenchymal cells that may contribute to the formation of dermal papillae, mesenchymal sheaths and dermal fibroblasts. Finally, we show that sonic hedgehog (shh)-/- mouse embryos have disrupted formation of dermal papillae. Such embryos fail to form pre-papilla aggregates of postmitotic PDGF-Ralpha-positive cells, suggesting that shh has a critical role in the assembly of the dermal papilla. (+info)
RP-1551s, a family of azaphilones produced by Penicillium sp., inhibit the binding of PDGF to the extracellular domain of its receptor.
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Nine azaphilones designated RP-1551-1, -2, -3, -4, -5, -6, -7, -M1, and -M2 were isolated from the culture broth of Penicillium sp. SPC-21609 as inhibitors of PDGF binding to its receptor. RP-1551s inhibit the binding of PDGF AA to the extracellular domain of PDGF alpha-receptor with IC50 values ranging from 0.1 to 2 microM without affecting PDGF BB binding to the extracellular domain of PDGF beta-receptor. PDGF binding was not restored after the PDGF alpha-receptor extracellular domain was washed in an attempt to remove the RP-1551-1 bound to the receptor. This result suggests that RP-1551-1 may irreversibly interact with the PDGF alpha-receptor. Since many azaphilone compounds possess high reactivity with an amino group, RP-1551-1 may prevent PDGF AA binding by reacting with amino groups on the alpha-receptor extracellular domain. (+info)
High-affinity binding of basic fibroblast growth factor and platelet-derived growth factor-AA to the core protein of the NG2 proteoglycan.
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NG2 is a transmembrane chondroitin sulfate proteoglycan that is expressed by immature progenitor cells in several developmental lineages and by some types of malignant cells. In vitro studies have suggested that NG2 participates in growth factor activation of the platelet-derived growth factor-alpha receptor. In this study the ability of recombinant NG2 core protein to interact with several different growth factors (epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-AA, PDGF-BB, vascular endothelial growth factor (VEGF)165 and transforming growth factor (TGF)-beta1) was investigated using two different assay systems: enzyme-linked immunosorbent assay-type solid-phase binding and an optical biosensor (BIAcore) system. High-affinity binding of bFGF and PDGF-AA to the core protein of NG2 could be demonstrated with both types of assays. Using both the BIAcore software analysis program and nonlinear regression analysis of the solid phase binding data, KD values in the low nanomolar range were obtained for binding of each of these growth factors to NG2. The results further indicate that NG2 contains at least two binding sites for each of these two growth factors. PDGF-BB, TGF-beta1, VEGF, and EGF exhibited little or no binding to NG2 in either type of assay. These data suggest that NG2 can have an important role in organizing and presenting some types of mitogenic growth factors at the cell surface. (+info)
Differential expression of platelet-derived growth factor-alpha receptor by Thy-1(-) and Thy-1(+) lung fibroblasts.
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Fibroblasts are heterogeneous with respect to surface markers, morphology, and participation in fibrotic responses. This study was undertaken to determine whether Thy-1(-) and Thy-1(+) rat lung fibroblasts, which have distinct and relevant phenotypes, differ in their proliferative responses to platelet-derived growth factor (PDGF) isoforms. Homogeneous populations of Thy-1(-) and Thy-1(+) fibroblasts were found to proliferate equally in the presence of PDGF-BB, but PDGF-AA-mediated proliferation occurred only in Thy-1(-) cells. This differential activity correlated with significantly higher expression of PDGF-alpha receptor in Thy-1(-) fibroblasts as shown by immunoblotting, immunofluorescence, and Northern blotting. There was a rapid increase in c-myc mRNA in Thy-1(-) but not in Thy-1(+) fibroblasts on stimulation with PDGF-AA and PDGF-BB. The PDGF-alpha receptor, which mediates signaling by all PDGF isoforms, has been implicated in numerous clinical and experimental forms of fibrosis and regulates lung morphogenesis. Differential expression of the PDGF-alpha receptor supports distinct roles for Thy-1(-) and Thy-1(+) fibroblast populations in developmental and fibrotic processes in the lung. (+info)