The platelet-derived growth factor (PDGF) family plays an important role in embryonic development, malignancy, wound healing, atherosclerosis, and fibrosis in multiple organs. It belongs to the best-characterized growth factor systems in normal and diseased kidneys, and there is accumulating evidence that members of the PDGF family are key players in the development of renal fibrosis independent of the underlying kidney disease. All components of the PDGF system, consisting of four isoforms (PDGF-A, -B, -C, -D) and two receptor chains (PDGFR-α and -β), are constitutively or inducibly expressed in most renal cells. They regulate multiple pathophysiologic events, ranging from cell proliferation and migration, extracellular matrix accumulation and production of pro- and anti-inflammatory mediators, to tissue permeability and hemodynamics. This review focuses on advances in defining the roles of different PDGF isoforms in the development of glomerulosclerosis and tubulointerstitial fibrosis. The recent identification of endogenous PDGF inhibitors offers additional novel therapeutic strategies.

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.

The appropriate regulation of the actin cytoskeleton is essential for cell movement, changes in cell shape, and formation of membrane protrusions like lamellipodia and filopodia. Moreover, several regulatory proteins affecting actin dynamics have been identified in the motile regions of cells. Here, we provide evidence for the involvement of SPIN90 in the regulation of actin cytoskeleton and actin comet tail formation. SPIN90 was distributed throughout the cytoplasm in COS-7 cells, but exposing the cells to platelet-derived growth factor (PDGF) caused a redistribution of SPIN90 to the cell cortex and the formation of lamellipodia (or membrane ruffles), both of which were dramatically inhibited in SPIN90-knockdown cells. In addition, the binding of the C terminus of SPIN90 with both the Arp2/3 complex (actin-related proteins Arp 2 and Arp 3) and G-actin activates the former, leading to actin polymerization in vitro. And when coexpressed with phosphatidylinositol 4-phosphate 5 kinase, SPIN90 was observed within actin comet tails. Taken these findings suggest that SPIN90 participates in reorganization of the actin cytoskeleton and in actin-based cell motility.

Treatment of quiescent Balb/3T3 clone A31-1-1 cells with 0.1-0.2 mM H2O2 in the presence of 1 microM insulin induced DNA synthesis 20-24 h later at almost the same level as that in cells treated with 10% serum. Treatment with 0.1-0.2 mM H2O2 alone did not induce DNA synthesis and was not toxic to the cells. Cell cycle analysis indicated that treatment with H2O2 plus insulin induced progression of the cell cycle from the quiescent state. The amounts of mRNA for competence family genes such as c-fos, KC and JE were increased by the addition of H2O2. Under these conditions H2O2 caused rapid phosphorylation of a protein of 78 kDa with a pI of 6.3 (p78). Phosphorylation of p78 increased on treatment with TPA and serum as well. Catalase reduced the increase in phosphorylation of p78 induced by TPA and serum. Endogenous production of H2O2 was observed within 10 min after treatment of quiescent cells with platelet derived growth factor (PDGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). These results indicate that H2O2 at certain concentrations mimics the action of competence factors on resting Balb/3T3 cells.

Human platelet-derived growth factor (PDGF) is mainly composed of two polypeptide chains (PDGF-AB). All three possible dimeric forms of PDGF--i.e., PDGF-AA, PDGF-BB and PDGF-AB--exist in nature. We have used two recombinant PDGF homodimers to determine the roles of each isoform in the activation of phosphatidylinositol turnover in vascular smooth muscle cells (VSMC) isolated from rat thoracic aorta, their mitogenic effect on VSMC, and their vasoconstrictor effect on intact strips of aortic vascular tissue. Three Ca2+-channel blockers, nifedipine, verapamil, and diltiazem, were used as antagonists for investigating the PDGF-dependent changes mediated by the homodimers. PDGF-BB had a greater efficacy than PDGF-AA on inositol 1,4,5-trisphosphate release, on the formation of diacylglycerol, and on Ca2+ mobilization, which was also associated with vasoconstrictor activity and effective mitogenicity. PDGF-AA, on the other hand, was more potent than PDGF-BB in stimulating protein kinase C. In all instances, the activation of the phosphatidylinositol turnover by the two homodimers was inhibited by the Ca2+-channel blockers.

This report describes the generation and characterization of a panel of monoclonal antibodies (MAb) to the catenin p120ctn. p120ctn (formerly p120cas) is a cadherin-binding protein with structural similarity to the classical catenins beta-catenin and plakoglobin. It was originally identified as a prominent Src substrate and subsequently as a substrate for the Platelet Derived Growth Factor (PDGF), Epidermal Growth Factor (EGF), and Colony Stimulating Factor-1 (CSF-1) receptor tyrosine kinases. To facilitate further study of p120 function, we have generated novel MAbs to both the N- and C-terminal ends of p120 and compared them to previously described antibodies to these regions.

Phospholipase C (PLC) is shown to comprise at least nine isoforms. These isoforms can be separated into three structurally related classes. Within a class the isozymes have similar enzymological properties. In the case of the PLC gamma class, both isoforms may be regulated by tyrosine phosphorylation. For PLC gamma 1 we show that the tyrosine phosphorylation sites are contained within the SH2/SH3 region or 'modulatory domain'. The overexpression of PLC gamma 1 in Rat-2 cells results in increased phosphatidylinositol breakdown in response to PDGF treatment, demonstrating that PLC gamma 1 mediates this response. We note that thrombin activates PLC gamma 1 in addition to other PLC isoforms.

Mononuclear cells generate a variety of hormone-like proteins termed growth factors that are instrumental in the evolution and resolution of inflammatory reactions. Many of these growth regulatory molecules have multifunctional properties. For example, the mononuclear cell-derived growth factors, platelet-derived growth factor (PDGF), and transforming growth factor beta (TGF-beta), are potent leukocyte chemoattractants. In addition, TGF-beta, a product of platelets, T lymphocytes, and monocytes, appears to induce the transcription of other monocyte-derived growth hormone genes. In this regard, picomolar concentrations of TGF-beta stimulate peripheral blood monocytes to transcribe the genes for PDGF (c-sis), basic fibroblast growth factor (FGF), interleukin 1 (IL-1), and tumor necrosis factor (TNF). Furthermore, levels of mRNA for TGF-beta, which is constitutively expressed in resting monocytes, are also increased by exogenous TGF-beta. Each of these monocyte products exhibits a plethora of biological activities on other cell types. T lymphocytes, in response to antigen, contribute to this network by secreting growth factors and lymphokines that regulate monocyte growth factor production.

In the course of the random screening of a pool of CIBA chemicals, the two pyrazolopyrimidines 1 and 2 have been identified as fairly potent inhibitors of the EGF-R tyrosine kinase. Using a pharmacophore model for ATP-competitive inhibitors interacting with the active site of the EGF-R protein tyrosine kinase (PTK), the class of the pyrazolo[3,4-d]pyrimidines was then optimized in an interactive process leading to a series of 4-(phenylamino)-1H-pyrazolo[3,4-d]-pyrimidines as highly potent inhibitors of the EGF-R tyrosine kinase. The most potent compounds 13, 14, 15, 17, 19, 22, 26, 28, and 30 of this series inhibited the EGF-R PTK with IC50 values below 10 nM. High selectivity toward a panel of nonreceptor tyrosine kinases (c-Src, v-Abl and serine/threonine kinases (PKC alpha, CDK1) was observed. In cells, EGF-stimulated cellular tyrosine phosphorylation was inhibited by compounds 13, 15, 19, 22, and 23 at IC50 values below 50 nM, whereas PDGF-induced tyrosine phosphorylation was not affected by concentrations up to 10 microM, thus indicating high selectivity for the inhibition of the ligand-activated EGF-R signal transduction pathway. Compounds 15 and 19 inhibited proliferation of the EGF-dependent MK cell line with IC50 values below 0.5 microM. In addition, two compounds, 9 and 11, showing satisfactory oral bioavailability in mice after oral administration, exhibited good in vivo efficacy at doses of 12.5 and 50 mg/kg in a nude mouse tumor model using xenografts of the EGF-R overexpressing A431 cell line. From SAR studies, a binding mode for 4-(phenylamino)-1H-pyrazolo[3,4-d]pyrimidines, especially for compound 15, at the ATP-binding site of the EGF-R tyrosine kinase is proposed. 4-(Phenylamino)-1H-pyrazolo[3,4-d]pyrimidines represent a new class of highly potent tyrosine kinase inhibitors which preferentially inhibit the EGF-mediated signal transduction pathway and have the potential for further evaluation as anticancer agents.

Enhanced activity of receptor tyrosine kinases such as the PDGF beta-receptor and EGF receptor has been implicated as a contributing factor in the development of malignant and nonmalignant proliferative diseases such as cancer and atherosclerosis. Several epidemiological studies suggest that green tea may prevent the development of cancer and atherosclerosis. One of the major constituents of green tea is the polyphenol epigallocathechin-3 gallate (EGCG). In an attempt to offer a possible explanation for the anti-cancer and anti-atherosclerotic activity of EGCG, we examined the effect of EGCG on the PDGF-BB-, EGF-, angiotensin II-, and FCS-induced activation of the 44 kDa and 42 kDa mitogen-activated protein (MAP) kinase isoforms (p44(mapk)/p42(mapk)) in cultured vascular smooth muscle cells (VSMCs) from rat aorta. VSMCs were treated with EGCG (1-100 microM) for 24 h and stimulated with the above mentioned agonists for different time periods. Stimulation of the p44(mapk)/p42(mapk) was detected by the enhanced Western blotting method using phospho-specific MAP kinase antibodies that recognized the Tyr204-phosphorylated (active) isoforms. Treatment of VSMCs with 10 and 50 microM EGCG resulted in an 80% and a complete inhibition of the PDGF-BB-induced activation of MAP kinase isoforms, respectively. In striking contrast, EGCG (1-100 microM) did not influence MAP kinase activation by EGF, angiotensin II, and FCS. Similarly, the maximal effect of PDGF-BB on the c-fos and egr-1 mRNA expression as well as on intracellular free Ca2+ concentration was completely inhibited in EGCG-treated VSMCs, whereas the effect of EGF was not affected. Quantification of the immunoprecipitated tyrosine-phosphorylated PDGF-Rbeta, phosphatidylinositol 3'-kinase, and phospholipase C-gamma1 by the enhanced Western blotting method revealed that EGCG treatment effectively inhibits tyrosine phosphorylation of these kinases in VSMCs. Furthermore, we show that spheroid formation of human glioblastoma cells (A172) and colony formation of sis-transfected NIH 3T3 cells in semisolid agar are completely inhibited by 20-50 microM EGCG. Our findings demonstrate that EGCG is a selective inhibitor of the tyrosine phosphorylation of PDGF-Rbeta and its downstream signaling pathway. The present findings may partly explain the anti-cancer and anti-atherosclerotic activity of green tea.

Transforming growth factor beta (TGF-beta) and platelet-derived growth factor A (PDGFAlpha) play a central role in tissue morphogenesis and repair, but their interplay remain poorly understood. The nuclear factor I C (NFI-C) transcription factor has been implicated in TGF-beta signaling, extracellular matrix deposition, and skin appendage pathologies, but a potential role in skin morphogenesis or healing had not been assessed. To evaluate this possibility, we performed a global gene expression analysis in NFI-C(-/-) and wild-type embryonic primary murine fibroblasts. This indicated that NFI-C acts mostly to repress gene expression in response to TGF-beta1. Misregulated genes were prominently overrepresented by regulators of connective tissue inflammation and repair. In vivo skin healing revealed a faster inflammatory stage and wound closure in NFI-C(-/-) mice. Expression of PDGFA and PDGF-receptor alpha were increased in wounds of NFI-C(-/-) mice, explaining the early recruitment of macrophages and fibroblasts. Differentiation of fibroblasts to contractile myofibroblasts was also elevated, providing a rationale for faster wound closure. Taken together with the role of TGF-beta in myofibroblast differentiation, our results imply a central role of NFI-C in the interplay of the two signaling pathways and in regulation of the progression of tissue regeneration.

Merkel cell carcinoma is a rare but very aggressive tumor of the skin. With current treatment options, Merkel cell carcinoma is associated with a high incidence of recurrence and metastasis. Targeted anticancer therapies such as receptor tyrosine kinase inhibitors and antisense oligonucleotides have been found to be a promising new type of treatment for various types of cancer. To evaluate whether the use of targeted therapies is a possible treatment option in Merkel cell carcinoma, we determined the expression of the target molecules c-kit, Mcl-1, Bmi-1, vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-receptor 2 (VEGF-R2), platelet-derived growth factor (PDGF)-alpha, PDGF-beta, epidermal growth factor receptor (EGFR) and Her-2/Neu in a tissue microarray of 32 samples of 29 patients with Merkel cell carcinoma. C-kit-positive samples were analyzed for mutations in exons 9 and 11. The tissue microarray was stained immunohistochemically with antibodies directed against the above-mentioned proteins, and an immunoreactivity score was calculated. DNA was extracted from c-kit-positive samples and was analyzed for exon 9 and 11 mutations using direct DNA sequencing. We found that c-kit (7%), Mcl-1 (88%), Bmi-1 (78%), VEGF-A (91%), VEGF-C (75%) VEGF-R2 (88%), PDGF-alpha (72%) and PDGF-beta (13%) were expressed in Merkel cell carcinomas. All samples showed a lack of EGFR and Her-2/Neu expression. Analysis of c-kit revealed no mutations. As VEGF-A, VEGF-C, VEGF-R2, PDGFs and c-kit are targets of new cytostatic agents used in the treatment of other cancers, inhibition by a multitargeted chemotherapy could be a very promising treatment option. High expression of Bmi-1 and Mcl-1 warrants further studies on the use of antisense oligonucleotides in Merkel cell carcinoma.

Angiogenesis is essential for primary tumor growth and metastatic dissemination. E2F1, frequently upregulated in advanced cancers, was recently shown to drive malignant progression. In an attempt to decipher the molecular events underlying this behavior, we demonstrate that the tumor cell-associated vascular endothelial growth factor-C/receptor-3 (VEGF-C/VEGFR-3) axis is controlled by E2F1. Activation or forced expression of E2F1 in cancer cells leads to the upregulation of VEGFR-3 and its ligand VEGF-C, whereas E2F1 depletion prevents their expression. E2F1-dependent receptor induction is crucial for tumor cells to enhance formation of capillary tubes and neovascularization in mice. We further provide evidence for a positive feedback loop between E2F1 and VEGFR-3 signaling to stimulate pro-angiogenic platelet-derived growth factor B (PDGF-B). E2F1 or VEGFR-3 knockdown results in reduced PDGF-B levels, while the coexpression synergistically upregulates promoter activity and endogenous protein expression of PDGF-B. Our findings delineate an as yet unrecognized function of E2F1 as enhancer of angiogenesis via regulation of VEGF-C/VEGFR-3 signaling in tumors to cooperatively activate PDGF-B expression. Targeting this pathway might be reasonable to complement standard anti-angiogenic treatment of cancers with deregulated E2F1.

Fibrosis is part of a tissue repair response to injury, defined as increased deposition of extracellular matrix. In some instances, fibrosis is beneficial; however, in the majority of diseases fibrosis is detrimental. Virtually all chronic progressive diseases are associated with fibrosis, representing a huge number of patients worldwide. Fibrosis occurs in all organs and tissues, becomes irreversible with time and further drives loss of tissue function. Various cells types initiate and perpetuate pathological fibrosis by paracrine activation of the principal cellular executors of fibrosis, i.e. stromal mesenchymal cells like fibroblasts, pericytes and myofibroblasts. Multiple pathways are involved in fibrosis, platelet-derived growth factor (PDGF)-signaling being one of the central mediators. Stromal mesenchymal cells express both PDGF receptors (PDGFR) α and β, activation of which drives proliferation, migration and production of extracellular matrix, i.e. the principal processes of fibrosis. Here, we review the role of PDGF signaling in organ fibrosis, with particular focus on the more recently described ligands PDGF-C and -D. We discuss the potential challenges, opportunities and open questions in using PDGF as a potential target for anti-fibrotic therapies.

c-Abl is a nuclear and cytoplasmic tyrosine kinase involved in a variety of cellular growth and differentiation processes. In contrast to its oncogenic counterparts, like BCR-Abl, c-Abl is not constitutively tyrosine phosphorylated and its catalytic activity is very low. Here we report tyrosine phosphorylation of endogenous c-Abl and a concomitant increase in catalytic activity. Using Abl -/- cells reconstituted with mutated c-Abl forms, we show that phosphorylation and activity depend on Tyr412 in the activation loop. Tyr412 is also required for stimulation by PDGF or by cotransfection of active Src. Phosphorylation of Tyr412 can occur autocatalytically by a trans-mechanism and cause activation of otherwise inactive c-Abl, suggesting a positive feedback loop on c-Abl activity. In the recent structure of the Abl catalytic domain bound to the STI-571 inhibitor, unphosphorylated Tyr412 in the activation loop points inward and appears to interfere with catalysis. We mutated residues involved in stabilizing this inhibited form of the activation loop and in positioning Tyr412. These mutations resulted in tyrosine phosphorylation and activation of c-Abl, as if relieving c-Abl from inhibition. Tyr412 is therefore necessary both for activity and for regulation of c-Abl, by stabilizing the inactive or the active conformation of the enzyme in a phosphorylation-dependent manner.

Elevation of intracellular Ca2+ at fertilization is essential for the initiation of development in the Xenopus egg, but the pathway between sperm-egg interaction and Ca2+ release from the egg's endoplasmic reticulum is not well understood. Here we show that injection of an inhibitory antibody against the type I IP(3) receptor reduces Ca2+ release at fertilization, indicating that the Ca2+ release requires IP(3). We then examine how IP(3) production is initiated. Xenopus eggs were injected with specific inhibitors of the activation of two phospholipase C isoforms, PLCgamma and PLCbeta. The Src-homology 2 (SH2) domains of PLCgamma were used to inhibit SH2-mediated activation of PLCgamma, and an antibody against G(q) family G-proteins was used to inhibit G(q)-mediated activation of PLCbeta. Though the PLCgamma SH2 domains inhibited platelet-derived growth factor (PDGF)-induced Ca2+ release in eggs with exogenously expressed PDGF receptors, they did not inhibit the Ca2+ rise at fertilization. Similarly, the G(q) family antibody blocked serotonin-induced Ca2+ release in eggs with exogenously expressed serotonin 2C receptors, but not the Ca2+ rise at fertilization. A mixture of PLCgamma SH2 domains and the G(q) antibody also did not inhibit the Ca2+ rise at fertilization. These results indicate that Ca2+ release at fertilization of Xenopus eggs requires type I IP(3)-gated Ca2+ channels, but not SH2 domain-mediated activation of PLCgamma or G(q)-mediated activation of PLCbeta.

The double-stranded RNA (dsRNA)-activated protein kinase PKR is an interferon (IFN)-induced enzyme that controls protein synthesis through phosphorylation of eukaryotic initiation factor 2alpha (eIF-2alpha). PKR also regulates signals initiated by diverse stimuli, including dsRNA, IFN-gamma, tumor necrosis factor-alpha, interleukin-1 and lipopolysaccharide, to different transcription factors, resulting in pro-inflammatory gene expression. Stat3 plays an essential role in promoting cell survival and proliferation by different growth factors, including platelet-derived growth factor (PDGF). Here we show that PKR physically interacts with Stat3 and is required for PDGF-induced phosphorylation of Stat3 at Tyr705 and Ser727, resulting in DNA binding and transcriptional activation. PKR-mediated phosphorylation of Stat3 on Ser727 is indirect and channeled through ERKS: Although PKR is pre-associated with the PDGF beta-receptor, treatment with PDGF only modestly activates PKR. However, the induction of c-fos by PDGF is defective in PKR-null cells. Taken together, these results establish PKR as an upstream regulator of activation of Stat3 and as a common mediator of both growth-promoting and growth-inhibitory signals.

Proliferation of mesangial cells requires platelet-derived growth factor receptor beta (PDGFR)-mediated signal transduction. We have previously shown that activation of phosphatidylinositol (PI) 3-kinase is necessary for PDGFR-induced DNA synthesis in these cells. The mechanism by which PI 3-kinase stimulates DNA synthesis is not known. One target of PI 3-kinase, Akt serine threonine kinase, regulates survival of many cells by inhibiting the actions of certain proapoptotic proteins. In this study, we investigated the role of Akt in PDGF-induced DNA synthesis in mesangial cells. PDGF increased Akt serine threonine kinase activity in a time- and PI 3-kinase-dependent manner. Expression of dominant negative Akt by adenovirus-mediated gene transfer blocked PDGF-induced activation of endogenous Akt in mesangial cells, resulting in complete inhibition of DNA synthesis. On the other hand, inhibition of MAPK attenuated PDGF-induced DNA synthesis only partially. Inhibition of Akt also attenuated PDGF-induced c-fos gene transcription, with concomitant inhibition of Elk-1-dependent transcription, indicating positive regulation of this early response gene by Akt. To further determine the role of Akt in PDGF-induced DNA synthesis, we investigated its effect on cyclin-dependent kinase 2 (CDK2). PDGF stimulated CDK2 activity in mesangial cells and decreased the level of p27(kip1) cyclin kinase inhibitor protein. Expression of dominant negative Akt increased p27(kip1) protein and resulted in inhibition of CDK2 activity. The increase in p27(kip1) expression in response to Akt kinase inhibition was due to increased transcription of the p27(kip1) gene. p27(kip1) transcription similarly was decreased by expression of constitutively active Akt kinase in mesangial cells. These data provide the first evidence that Akt kinase regulates PDGF-induced DNA synthesis by regulating CDK2 activity and define Akt-mediated inhibition of transcription of p27(kip1) as one of the mechanisms for PDGF-induced DNA synthesis in mesangial cells.

The proteolytic activity of Furin responsible for processing full length Notch-1 (p300) plays a critical role in Notch signaling. The amplitude and duration of Notch activity can be regulated at various points in the pathway, but there has been no report regarding regulation of the Notch-1-Furin interaction, despite its importance. In the present study, we found that the Notch-1-Furin interaction is regulated by the non-receptor tyrosine kinase, c-Src. c-Src and Notch-1 are physically associated, and this association is responsible for Notch-1 processing and activation. We also found that growth factor TGF-α, an EGFR ligand, and PDGF-BB, a PDGFR ligand, induce the Notch-1-Furin interaction mediated by c-Src. Our results support three new and provocative conclusions: (1) The association between Notch-1 and Furin is a well-regulated process; (2) Extracellular growth factor signals regulate this interaction, which is mediated by c-Src; (3) There is cross-talk between the plasma growth factor receptor-c-Src and Notch pathways. Co-localization of Notch-1 and c-Src was confirmed in xenograft tumor tissues and in the tissues of pancreatic cancer patients. Our findings have implications for the mechanism by which the Notch and growth factor receptor-c-Src signaling pathways regulate carcinogenesis and cancer cell growth.

Addition of mouse interferon-alpha/beta (IFN) to confluent, quiescent BALB/c 3T3 (clone A31) mouse fibroblasts resulted in a block or delay in serum-induced activation of the cell cycle. It was necessary to add IFN within 6 hours after serum stimulation to inhibit nuclear labeling with [3H]thymidine. This is consistent with the time required for platelet-derived growth factor (PDGF) to induce cells to become competent to respond to additional growth factors present in platelet-poor plasma. Simultaneous addition of IFN with PDGF inhibited the PDGF-induced synthesis of a 29-kilodalton and a 35-kilodalton protein that normally occurs within 1 hour after PDGF addition. IFN also suppressed the general increase in protein synthesis that occurs by the fifth hour after PDGF addition. These results show that IFN antagonizes the action of PDGF, thereby interfering with the activation of Go cells for G1 traverse and S-phase entry.

The molecular mechanism(s) by which high glucose induces fibronectin expression via G-protein activation in the kidney are largely unknown. This investigation describes the effect of high glucose (HG) on a small GTP-binding protein, Rap1b, expression and activation, and the relevance of protein kinase C (PKC) and Raf pathways in fibronectin synthesis in cultured renal glomerular mesangial cells (MCs). In vivo experiments revealed a dose-dependent increase in Rap1b expression in glomeruli of diabetic rat kidneys. Similarly, in vitro exposure of MCs to HG led to an up-regulation of Rap1b with concomitant increase in fibronectin (FN) mRNA and protein expression. The up-regulation of Rap1b mRNA was mitigated by the PKC inhibitors, calphostin C, and bisindolymaleimide, while also reducing HG- induced FN expression in non-transfected MCs. Overexpression of Rap1b by transfection with pcDNA 3.1/Rap1b in MCs resulted in the stimulation of FN synthesis; however, the PKC inhibitors had no significant effect in reducing FN expression in Rap1b-transfected MCs. Transfection of Rap1b mutants S17N (Ser ->> Asn) or T61R (Thr ->> Arg) in MCs inhibited the HG-induced increased FN synthesis. B-Raf and Raf-1 expression was investigated to assess whether Rap1b effects are mediated via the Raf pathway. B-Raf, and not Raf-1, expression was increased in MCs transfected with Rap1b. HG also caused activation of Rap1b, which was largely unaffected by anti-platelet-derived growth factor (PDGF) antibodies. HG-induced activation of Rap1b was specific, since Rap2b activation and expression of Rap2a and Rap2b were unaffected by HG. These findings indicate that hyperglycemia and HG cause an activation and up-regulation of Rap1b in renal glomeruli and in cultured MCs, which then stimulates FN synthesis. This effect appears to be PKC-dependent and PDGF-independent, but involves B-Raf, suggesting a novel PKC-Rap1b-B-Raf pathway responsible for HG-induced increased mesangial matrix synthesis, a hallmark of diabetic nephropathy.

Platelet-derived growth factor (PDGF), a key mitogen for liver fat-storing cells (FSC), is a dimeric molecule that occurs as homodimers or heterodimers of related polypeptide chains (PDGF-BB, -AB, and -AA). In chronic inflammation of the liver lobule, any of the three dimeric forms of PDGF derived from multiple sources could potentially interact with FSC. We explored the effects of the three different PDGF isoforms on DNA synthesis and early signal transduction pathways potentially related to PDGF mitogenicity in rat liver FSC. PDGF-BB homodimer and -AB heterodimer induced a marked increase in DNA synthesis, whereas the effect of PDGF-AA homodimer was considerably lower. Moreover, the mitogenicity of each isoform proportionally correlated with their effects on phosphoinositide turnover and intracellular Ca2+. Both the PDGF-BB and -AB dimers likely interact with the PDGF-beta-receptor, although PDGF-AB requires at least one alpha-receptor. The low responsiveness to PDGF-AA could not be accounted for by downregulation of the PDGF-alpha-receptor because FSC expressed very low levels of PDGF-A- and B-chain mRNAs and did not secrete detectable amounts of PDGF activity in the conditioned media. In addition, preincubation of FSC with suramin, a potent inhibitor of PDGF binding to its receptor, failed to increase PDGF-AA-induced DNA synthesis. These results are consistent with a predominant expression of PDGF-beta-receptor in liver FSC, that is linked to phospholipase C activation.

Platelet-derived growth factor (PDGF) has been implicated in promoting survival and proliferation of immature neurons, and even protecting neurons from gp120-induced cytotoxicity. However, the mechanisms involved in neuroprotection are not well understood. In the present study we demonstrate the role of phosphatidylinositol 3-kinase (PI3K)/Akt signaling in PDGF-mediated neuroprotection. Pharmacological inhibition of PI3K greatly reduced the ability of PDGF-BB to block gp120 IIIB-mediated apoptosis and cell death in human neuroblastoma cells. The role of Akt in PDGF-mediated protection was further corroborated using a dominant-negative mutant of Akt, which was able to block the protective effect of PDGF. We next sequentially examined the signals downstream of Akt in PDGF-mediated protection in human neuroblastoma cells. In cells pretreated with PDGF prior to gp120 there was increased phosphorylation of both GSK-3beta and Bad, an effect that was inhibited by PI3-kinase inhibitor. Nuclear translocation of NF-kappaB, which lies downstream of GSK-3beta, however, remained unaffected in cells treated with PDGF. In addition to inducing phosphorylation of Bad, PDGF-mediated protection also involved down-regulation of the proapoptotic protein Bax. Furthermore, PDGF-mediated protection also involved the inhibition of gp120-induced release of mitochondrial cytochrome C. Our findings thus underscore the roles of both PI3K/Akt and Bcl family pathways in PDGF-mediated neuroprotection.

Tubulointerstitial fibrosis is a major characteristic of progressive renal diseases. Platelet-derived growth factor (PDGF) is a family of growth regulatory molecules consisting of PDGF-A and -B, along with the newly discovered PDGF-C and -D. They signal through cell membrane receptors, PDGF receptor alpha (PDGF-Ralpha) and receptor beta (PDGF-Rbeta). Involvement of PDGF-B and PDGF-Rbeta in the initiation and progression of renal fibrosis has been well documented. The authors studied the localization of PDGF ligands and receptors by immunohistochemistry, with emphasis on the role of PDGF-D in murine renal fibrosis induced by unilateral ureteral obstruction (UUO). In mice with UUO, de novo expression of PDGF-D was detected in interstitial cells at day 4, which increased to maximal expression at day 14. Increased expression of PDGF-B by interstitial cells and in some tubules was observed after day 4. The diseased mice did not show augmentation of PDGF-A or PDGF-C proteins in the areas of fibrosis. PDGF-Ralpha and -Rbeta protein expression was increased in interstitial cells after day 4 and reached maximal expression at day 14. Human renal nephrectomies (n = 10) of chronic obstructive nephropathy demonstrated similar de novo expression of PDGF-D in interstitial cells, correlating with expression of PDGF-Rbeta and PDGF-B, as it did in the murine model. These observations suggest that PDGF-D plays an important role in the pathogenesis of tubulointerstitial injury through binding of PDGF-Rbeta in both human obstructive nephropathy and the corresponding murine model of UUO.