Previous studies have identified several cytokines as inducers of tenascin-C (TN-C) expression in various tissue culture systems. However, the signaling pathways of the regulation of TN-C expression are almost unknown. In this study, we clarified the molecular mechanism(s) underlying the regulation of the TN-C gene by platelet derived growth factor (PDGF) in cultured human dermal fibroblasts. PDGF induced the expression of TN-C protein as well as mRNA in a dose-dependent manner. Actinomycin D, an RNA synthesis inhibitor, significantly blocked the PDGF-mediated upregulation of TN-C mRNA expression, whereas cycloheximide, a protein synthesis inhibitor, did not. The PDGF-mediated induction of TN-C expression was inhibited by the treatment of fibroblasts with a selective phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, or LY294002. These results suggest that PDGF induced the expression of TN-C at a transcriptional level via phosphoinositide3-kinase/Akt signaling pathways. We performed serial 5' deletions and a transient transfection analysis to define the region in the TN-C promoter mediating the responsiveness to PDGF. Overexpression of Sp1, Ets1, or Ets2 activated the TN-C promoter and superinduced TN-C promoter activity stimulated by PDGF, whereas overexpression of Fli1 inhibited the effects of PDGF on TN-C expression. Mutation of the Sp1/3 binding sites or Ets binding sites in the TN-C promoter region responsible to PDGF abrogated the PDGF-inducible promoter activity. Immunoprecipitation analysis revealed that Sp1, Ets1, and Ets2 form a transcriptionally active complex. On the other hand, the interaction of Fli1 with Sp1 decreased after PDGF treatment. These results suggest that the upregulation of TN-C expression by PDGF involves Ets family transcription factors, co-operating with Sp1.

We have used a proteomic approach using mass spectrometry to identify signaling molecules involved in receptor tyrosine kinase signaling pathways. Using affinity purification by anti-phosphotyrosine antibodies to enrich for tyrosine phosphorylated proteins, we have identified a novel signaling molecule in the epidermal growth factor receptor signaling pathway. This molecule, designated Odin, contains several ankyrin repeats, two sterile alpha motifs and a phosphotyrosine binding domain and is ubiquitously expressed. Using antibodies against endogenous Odin, we show that it undergoes tyrosine phosphorylation upon addition of growth factors such as EGF or PDGF but not by cytokines such as IL-3 or erythropoietin. Immunofluorescence experiments as well as Western blot analysis on subcellular fractions demonstrated that Odin is localized to the cytoplasm both before and after growth factor treatment. Deletion analysis showed that the phosphotyrosine binding domain of Odin is not required for its tyrosine phosphorylation. Overexpression of Odin, but not an unrelated adapter protein, Grb2, inhibited EGF-induced activation of c-Fos promoter. Microinjection of wild-type or a mutant version lacking the PTB domain into NIH3T3 fibroblasts inhibited PDGF-induced mitogenesis. Taken together, our results indicate that Odin may play a negative role in growth factor receptor signaling pathways.

We have previously reported that antibodies to phosphotyrosine recognize the phosphorylated forms of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors (Zippel et al., Biochim. Biophys. Acta 881:54-61, 1986, and Sturani et al., Biochem. Biophys. Res. Commun. 137:343-350, 1986). In this report, the time course of receptor phosphorylation is investigated. In normal human fibroblasts, ligand-induced phosphorylation of PDGF and EGF receptors is followed by rapid dephosphorylation. However, in A431 cells the tyrosine-phosphorylated form of EGF receptor persists for many hours after EGF stimulation, allowing a detailed analysis of the conditions affecting receptor phosphorylation and dephosphorylation. In A431 cells, the number of receptor molecules phosphorylated on tyrosine was quantitated and found to be about 10% of total EGF receptors. The phosphorylated receptor molecules are localized on the cell surface, and they are rapidly dephosphorylated upon removal of EGF from binding sites by a short acid wash of intact cells and upon a mild treatment with trypsin. ATP depletion also results in rapid dephosphorylation, indicating that continuous phosphorylation-dephosphorylation reactions occur in the ligand-receptor complex at steady state. Phorbol 12-myristate 13-acetate added shortly before EGF reduces the rate and the final extent of receptor phosphorylation. Moreover, it also reduces the amount of phosphorylated receptors if it is added after EGF. Down-regulation of protein kinase C by chronic treatment with phorbol dibutyrate increases the receptor phosphorylation induced by EGF, suggesting a homologous feedback regulation of EGF receptor functions.

Transcriptional regulation requires co-ordinated action of transcription factors, co-activator complexes and general transcription factors to access specific loci in the dense chromatin structure. In the present study we demonstrate that the transcriptional co-regulator SPBP [stromelysin-1 PDGF (platelet-derived growth factor)-responsive element binding protein] contains two independent chromatin-binding domains, the SPBP-(1551-1666) region and the C-terminal extended PHD [ePHD/ADD (extended plant homeodomain/ATRX-DNMT3-DNMT3L)] domain. The region 1551-1666 is a novel core nucleosome-interaction domain located adjacent to the AT-hook motif in the DNA-binding domain. This novel nucleosome-binding region is critically important for proper localization of SPBP in the cell nucleus. The ePHD/ADD domain associates with nucleosomes in a histone tail-dependent manner, and has significant impact on the dynamic interaction between SPBP and chromatin. Furthermore, SPBP and its homologue RAI1 (retinoic-acid-inducible protein 1), are strongly enriched on chromatin in interphase HeLa cells, and both proteins display low nuclear mobility. RAI1 contains a region with homology to the novel nucleosome-binding region SPBP-(1551-1666) and an ePHD/ADD domain with ability to bind nucleosomes. These results indicate that the transcriptional co-regulator SPBP and its homologue RAI1 implicated in Smith-Magenis syndrome and Potocki-Lupski syndrome both belong to the expanding family of chromatin-binding proteins containing several domains involved in specific chromatin interactions.

The three isoforms of PDGF bind with different affinities to two related tyrosine kinase receptors, denoted the PDGF alpha- and beta-receptors. Ligand binding induces receptor dimerization, creating receptor homo- or heterodimers. Dimerization is accompanied by, and might be a prerequisite for, receptor autophosphorylation and kinase activation. Receptor autophosphorylation serves to regulate the kinase activity and to create binding sites on the receptor molecule for downstream signalling components. The activities of the signalling components are ultimately manifested as specific biological responses. All the currently described PDGF receptor-binding components, e.g. phospholipase C-gamma, members of the src family of cytoplasmic tyrosine kinases, the rasGT-Pase activating protein and p85, the regulatory subunit of phosphatidylinositol 3' kinase, contain a conserved src homology 2-domain, through which the association with the receptor takes place. The receptor-binding components appear to either possess an intrinsic enzymatic activity, or they function as adaptors, which may complex with catalytically active components. For most receptor-binding components, there is insufficient understanding of how binding to the receptor affects the catalytic function. Certain of these components become tyrosine-phosphorylated, i.e. they are substrates for the receptor tyrosine kinase. Moreover, the change in subcellular localization, which most of the receptor binding components undergo in conjunction with receptor binding, could play a critical role. The current efforts of many laboratories are aimed at delineating different PDGF receptor signal transduction pathways and what roles the different receptor-binding components play in the establishment of these pathways.

The structure of phospholipase Cgamma1 (PLC-gamma1) contains two SH2 domains and one SH3 domain. While the function of the SH2 domains in PLC-gamma1 are well described, to date no growth factor-dependent function for the SH3 domain has been presented. To assess SH3 domain function in the context of the full-length PLC-gamma1, this domain was deleted and the mutant was stably expressed in Plcg1 null mouse embryonic fibroblasts. Following EGF treatment of cells, the PLC-gamma1DeltaSH3 mutant displayed the same increased level of tyrosine phosphorylation and association with EGF receptor as wild-type PLC-gamma1. Also, the SH3 mutant demonstrated membrane translocation and mediated the mobilization of intracellular Ca(2+) in response to EGF. c-Cbl is shown to associate with tyrosine phosphorylated PLC-gamma1 in an EGF-dependent manner, but no association was detected with the PLC-gamma1DeltaSH3 mutant. Interestingly, PDGF, which also tyrosine phosphorylates PLC-gamma1, failed to induce c-Cbl association with PLC-gamma1 and also provoked no c-Cbl tyrosine phosphorylation. This suggests that c-Cbl tyrosine phosphorylation is necessary for its interaction with PLC-gamma1. Evidence of a direct association of c-Cbl with PLC-gamma1 was provided by pull-down and overlay experiments, using glutathione S-transferase fusion proteins that contain the SH3 domain of PLC-gamma1. The data, therefore, show an EGF-inducible direct association of PLC-gamma1 with c-Cbl in vivo that is mediated by the SH3 domain of PLC-gamma1.

OBJECTIVE

Proliferative vitreoretinopathy (PVR) is the primary cause of failure of retinal reattachment surgery. Growth factors such as platelet-derived growth factor (PDGF) are strongly associated with PVR. Of the five PDGF family members, PDGF-C predominates in the vitreous of experimental and clinical PVR. PDGF-C is secreted as a latent protein that requires proteolytic processing for activation. Although tissue plasminogen activator (tPA) is primarily responsible for processing PDGF-C in cultured cells, it constitutes a minority of the processing activity in the vitreous of experimental animals and in patients with PVR. Identifying the major PDGF-C processing protease was the purpose of this study.

METHODS

The presence of serum proteins in the vitreous was detected by Coomassie blue staining and Western blotting. PDGF-C processing activity was detected in an in vitro processing assay using either native or recombinant PDGF-C as the substrate. Plasmin activity was blocked using alpha(2)-plasmin inhibitor. Phosphorylation of the PDGF receptor (PDGFR) was monitored by antiphosphotyrosine Western blotting. Vitreous specimens were collected from experimental rabbits or from patients undergoing vitrectomy to repair retinal detachment or for other reasons.

RESULTS

A number of prominent serum proteins (albumin and IgG) were detected in the vitreous of all patients undergoing retinal surgery. The level of these proteins markedly increased in the vitreous of rabbits as they developed PVR. These observations suggested that serum-borne proteases are also likely to be present in the vitreous. Indeed, plasmin (a protease capable of processing PDGF-C) was present in the vitreous from PVR rabbits and retinal surgery patients. Plasmin was dramatically more effective than tPA in processing PDGF-C in an in vitro assay. Blocking plasmin activity eliminated most of the processing activity in the vitreous of patients and rabbits with PVR.

CONCLUSIONS

Plasmin was the major PDGF-C processing protease in the vitreous of PVR rabbits and patients undergoing retinal surgery. Blocking plasmin prevented the generation of active PDGF-C, which is the major PDGF isoform relevant for PVR. These observations are the first report of an in vivo protease responsible for processing PDGF-C. In addition, plasmin was identified as a novel therapeutic target for patients with PVR.

Upon stimulation of cells with platelet-derived growth factor (PDGF), phospholipase C-gamma1 (PLC-gamma1) binds to the tyrosine-phosphorylated PDGF receptor through one or both of its Src homology 2 (SH2) domains, is phosphorylated by the receptor kinase, and is thereby activated to hydrolyze phosphatidylinositol 4, 5-bisphosphate. Association of PLC-gamma1 with the insoluble subcellular fraction is also enhanced in PDGF-stimulated cells. The individual roles of the two SH2 domains of PLC-gamma1 in mediating the interaction between the enzyme and the PDGF receptor have now been investigated by functionally disabling each domain. A critical Arg residue in each SH2 domain was mutated to Ala. Both wild-type and mutant PLC-gamma1 proteins were transiently expressed in a PLC-gamma1-deficient fibroblast cell line, and these transfected cells were stimulated with PDGF. The mutant protein in which the COOH-terminal SH2 domain was disabled bound to the PDGF receptor. Accordingly, it was phosphorylated by the receptor, catalyzed the production of inositol phosphates, and mobilized intracellular calcium to extents similar to (but slightly less than) those observed with the wild-type enzyme. In contrast, the mutant in which the NH(2)-terminal SH2 domain was impaired did not bind to the PDGF receptor and consequently was neither phosphorylated nor activated. These results suggest that the NH(2)-terminal SH2 domain, but not the COOH-terminal SH2 domain, of PLC-gamma1 is required for PDGF-induced activation of PLC-gamma1. Functional impairment of the SH2 domains did not affect the PDGF-induced redistribution of PLC-gamma1, suggesting that recruitment of PLC-gamma1 to the particulate fraction does not involve the SH2 domains.

The somatostatin receptor subtype sst2A is highly expressed, non-mutated and functionally active in gliomas. After stimulation of cultivated human U343 glioma cells with somatostatin, octreotide (sst2-, sst3- and sst5-selective peptide agonist) or the sst2-selective non-peptide agonist L-054,522 multiple signal transduction pathways are induced: elevated cAMP levels are reduced, protein tyrosine phosphatases (especially SHP2) are activated and mitogen-activated protein kinases are inhibited. Stimulation of the phosphatases resulted in dephosphorylation of activated receptors for EGF and PDGF (epidermal and platelet-derived growth factor), and as a consequence the mitogen-activated protein kinases ERK 1 and 2 (p42/p44) were de-phosphorylated in co-stimulation experiments. Furthermore, somatostatin or sst2-selective agonists reduced EGF-stimulated expression of the AP-1 complex (c-jun/c-jun) on the transcriptional and translational level. These experiments show that the interaction of stimulatory and inhibitory receptors are important mechanisms for the regulation of signal cascades and gene expression.

Upon binding of platelet-derived growth factor (PDGF), PDGF receptor is autophosphorylated at tyrosine residues in its cytoplasmic region, which induces the activation of diverse intracellular signaling pathways such those involving Ras-ERK, c-Src, and Rap1-Rac. Signaling through activated Ras-ERK promotes cell cycle and cell proliferation. The sequential activation of Rap1 and Rac affects cellular morphology and induces the formation of leading-edge structures, including lamellipodia, peripheral ruffles, and focal complexes, resulting in the enhancement of cell movement. In addition to the promotion of cell proliferation, the Ras-ERK signaling is involved in the regulation of cellular morphology. Here, we showed a novel role of afadin in the regulation of PDGF-induced intracellular signaling and cellular morphology in NIH3T3 cells. Afadin was originally identified as an actin filament-binding protein, which binds to a cell-cell adhesion molecule nectin and is involved in the formation of cell-cell junctions. When afadin was tyrosine-phosphorylated by c-Src activated in response to PDGF, afadin physically interacted with and increased the phosphatase activity of Src homology 2 domain-containing phosphatase-2 (SHP-2), a protein-tyrosine phosphatase that dephosphorylates PDGF receptor, leading to the prevention of hyperactivation of PDGF receptor and the Ras-ERK signaling. In contrast, knockdown of afadin or SHP-2 induced the hyperactivation of PDGF receptor and Ras-ERK signaling and consequently suppressed the formation of leading-edge structures. Thus, afadin plays a critical role in the proper regulation of the PDGF-induced activation of PDGF receptor and signaling by Ras-ERK. This effect, which is mediated by SHP-2, impacts cellular morphology.

Fibroblast growth factor (FGF)-1 and PDGF-B-like factors have been implicated in the pathobiology of RA and animal models of this disease. Since the receptors for FGF-1 and PDGF are tyrosine kinases, we examined the expression of tyrosine phosphorylated proteins (phosphotyrosine, P-Tyr) in synovial tissues from patients with RA and osteoarthritis (OA), and rats with streptococcal cell wall (SCW) and adjuvant arthritis (AA). Synovia from patients with RA and LEW/N rats with SCW and AA arthritis, in contrast to controls, stained intensely with anti-P-Tyr antibody. The staining colocalized with PDGF-B and FGF-1 staining. Comparative immunoblot analysis showed markedly enhanced expression of a 45-kD P-Tyr protein in the inflamed synovia. Treatment with physiological concentrations of dexamethasone suppressed both arthritis and P-Tyr expression in AA. P-Tyr was only transiently expressed in athymic nude Lewis rats and was not detected in relatively arthritis-resistant F344/N rats. These data suggest that (a) FGF-1 and PDGF-B-like factors are upregulated and may induce tyrosine phosphorylation of proteins in vivo in inflammatory joint diseases, (b) persistent high level P-Tyr expression is T lymphocyte dependent, correlates with disease severity, and is strain dependent in rats, (c) corticosteroids, in physiological concentrations, downregulate P-Tyr expression in these lesions.

In studies aimed at identifying and characterizing pp60c-src substrates that participate in the enhanced mitogenic response to epidermal growth factor (EGF) observed in murine C3H10T1/2 fibroblasts overexpressing c-src, we have identified a 75-kDa protein (p75) whose properties are consistent with those expected of such a substrate. We present evidence to show that p75 is immunologically related to a recently described, cytoskeleton-associated, pp60v-src substrate [Wu et al. (1991). Mol. Cell. Biol., 11, 5113-5124), and that its phosphotyrosine content is increased cooperatively by c-src overexpression and EGF stimulation. p75 is rapidly (within 2 min) phosphorylated on tyrosine upon EGF treatment and undergoes a second, prolonged phase of tyrosyl phosphorylation from 7 to 21 h after EGF addition, suggesting that tyrosyl phosphorylation of p75 is important for late as well as early events following EGF receptor activation. Enhanced tyrosyl phosphorylation of p75 is also seen when cells overexpressing c-src are treated with platelet-derived growth factor (PDGF), but significantly less phosphorylation is observed with insulin and fibroblast growth factor (FGF). Both basal and EGF-induced tyrosyl phosphorylation of p75 are reduced in cells overexpressing mutated forms of c-src (unmyristylated, or kinase deficient) as compared with wild-type c-src overexpressers, indicating the dependence of the enhanced tyrosyl phosphorylation on membrane-associated, enzymatically active pp60c-src. In cellular fractionation experiments p75 partitions with the cytosol, while immunofluorescence studies reveal a striking colocalization with pp60c-src at the plasma membrane and in the perinuclear region. Partial co-staining of p75 and actin occurs at the cell's periphery. These data provide evidence for p75 being a direct substrate of pp60c-src. The possible role of p75 in the enhanced response to EGF seen in c-src overexpressers is discussed.

Cultured vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats express both alpha and beta isoforms of the platelet-derived growth factor (PDGF) receptors at high levels (100,000 and 240,000 sites/cell, respectively). In this cell type, PDGF-BB elicited a mitogenic response; however, PDGF-AA increased only protein synthesis without activating DNA synthesis. Protein kinase C (PKC) was activated by PDGF-AA as well as PDGF-BB with concomitant translocation from cytosol to membrane fractions. However, the hypertrophic effect of PDGF-AA was not affected by depletion of cellular PKC, whereas the mitogenic action of PDGF-BB was partially attenuated by the depletion. Following incubation with PDGF-AA or -BB, phospholipase C-gamma 1 (PLC-gamma 1) and phosphatidylinositol 3-kinase were tyrosine phosphorylated; however, the phosphorylation of Ras-GTPase-activating protein was induced only by PDGF-BB. Both PDGF isoforms resulted in a prompt and transient increase in the level of 1,2-diacylglycerol (DAG), presumably through the action of PLC-gamma 1. After returning to basal levels, the rate of DAG synthesis steadily increased for at least 15 min due to activation of phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC). Incubation with PDGF-BB-activated phospholipase D (PLD) in a PKC-dependent manner resulting in the formation of phosphatidic acid (PA). PA was also formed by the sequential reactions of PC-PLC and DAG kinase in the PDGF-BB-stimulated VSMC, and these sequential reactions were not affected by PKC depletion. In contrast, PDGF-AA stimulation did not result in increased PA synthesis as neither PLD nor DAG kinase activities were affected. PA may be a significant second messenger in the activation of DNA synthesis by PDGF-BB. These results indicate that signaling mechanisms of the PDGF-alpha and -beta receptors in VSMC are distinctly different in signal transduction in VSMC and that the alpha receptor promotes cellular hypertrophy (but not hyperplasia), whereas a mitogenic response is mediated only through the beta receptor.

The cyclin-dependent kinase inhibitor p21(Cip1) is up-regulated in response to mitogenic stimulation in various cells. PPARgamma ligands troglitazone (TRO, 10 microm) and rosiglitazone (RSG, 10 microm) attenuated the induction of p21(Cip1) protein by platelet-derived growth factor (PDGF) and insulin without affecting cognate mRNA levels in rat aortic smooth muscle cells (RASMC). The protein kinase Cdelta (PKCdelta) inhibitor rottlerin also blocked the induction of p21(Cip1) protein, whereas the conventional PKC isotype inhibitor Gö 6976 had no effect. Kinetic studies using the protein synthesis inhibitor cycloheximide showed that TRO, RSG, and rottlerin shortened the half-life of p21(Cip1) protein. TRO, RSG, and rottlerin inhibited PDGF-induced expression of p21(Cip1), but they did not affect insulin-induced expression of p21(Cip1). Both ligands inhibited PKCdelta enzymatic activity in PDGF-stimulated RASMC but not in insulin-stimulated cells. Adenovirus-mediated overexpression of PKCdelta rescued the down-regulation of p21(Cip1) expression both by TRO and RSG in PDGF-treated RASMC. These data suggested that the PKCdelta pathway plays a critical role in PDGF-induced expression of p21(Cip1) in RASMC and may be the potential target for PPARgamma ligand effects. Src kinase-dependent tyrosine phosphorylation of PKCdelta was decreased substantially by TRO and RSG. Tyrosine phosphorylation and activation of c-Src in response to PDGF were unaffected by either PPARgamma ligand. Protein-tyrosine-phosphatase inhibitors sodium orthovanadate and dephostatin prevented PPARgamma ligand effects on PKCdelta tyrosine phosphorylation and enzymatic activity. Both inhibitors also reversed PPARgamma ligand effects on p21(Cip1) expression in PDGF-treated RASMC. PPARgamma ligands enhanced protein-tyrosine-phosphatase activity in RASMC, which may be the mechanism for decreased PKCdelta tyrosine phosphorylation and activity. PPARgamma ligands regulate p21(Cip1) at a post-translational level by blocking PKCdelta signaling and accelerating p21(Cip1) turnover.

Human Platelet Derived Growth Factors (PDGF) are potent mitogens for mesenchymal cells and encoded by two related genes, the A- (or 1-) and B- (or 2-) chain. The latter is known as the human homolog (c-sis) of the v-sis oncogene. We investigated the expression and cytokine-mediated regulation of PDGF A- and B-chain mRNA in endoderm-derived cells, i.e. cultured human pancreatic adenocarcinoma cells. Northern blot analysis revealed that out of 14 cells lines 11 were positive for the A-chain and 10 for the B-chain. Tumor Necrosis Factor (TNF) -alpha and -beta, but not Interferon (IFN) -gamma, drastically upregulate the mRNA levels for PDGF B-chain and for the A-chain in a dose-dependent manner in nearly every pancreatic tumor cell line investigated (n = 6). With respect to the signal pathway stimulated by TNF, no evidence emerged for an activation of protein kinase A. The inhibition of protein kinase C by staurosporine (in the absence or presence of TNF) as well as its stimulation by PMA resulted in an increased mRNA level for the B-chain, indicating a functional role of PKC in this system. Furthermore, time course experiments and Cycloheximide treatment showed that the A- and B-chain mRNA are regulated by different mechanisms in transformed epithelial cells. Irrespective of these differences, the sum of their biological functions may contribute to the phenomenon of desmoplasia in pancreatic tumors by epithelial/mesenchymal interactions.

The regulation of amphiregulin, an epidermal growth factor (EGF) family member, and its effect on vascular smooth muscle cells (VSMC) were examined. Amphiregulin mRNA was upregulated by amphiregulin itself as well as alpha-thrombin. Amphiregulin caused an approximate 3-fold increase in DNA synthesis. Its effect on growth was compared with those of other mitogens, and was found to be approximately 3.5-, 2.4-, and 1.0-fold greater than those of endothelin-I (ET-I), alpha-thrombin, and platelet-derived growth factor-AB (PDGF-AB), respectively. As evidenced by Western blot analysis, amphiregulin stimulated the phosphorylation of p42/p44-mitogen-activated protein kinase (MAPK), p38-MAPK, c-Jun NH2-terminal protein kinase (JNK), and Akt/protein kinase B (PKB), respectively. By statistical analysis, the amphiregulin-induced growth effect was significantly decreased by the MAP kinase/ extracellular regulated kinase kinase-1 (MEK-1) inhibitor PD98059, p38-MAPK inhibitor SB203580, and phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor wortmannin, respectively, but was not decreased by JNK inhibitor SP600125. These results suggest that amphiregulin is the most potent mitogen of the mitogens tested, and its growth effect is mediated at least in part through the p42/p44-MAPK, p38-MAPK, and PI-3 kinase-Akt/PKB pathways in VSMC.

Protein kinase signaling cascades control most aspects of cellular function. The ATP binding domains of signaling protein kinases are the targets of most available inhibitors. These domains are highly conserved from mammals to flies. Herein we describe screening of a library of small molecule inhibitors of protein kinases for their ability to increase Drosophila lifespan. We developed an assay system which allowed screening using the small amounts of materials normally present in commercial chemical libraries. The studies identified 17 inhibitors, the majority of which targeted tyrosine kinases associated with the epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) receptors, G-protein coupled receptor (GPCR), Janus kinase (JAK)/signal transducer and activator of transcription (STAT), the insulin and insulin-like growth factor (IGFI) receptors. Comparison of the protein kinase signaling effects of the inhibitors in vitro defined a consensus intracellular signaling profile which included decreased signaling by p38MAPK (p38), c-Jun N-terminal kinase (JNK) and protein kinase C (PKC). If confirmed, many of these kinases will be novel additions to the signaling cascades known to regulate metazoan longevity.

The ability of mitogens to rapidly induce tyrosine phosphorylation of cellular proteins has been taken as evidence of participation in subsequent signaling pathways. SSeCKS, a major protein kinase C (PKC) substrate with protein scaffolding and tumor suppressive properties, becomes tyrosine phosphorylated in NIH3T3 and rodent embryo fibroblasts after short-term treatment with epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or fetal calf serum in the presence of pervanadate, but not by treatment with insulin or insulin-like growth factor-1. The relative phosphotyrosine level on SSeCKS was higher in actively dividing cells than in confluent cultures. Tyrosine phosphorylation of SSeCKS was apparent in cells deficient in Src, Fyn, Yes, or Abl tyrosine kinases or in NIH3T3 cells expressing a temperature-sensitive v-Src allele, but not in FAK-deficient embryo fibroblasts. Purified FAK or Src enzyme failed to directly phosphorylate SSeCKS in vitro. EGF failed to induce SSeCKS tyrosine phosphorylation in FAK-/- fibroblasts, indicating that the EGF receptor is probably not the direct kinase of SSeCKS. Phosphorylation under these conditions was rescued by the transient reexpression of wt-FAK but not FAK mutated at Y397, a major autophosphorylation and SH2-based docking site. Adhesion of FAK+/+ cells to fibronectin failed to significantly induce SSeCKS tyrosine phosphorylation although FAK was activated, suggesting that SSeCKS phosphorylation is mediated through a growth factor receptor-FAK rather than an integrin-FAK pathway. Moreover, PDGF could induce SSeCKS tyrosine phosphorylation in the absence of FAK activation, suggesting a role for FAK SH2-based docking rather than kinase activity. Immunofluorescence analysis showed that in FAK-/- cells, SSeCKS costains along F-actin stress fibers, in contrast to FAK+/+ cells, where most SSeCKS stains at the cell edge and along a cortical cytoskeletal matrix. This correlated with increased coprecipitation of SSeCKS with biotin-phalloidin-bound F-actin from FAK-/- compared to FAK+/+ cell lysates. Similarly, bacterially expressed, unphosphorylated SSeCKS cosedimented with F-actin in ultracentrifugation assays. These data suggest that mitogen-induced, FAK-dependent tyrosine phosphorylation of SSeCKS modulates its binding to the actin-based cytoskeleton, suggesting a role for SSeCKS in mitogen-induced cytoskeletal reorganization.

OBJECTIVE

During the development of atherosclerotic plaques, vascular smooth muscle cells (VSMCs) migrate from the media to the intima through the basement membrane and interstitial collagenous matrix, and proliferate to form neointima. Here, we investigate the mechanism of VSMC migration and proliferation caused by aggretin, a snake venom integrin alpha2beta1 agonist.

METHODS

Cultures of rat and human VSMCs were treated with aggretin and the signal transduction pathways induced by this agonist were examined by Western blotting, immunoprecipitation and electrophoretic mobility shift assay techniques.

RESULTS

Aggretin-induced VSMC proliferation was blocked by a monoclonal antibody (mAb) against integrin alpha2 (AII2E10) or against the platelet-derived growth factor receptor (PDGFR)-beta. Proliferation was also blocked by inhibition of the tyrosine kinase Src with PP2, phospholipase C (PLC) with U73122, extracellular signal-regulated kinase (ERK) with PD98059 or nuclear factor-kappa B (NF-kB) activation with pyrrolidine dithiocarbamate (PDTC). VSMC migration towards immobilized aggretin was increased in a modified Boyden chamber and this effect was blocked by alpha2beta1-Src-PLC-MAPK axis inhibitors, but not by PDTC, PDGFR-beta mAb, or a phosphoinositide-3 kinase inhibitor, LY294002. Aggretin stimulated the phosphorylation of PDGFR-beta, Src and ERK in a time-dependent manner. NF-kB translocation and platelet-derived growth factor (PDGF)-BB production were also observed. The ERK activation, NF-kB translocation and PDGF-BB production were blocked by PP2, U73122 and PD98059.

CONCLUSIONS

Aggretin induces VSMC proliferation and migration mainly through binding to integrin alpha2beta1, and subsequently activates Src, PLC and ERK pathways, inducing NF-kB activation and PDGF production.

BACKGROUND

Thrombocytopenia is a marked feature of chronic liver disease and cirrhosis. We tried to clarify whether an accumulation of platelets in the liver contributes to thrombocytopenia and liver fibrosis in chronic liver disease.

METHODS

Thirty-eight patients who underwent hepatectomy for hepatocellular carcinoma (HCC) with hepatitis C virus infection were included. The locations of platelets and Kupffer cells and the expression of platelet-derived growth factor (PDGF) receptor-β and smooth muscle actin (SMA) were identified by immunohistochemistry. Perisinusoidal mesenchymal cells that express PDGF receptor-β and SMA were interpreted as transformed hepatic stellate cells (HSCs).

RESULTS

Patients with cirrhosis had a more extensive platelet area in the liver compared to controls (5601 ± 5611 vs. 564 ± 361 μm(2), p = 0.02), although the blood platelet count significantly decreased along with the progression of liver fibrosis. In cirrhotic liver, most platelets were present in the sinusoidal space of the periportal area with inflammation, where HSCs expressing PDGF receptor-β were frequently observed. In addition, the platelet and Kupffer cell areas were significantly smaller in cancerous tissue than those in noncancerous tissues (platelet area: 492 ± 823 vs. 3643 ± 4055 μm(2), p = 0.001; Kupffer cell area: 450 ± 841 vs. 3012 ± 3051 μm(2), p = 0.001).

CONCLUSIONS

The accumulation of platelets in the liver with chronic hepatitis may be involved in thrombocytopenia and liver fibrosis through the activation of HSCs. In addition, our findings also indicate that both platelets and Kupffer cells decrease in HCC tissues.

Sinusoidal obstruction syndrome (SOS; formerly veno-occlusive disease) is a well-established complication of hematopoietic stem cell transplantation, pyrrolizidine alkaloid intoxication, and widely used chemotherapeutic agents such as oxaliplatin. It is associated with substantial morbidity and mortality. Pathogenesis of SOS in humans is poorly understood. To explore its molecular mechanisms, we used Affymetrix U133 Plus 2.0 microarrays to investigate the gene expression profile of 11 human livers with oxaliplatin-related SOS and compared it to 12 matched controls. Hierarchical clustering analysis showed that profiles from SOS and controls formed distinct clusters. To identify functional networks and gene ontologies, data were analyzed by the Ingenuity Pathway Analysis Tool. A total of 913 genes were differentially expressed in SOS: 613 being upregulated and 300 downregulated. Reverse transcriptase-PCR results showed excellent concordance with microarray data. Pathway analysis showed major gene upregulation in six pathways in SOS compared with controls: acute phase response (notably interleukin 6), coagulation system (Serpine1, THBD, and VWF), hepatic fibrosis/hepatic stellate cell activation (COL3a1, COL3a2, PDGF-A, TIMP1, and MMP2), and oxidative stress. Angiogenic factors (VEGF-C) and hypoxic factors (HIF1A) were upregulated. The most significant increase was seen in CCL20 mRNA. In conclusion, oxaliplatin-related SOS can be readily distinguished according to morphologic characteristics but also by a molecular signature. Global gene analysis provides new insights into mechanisms underlying chemotherapy-related hepatotoxicity in humans and potential targets relating to its diagnosis, prevention, and treatment. Activation of VEGF and coagulation (vWF) pathways could partially explain at a molecular level the clinical observations that bevacizumab and aspirin have a preventive effect in SOS.

Gliomas are the most common and deadly form of malignant primary brain tumors. Loss of the tumor-suppressor PTEN and activation of the receptor tyrosine kinases (RTKs) EGF receptor, c-Met, PDGF receptor and VEGF receptor are among the most common molecular dysfunctions associated with glioma malignancy. PTEN interacts with RTK-dependent signaling at multiple levels. These include the ability of PTEN to counteract PI3K activation by RTKs, as well as possible effects of PTEN on RTK activation of the MAPK pathway and RTK-dependent gene-expression regulation. Consequently, PTEN expression affects RTK-induced malignancy. Importantly, the PTEN status was recently found to be critical for the outcome of RTK-targeted clinical therapies that have been developed recently. Combining RTK-targeted therapies with therapies aimed at counteracting the effects of PTEN loss, such as mTOR inhibition, might also have therapeutic advantage. This article reviews the known molecular and functional interactions between PTEN and RTK pathways and their implications for glioma therapy.

BACKGROUND

Periodontal regeneration requires a coordinated series of events that includes not only the recruitment of periodontal ligament (PDL)-specific cells, but vascular cells as well. The mechanisms of action of enamel matrix derivative (EMD) are poorly understood, and its effects on vascular cells are unknown. The objective of this study was to examine the extent to which EMD affects angiogenesis and PDL cell recruitment.

METHODS

The effects of EMD on human microvascular endothelial cells (HMVECs) were determined by examining proliferation, chemotaxis, angiogenesis, and migration. Proliferation was determined using water-soluble tetrazolium salt (WST)-1 reagent. Chemotaxis was determined using microporous-culture well inserts. Angiogenesis was assessed on plates containing matrigel. The effects of HMVECs on the migration of PDL cells were assessed by evaluating PDL cell outgrowth from collagen gels cultured in the presence of HMVECs on fibrin matrix and surrounded by fibronectin-containing fibrin clots at 24 hours. Effects of EMD on PDL expression of vascular endothelial cell (VEGF) types (A, B, C, and D) and isoforms were determined using reverse transcription-polymerase chain reaction (RT-PCR). Production of VEGF, platelet-derived growth factor (PDGF)-AA, PDGF-BB, PDGF-AB, and transforming growth factor (TGF)-beta1 by EMD-stimulated PDL cells was assessed quantitatively in conditioned media using specific enzyme-linked immunosorbent assays (ELISAs).

RESULTS

EMD at concentrations <50 microg/ml resulted in significant (P <0.05) stimulation of HMVEC proliferation. Compared to baseline, EMD also stimulated a 100% increase in HMVEC chemotaxis when PDL cells were present (P <0.05). All doses of EMD tested (25, 50, and 100 microg/ml) increased angiogenesis in vitro. HMVECs, in combination with EMD at a concentration of 100 microg/ml, stimulated a 750% increase in migration of PDL cells from collagen gels into fibrin clots compared to controls when neither was present. RT-PCR results indicated that PDL cells expressed VEGF-A, -B, and -C and multiple isoforms of VEGF-A, including VEGF(121), VEGF(165), and VEGF(189), whether or not EMD was present in the culture media. ELISAs determined a 400% increase in VEGF concentration by PDL C cells in EMD-stimulated conditioned media and a similar increase in TGF-beta(1)-stimulated media.

CONCLUSIONS

It is likely that EMD stimulates angiogenesis directly by stimulating endothelial cells and indirectly by stimulating the production of angiogenic factors (VEGF) by PDL cells. Importantly, the data are consistent with the concept that EMD enhances bidirectional communication between HMVEC and PDL cells during angiogenesis associated with healing.

Platelet-derived growth factor (PDGF) and its cognate receptor are widely expressed on melanomas. Coexpression of the growth factor and receptor suggests their role in autocrine or paracrine growth mechanisms. Imatinib mesylate was previously reported to have specific activity in inhibiting select tyrosine kinase receptors, including PDGF and c-Kit. Melanoma cells express abundant levels of the PDGF receptor (PDGFR). Nevertheless, c-Kit expression is progressively lost as the cells take on a more highly metastatic phenotype. To investigate the potential of imatinib mesylate as a therapy for melanoma, we studied its effect on the growth of melanoma cells using an in vivo mouse model. Melanoma cells with high malignant potential (PDGFR-positive, c-Kit-negative) or low malignant potential (PDGFR-positive, c-Kit-positive) were injected subcutaneously into athymic nude mice. Mice were treated with imatinib mesylate (100 mg/kg three times weekly) or with phosphate-buffered saline for 4 to 6 wk. PDGFR-alpha and -beta were expressed on all melanoma cell lines tested. The level of PDGFR expression correlated with the metastatic potential of the melanoma cells: higher levels of PDGFR-alpha were expressed on cells with higher metastatic potential, and higher levels of PDGFR-beta were expressed on cells with lower metastatic potential. There was no significant difference in tumor size between treated and control mice. Immunohistochemical studies demonstrated inhibition of PDGFR phosphorylation on the tumors from mice treated with imatinib mesylate but not from control mice, suggesting that the receptors were functional and that the concentration of drug used was appropriate. Our data demonstrated that imatinib mesylate blocked both PDGFR-alpha and PDGFR-beta in vivo. It did not, however, affect the growth of melanoma cells expressing PDGFR, regardless of whether the cells expressed c-Kit.