Platelet-derived growth factor BB (PDGF) and PDGF receptor-beta (PDGFR) play critical roles in mesangial cell proliferation during embryonic development and in mesangioproliferative glomerulonephritis. We have shown previously that phosphatidylinositol (PI) 3 kinase/Akt and Erk1/2 mitogen-activated protein kinase (MAPK) contribute to PDGF-dependent proliferation of mesangial cells, but the mechanism by which these two enzyme cascades are activated by PDGFR signaling is not precisely known. We examined the role of c-Src tyrosine kinase in this process. PDGF increased phosphorylation of c-Src in a time-dependent manner indicating its activation. A pharmacologic inhibitor of c-Src, PP1, blocked PDGF-induced DNA synthesis with concomitant inhibition of c-Src phosphorylation. Immune-complex kinase assays of c-Src and PDGFR demonstrated inhibition of c-Src tyrosine kinase activity by PP1, without an effect on PDGFR tyrosine phosphorylation. Both PP1 and expression of dominant negative c-Src inhibited PDGF-induced PI 3 kinase, resulting in attenuation of Akt kinase activity. Expression of constitutively active c-Src increased Akt activity to the same extent as with PDGF. Constitutively active c-Src augmented PDGF-induced Akt activity, thus contributing to Akt signaling. Inhibition of c-Src tyrosine kinase blocked PDGF-stimulated MAPK activity and resulted in attenuation of c-fos gene transcription with concomitant prevention of Elk-1 transactivation. Furthermore, inhibition of c-Src increased p27(Kip1) cyclin kinase inhibitor, and attenuated PDGF-induced pRb phosphorylation and CDK2 activity. These data provide the first evidence in mesangial cells that PDGF-activated c-Src tyrosine kinase relays signals to PI 3 kinase/Akt and MAPK. Furthermore our results demonstrate that c-Src integrates signals into the nucleus to activate CDK2, which is required for DNA synthesis.

BACKGROUND

The migration of vascular smooth muscle cells (VSMCs) is a key event in the pathogenesis of many vascular diseases. We have previously shown that VSMC migration in response to platelet-derived growth factor (PDGF) is suppressed when cultured cells are growth-arrested and induced to differentiate. The present study was undertaken to elucidate the mechanism of this suppression.

RESULTS

While both proliferating and growth-arrested VSMCs upregulated expression of the immediate early response genes, c-fos and JE (monocyte chemoattractant protein 1), growth-arrested VSMCs exhibited much smaller changes in intracellular calcium in response to PDGF and failed to activate the calcium/calmodulin-dependent protein kinase II (CaM kinase II). Blocking calcium-calmodulin interactions (50 mumol/L W7) or the activation of CaM kinase II (10 mumol/L KN62) in proliferating cells blocked their migration by more than 90%, whereas inhibition of protein kinase C activation had no significant effect on migration. Pretreatment of growth-arrested VSMCs with the calcium ionophore ionomycin resulted in an approximately 2.5-fold activation of CaM kinase II and increased migration of growth-arrested cells to 84 +/- 6% that of proliferating cells. These effects of ionomycin were blocked by inhibitors of CaM kinase II. Constitutively activated (ie, calcium/calmodulin-independent) CaM kinase II introduced by gene transfection into growth-arrested cells significantly increased migration toward PDGF from < 20% to>> 70% that of proliferating cells.

CONCLUSIONS

These results demonstrate that activation of CaM kinase II is required for VSMC migration, that its activation in response to PDGF is suppressed in growth-arrested VSMCs, and that this suppression of CaM kinase II activation is responsible, in large part, for the failure of growth-arrested VSMCs to migrate toward PDGF.

The early response to vascular injury is characterized by migration of inflammatory cells, including monocytes, and platelets to the damaged vessel wall. These inflammatory cells may serve as a source of growth factors and cytokines that stimulate vascular smooth muscle cell (VSMC) migration and proliferation associated with intimal hyperplasia. JE is a platelet-derived growth factor (PDGF)-inducible "early" gene that encodes a monocyte chemoattractant and, as such, could play an important role in inflammation. We now report that JE mRNA levels are increased in intact aorta after balloon injury. The time course of this increase, with maximal levels at 4 hours, is similar to that seen in PDGF-treated cultured rat aortic VSMCs. The accumulation of JE mRNA in cultured VSMCs is accompanied by a marked increase in the secretion of JE protein. The elevation of JE mRNA levels in VSMCs shows specificity for PDGF, because angiotensin II, alpha-thrombin, and epidermal growth factor fail to increase JE mRNA levels. In contrast to 3T3 fibroblasts, the accumulation of JE mRNA in VSMCs in response to PDGF is predominantly due to an increase in JE mRNA stability. The accumulation of JE mRNA in VSMCs stimulated by PDGF appears to occur via a novel pathway(s) independent of Ca2+ mobilization, Na(+)-H+ exchange, protein kinase C activation, or elevation in cAMP levels. These findings suggest that VSMCs may take part in the early inflammatory response after injury through the production of JE, a potent monocyte chemoattractant. Finally, our data suggest that JE may be a marker for PDGF-specific effects on VSMCs, both in vitro and in vivo. Thus, in addition to direct effects on VSMC growth and migration, PDGF may play a role in the early inflammatory response after vascular injury by inducing chemoattractants, such as that encoded by JE.

OBJECTIVE

Pirfenidone has been recently shown to reduce dimethynitrosamine-induced liver fibrosis in the rat, but no information are available on the effect of this drug on cultured hepatic stellate cells (HSC).

METHODS

HSC proliferation was evaluated by measuring bromodeoxyuridine incorporation; PDGF-receptor autophosphorylation, extracellular signal-regulated kinase (ERK1/2) and pp70(S6K) activation were evaluated by western blot; protein kinase C activation was evaluated by western blot and by ELISA; type I collagen accumulation and alpha1(I) procollagen mRNA expression were evaluated by ELISA and northern blot, respectively.

RESULTS

Pirfenidone significantly inhibited PDGF-induced HSC proliferation, starting at a concentration of 1 microM, with a maximal effect at 1000 microM, without affecting HSC viability and without inducing apoptosis. The inhibition of PDGF-induced HSC proliferation was associated neither with variations in PDGF-receptor autophosphorylation, or with ERK1/2 and pp70(S6K) activation. On the other hand, pirfenidone was able to inhibit PDGF-induced activation of the Na(+)/H(+) exchanger, which is involved in PDGF-induced HSC proliferation in HSC, with a maximal effect at 1000 microM and inhibited PDGF-induced protein kinase C activation. Pirfenidone 100 and 1000 microM inhibited type I collagen accumulation in the culture medium induced by transforming growth factor(beta1) by 54% and 92%, respectively, as well as TGF(beta1)-induced alpha1(I) procollagen mRNA expression.

RESULTS

Pirfenidone could be a new candidate for antifibrotic therapy in chronic liver diseases.

Platelet-derived growth factor (PDGF) has been previously shown to be homologous to the transforming gene of simian sarcoma virus (v-sis), and inappropriate expression of the cellular counterpart of the v-sis gene (c-sis) has been implicated in the generation of mesenchymal tumors. The U-2 OS human osteosarcoma line was shown to contain multiple c-sis transcripts. Immunoprecipitation experiments with antiserum to PDGF identified a variety of polypeptides ranging in size from 18,000 to 165,000 daltons that were immunoprecipitated specifically from U-2 OS cell extracts. The osteosarcoma also was shown to secrete a 29,000-dalton protein having the serological and structural characteristics of PDGF.

The aberrant transcription factors associated with many human malignancies function by deregulation of tumorigenic pathways. However, identification of these pathways has come slowly. Virtually all cases of Ewing's Sarcoma and peripheral Primitive Neuroectodermal Tumor (PNET) are associated with aberrant transcription factors which fuse amino-terminal EWS with the DNA binding moiety of an ETS transcription factor (FLI-1 in 90% of cases). Attempts to identify the downstream targets of these chimeras in the Ewing Family Tumors (EFT) on the basis of differential gene regulation have produced little association with tumor biology. As an alternative approach, we have used highly efficient retroviral systems to biologically screen cDNA derived from cells transformed by EWS/FLI-1. We have identified the recently described PDGF-C as target of EWS/ETS transcriptional deregulation. This transcriptional deregulation is specific to EWS/FLI. PDGF-C possesses substantial biologic activity in vitro and in vivo. It is expressed in EFT cell lines and in primary tumors. Within these EFT cell lines, PDGF-C expression is dependent upon EWS/FLI activity. These results suggest that PDGF-C may be a significant mediator of EWS/FLI driven oncogenesis.

The Ewing's sarcoma family of tumors (ESFT) contain a translocation, t(11;22), which results in the novel oncogenic fusion protein EWS/FLI1. Platelet-derived growth factors (PDGF) and their receptors (PDGFR) are involved in the induction and proliferation of numerous solid tumors and are the potential candidates for novel targeted antitumor therapy. Since a relation was reported between PDGF-C and EWS/FLI1, we sought to characterize the PDGF signaling pathway in ESFT. Eight out of nine ESFT cell lines were found to express significant levels of beta-PDGFR. Interestingly, none of the tested cell lines expressed alpha-PDGFR, which is the receptor isotype required for PDGF-C binding. By immunohistochemical staining 47 of 52 (90.4%) archival tumor samples from patients with ESFT were positive for beta-PDGFR. ESFT cell lines were treated with PDGF-AA or PDGF-BB ligands to evaluate downstream signaling. Autophosphorylation of beta-PDGFR and tyrosine phosphorylation of PLC-gamma, PI3Kp85 and Shc were detected only in PDGF-BB-stimulated cells that express beta-PDGFR. Receptor function was further evaluated using chemotaxis assays that showed TC-32 cell migration towards PDGF-BB. A specific PDGFR kinase inhibitor AG1295 blocked beta-PDGFR activation, downstream signaling, growth in cell culture and chemotaxis of TC-32 cells. AG1295 also delayed tumor formation and prolonged survival in an ESFT animal model. We conclude that ESFT express beta-PDGFR and that this is a functional and potentially crucial signaling pathway. Therefore, beta-PDGFRs may provide a novel therapeutic target in ESFT that can be utilized to design better treatment modalities.

BACKGROUND

Masitinib is a tyrosine kinase inhibitor targeting stem cell factor receptor (c-kit) and platelet-derived growth factor (PDGF) receptor, which are expressed on several cell types including mast cells and bronchial structural cells, respectively. We hypothesized that c-kit and PDGF receptor inhibition may decrease bronchial inflammation and interfere with airway remodeling, which are crucial features of severe asthma.

OBJECTIVE

The primary endpoint was the percent change from baseline in oral corticosteroids after 16 weeks of treatment. Change in asthma control (asthma control questionnaire), exacerbation rate, pulmonary function tests, rescue medication requirement and safety were secondary endpoints.

METHODS

A 16-week randomized, dose-ranging (3, 4.5, and 6 mg/kg/day), placebo-controlled study was undertaken in 44 patients with severe corticosteroid-dependent asthma who remained poorly controlled despite optimal asthma management.

RESULTS

At 16 weeks of treatment, a comparable reduction in oral corticosteroids was achieved with masitinib and placebo (median reduction of -78% and -57% in the masitinib and placebo arms, respectively). Despite this similar reduction, the Asthma Control Questionnaire score was significantly better in the masitinib arm as compared to placebo with a reduction by 0.99 unit at week 16 (P < 0.001) vs 0.43 unit in the placebo arm. Masitinib therapy was associated with more transient skin rash and edema.

CONCLUSIONS

Masitinib, a c-kit and PDGF-receptor tyrosine kinase inhibitor, may represent an innovative avenue of treatment in corticosteroid-dependent asthma. These preliminary results warrant further long-term clinical studies in severe asthma

We have investigated the roles of the phosphotyrosine phosphatase Syp (also called SH-PTP2), phospholipase C (PLC) gamma1, rasGTPase Activating Protein (rasGAP) and the adapter molecules Nck and Shc in the mitogenic response induced by PDGF in fibroblasts. Two separate approaches were used to inhibit the biological activity of these signalling proteins in vivo. Either glutathione S-transferase (GST) fusion proteins containing the SH2 domains of these proteins, or antibodies specific for these polypeptides, were microinjected into cells. GST-SH2 fusion proteins are expected to act as dominant inhibitors by competing for physiological SH2-mediated interactions, while microinjected antibodies can directly block protein functions. Inhibition of PLCgamma, Syp, Shc and Nck signals blocked PDGF-stimulated cells in G1 showing a requirement for these proteins for S-phase entry. Inhibition of rasGAP, in contrast, had no effect on S-phase entry. We next examined which of these signals were required for PDGF-induced cFos expression, a Ras-dependent event important for signalling. By using the same approaches with cells expressing beta-galactosidase under the control of a c-fos promoter, we showed that PLCgamma, Syp and Shc were necessary for ligand-induced cFos expression whereas Nck and phosphatidylinositol 3-kinase alpha were not. From these results we concluded that PDGF generates Ras-dependent and Ras-independent pathways important for DNA synthesis.

Analysis of Tenascin-C (TN-C) knockout mice revealed novel roles for this extracellular matrix (ECM) protein in regulation of the developmental programme of oligodendrocyte precursor cells (OPCs), their maturation into myelinating oligodendrocytes and sensitivity to growth factors. A major component of the ECM of developing nervous tissue, TN-C was expressed in zones of proliferation, migration and morphogenesis. Examination of TN-C knockout mice showed roles for TN-C in control of OPC proliferation and migration towards zones of myelination [E. Garcion et al. (2001) Development, 128, 2485-2496]. Extending our studies of TN-C effects on OPC development we found that OPCs can endogenously express TN-C protein. This expression covered the whole range of possible TN-C isoforms and could be strongly up-regulated by leukaemia inhibitory factor and ciliary neurotrophic factor, cytokines known to modulate OPC proliferation and survival. Comparative analysis of TN-C knockout OPCs with wild-type OPCs reveals an accelerated rate of maturation in the absence of TN-C, with earlier morphological differentiation and precocious expression of myelin basic protein. TN-C knockout OPCs plated on poly-lysine displayed higher levels of apoptosis than wild-type OPCs and there was also an earlier loss of responsiveness to the protective effects of platelet-derived growth factor (PDGF), indicating that TN-C has anti-apoptotic effects that may be associated with PDGF signalling. The existence of mechanisms to compensate for the absence of TN-C in the knockout is indicated by the development of oligodendrocytes derived from TN-C knockout neurospheres. These were present in equivalent proportions to those found in wild-type neurospheres but displayed enhanced myelin membrane formation.

Pancreatic cancer is often associated with an intense production of interstitial collagens, known as the desmoplastic reaction. To understand more about desmoplasia in pancreatic cancer, the expression of mRNA for type I and III collagens and potent desmoplastic inducing growth factors transforming growth factor-beta (TGF-beta), connective tissue growth factor (CTGF), acidic and basic fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) A and C and epidermal growth factor (EGF) was analysed by quantitative RT-PCR. Expression of both collagens in 23 frozen primary pancreatic cancer nodules was significantly higher than that in 15 non-neoplastic pancreatic tissues. The expressions of mRNAs for TGF-beta, acidic FGF, basic FGF and PDGF C were likewise higher in surgical cancer nodules, while that of CTGF, PDGF A and EGF were not. Among these growth factors, the expression of TGF-beta mRNA showed the most significant correlation with that of collagens (P<0.0001). By immunohistochemistry, TGF-beta showed faint cytoplasmic staining in cancer cells. In contrast, isolated cells, mainly located on the invasive front surrounding cancerous nests, were prominently and strongly stained. These TGF-beta-positive cells contained a segmented nucleus, were negative for anti-macrophage (CD68) and positive for anti-granulocyte antibodies, indicating their granulocytic nature. In conclusion, TGF-beta seemed to play a major role among the various growth factors in characteristic overproduction of collagens in pancreatic cancer. Moreover, the predominant cells that express TGF-beta were likely to be infiltrated granulocytes (mostly are neutrophils) and not pancreatic cancer cells.

Cediranib is a potent inhibitor of the VEGF receptor (VEGFR)-2 and VEGFR-3 tyrosine kinases. This study assessed the activity of cediranib against the VEGFR-1 tyrosine kinase and the platelet-derived growth factor receptor (PDGFR)-associated kinases c-Kit, PDGFR-α, and PDGFR-β. Cediranib inhibited VEGF-A-stimulated VEGFR-1 activation in AG1-G1-Flt1 cells (IC(50) = 1.2 nmol/L). VEGF-A induced greatest phosphorylation of VEGFR-1 at tyrosine residues Y1048 and Y1053; this was reversed by cediranib. Potency against VEGFR-1 was comparable with that previously observed versus VEGFR-2 and VEGFR-3. Cediranib also showed significant activity against wild-type c-Kit in cellular phosphorylation assays (IC(50) = 1-3 nmol/L) and in a stem cell factor-induced proliferation assay (IC(50) = 13 nmol/L). Furthermore, phosphorylation of wild-type c-Kit in NCI-H526 tumor xenografts was reduced markedly following oral administration of cediranib (≥1.5 mg/kg/d) to tumor-bearing nude mice. The activity of cediranib against PDGFR-β and PDGFR-α was studied in tumor cell lines, vascular smooth muscle cells (VSMC), and a fibroblast line using PDGF-AA and PDGF-BB ligands. Both receptor phosphorylation (IC(50) = 12-32 nmol/L) and PDGF-BB-stimulated cellular proliferation (IC(50) = 32 nmol/L in human VSMCs; 64 nmol/L in osteosarcoma cells) were inhibited. In vivo, ligand-induced PDGFR-β phosphorylation in murine lung tissue was inhibited by 55% following treatment with cediranib at 6 mg/kg but not at 3 mg/kg or less. In contrast, in C6 rat glial tumor xenografts in mice, ligand-induced phosphorylation of both PDGFR-α and PDGFR-β was reduced by 46% to 61% with 0.75 mg/kg cediranib. Additional selectivity was showed versus Flt-3, CSF-1R, EGFR, FGFR1, and FGFR4. Collectively, these data indicate that cediranib is a potent pan-VEGFR kinase inhibitor with similar activity against c-Kit but is significantly less potent than PDGFR-α and PDGFR-β.

Airway smooth muscle (ASM) accumulation and enrichment of the extracellular matrix (ECM) with type I collagen and fibronectin are major pathologic features of airway remodeling in asthma. These ECM components confer enhanced ASM proliferation in vitro, but a requirement for specific integrin ECM receptors has not been examined. Here, we examined the mitogen platelet-derived growth factor (PDGF)-BB on beta1-integrin expression on human ASM cells cultured on these ECM substrates and defined the involvement of specific integrins in cell attachment and proliferation using integrin-neutralizing antibodies. PDGF-BB-dependent proliferation was enhanced two- to threefold by monomeric type I collagen or fibronectin and to a lesser extent by vitronectin; other interstitial ECM components (fibrillar type I and III collagen and tenascin-C) had no effect. Except for increased alpha3 expression induced by PDGF-BB and monomeric type I collagen or fibronectin, alpha1, alpha2, alpha4, alpha5, alphav, and alphavbeta3 integrins were unchanged compared with unstimulated cells on plastic. Blocking antibodies revealed alpha2beta1- and alphavbeta3-mediated attachment to monomeric type I collagen, whereas attachment to fibronectin required alpha5beta1. In contrast, enhancement of PDGF-BB-dependent proliferation by either monomeric type I collagen or fibronectin required alpha2beta1, alpha4beta1, and alpha5beta1 integrins. These data suggest multiple beta1-integrins regulate enhanced ASM proliferative responses.

During the formation of granulation tissue in a dermal wound, platelets, monocytes and other cellular blood constituents release various peptide growth factors to stimulate fibroblasts to migrate into the wound site and proliferate, in order to reconstitute the various connective tissue components. The effect on fibroblast migration and proliferation of these growth factors, and of Solcoseryl (HD), a deproteinized fraction of calf blood used to normalize wound granulation and scar tissue formation, was quantified in vitro. The presence of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta) and hemodialysate (HD) increased the number of cells in the denuded area, i.e., in the "wound space" of an artificially ruptured monolayer of LM-fibroblasts (mouse lung fibroblasts). When cell proliferation was blocked with Mitomycin C, in the first 24 h all factors, i.e., bFGF, PDGF, TGF-beta and HD, promoted cell migration, whereas after 48 h it became obvious that each factor stimulated both migration and proliferation, each in a characteristic way. The effects were significant and more distinct after 48 h, following the order: PDGF (46%) approximately bFGF (87%)>> HD (45%) approximately TGF-beta (40%)>> control (62%). The relative contributions of migration after inhibiting proliferation are given in brackets. The modulatory activity of HD was localized in its hydrophilic fraction. It was destroyed by acid hydrolysis. Furthermore, this activity could be blocked by protamine sulfate, an inhibitor blocking peptide growth factor receptor binding.

K-Ras-negative fibroblasts are defective in their steady-state expression of MMP-2. This occurs through c-K(B)-Ras dependent regulation of basal levels of AKT activity. In this report, we have extended those studies to demonstrate that in the absence of K-Ras expression, PDGF-BB fails to induce significant AKT activation, although this was not the case in N-Ras-negative cells. This phenotype was directly linked to PDGF-dependent cell migration. All of the independently immortalized K-Ras-negative cells failed to migrate upon the addition of PDGF. Only ectopic expression of c-K(B)-Ras, not c-K(A)-Ras nor oncogenic N-Ras, could restore both PDGF-dependent AKT activation and cell migration. Since most Ras binding partners can interact with all Ras isoforms, the specificity of PDGF-dependent activation of AKT and enhanced cell migration suggests that these outcomes are likely to be regulated through a c-K(B)-Ras-specific binding partner. Others have published that of the four Ras isoforms, only K(B)-Ras can form a stable complex with calmodulin (CaM). Along those lines, we provide evidence that 1) PDGF addition results in increased levels of a complex between c-K(B)-Ras and CaM and 2) the biological outcomes that are strictly dependent on c-K(B)-Ras (AKT activation and cell migration) are blocked by CaM antagonists. The PDGF-dependent activation of ERK is unaffected by the absence of K(B)-Ras and presence of CaM antagonists. This is the first example of a linkage between a specific biological outcome, cell migration, and the activity of a single Ras isoform, c-K(B)-Ras.

The primary response transcription factor, early growth response-1 (Egr-1), is rapidly activated by a variety of extracellular stimuli. Egr-1 binds to a sequence found in the promoters of genes involved in vascular injury, such as PDGF-A and tissue factor, and trans-activates their expression in endothelial cells in response to fluid shear stress. Here we show that egr-1 mRNA is increased after 30 min of flow in human aortic endothelial cell and HeLa cell cultures. Transient transfection of HeLa cells with reporter gene constructs driven by the murine or human egr-1 5' flanking sequence revealed a five- and ninefold induction, respectively, in transcriptional activity after exposure to a shear stress of 5 dynes/cm2 for 3 h. Deletion of sequences in the murine promoter containing two AP1 sites and an inhibitory Egr-1 binding sequence, did not reduce shear stress inducibility. However, progressive deletion of five serum response elements, reduced both the basal promoter activity and its capacity to be activated by shear stress. Further examination indicated that the three upstream serum response elements are predominantly responsible for shear stress activation of the egr-1 promoter. Treatment of cells with PD98059, a specific inhibitor of mitogen-activated protein kinase-1 inhibited shear stress activation of egr-1. We suggest that egr-1 activation by shear stress involves activation of Elk-1 but not c-jun activity. These data, which are consistent with previous findings for shear mediated signaling via the mitogen-activated protein kinase cascade, now implicate shear modulation of the Egr-1 transcription factor in this pathway.

NF-kappaB is a family of transcription factors that have been shown to be elevated in a variety of tumor types and in some cases central to their survival and growth. Here we present evidence that U-87 MG and U-118 MG growth is regulated by NF-kappaB and controlled by PDGF. NF-kappaB activity was suppressed by a dominant negative mutant of the human PDGF type beta receptor and PDGF-B chain neutralizing antibodies. Creation of cell lines that had inducible expression of shRNAs directed against either c-Rel or RelA inhibited growth almost 90% indicating that NF-kappaB plays a central role in glioblastoma growth.

Stimulation of fibroblast growth factor receptor-1 (FGFR-1) is known to result in phosphorylation of tyrosine 766 and the recruitment and subsequent activation of phospholipase C-gamma (PLC-gamma). To assess the role of tyrosine 766 in endothelial cell function, we generated endothelial cells expressing a chimeric receptor, composed of the extracellular domain of the PDGF receptor-alpha and the intracellular domain of FGFR-1. Mutation of tyrosine 766 to phenylalanine prevented PLC-gamma activation and resulted in a reduced phosphorylation of FRS2 and reduced activation of the Ras/MEK/MAPK pathway relative to the wild-type chimeric receptor. However, FGFR-1-mediated MAPK activation was not dependent on PKC activation or intracellular calcium, both downstream mediators of PLC-gamma activation. We report that the adaptor protein Shb is also able to bind tyrosine 766 in the FGFR-1, via its SH2 domain, resulting in its subsequent phosphorylation. Overexpression of an SH2 domain mutant Shb caused a dramatic reduction in FGFR-1-mediated FRS2 phosphorylation with concomitant perturbment of the Ras/MEK/MAPK pathway. Expression of the chimeric receptor mutant and the Shb SH2 domain mutant resulted in a similar reduction in FGFR-1-mediated mitogenicity. We conclude, that Shb binds to tyrosine 766 in the FGFR-1 and regulates FGF-mediated mitogenicity via FRS2 phosphorylation and the subsequent activation of the Ras/MEK/MAPK pathway.

The intermediate conductance calcium-activated potassium channel KCa3.1 contributes to a variety of cell activation processes in pathologies such as inflammation, carcinogenesis, and vascular remodeling. We examined the electrophysiological and transcriptional mechanisms by which KCa3.1 regulates vascular smooth muscle cell (VSMC) proliferation. Platelet-derived growth factor-BB (PDGF)-induced proliferation of human coronary artery VSMCs was attenuated by lowering intracellular Ca(2+) concentration ([Ca(2+)]i) and was enhanced by elevating [Ca(2+)]i. KCa3.1 blockade or knockdown inhibited proliferation by suppressing the rise in [Ca(2+)]i and attenuating the expression of phosphorylated cAMP-response element-binding protein (CREB), c-Fos, and neuron-derived orphan receptor-1 (NOR-1). This antiproliferative effect was abolished by elevating [Ca(2+)]i. KCa3.1 overexpression induced VSMC proliferation, and potentiated PDGF-induced proliferation, by inducing CREB phosphorylation, c-Fos, and NOR-1. Pharmacological stimulation of KCa3.1 unexpectedly suppressed proliferation by abolishing the expression and activity of KCa3.1 and PDGF β-receptors and inhibiting the rise in [Ca(2+)]i. The stimulation also attenuated the levels of phosphorylated CREB, c-Fos, and cyclin expression. After KCa3.1 blockade, the characteristic round shape of VSMCs expressing high l-caldesmon and low calponin-1 (dedifferentiation state) was maintained, whereas KCa3.1 stimulation induced a spindle-shaped cellular appearance, with low l-caldesmon and high calponin-1. In conclusion, KCa3.1 plays an important role in VSMC proliferation via controlling Ca(2+)-dependent signaling pathways, and its modulation may therefore constitute a new therapeutic target for cell proliferative diseases such as atherosclerosis.

OBJECTIVE

Collateral vessels restore only about 40% of the maximum dilatory reserve after femoral artery occlusion, whereas complete normalization is reached by increased fluid shear stress (FSS). We studied the role of known potent angiogenic growth factors (separately or in combination) in arteriogenesis by determining their expression in FSS-stimulated collaterals and close-to-collateral infusion of growth factor peptides in a rabbit model of femoral artery occlusion.

METHODS

Values of maximum collateral conductance (C(max)) and post mortem angiograms were compared to those achievable by high FSS. mRNA levels of growth factor ligands and receptors were determined in FSS-stimulated collaterals.

RESULTS

Seven days after vessel occlusion, FSS-stimulated legs showed a C(max) not significantly different from that of not occluded femoral arteries. Arteriogenesis was significantly less enhanced after growth factor treatment (MCP-1 86%, Ad5.1-FGF-4 75%, bFGF 72%, PDGF 64%, VEGF 50% of C(max) after FSS stimulation). RT-PCR showed no differential expression of FGF receptors, but an up-regulation of VEGF-receptor-2.

CONCLUSIONS

The most potent known angiogenic growth factors at high pharmacological doses reach only a fraction of the maximum conductance obtained by high FSS. Arteriogenesis differs from angiogenesis, so the main focus to markedly improve arteriogenesis should be put on the underlying mechanisms of shear stress.

To investigate expression, subcellular localization and mechanisms of translocation of phosphatidylcholine-specific phospholipase C (PC-PLC) during the cell proliferative response, biochemical, immunoblotting, and immunofluorescence analyses were performed on quiescent and mitogen-stimulated NIH-3T3 fibroblasts. Platelet-derived growth factor (PDGF), insulin and 12-O-tetradecanoylphorbol-13-acetate induced, in 10-60 min, PC-PLC translocation from a perinuclear cytoplasmic area to the plasma membrane. Following cell exposure to PDGF (60 min), the overall PC-PLC expression increased up to 2-3x, while the enzyme activity increased 5x in total cell lysates, 2x in the plasma membrane, and 4x in the nucleus; moreover, confocal laser scanning microscopy showed a progressive externalization of PC-PLC on the outer plasma membrane surface and its accumulation in the nuclear matrix. Pre-incubation of cells with the PC-PLC inhibitor tricyclodecan-9-yl potassium xanthate (D609), before PDGF-stimulation, not only reduced the enzyme activity in total cell lysates as well as in plasma membrane and nuclear fractions, but also blocked the mechanisms of PC-PLC subcellular redistribution. These effects were associated with a D609-induced long-lasting cell cycle block in Go.

The neuroprotective effects of platelet-derived growth factor (PDGF) and the major signaling pathways involved in these were examined using primary cultured mouse cortical neurons subjected to H(2)O(2)-induced oxidative stress. The specific function of the PDGF beta-receptor (PDGFR-beta) was examined by the selective deletion of the corresponding gene using the Cre-loxP system in vitro. In wild-type neurons, PDGF-BB enhanced the survival of these neurons and suppressed H(2)O(2)-induced caspase-3 activation. The prosurvival effect of PDGF-AA was less than that of PDGF-BB. PDGF-BB highly activated Akt, extracellular signal-regulated kinase (ERK), c-jun amino-terminal kinase (JNK) and p38. PDGF-AA activated these molecules at lesser extent than PDGF-BB. In particular, PDGF-AA induced activation of Akt was at very low level. The neuroprotective effects of PDGF-BB were antagonized by inhibitors of phosphatidylinositol 3-kinase (PI3-K), mitogen-activated protein kinase kinase (MEK), JNK and p38. The PDGFR-beta-depleted neurons showed increased vulnerability to oxidative stress, and less responsiveness to PDGF-BB-induced cytoprotection and signal activation, in which Akt activation was most strongly suppressed. After all, these results demonstrated the neuroprotective effects of PDGF and the signaling pathways involved against oxidative stress. The effects of PDGF-BB were more potent than those of PDGF-AA. This might be due to the activation and additive effects of two PDGFRs after PDGF-BB stimulation. Furthermore, the PI3-K/Akt pathway that was deduced to be preferentially activated by PDGFR-beta may explain the potent effects of PDGF-BB.

Although immunoreactive somatomedin (IR-SM) secretion by cultured human fibroblasts has been reported, the hormonal control of IR-SM production is not well defined. This study concerns the effects of several hormones and growth factors on IR-SM production by cultured human fibroblasts. Platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) had concentration-dependent stimulatory effects on IR-SM production when added to confluent quiescent cultures. When PDGF, FGF, macrophage growth factor (MGF), or epidermal growth factor (EGF) was added transiently for 5 h and the cells subsequently incubated in SM-C deficient, platelet-poor plasma (SM-C-deficient PPP), persistent stimulation of IR-SM production occurred. In contrast, transient exposure to other known stimuli of IR-SM production, such as human GH, T4, and insulin, resulted in no stimulation. Continuous exposure of quiescent, high density fibroblast cultures to EGF, insulin, hydrocortisone, or T4 incubated in the presence of serum-free medium alone resulted in no significant stimulation. During simultaneous incubation with PDGF and SM-C-deficient PPP, however, hydrocortisone, T4, EGF, and insulin produced concentration-dependent increases in IR-SM production. After transient exposure to PDGF, only hydrocortisone and T4 were stimulatory in the presence of SM-C-deficient PPP. We conclude that competence factors, such as PDGF and FGF, are potent stimuli of IR-SM production in the presence of serum-free medium, and that this hormonal signal can be remembered by the cell even after withdrawal of the growth factor. In contrast, progression factors, such as hydrocortisone, T4, and insulin, are effective only in nonquiescent cells. EGF and insulin require simultaneous incubation with PDGF. The PDGF-stimulated cell, therefore, responded differently to hormonal stimuli of IR-SM production than did the quiescent cell.

Ito cells play a pivotal role in the development of liver fibrosis associated with chronic liver diseases. During this process, Ito cells acquire myofibroblastic features, proliferate, and synthesize fibrosis components. Considering the reported mitogenic properties of endothelin-1 (ET-1), we investigated its effects on the proliferation of human Ito cells in their myofibroblastic phenotype. Both ET receptor A (ETA: 20%) and ET receptor B (ETB: 80%) binding sites were identified, using a selective ETA antagonist, BQ 123, and a selective ETB agonist, sarafotoxin S6C (SRTX-C). ET-1 did not stimulate proliferation of myofibroblastic Ito cells. In contrast, ET-1 inhibited by 60% DNA synthesis and proliferation of cells stimulated with either human serum or platelet-derived growth factor -BB (PDGF-BB). PD 142893, a nonselective ETA/ETB antagonist totally blunted this effect. SRTX-C was as potent as ET-1, while BQ 123 did not affect ET-1-induced growth inhibition. Analysis of the intermediate steps leading to growth-inhibition by ET-1 revealed that activation of mitogen-activated protein kinase by serum or PDGF-BB was decreased by 50% in the presence of SRTX-C. In serum-stimulated cells, SRTX-C reduced c-jun mRNA expression by 50% whereas c-fos or krox 24 mRNA expression were not affected. We conclude that ET-1 binding to ETB receptors causes a potent growth inhibition of human myofibroblastic Ito cells, which suggests that this peptide could play a key role in the negative control of liver fibrogenesis. Our results also point out that, in addition to its well known promitogenic effects, ET-1 may also exert negative control of growth on specific cells.