Growth factor therapy is an emerging treatment modality that enhances tissue vascularization, promotes healing and regeneration and can treat a variety of inflammatory diseases. Both recombinant human growth factor proteins and their gene therapy are in human clinical trials to heal chronic wounds. As platelet-derived growth factor-bb (PDGF-BB) and fibroblast growth factor-2 (FGF-2) are known to induce chemotaxis, proliferation, differentiation, and matrix synthesis, we investigated a non-viral means for gene delivery of these factors using the cationic polysaccharide chitosan. Chitosan is a polymer of glucosamine and N-acetyl-glucosamine, in which the percentage of the residues that are glucosamine is called the degree of deacetylation (DDA). The purpose of this study was to express PDGF-BB and FGF-2 genes in mice using chitosan-plasmid DNA nanoparticles for the controlled delivery of genetic material in a specific, efficient, and safe manner. PDGF-BB and FGF-2 genes were amplified from human tissues by RT-PCR. To increase the secretion of FGF-2, a recombinant 4sFGF-2 was constructed bearing eight amino-acid residues of the signal peptide of FGF-4. PCR products were inserted into the expression vector pVax1 to produce recombinant plasmids pVax1-4sFGF2 and pVax1-PDGF-BB, which were then injected into BALB/C mice in the format of polyelectrolyte nanocomplexes with specific chitosans of controlled DDA and molecular weight, including 92-10, 80-10, and 80-80 (DDA-number average molecular weight or M(n) in kDa). ELISA assays on mice sera showed that recombinant FGF-2 and PDGF-BB proteins were efficiently expressed and specific antibodies to these proteins could be identified in sera of injected mice, but with levels that were clearly dependent on the specific chitosan used. We found high DDA low molecular weight chitosans to be efficient protein expressors with minimal or no generation of neutralizing antibodies, while lowering DDA resulted in greater antibody levels and correspondingly lower levels of detected recombinant protein. Histological analyses corroborated these results by revealing greater inflammatory infiltrates in lower DDA chitosans, which produced higher antibody titers. We found, in general, a more efficient delivery of the plasmids by subcutaneous than by intramuscular injection. Specific chitosan carriers were identified to be either efficient non-toxic therapeutic protein delivery systems or vectors for DNA vaccines.

Inhibiting tyrosine kinases has recently emerged as a therapeutic modality in several forms of neoplasia. The tyrosine kinase inhibitor STI571 (IMATINIB MESYLATE; GLEEVEC; GLIVEC) is a case in point as it has shown promise in the treatment of malignancies expressing the BCR/ABL fusion protein. In addition to BCR/ABL, STI571 inhibits the tyrosine kinase moieties of several cell surface receptors including the platelet-derived growth factor (PDGF) receptors and c-Kit. Previous work demonstrated that c-Kit activation supports migration, invasion and, survival of certain colorectal carcinoma cells including DLD-1. Here we describe that blocking c-Kit with STI571 inhibits these malignant traits not only in DLD-1 cells but also in two early passage colorectal carcinoma cell strains. Specifically, STI571 inhibited anchorage-independent colony formation and cell scattering in semi-solid medium. Furthermore, it enhanced apoptosis susceptibility and abrogated invasion of DLD-1 cells through Matrigel. In addition, STI571 treatment affected the balance of the Bcl-2 family of apoptosis regulators on favor of a pro-apoptotic phenotype. Specifically, STI571 treatment of DLD-1 cells was associated with lower levels of Bcl-2 expression accompanied by de novo expression of Bcl-xS. Finally, STI571 acted as a chemosensitizing agent in DLD-1 cells when used in combination with 5-fluorouracil.

Stimulation of quiescent Balb/c 3T3 fibroblasts into S phase requires the synergistic action of platelet-derived growth factor (PDGF) and progression factors found in platelet-poor plasma (PPP). Traverse of the G1/S phase boundary and the initiation of DNA replication require functional cyclin E-cyclin-dependent kinase (Cdk) 2 and cyclin A-Cdk2 complexes; however, the mechanisms by which PDGF and PPP regulate Cdk2 activation are not known. Density-arrested fibroblasts contain low levels of cyclins E and A, and high levels of the Cdk inhibitor p27kip1. Exposure of PDGF, which stimulates cell cycle entry but not progression through G1, induces the formation of cyclin D1-Cdk4 complexes that bind p27kip1 and titrate the pool of Kip1 available to inhibit Cdk2. In addition, PDGF stimulates a moderate transient reduction in the abundance of p27kip1 protein. However, limited expression of cyclin E and cyclin A is observed after PDGF treatment, and in the absence of PPP, p27 levels are sufficient to bind and inactivate existing cyclin-Cdk complexes. Although plasma does not significantly increase the proportion of Kip1 bound to cyclin D1-Cdk4, stimulation of PDGF-treated cells with plasma does overcome the threshold inhibition of p27kip1 by further increasing the expression of cyclins E and A and decreasing the amount of Kip1 over a prolonged time period. Our results indicate that the distinct mitogenic activities of PDGF and PPP differentially influence the activation of cyclin E- and cyclin A-associated kinases that ultimately regulate entry into S phase.

Comparative analysis of cosmid clones containing the human and feline c-sis genetic regions revealed the similar structural organization of these areas in the two species. The areas shared seven different genetic regions in and around the c-sis locus and of these was related to v-sis. Another region, 1.9 kbp in size and located about 8 kbp upstream of the v-sis homologous region in the human genome, also hybridized to the main c-sis transcriptional product of 3.5 kb. Comparison with a recently described c-sis cDNA clone (Collins et al., Nature 316, 748-750 (1985)) revealed that the 1.9 kbp DNA region contained a large 5' c-sis exon of at least 1050 bp. In this exon, the presumed initiation site of the predicted PDGF-2 containing precursor protein was located and appeared to be preceded by a large untranslated region. In the region immediately upstream of this exon, a TATA box and a consensus sequence for a potential Sp1 binding site were found at similar positions in both species. This region also exhibited promoter activity when tested in an assay in which coding sequences of bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA: chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) were placed under its control. The five other DNA regions were found upstream and downstream of the human c-sis transcription unit and also in an intron. Four of them contained repetitive sequences. Hybridization analysis of human and feline c-sis containing cosmid clones with a mixed synthetic nucleotide probe, which corresponded to sequences encoding amino acid residues 2-7 of chain 1 of platelet-derived growth factor (PDGF-1), suggested that the c-sis cosmid clones did not include PDGF-1-specific genetic sequences.

Ectodomain cleavage (shedding) of transmembrane proteins by metalloproteases (MMP) generates numerous essential signaling molecules, but its regulation is not totally understood. CD44, a cleaved transmembrane glycoprotein, exerts both antiproliferative or tumor-promoting functions, but whether proteolysis is required for this is not certain. CD44-mediated contact inhibition and cellular proliferation are regulated by counteracting CD44 C-terminal interacting proteins, the tumor suppressor protein merlin (NF2) and ERM proteins (ezrin, radixin, moesin). We show here that activation or overexpression of constitutively active merlin or downregulation of ERMs inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced [as well as serum, hepatocyte growth factor (HGF), or platelet-derived growth factor (PDGF)] CD44 cleavage by the metalloprotease ADAM10, whereas overexpressed ERM proteins promoted cleavage. Merlin- and ERM-modulated Ras or Rac activity was not required for this function. However, latrunculin (an actin-disrupting toxin) or an ezrin mutant which is unable to link CD44 to actin, inhibited CD44 cleavage, identifying a cytoskeletal C-terminal link as essential for induced CD44 cleavage. Cellular migration, an important tumor property, depended on CD44 and its cleavage and was inhibited by merlin. These data reveal a novel function of merlin and suggest that CD44 cleavage products play a tumor-promoting role. Neuregulin, an EGF ligand released by ADAM17 from its pro-form NRG1, is predominantly involved in regulating cellular differentiation. In contrast to CD44, release of neuregulin from its pro-form was not regulated by merlin or ERM proteins. Disruption of the actin cytoskeleton however, also inhibited NRG1 cleavage. This current study presents one of the first examples of substrate-selective cleavage regulation.

CONCLUSIONS

Investigating transmembrane protein cleavage and their regulatory pathways have provided new molecular insight into their important role in cancer formation and possible treatment.

Platelet-derived growth factor (PDGF) has been implicated in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH), but the mechanism remains unknown. The purpose of this study was to assess whether imatinib mesylate (imatinib), an inhibitor of the tyrosine kinases of PDGF receptors (PDGFRs), prevents cerebral vasospasm after SAH in rats, and to elucidate if tenascin-C (TNC), a matricellular protein, is involved in the mechanism. Imatinib (10 or 50 mg/kg body weight) was administered intraperitoneally to rats undergoing SAH by endovascular perforation, and the effects were evaluated by neurobehavioral tests and India-ink angiography at 24-72 h post-SAH. Western blotting and immunohistochemistry were performed to explore the underlying mechanisms in cerebral arteries at 24h post-SAH. Recombinant TNC was administered intracisternally to imatinib-treated SAH rats, and the effects were evaluated by neurobehavioral tests, India-ink angiography and immunohistochemistry at 24 h post-SAH. Both dosages of imatinib significantly prevented post-SAH neurological impairments and vasospasm at 24-72 h. SAH caused PDGFR-β upregulation, PDGFR activation, mitogen-activated protein kinase activation, and TNC upregulation in the spastic cerebral arteries, all of which were significantly suppressed by imatinib treatment. Recombinant TNC reversed the anti-vasospastic effects and protein expression changes by imatinib. This study suggests that imatinib prevents cerebral vasospasm at least partly via inhibiting the upregulation of TNC, implying that TNC may be a new therapeutic target for post-SAH vasospasm.

Homodimers of the platelet-derived growth factor (PDGF) A-chain are strong mitogens for cells of mesenchymal origin. Differences in the levels of expression of the PDGF A-chain gene have been reported in both normal and transformed cell lines, suggesting that transcription of the PDGF A-chain gene is highly regulated. We have now identified two S1-hypersensitive sites which flank a 13-base pair oligo(dG).oligo(dC) sequence located 70-82 base pairs upstream of the transcription initiation site. Three lines of evidence suggest that these S1-sensitive sites contribute to optimum promoter activity. Nuclear protein(s) binding to these sites were detected in gel mobility shift assays. Deletion of the S1-sensitive sites results in a 2-3-fold decrease in the transcriptional activity and eliminated sensitivity to S1 nuclease. Deletions in the oligo(dG).oligo(dC) motif also eliminated sensitivity to S1 and resulted in a 2.5-fold decrease of the promoter activity in the stable transfection assays. The results suggest that the highly G+C-rich region in the PDGF A-chain gene promoter locally induces the formation of non-B-form DNA under torsional stress which appears to be important in the transcriptional regulation of the PDGF A-chain gene in vivo.

This study examined the role of mitogen-activated protein (MAP) kinase in PDGF-BB-induced proliferation and gene expression of human mesangial cells (MC). PDGF-BB stimulation of MC increased mRNA for transforming growth factor-beta1 (TGF-beta1), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1) and increased the cell numbers. To inhibit activation of extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38, MC were infected with recombinant adenovirus containing dominant-negative mutants of ERK, JNK, and p38 (Ad-DN-ERK, Ad-DN-JNK, Ad-DN-p38, respectively), respectively. Infection of MC with Ad-DN-ERK or Ad-DN-JNK inhibited PDGF-BB-induced increase in [(3)H]thymidine incorporation and cell numbers, whereas Ad-DN-p38 did not. Ad-DN-ERK inhibited MCP-1 and PAI-1 mRNA expression in MC, but not TGF-beta1. Ad-DN-JNK and Ad-DN-p38 inhibited TGF-beta1 and MCP-1 mRNA expression, but not PAI-1. The inhibition of activator protein-1 (AP-1) in MC, by adenovirus containing dominant-negative mutant of c-Jun (Ad-DN-c-Jun), inhibited PDGF-BB-induced cell proliferation and TGF-beta1, MCP-1, and PAI-1 expressions. Furthermore, Ad-DN-JNK or Ad-DN-p38, but not Ad-DN-ERK, attenuated PDGF-BB-induced AP-1 activation in MC, indicating the involvement of JNK and p38 in AP-1 activation. Our results indicated that ERK and JNK, but not p38, participated in PDGF-BB-induced MC proliferation. PDGF-BB-induced expression of TGF-beta1 was mediated by JNK and p38, MCP-1 expression was through ERK, JNK, and p38, whereas PAI-1 expression was due to only ERK. AP-1 activation, which was partially due to JNK and p38 activations, was involved in MC proliferation and these three gene expressions. Thus, three MAP kinases seem to contribute to progression of glomerular disease via different molecular mechanisms.

Protein kinase C (PKC) is implicated in the regulation of smooth muscle contractility and growth. We have previously described the pattern of isoform expression of PKC in canine airway smooth muscle. This study identified the isoforms present in human cultured airway smooth muscle cells and also addressed the question of whether mitogenesis in these cells is associated with changes in a specific isoform, PKC-zeta. Western blot analysis revealed the presence of PKC-alpha, -betaI, and -betaII of the conventional group; PKC-delta, -theta, -epsilon, and -eta of the novel group; and PKC-zeta, -mu, and -iota of the atypical group. There was a significant increase in density of the Western blot for PKC-zeta in cells proliferating in response to 10% fetal bovine serum (FBS) to 372 +/- 115% of control values (P < 0.05; n = 3 patients) in the cytosolic fraction. Platelet-derived growth factor (PDGF) produced increases in PKC-zeta in both the cytosolic and membrane fractions to 210 +/- 49 and 443 +/- 227%, respectively, of control values (P < 0.05; n = 4 patients). There was no change in expression of PKC-alpha, -betaI, -betaII, -theta, -epsilon, -eta, -delta, or -iota in response to the same stimuli. PGE2 (1 microM) added to the cells 30 min before PDGF reduced incorporation of [3H]thymidine from 5,580 +/- 633 (SE) to 3, 980 +/- 126 dpm (P < 0.05; n = 3 patients) and, in addition, reduced expression of PKC-zeta in the membrane fraction as determined by Western blotting from 266 +/- 66 to 110 +/- 4% of control values (P < 0.05; n = 3 patients). PKC-zeta activity in stimulated cells (10% FBS), as assessed by immunoprecipitation and phosphorylation of glycogen synthase peptide, was approximately 3-fold greater than that in unstimulated cells, and the amount of PKC-zeta protein correlated with isoenzyme activity (r2 = 0.91; P < 0.02; n = 4 patients). In conclusion, this study 1) provides the first description of which isoforms of PKC are present in human cultured airway smooth muscle cells and 2) shows that proliferation of these cells is associated with upregulation of PKC-zeta. Whether activation of PKC-zeta is a primary or secondary event in airway smooth muscle cell proliferation remains to be determined.

The role of Ca2+ influx in the regulation of the sarco-endoplasmic reticulum Ca2+ATPases (SERCA) associated with intracellular Ca2+ pools was investigated during smooth muscle cell (SMC) proliferation induced by platelet-derived growth factor (PDGF). We first defined that the previously described up-regulation of the SERCA2a isoform found in vascular SMC after a 24-h stimulation with PDGF (Magnier, C. , Papp, B., Corvazier, E., Bredoux, R., Wuytack, F., Eggermont, F., Maclouf, J., and Enouf, J. (1992) J. Biol. Chem. 267, 15808-15815) was precisely associated with SMC entry into S phase as it appeared linked with [3H]thymidine incorporation. This was further confirmed by testing the effect of transforming growth factor-beta1, which inhibited both aortic SMC proliferation associated with G1 cell cycle arrest and PDGF-induced SERCA2a up-stimulation. Then, we tested the role of Ca2+ influx by using SR 33805, a new Ca2+ channel blocker, which was characterized with regard to the voltage Ca2+ channel blocker nifedipine and the capacitative entry Ca2+ blocker SKF 96365. SR 33805 was found to be the most potent inhibitor of both PDGF-induced SMC proliferation and the associated rise in intracellular Ca2+ concentration with ICCA2a and SERCA2b isoforms, in terms of activity and expression, we could determine that PDGF-induced stimulation of total SERCA activity (detected by formation of the phosphorylated intermediate, E approximately P) and of SERCA2a expression (Western blotting) were abolished when extracellular Ca2+ entry was prevented by SR 33805. This study demonstrates that SERCA2a up-regulation is: 1) related to the G1/S transition step of cell cycle and 2) dependent on Ca2+ entry during PDGF-induced SMC proliferation.

Bradykinin activates adenylate cyclase via a pathway that involves the 'up-stream' regulation of phospholipase D (PLD)-catalysed hydrolysis of phosphatidylcholine and activation of protein kinase C (PKC) in airway smooth muscle [Stevens, Pyne, Grady and Pyne (1994) Biochem. J. 297, 233-239]. Coincident signal (Gs alpha and PKC) amplification of the cyclic AMP response can be completely attenuated either by diverting PLD-derived phosphatidate or by inhibiting PKC. In this regard, the coincident signal detector type II adenylate cyclase is expressed as a 110/112 kDa polypeptide in these cells. PKC alpha is not involved in the activation of adenylate cyclase, since a B2-receptor antagonist (NPCCyclic AMP formation can also be activated by platelet-derived growth factor (PDGF), via a PKC-dependent pathway, although the magnitude of the response is less than that elicited by bradykinin. Nevertheless, these results indicate that multiple receptor types employ PKC to initiate cyclic AMP signals. PDGF (10 ng/ml) elicited the marked sustained activation of extracellular-signal-regulated kinase-2 (ERK-2), whereas bradykinin (1 microM) provoked only modest transient activation of ERK-2. Deoxyadenosine (0.1 mM), a P-site inhibitor of adenylate cyclase, blocked bradykinin-stimulated cyclic AMP formation and converted the activation of ERK-2 into a sustained response. Thus the PKC-stimulated cyclic AMP response can limit the activation of ERK-2 in response to bradykinin. These studies indicate that the integration of distinct signal pathways by adenylate cyclase can determine the kinetics of ERK activation, an enzyme that appears to be important for mitogenic progression.

Tissue factor (TF) is the cellular receptor for factor FVIIa (FVIIa), and the complex is the principal initiator of blood coagulation. The effects of FVIIa binding to TF on cell migration and signal transduction of human fibroblasts, which express high amounts of TF, were studied. Fibroblasts incubated with FVIIa migrated toward a concentration gradient of PDGF-BB at approximately 100 times lower concentration than do fibroblasts not ligated with FVIIa. Anti-TF antibodies inhibited the increase in chemotaxis induced by FVIIa/TF. Moreover, a pronounced suppression of chemotaxis induced by PDGF-BB was observed with active site-inhibited FVIIa (FFR-FVIIa). The possibility that hyperchemotaxis was induced by a putative generation of FXa and thrombin activity was excluded. FVIIa/TF did not induce increased levels of PDGF beta-receptors on the cell surface. Thus, the hyperchemotaxis was not a result of this mechanism. FVIIa induced the production of inositol-1,4, 5-trisphosphate to the same extent as PDGF-BB; the effects of FVIIa and PDGF-BB were additive. FFR-FVIIa did not induce any release of inositol-1,4,5,-trisphosphate. Thus, binding of catalytically active FVIIa to TF can, independent of coagulation, modulate cellular responses, such as chemotaxis.

The mode of phospholipase C activation initiated with platelet-derived growth factor (PDGF) has been studied in comparison with that initiated with vasopressin and bombesin in a rat fibroblast line, WFB. Stimulation of WFB cells by PDGF, vasopressin, and bombesin elicites rapid hydrolysis of polyphosphoinositides and an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i). On stimulation by PDGF, there was a lag period of about 10 s before an increase in [Ca2+]i. No measurable lag period was observed in the [Ca2+]i response induced by vasopressin or bombesin. Pretreatment of WFB cells with phorbol 12-myristate 13-acetate profoundly inhibited inositol phosphate formation evoked by vasopressin and bombesin, but enhanced to some extent inositol phosphate formation stimulated by PDGF. In membranes prepared from WFB cells, GTP markedly augmented inositol polyphosphate formation induced by vasopressin and bombesin. It was not successful in showing the PDGF-stimulated formation of inositol phosphates in the membrane preparation. The effects of GTP, guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) on polyphosphoinositide hydrolysis stimulated by growth factors were studied in WFB cells made permeable to nucleotides by treatment with either saponin or Pseudomonas aeruginosa cytotoxin. PDGF, vasopressin, and bombesin elicited inositol phosphate production in the permeabilized WFB cells in the absence of added GTP. GDP beta S, a competitive inhibitor of GTP-binding proteins (G-proteins), markedly reduced the bombesin- and vasopressin-stimulated production of inositol phosphates. However, the PDGF-stimulated production of inositol phosphates was not affected by the addition of GDP beta S. GTP gamma S, an agonist of G-proteins, largely enhanced the vasopressin- and bombesin-stimulated hydrolysis of inositol lipids when added at 10-100 microM. In the presence of GTP gamma S, the PDGF-stimulated hydrolysis of inositol lipids was not enhanced, but was reduced: 100 microM GTP gamma S reduced the stimulated hydrolysis to about a half of the control level. Only GTP gamma S, and no other nucleoside triphosphates, was found to have these effects. Activation of G-proteins in WFB cells by fluoroaluminate resulted in the inhibition of inositol phosphate production elicited with not only PDGF, but also with vasopressin and bombesin. These results indicate that a G-protein couples vasopressin and bombesin receptors to the activation of phospholipase C. Moreover, these results suggest that coupling of the PDGF receptor to phospholipase C is not mediated through a G-protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Endothelin, a 17-DKa peptide originally described as a potent vasoconstrictor, also stimulates the release of important regulators of glomerular hemodynamics such as atrial natriuretic factor and renin. In the present study we investigated the role of endothelin in the release of another potent vasoconstrictor and mitogen of human mesangial cells, the platelet-derived growth factor. Endothelin stimulated PDGF release at 12 hours and the effect was sustained for 36 hours. This effect was associated with the enhanced induction of mRNAs encoding PDGF A- and B-chain. Endothelin also induced mitogenesis in human mesangial cells which was accompanied by activation of phospholipase C with increased inositol phosphate turnover. These data suggest a mechanism by which endothelin may regulate mesangial cell function in disease states.

The gene product of the v-sis gene is closely related to the B chain of platelet-derived growth factor (PDGF). However, v-sis also encodes additional amino acids at its N and C termini, which are not represented in the sequence data of PDGF. We have constructed a series of N-terminal deletion mutants in the v-sis gene to define the minimum region required for transformation. These mutants were assayed for biological activity by using retroviral expression vectors which donate a signal sequence, required for translocation across the rough endoplasmic reticulum, to the mutant gene product. The minimal transforming region of the v-sis gene product defined by this analysis has 15 residues missing at the N terminus when compared with the PDGF-B chain. There are only two residues separating the closest transforming and nontransforming gene products. Mutant gene products lacking both the basic dipeptide processing site and the N-linked glycosylation site were found to be biologically active, indicating the dispensability of those processing steps. These results delimit the minimal transforming region of the v-sis gene product to residues 127 through 214, a total of 21 residues smaller than the PDGF-B chain.

Phosphatidylinositol 3-kinase (PI 3-K) is a lipid and protein kinase which associates with the activated platelet-derived growth factor (PDGF) receptor and other tyrosine kinases. We studied the effects of wortmannin, a selective inhibitor of PI 3-K, on the activation of extracellular-signal regulated kinase (ERK) by PDGF in cultured hepatic stellate cells, mesenchymal cells responsible for extracellular matrix synthesis within the liver. Incubation with 100 nM wortmannin, a dose which almost completely blocks PI 3-K, resulted in 50% reduction of ERK activity. Direct inhibition of ERK by wortmannin could not be considered responsible for this effect, since wortmannin did not inhibit ERK activity in vitro. Rather, inhibition of PI 3-K acts on the kinase cascade that leads to ERK activation, since PDGF-dependent phosphorylation of ERK was found to be reduced after incubation with wortmannin. Wortmannin also inhibited the increase in c-fos mRNA induced by PDGF, which is dependent on ERK activation. The results of this study show that in hepatic stellate cells PI 3-K is involved in ERK activation, although it is not necessary. These data indicate cross-talk between PI 3-K and the Ras/ERK pathway in PDGF-stimulated cells.

alpha-Thrombin, a G-protein-coupled receptor agonist, is mitogenic for neonatal vascular smooth muscle (VSM) cells, but it also causes secretion of the tyrosine kinase-coupled receptor agonist platelet-derived growth factor (PDGF). In order to determine the role of growth factors with tyrosine kinase-coupled receptors in thrombin's mitogenic signal transduction cascade, the synergistic effect of basic fibroblast growth factor (bFGF) in this system was examined. While bFGF itself is a growth factor for VSM cells, it causes a 1.7-fold synergistic effect when added together with thrombin. Herbimycin A, a specific tyrosine kinase inhibitor, both decreases thrombin-induced mitogenesis by greater than 90% and abolishes tyrosine phosphorylation of phospholipase C (PLC)-gamma-1. The magnitude and time course of the increase in intracellular free calcium concentration in response to thrombin is comparable in both the presence and absence of herbimycin A. These results provide evidence that herbimycin A specifically inhibits PLC-gamma-1 tyrosine phosphorylation without affecting VSM cell viability or calcium release. Furthermore, tyrosine phosphorylation is a necessary step in thrombin's mitogenic signal transduction cascade, but it is not essential for thrombin-induced release of calcium from intracellular stores. These data suggest that a tyrosine kinase, possibly supplied by the bFGF receptor, plays an essential role in thrombin-induced mitogenesis.

We have examined the state of tyrosine phosphorylation of ligand-bound, internalized platelet-derived growth factor (PDGF) beta-receptors. Analysis by immunofluorescence staining of cells stimulated with PDGF-BB at 37 degrees C indicated colocalization of phosphotyrosine, PDGF beta-receptors, and PDGF-BB in endosome-like vesicles. Treatment of cells with an acidic buffer, which removed cell surface-bound PDGF-BB, led to a considerable decrease in phosphorylation and kinase activity of cell surface localized PDGF beta-receptors, but not of internalized receptors. Immunoprecipitations using antisera against phosphotyrosine and the PDGF beta-receptor from metabolically labeled cells showed that a major fraction of the tyrosine-phosphorylated pool of receptors were still accessible to the acid buffer treatment after 10 min of incubation of the cells at 37 degrees C. Under these conditions, about 20-25% of the total pool of tyrosine-phosphorylated receptors were intracellular, since they remained tyrosine phosphorylated after the acid buffer treatment. A considerable pool of tyrosine-phosphorylated, internalized receptors, after 10 min of incubation of the cells at 37 degrees C, could also be detected by immunoblotting analysis, using antisera against the PDGF beta-receptor and phosphotyrosine. Analysis by in vitro kinase assays of immunoprecipitated PDGF beta-receptors, obtained from PDGF-BB-stimulated cells different times after acid wash, showed that the internalized receptors retained kinase activity. These data suggest that a pool of internalized PDGF beta-receptors remain active and may participate in signalling a considerable time after internalization.

Protein tyrosine kinases play an important role in cellular proliferation and differentiation of various cell types. To identify potential tyrosine kinases involved in glomerular functions we have utilized the polymerase chain reaction (PCR) and degenerate oligonucleotides for isolation of such genes from isolated glomeruli, cultured mesangial cell, and glomerular endothelial cells. Sequence analysis of PCR-amplified cDNAs resulted in the isolation of 24 tyrosine kinases. Here we describe for the first time the constitutive expression of 15 tyrosine kinases, tyro-1, tyro-4, tyro-6, hyk, Ptk-3, Ryk, tie, yes, lyn, tec, Jak1, Jak2, Jak3, c-abl, and flk, in renal glomeruli. In addition, Flt-1, an endothelial cell-specific receptor for vascular endothelial growth factor (VEGF), is expressed in renal mesangial cells and its gene expression is up-regulated upon the stimulation of platelet-derived growth factor (PDGF) with the concomitant up-regulation of VEGF. These data suggest that possible involvement of VEGF/Flt-1 system in cytokine-induced mesangial cell proliferations.

The human platelet-derived growth factor (PDGF) receptor complementary DNA was cloned and expressed by transfection of Chinese hamster ovary (CHO) fibroblasts. The ability of CHO cells expressing the human receptor complementary DNA (CHO-HR5) to interact with different recombinant forms of PDGF (AA and BB homodimers) was tested. Both forms of PDGF bind to the transfected receptor, stimulate the receptor tyrosine kinase activity, and elicit a mitogenic response in a manner that was indistinguishable from the responses of Balb/c 3T3 cells to AA and BB forms of PDGF can be attributed to a single type of receptor and show that the AA form, like the BB form, is a true mitogen.

BACKGROUND

We postulated that in mesangial cells exposed to high glucose, protein kinase C-zeta (PKC-zeta) is necessary for the generation of reactive oxygen species (ROS) by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and that the requirement of PKC-zeta for filamentous (F)-actin disassembly may involve ROS. To identify signaling mechanisms relevant to PKC-zeta activation and ROS generation, including phosphoinositide 3 kinase (PI3 kinase), we examined mesangial cell stimulation with platelet-derived growth factor (PDGF).

METHODS

In primary rat mesangial cells cultured in 5.6 mmol/L or 30 mmol/L d-glucose, PKC-zeta expression was identified with immunoblotting and activity was analyzed in cell membrane immunoprecipitates and by confocal immunofluorescence imaging. ROS generation was measured by dichlorofluorescein fluorescence using confocal microscopy and was inhibited by transfection of antisense against NADPH subunits p22(phox) or p47(phox) or with Tempol. F-actin disassembly was observed by dual-channel confocal fluorescence imaging. PI3 kinase activity was detected by immunoblotting of phosphorylated Akt.

RESULTS

In high glucose, generation of NADPH oxidase-dependent ROS was dependent on PKC-zeta. Conversely, sustained PKC-zeta activity was dependent on ROS generation, suggesting a positive feedback. PKC-zeta-dependent F-actin disassembly in high glucose required ROS generation. PDGF stimulated NADPH oxidase generation of ROS through a PKC-zeta mechanism that was independent of Akt phosphorylation and remained unchanged in high glucose.

CONCLUSIONS

In high glucose, mesangial cell PKC-zeta is required for ROS generation from NADPH oxidase similar to PDGF stimulation of PKC-zeta-dependent ROS generation through a pathway independent of PI3 kinase. F-actin disassembly in high glucose also requires ROS. A positive feedback loop occurs between ROS and the activation of PKC-zeta in high glucose.

Using a cDNA microarray screening approach, we have identified seven novel thrombin-responsive genes in human umbilical vein endothelial cells that were verifiable by Northern blot analysis. Among them CL-100, a dual-specificity phosphatase also known as MAP kinase phosphatase-1 (MKP-1), showed greatest induction by thrombin. Steady-state levels of CL-100 mRNA induction by thrombin peaked at 1 h and declined rapidly (t1/2 approximately 45 min). Induction by thrombin was protease-activated receptor-1-mediated, protein synthesis-independent, and transcriptionally regulated. Metabolic labeling followed by immunoprecipitation verified that the thrombin-induced CL-100 mRNA was translated into protein. We found that both Src-kinase and p42/p44 ERK activity are critical for thrombin-induced CL-100 expression, whereas phosphatidylinositol 3-kinase and protein kinase C activity were not required. Antisense-mediated inhibition of CL-100 was shown to prolong thrombin-induced ERK activity in endothelial cells, concomitant with an inhibition in thrombin-induced PDGF-A (platelet-derived growth factor A) and PDGF-B gene expression and an up-regulation in thrombin-induced VCAM-1 and E-selectin gene expression. Inhibition of ERK activation by PD98059 in endothelial cells was shown to potentiate thrombin-induced expression of PDGF-B (approximately 3-fold) while inhibiting thrombin-induced VCAM-1 and E-selectin gene expression by 60 and 70%, respectively. These results suggested that induced expression of the CL-100 phosphatase and its subsequent regulation of ERK activity play a key regulatory role in the thrombin signaling pathway and in the transcriptional regulation of pathologically important "endothelial cell activation genes."

The distinct effects of cytokines on cellular growth and differentiation suggest that specific signaling pathways mediate these diverse biological activities. Fibroblast growth factors (FGFs) are well-established inhibitors of skeletal muscle differentiation and may operate via activation of specific signaling pathways distinct from recently identified mitogen signaling pathways. We examined whether platelet-derived growth factor (PDGF)-activated signaling pathways are sufficient to mediate FGF-dependent repression of myogenesis by introducing the PDGF beta receptor into a mouse skeletal muscle cell line. Addition of PDGF-BB to cells expressing the PDGF beta receptor activated the PDGF beta receptor tyrosine kinase, stimulated mitogen-activated protein (MAP) kinase, and increased the steady-state levels of junB and c-fos mRNAs. Despite the activation of these intracellular signaling molecules, PDGF beta receptor activation elicited no detectable effect on cell proliferation or differentiation. In contrast to PDGF-BB, addition of FGF-2 to myoblasts activated signaling pathways that resulted in DNA synthesis and repression of differentiation. Because of the low number of endogenous FGF receptors expressed, FGF-stimulated signaling events, including tyrosine phosphorylation and activation of MAP kinase, could be detected only in cells expressing higher levels of a transfected FGF receptor cDNA. As the PDGF beta receptor- and FGF receptor-stimulated signaling pathways yield different biological responses in these skeletal muscle cells, we hypothesize that FGF-mediated repression of skeletal muscle differentiation activates signaling pathways distinct from those activated by the PDGF beta receptor. Activation of PDGF beta receptor tyrosine kinase activity, stimulation of MAP kinase, and upregulation of immediate-early gene expression are not sufficient to repress skeletal muscle differentiation.

We tested the hypothesis that Src family kinases (SFK) contribute to c-Cbl-mediated degradation of the platelet-derived growth factor (PDGF) alpha receptor (alphaPDGFR). Using either a receptor mutant that does not engage SFKs (F72/74), or cells that that lack SFKs, we found that SFKs contributed to degradation of the alphaPDGFR. Overexpression of c-Cbl also reduced the receptor half-life, but only if the receptor was able to engage SFKs. In cultured cells, prolonging the half-life of the receptor correlated with enhanced signaling and more efficient S phase entry, whereas accelerating receptor degradation had the opposite effect. Consistent with these tissue culture findings, there was a statistically significant increase in the onset of a proliferative retinal disease when animals were injected with cells expressing the F72/74 receptor, as compared with cells expressing the WT receptor. Our findings suggest that SFKs cooperate with c-Cbl to negatively regulate the alphaPDGFR, and that the SFK/c-Cbl suppression of alphaPDGFR output is relevant to the onset and progression of a proliferative disease.