Tissue plasminogen activator (tPA) is a serine protease involved in the degradation of blood clots through the activation of plasminogen to plasmin. Here we report on the identification of tPA as a specific protease able to activate platelet-derived growth factor C (PDGF-C). The newly identified PDGF-C is secreted as a latent dimeric factor (PDGF-CC) that upon proteolytic removal of the N-terminal CUB domains becomes a PDGF receptor alpha agonist. The CUB domains in PDGF-CC directly interact with tPA, and fibroblasts from tPA-deficient mice fail to activate latent PDGF-CC. We further demonstrate that growth of primary fibroblasts in culture is dependent on a tPA-mediated cleavage of latent PDGF-CC, generating a growth stimulatory loop. Immunohistochemical analysis showed similar expression patterns of PDGF-C and tPA in developing mouse embryos and in tumors, indicating both autocrine and paracrine modes of activation of PDGF receptor-mediated signaling pathways. The identification of tPA as an activator of PDGF signaling establishes a novel role for the protease in normal and pathological tissue growth and maintenance, distinct from its well-known role in plasminogen activation and fibrinolysis.

The cancer drug imatinib inhibits the tyrosine kinases c-Abl, c-Kit, and the PDGF receptor. Imatinib is less effective against c-Src, which is difficult to understand because residues interacting with imatinib in crystal structures of Abl and c-Kit are conserved in c-Src. The crystal structure of the c-Src kinase domain in complex with imatinib closely resembles that of Abl*imatinib and c-Kit*imatinib, and differs significantly from the inactive "Src/CDK" conformation of the Src family kinases. Attempts to increase the affinity of c-Src for imatinib by swapping residues with the corresponding residues in Abl have not been successful, suggesting that the thermodynamic penalty for adoption of the imatinib-binding conformation by c-Src is distributed over a broad region of the structure. Two mutations that are expected to destabilize the inactive Src/CDK conformation increase drug sensitivity 15-fold, suggesting that the free-energy balance between different inactive states is a key to imatinib binding.

Heparin-binding EGF-like growth factor (HB-EGF), but not EGF, binds to cell surface heparan sulfate proteoglycan (HSPG). This was demonstrated in (a) the binding of 125I-HB-EGF to mutant CHO cells deficient in HS production was diminished by 70% compared to wild-type CHO cells, (b) the binding of 125I-HB-EGF to CHO cells and bovine aortic smooth muscle cells (BASMC) was diminished 80% by heparitinase or chlorate treatment, and (c) 125I-EGF did not bind to CHO cells and its binding to BASMC was not diminished at all by heparitinase and only slightly by chlorate treatment. Accordingly, the role of HB-EGF interactions with HSPG in modulating bioactivity was examined. Heparitinase or chlorate treatment of BASMC diminished the ability of HB-EGF to stimulate BASMC migration by 60-80%. A similar inhibition of migration occurred when BASMC were treated with a synthetic peptide (P21) corresponding to the sequence of the putative heparin-binding domain of HB-EGF. As a control for BASMC viability, and for specificity, it was found that heparitinase and P21 did not inhibit at all and chlorate inhibited only slightly the stimulation of BASMC migration by PDGF AB. Since heparitinase, chlorate, and P21 treatment also diminished by 70-80% the cross-linking of 125I-HB-EGF to the EGF receptor, it was concluded that the interaction of HB-EGF, via its heparin-binding domain, with cell surface HSPG was essential for its optimal binding to the EGF receptor on BASMC and hence for its optimal ability to stimulate migration.

Signals transmitted from mesenchyme to epithelia or vice versa constitute the basis of reciprocal epithelial-mesenchymal interactions. As a first step toward understanding epithelial-mesenchymal interactions on the ocular surface where the transit amplifying cell-containing corneal epithelium is anatomically separated from the stem cell-containing limbal epithelium, we sought to characterize the expression patterns of cytokines and their receptors by primary epithelial and early-passaged fibroblast cultures of human cornea and limbus. Northern hybridization with oligonucleotide and cDNA probes to a total of 25 cytokines and 12 of their receptors revealed that the positively expressed cytokines could be divided into the following four patterns. Type I: TGF-alpha, IL-1 beta, and PDGF-B were expressed exclusively by epithelial cells but their respective receptors EGFR and IL-1R were predominantly and PDGFR-beta was exclusively expressed by fibroblasts. Type II: IGF-I, TGF-beta 1, -beta 2, LIF, and bFGF, and their receptors were expressed by both epithelial cells and fibroblasts. FGFR-1 (flg) and FGFR-2 (bek) were expressed more by fibroblasts and bFGF was expressed more by corneal than limbal epithelial cells. Type III: keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) were expressed exclusively by fibroblasts and their respective receptors, KGFR and c-met, were predominantly expressed by epithelial cells. Combined with RT-PCR, the quantity of KGF and KGFR transcripts was highest in limbal fibroblasts and epithelial cells, respectively. In contrast, the quantity of HGF and HGFR (c-met) transcripts was highest in corneal fibroblasts and epithelial cells, respectively. Type IV: M-CSF and IL-8 were expressed by fibroblasts and/or epithelial cells but their receptors were not expressed by epithelial cells nor fibroblasts, but by immune or inflammatory cells. In addition to these potential paracrine actions, autocrine actions mediated by TGF-alpha/EGFR, IL-1 beta/IL1-R, and bFGF/FGFR-1 were more expressed by corneal than limbal epithelial cells. Immunofluorescence staining on human corneoscleral cryosections confirmed that EGFR and bFGF were not expressed by the limbal basal epithelium, but expressed strongly by the corneal epithelium, a pattern consistent with Northern hybridization. These results indicate that ocular surface epithelial cells and fibroblasts can express a myriad of cytokines, among which the first three patterns constitute the network of potential epithelial-mesenchymal cytokine dialogues. The difference of certain cytokine expression between corneal and limbal regions suggests that this network participates in normal epithelial growth and differentiation, and plays an important role in wound healing.

Platelets contain a polypeptide growth factor that stimulates the replication of normal connective tissue cells; this platelet-derived growth factor (PDGF) is released during the clotting process. Human platelets from normal volunteers were disrupted by nitrogen cavitation, and the subcellular organelles were fractionated by ultracentrifugation through a 30%--60% sucrose gradient. Electron microscopy revealed that fraction 7 (density 1.23 g/liter) contained the largest number of alpha granules. The specific activity of platelet fibrinogen, an alpha-granule marker, was also highest in this fraction. The subcellular fractions were assay for the presence of PDGF and for beta-thromboglobulin. PDGF was assayed quantitatively by the stimulation of DNA synthesis in confluent growth-arrested BALB/c-3T3 cells, whereas the concentration of beta-thromboglobulin was determined by radioimmunoassay. The highest concentrations of both PDGF and beta-thromboglobulin were found in the alpha-granule fraction. In contrast, beta-glucuronidase, a lysosomal enzyme, was more diffusely distributed and had its highest specific activity in fractions of lower density than those for PDGS, beta-thromboglobulin, or fibrinogen. The data demonstrate that the alpha granules of platelets provide a unique delivery system for PDGF, a polypeptide hormone with growth-promoting activity for connective tissue cells.

Vascular smooth muscle cell (VSMC) proliferation and migration are responses to arterial injury that are highly important to the processes of restenosis and atherosclerosis. In the arterial balloon injury model in the rat, platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) are induced in the vessel wall and regulate these VSMC activities. Novel insulin sensitizing agents, thiazolidinediones, have been demonstrated to inhibit insulin and epidermal growth factor-induced growth of VSMCs. We hypothesized that these agents might also inhibit the effect of PDGF and bFGF on cultured VSMCs and intimal hyperplasia in vivo. Troglitazone (1 microM), a member of the thiazolidinedione class, produced a near complete inhibition of both bFGF-induced DNA synthesis as measured by bromodeoxyuridine incorporation (6.5+/-3.9 vs. 17.6+/-4.3% cells labeled, P < 0.05) and c-fos induction. This effect was associated with an inhibition (by 73+/-4%, P < 0.01) by troglitazone of the transactivation of the serum response element, which regulates c-fos expression. Inhibition of c-fos induction by troglitazone appeared to occur via a blockade of the MAP kinase pathway at a point downstream of MAP kinase activation by MAP kinase kinase. At this dose, troglitazone also inhibited PDGF-BB-directed migration of VSMC (by 70+/-6%, P < 0.01). These in vitro effects were operative in vivo. Quantitative image analysis revealed that troglitazone-treated rats had 62% (P < 0.001) less neointima/media area ratio 14 d after balloon injury of the aorta compared with injured rats that received no troglitazone. These results suggest troglitazone is a potent inhibitor of VSMC proliferation and migration and, thus, may be a useful agent to prevent restenosis and possibly atherosclerosis.

The rapid induction of the proto-oncogene c-fos by growth factors and other bioactive agents, and the recent evidence that the c-fos protein (Fos) is associated with transcriptional complexes, suggests that Fos may represent an integral part of an intracellular messenger pathway that triggers changes in gene expression and ultimately phenotypic alterations. This report examines the role of c-fos in growth factor stimulation of transin, a matrix-degrading secreted metalloproteinase. Platelet-derived growth factor (PDGF) stimulation of transin RNA was blocked by a selective reduction in Fos synthesis with antisense c-fos mRNA, whereas epidermal growth factor (EGF) stimulation of transin occurred despite an equivalent inhibition of Fos levels. The stimulatory effect of both PDGF and EGF on transin transcription involved factors recognizing the sequence TGAGTCA, which is found in the transin promoter and is reported to be a binding site for the transcriptional factor Jun/AP-1 and for associated Fos and Fos-related complexes. Thus both Fos-dependent and Fos-independent pathways exist for growth factor regulation of gene expression, and both effects may be mediated through the same cis-acting transcription element.

In the present study, we have identified several proteins in Swiss 3T3 cells that are phosphorylated on tyrosine in response to platelet-derived growth factor (PDGF) and exhibit an unusual bell-shaped dose-response curve with a maximum at 5 ng/ml platelet-derived growth factor (PDGF). These proteins include two that are associated with focal adhesions, namely the focal adhesion kinase (p125FAK), a novel cytosolic tyrosine kinase, and paxillin. At low concentrations of PDGF (1-5 ng/ml), these proteins are the predominant tyrosine-phosphorylated species. At 30 ng/ml PDGF, however, there was no stimulation of their phosphorylation over control levels. In contrast, tyrosine phosphorylation of previously described substrates of the PDGF receptor tyrosine kinase, namely the p21ras GTPase-activating protein, p120, phosphatidyl inositol 3' kinase, and phospholipase C gamma exhibited sigmoidal dose-response curves with PDGF and were all efficiently phosphorylated on tyrosine at 30 ng/ml PDGF. Cytochalasin D, which disrupts the actin cytoskeleton, completely inhibited the tyrosine phosphorylation of p125FAK and paxillin by PDGF. Examination of the actin cytoskeleton after stimulation of cells with different concentrations of PDGF revealed that at 5 ng/ml PDGF, actin appears in stress fibers and in membrane ruffles, while at 30 ng/ml, PDGF disrupts the actin cytoskeleton. Bombesin stimulates actin stress fiber formation with no evidence of disruption of stress fibers at high concentrations. When cells were stimulated with bombesin (10 nM) in the presence of 30 ng/ml PDGF, however, the actin cytoskeleton was completely disrupted. Further, the tyrosine phosphorylation of both p125FAK and paxillin induced by bombesin (10 nM) was completely prevented when cells were stimulated with bombesin in the presence of 30 ng/ml PDGF. We propose that the inhibitory limb in the bell-shaped dose-response curve of PDGF and the novel cross-talk between PDGF and bombesin on tyrosine phosphorylation may be explained by the ability of PDGF at 30 ng/ml to disrupt the actin cytoskeleton.

In fibroblasts, insulin is a weak mitogen and does not induce expression of c-fos, c-jun or p33. However, increasing the expression levels of either normal p21Hras or the insulin receptor, but not mutant p21Hras, enables insulin to induce the expression of these genes. In cells expressing elevated levels of insulin receptor, this process involves a rapid increase in p21rasGTP levels (from 20% to 70% GTP as a percentage of total guanine nucleotides). No increase in p21rasGTP levels was observed after PDGF and EGF stimulation of cells expressing high levels of the cognate receptor, stressing the specificity of the insulin-induced increase. We conclude that in fibroblasts, p21ras is an intermediate of the insulin signal transduction pathway involved in the regulation of gene expression and mitogenicity.

Overexpression of a TPA-insensitive PKC member, an atypical protein kinase C (aPKClambda), results in an enhancement of the transcriptional activation of TPA response element (TRE) in cells stimulated with epidermal growth factor (EGF) or platelet-derived growth factor (PDGF). EGF or PDGF also caused a transient increase in the in vivo phosphorylation level and a change in the intracellular localization of aPKClambda from the nucleus to the cytosol, indicating the activation of aPKClambda in response to this growth factor stimulation. These immediate signal-dependent changes in aKPClambda were observed for a PDGF receptor add-back mutant (Y40/51) that possesses only two of the five major autophosphorylation sites and binds PI3-kinase, and were inhibited by wortmannin, an inhibitor of PI3-kinase. Furthermore, an N-terminal fragment of the catalytic subunit of PI3-kinase, p110alpha, inhibited aPKClambda-dependent activation of TRE in Y40/51 cells stimulated with PDGF. Overexpression of p110alpha resulted in an enhancement of TRE expression in response to PDGF and the regulatory domain of aPKClambda inhibited this TRE activation in Y40/51 cells. These results provide the first in vivo evidence supporting the presence of a novel signalling pathway from receptor tyrosine kinases to aPKClambda through PI3-kinase.

Platelet-derived growth factor (PDGF) is thought to mediate the proliferation of smooth muscle cells in injured arteries, and may be involved in the pathogenesis of atherosclerosis. PDGF-like molecules from non-platelet sources may also play a role in the regulation of cell activity in other circumstances. Transformation of cells by a wide range of oncongenic agents appears to activate a cellular gene encoding a PDGF-like molecule, possibly accounting for the ability of transformed cells to grow without addition of exogenous mitogens. We show here that a molecule (PDGF-c) which can compete with 125I-PDGF for binding to PDGF receptors is secreted by cultured rat aortic smooth muscle cells (rASMC) isolated from 13 to 18-day-old rats (pups) but not from three-month-old animals (adults). Thus, production of PDGF-c appears to be developmentally regulated and may be a factor in the more rapid proliferation of rASMC and synthesis of connective tissue components which occurs during growth of the aorta in vivo.

Platelet-derived growth factor (PDGF) stimulates expression of a "competence" gene family in Balb/c-3T3 cells. The competence family contains the c-myc and c-fos genes together with several functionally uncharacterized genes (JE, KC, and r-fos) that have been isolated as cDNA clones. We show that double-stranded ribonucleic acid is a potent inducer of the competence gene family. Infection with vesicular stomatitis virus also induces expression of this gene family. Conversely, PDGF stimulates expression of genes hitherto characterized as responsive to double-stranded ribonucleic acids, including the beta-fibroblast interferon and (2'-5')-oligoadenylate synthetase genes. These PDGF-inducible genes could conceivably function in a feedback loop to control 3T3 cell growth. Some of the genes, such as c-fos and c-myc, are induced quickly by PDGF and may initiate a round of cell division. Others, such as beta-fibroblast interferon and (2'-5')-oligoadenylate synthetase, are induced more slowly and may function as feedback inhibitors of the growth response to PDGF.

Lysophosphatidic acid (LPA) is a natural phospholipid with multiple biological functions. We show here that LPA induces phosphorylation and inactivation of glycogen synthase kinase 3 (GSK-3), a multifunctional serine/threonine kinase. The effect of LPA can be reconstituted by expression of Edg-4 or Edg-7 in cells lacking LPA responses. Compared to insulin, LPA stimulates only modest phosphatidylinositol 3-kinase (PI3K)-dependent activation of protein kinase B (PKB/Akt) that does not correlate with the magnitude of GSK-3 phosphorylation induced by LPA. PI3K inhibitors block insulin- but not LPA-induced GSK-3 phosphorylation. In contrast, the effect of LPA, but not that of insulin or platelet-derived growth factor (PDGF), is sensitive to protein kinase C (PKC) inhibitors. Downregulation of endogenous PKC activity selectively reduces LPA-mediated GSK-3 phosphorylation. Furthermore, several PKC isotypes phosphorylate GSK-3 in vitro and in vivo. To confirm a specific role for PKC in regulation of GSK-3, we further studied signaling properties of PDGF receptor beta subunit (PDGFRbeta) in HEK293 cells lacking endogenous PDGF receptors. In clones expressing a PDGFRbeta mutant wherein the residues that couple to PI3K and other signaling functions are mutated with the link to phospholipase Cgamma (PLCgamma) left intact, PDGF is fully capable of stimulating GSK-3 phosphorylation. The process is sensitive to PKC inhibitors in contrast to the response through the wild-type PDGFRbeta. Therefore, growth factors, such as PDGF, which control GSK-3 mainly through the PI3K-PKB/Akt module, possess the ability to regulate GSK-3 through an alternative, redundant PLCgamma-PKC pathway. LPA and potentially other natural ligands primarily utilize a PKC-dependent pathway to modulate GSK-3.

Angiotensin (Ang) II stimulates hypertrophic growth of vascular smooth muscle cells (VSMC). Accompanying this growth is the induction of the expression of growth-related protooncogenes (c-fos, c-jun, and c-myc), as well as the synthesis of the autocrine growth factors, such as PDGF-A and TGF-beta 1. In this study, we demonstrate further that Ang II also induces the synthesis of basic fibroblast growth factor (bFGF), a potent mitogen for VSMC. To examine how these factors interact to modulate the growth response of VSMC to Ang II, we used antisense oligomers to determine the relative contribution of these three factors. Treatment of confluent, quiescent smooth muscle cells with specific antisense oligomers complementary to bFGF, PDGF-A, and TGF-beta 1 efficiently inhibited the syntheses of these factors. Our results demonstrate that in these VSMC, TGF-beta 1 affects a key antiproliferative action, modulating the mitogenic properties of bFGF. Autocrine PDGF exerts only a minimal effect on DNA synthesis. An imbalance in these signals activated by Ang II may result in abnormal VSMC growth leading to the development of vascular disease.

We investigated the interaction of phospholipase C-gamma (PLC-gamma) with wild-type and mutant forms of the platelet-derived growth factor (PDGF) beta-receptor both in vivo and in vitro. After PDGF treatment of CHO cell lines expressing wild-type or either of two mutant (delta Ki and Y825F) PDGF receptors, PLC-gamma became tyrosine phosphorylated and associated with the receptor proteins. The receptor association and tyrosine phosphorylation of PLC-gamma correlated with the ability of these receptors to mediate ligand-induced phosphatidylinositol turnover. However, both the delta Ki and Y825F mutant receptors were deficient in transmitting mitogenic signals, suggesting that the PDGF-induced tyrosine phosphorylation and receptor association of PLC-gamma are not sufficient to account for the growth-stimulatory activity of PDGF. Wild-type and delta Ki mutant PDGF receptor proteins expressed with recombinant baculovirus vectors also associated in vitro with mammalian PLC-gamma. However, baculovirus-expressed c-fms, v-fms, c-src, and Raf-1 proteins failed to associate with PLC-gamma under similar conditions. Phosphatase treatment of the baculovirus-expressed PDGF receptor greatly decreased its association with PLC-gamma. This requirement for receptor phosphorylation was also observed in vivo, where PLC-gamma could not associate with a mutant PDGF receptor (K602A) defective in autophosphorylation. PLC-gamma also coimmunoprecipitated with two other putative receptor substrates, the serine-threonine kinase Raf-1 and the 85-kilodalton phosphatidylinositol-3' kinase, presumably through its association with the ligand-activated receptor. Furthermore, baculovirus-expressed Raf-1 phosphorylated purified PLC-gamma in vitro at sites which showed increased serine phosphorylation in vivo in response to PDGF. These results suggest that PDGF directly influences PLC activity by inducing the association of PLC-gamma with a receptor signaling complex, resulting in increased tyrosine and serine phosphorylation of PLC-gamma.

Imatinib is a tyrosine kinase inhibitor that suppresses the growth of bcr-abl-expressing chronic myeloid leukemia (CML) progenitor cells by blockade of the adenosine triphosphate (ATP)-binding site of the kinase domain of bcr-abl. Imatinib also inhibits the c-abl, platelet-derived growth factor (PDGF) receptor, abl-related gene (ARG) and stem-cell factor (SCF) receptor tyrosine kinases, and has been used clinically to inhibit the growth of malignant cells in patients with CML and gastrointestinal stromal tumors (GISTs). Although initially considered to have minimal effects of normal hematopoiesis, recent studies show that imatinib also inhibits the growth of some nonmalignant hematopoietic cells, including monocyte/macrophages. This inhibition could not be attributed to the known activity profile of imatinib. Here, we demonstrate for the first time that imatinib targets the macrophage colony-stimulating factor (M-CSF) receptor c-fms. Phosphorylation of c-fms was inhibited by therapeutic concentrations of imatinib, and this was not due to down-regulation in c-fms expression. Imatinib was also found to inhibit M-CSF-induced proliferation of a cytokine-dependent cell line, further supporting the hypothesis that imatinib affects the growth and development of monocyte and/or macrophages through inhibition of c-fms signaling. Importantly, these results identify an additional biologic target to those already defined for imatinib. Imatinib should now be assessed for activity in diseases where c-fms activation is implicated, including breast and ovarian cancer and inflammatory conditions.

This review intends to summarize the vast literature on K-Cl cotransport (COT) regulation from a functional and genetic viewpoint. Special attention has been given to the signaling pathways involved in the transporter's regulation found in several tissues and cell types, and more specifically, in vascular smooth muscle cells (VSMCs). The number of publications on K-Cl COT has been steadily increasing since its discovery at the beginning of the 1980s, with red blood cells (RBCs) from different species (human, sheep, dog, rabbit, guinea pig, turkey, duck, frog, rat, mouse, fish, and lamprey) being the most studied model. Other tissues/cell types under study are brain, kidney, epithelia, muscle/smooth muscle, tumor cells, heart, liver, insect cells, endothelial cells, bone, platelets, thymocytes and Leishmania donovani. One of the salient properties of K-Cl-COT is its activation by cell swelling and its participation in the recovery of cell volume, a process known as regulatory volume decrease (RVD). Activation by thiol modification with N-ethylmaleimide (NEM) has spawned investigations on the redox dependence of K-Cl COT, and is used as a positive control for the operation of the system in many tissues and cells. The most accepted model of K-Cl COT regulation proposes protein kinases and phosphatases linked in a chain of phosphorylation/dephosphorylation events. More recent studies include regulatory pathways involving the phosphatidyl inositol/protein kinase C (PKC)-mediated pathway for regulation by lithium (Li) in low-K sheep red blood cells (LK SRBCs), and the nitric oxide (NO)/cGMP/protein kinase G (PKG) pathway as well as the platelet-derived growth factor (PDGF)-mediated mechanism in VSMCs. Studies on VSM transfected cells containing the PKG catalytic domain demonstrated the participation of this enzyme in K-Cl COT regulation. Commonly used vasodilators activate K-Cl COT in a dose-dependent manner through the NO/cGMP/PKG pathway. Interaction between the cotransporter and the cytoskeleton appears to depend on the cellular origin and experimental conditions. Pathophysiologically, K-Cl COT is altered in sickle cell anemia and neuropathies, and it has also been proposed to play a role in blood pressure control. Four closely related human genes code for KCCs (KCCCC genes in terms of transcriptional and post-transcriptional regulation. A few reports indicate that the NO/cGMP/PKG signaling pathway regulates KCCCCCs at the post-transcriptional level. However, the detailed mechanisms of post-transcriptional regulation of KCC genes and of regulation of KCCCCCl COT field is expected to expand further over the next decades, as new isoforms and/or regulatory pathways are discovered and its implication in health and disease is revealed.

We have characterized platelet-derived growth factor (PDGF) C, a novel growth factor belonging to the PDGF family. PDGF-C is a multidomain protein with the N-terminal region homologous to the extracellular CUB domain of neuropilin-1, and the C-terminal region consists of a growth factor domain (GFD) with homology to vascular endothelial growth factor (25%) and PDGF A-chain (23%). A serum-sensitive cleavage site between the two domains allows release of the GFD from the CUB domain. Competition binding and immunoprecipitation studies on cells bearing both PDGF alpha and beta receptors reveal a high affinity binding of recombinant GFD (PDGF-CC) to PDGF receptor-alpha homodimers and PDGF receptor-alpha/beta heterodimers. PDGF-CC exhibits greater mitogenic potency than PDGF-AA and comparable or greater mitogenic activity than PDGF-AB and PDGF-BB on several mesenchymal cell types. Analysis of PDGF-CC in vivo in a diabetic mouse model of delayed wound healing showed that PDGF-CC significantly enhanced repair of a full-thickness skin excision. Together, these studies describe a third member of the PDGF family (PDGF-C) as a potent mitogen for cells of mesenchymal origin in in vitro and in vivo systems with a binding pattern similar to PDGF-AB.

Insulin-dependent phosphorylation of Akt target AS160 is required for GLUT4 translocation. Insulin and platelet-derived growth factor (PDGF) (Akt activators) or activation of conventional/novel (c/n) protein kinase C (PKC) and 5' AMP-activated protein kinase (AMPK) all promote a rise in membrane GLUT4 in skeletal muscle and cultured cells. However, the downstream effectors linking these pathways to GLUT4 traffic are unknown. Here we explore the hypothesis that AS160 is a molecular link among diverse signaling cascades converging on GLUT4 translocation. PDGF and insulin increased AS160 phosphorylation in CHO-IR cells. Stimuli that activate c/n PKC or AMPK also elevated AS160 phosphorylation. We therefore examined if these signaling pathways engage AS160 to regulate GLUT4 traffic in muscle cells. Nonphosphorylatable AS160 (4P-AS160) virtually abolished the net surface GLUT4myc gains elicited by insulin, PDGF, K(+) depolarization, or 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside but partly, yet significantly, inhibited the effects of 4-phorbol-12-myristate-13-acetate. However, the hypertonicity or 2,4-dinitrophenol-dependent gains in surface GLUT4myc were unaffected by 4P-AS160. RK-AS160 (GTPase-activating protein [GAP] inactive) or 4PRK-AS160 (GAP inactive, nonphosphorylatable) had no effect on surface GLUT4myc elicited by all stimuli. Collectively, these results indicate that activation of Akt, c/n PKC, or alpha2-AMPK intersect at AS160 to regulate GLUT4 traffic, as well as highlight the potential of AS160 as a therapy target to increase muscle glucose uptake.

Autophosphorylation of the platelet-derived growth factor (PDGF) receptor triggers intracellular signaling cascades as a result of recruitment of Src homology 2 domain-containing enzymes, including phosphatidylinositol 3-kinase (PI3K), the GTPase-activating protein of Ras (GAP), the protein-tyrosine phosphatase SHP-2, and phospholipase C-gamma1 (PLC-gamma1), to specific phosphotyrosine residues. The roles of these various effectors in PDGF-induced generation of H(2)O(2) have now been investigated in HepG2 cells expressing various PDGF receptor mutants. These mutants included a kinase-deficient receptor and receptors in which various combinations of the tyrosine residues required for the binding of PI3K (Tyr(740) and Tyr(751)), GAP (Tyr(771)), SHP-2 (Tyr(1009)), or PLC-gamma1 (Tyr(1021)) were mutated to Phe. PDGF failed to increase H(2)O(2) production in cells expressing either the kinase-deficient mutant or a receptor in which the two Tyr residues required for the binding of PI3K were replaced by Phe. In contrast, PDGF-induced H(2)O(2) production in cells expressing a receptor in which the binding sites for GAP, SHP-2, and PLC-gamma1 were all mutated was slightly greater than that in cells expressing the wild-type receptor. Only the PI3K binding site was alone sufficient for PDGF-induced H(2)O(2) production. The effect of PDGF on H(2)O(2) generation was blocked by the PI3K inhibitors LY294002 and wortmannin or by overexpression of a dominant negative mutant of Rac1. These results suggest that a product of PI3K is required for PDGF-induced production of H(2)O(2) in nonphagocytic cells, and that Rac1 mediates signaling between the PI3K product and the putative NADPH oxidase.

In this paper we address the linking of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (FGF-2) to intracellular signaling molecules in oligodendrocyte progenitors. It is demonstrated that both growth factors activate downstream targets similar to those shown for protein kinase C (PKC) activation. Yet, neither the arrest of terminal oligodendrocyte differentiation nor the proliferation induced by PDGF or FGF-2 can be antagonized by inhibition of PKC. Rather, p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, and pp70 S6 kinase were found to be necessary for the mitogenic activity of PDGF and FGF-2. Paradoxically, these kinases were also necessary for the onset of oligodendrocyte differentiation in control cells. In addition, cAMP-dependent kinase A (PKA) activation inhibited the mitogenic response of oligodendrocyte progenitors to FGF-2. Taken together, the molecular mechanism that controls oligodendrocyte lineage progression is operated by at least two signal pathways, which interfere either with proliferation and/or differentiation of oligodendrocyte progenitors.

Exposure of serum-deprived 3T3-L1 fibroblasts to phorbol 12-myristate 13-acetate (PMA), synthetic diacylglycerols, platelet-derived growth factor (PDGF), or pituitary fibroblast growth factor (FGF) resulted in stimulated phosphorylation of an acidic, multicomponent, soluble protein of Mr 80,000. Phosphorylation of this protein was promoted to a lesser extent by epidermal growth factor; however, neither insulin nor dibutyryl cAMP was effective. Phosphoamino acid analysis and peptide mapping of the Mr 80,000 32P-protein after exposure of fibroblasts to PDGF revealed identical patterns to those obtained with PMA or diacylglycerols. In contrast to the Mr 80,000 protein, proteins of Mr 22,000 (and pI 4.4) and Mr 31,000 were also phosphorylated in response to insulin as well as to PMA, diacylglycerols, epidermal growth factor, PDGF, and FGF in these cells. Similar findings were noted in fully differentiated 3T3-L1 adipocytes. Preincubation of the cells with high concentrations of active phorbol esters abolished specific [3H]phorbol 12,13-dibutyrate binding, protein kinase C activity, and immunoreactivity and also prevented stimulated phosphorylation of the Mr 80,000 protein by PMA, diacylglycerols, PDGF, or FGF, supporting the contention that this effect was mediated through protein kinase C. The stimulated phosphorylation of the Mr 22,000 and 31,000 proteins in response to PMA was also abolished by such pretreatment. In contrast, the ability of insulin, PDGF, and FGF to promote phosphorylation of the Mr 22,000 and 31,000 proteins was unaffected in the protein kinase C-deficient cells. We conclude that PDGF and FGF may exert some of their effects on these cells through at least two distinct pathways of protein phosphorylation, phorbol ester-like (P) activation of protein kinase C, and an insulin-like (I) pathway exemplified by phosphorylation of the Mr 22,000 and 31,000 proteins.

To identify kinases that regulate integrin recycling, we have immunoprecipitated alphavbeta3 integrin from NIH 3T3 fibroblasts in the presence and absence of primaquine (a drug that inhibits receptor recycling and leads to accumulation of integrins in endosomes) and screened for co-precipitating kinases. Primaquine strongly promoted association of alphavbeta3 integrin with PKD1, and fluorescence microscopy indicated that integrin and PKD1 associate at a vesicular compartment that is downstream of a Rab4-dependent transport step. PKD1 association was mediated by the C-terminal region of the beta3 integrin cytodomain, and mutants of beta3 that were unable to recruit PKD1 did not recycle in a PDGF-dependent fashion. Furthermore, suppression of endogenous PKD1 levels by RNAi, or overexpression of catalytically inactive PKD1 inhibited PDGF-dependent recycling of alphavbeta3 from early endosomes to the plasma membrane and blocked recruitment of alphavbeta3 to newly formed focal adhesions during cell spreading. These data indicate that PKD1 influences cell migration by directing vesicular transport of the alphavbeta3 integrin heterodimer.

First trimester human placentas actively express the sis proto-oncogene, the structural gene for the B chain of platelet-derived growth factor (PDGF). Using the in situ hybridization technique, the 4.2 kb c-sis transcript has been localized to the cytotrophoblastic component, especially the highly proliferative and invasive cytotrophoblastic shell, paralleling the distribution of c-myc transcripts in early placenta. Explants of first trimester placenta release significant levels of PDGF-like activity into the medium under apparent developmental control. Moreover, cultured trophoblasts display abundant high-affinity PDGF receptors and respond to exogenous authentic PDGF by an activation of the c-myc gene and DNA synthesis. The developing human placenta may therefore represent a case of autocrine growth regulation in a normal tissue, in which cells bearing receptors for a growth factor can also synthesize and respond to that factor.