Cutaneous melanoma is the most aggressive skin cancer; it is highly metastatic and responds poorly to current therapies. The expression of platelet-derived growth factor receptors (PDGF-Rs) is reported to be reduced in metastatic melanoma compared with benign nevi or normal skin; we then hypothesized that PDGF-Ralpha may control growth of melanoma cells. We show here that melanoma cells overexpressing PDGF-Ralpha respond to serum with a significantly lower proliferation compared with that of controls. Apoptosis, cell cycle arrest, pRb dephosphorylation, and DNA synthesis inhibition were also observed in cells overexpressing PDGF-Ralpha. Proliferation was rescued by PDGF-Ralpha inhibitors, allowing to exclude nonspecific toxic effects and indicating that PDGF-Ralpha mediates autocrine antiproliferation signals in melanoma cells. Accordingly, PDGF-Ralpha was found to mediate staurosporine cytotoxicity. A protein array-based analysis of the mitogen-activated protein kinase pathway revealed that melanoma cells overexpressing PDGF-Ralpha show a strong reduction of c-Jun phosphorylated in serine 63 and of protein phosphatase 2A/Balpha and a marked increase of p38gamma, mitogen-activated protein kinase kinase 3, and signal regulatory protein alpha1 protein expression. In a mouse model of primary melanoma growth, infection with the Ad-vector overexpressing PDGF-Ralpha reached a significant 70% inhibition of primary melanoma growth (P < .001) and a similar inhibition of tumor angiogenesis. All together, these data demonstrate that PDGF-Ralpha strongly impairs melanoma growth likely through autocrine mechanisms and indicate a novel endogenous mechanism involved in melanoma control.

Autocrine expression of a growth factor and its receptor in the same cell raises the possibility of an intracellular receptor-ligand interaction within the cell, in addition to a receptor-ligand interaction at the cell surface. We have constructed a NIH3T3 cell line which contains the v-sis gene under the inducible control of the Drosophila melanogaster hsp70 promoter. Expression of both v-sis RNA and protein is rapidly induced by a short period of heat-shock. We have analyzed the cellular site of interaction between the v-sis protein and the platelet-derived growth factor receptor in these cells. Autophosphorylation of the PDGF receptor and induction of the c-fos gene were found to occur at 45 and 50 min, respectively, after heat-induced synthesis of the v-sis protein. Monensin treatment of the heat-induced cells prevented autophosphorylation of the mature PDGF receptor and also prevented subsequent induction of c-fos. Autophosphorylation of the PDGF receptor and c-fos induction were also prevented by the addition of suramin to the medium. These results demonstrate that autocrine stimulation, as monitored by c-fos induction and by PDGF receptor autophosphorylation, requires an interaction between the v-sis protein and the PDGF receptor that occurs at the cell surface, rather than an intracellular location.

OBJECTIVE

The lysophospholipid mediator sphingosine-1-phosphate (S1P) activates G protein-coupled receptors (GPCRs) to induce potent inhibition of platelet-derived growth factor (PDGF)-induced Rac activation and, thereby, chemotaxis in rat vascular smooth muscle cells (VSMCs). We explored the heterotrimeric G protein and the downstream mechanism that mediated S1P inhibition of Rac and cell migration in VSMCs.

RESULTS

S1P inhibition of PDGF-induced cell migration and Rac activation in VSMCs was abolished by the selective S1P(2) receptor antagonist JTE-013. The C-terminal peptides of Galpha subunits (Galpha-CTs) act as specific inhibitors of respective G protein-GPCR coupling. Adenovirus-mediated expression of Galpha(12)-CT, Galpha(13)-CT, and Galpha(q)-CT, but not that of Galpha(s)-CT or LacZ or pertussis toxin treatment, abrogated S1P inhibition of PDGF-induced Rac activation and migration, indicating that both G(12/13) and G(q) classes are necessary for the S1P inhibition. The expression of Galpha(q)-CT as well as Galpha(12)-CT and Galpha(13)-CT also abolished S1P-induced Rho stimulation. C3 toxin, but not a Rho kinase inhibitor or a dominant negative form of Rho kinase, abolished S1P inhibition of PDGF-induced Rac activation and cell migration. The angiotensin II receptor AT(1), which robustly couples to G(q), did not mediate either Rho activation or inhibition of PDGF-induced Rac activation or migration, suggesting that activation of G(q) alone was not sufficient for Rho activation and resultant Rac inhibition. However, the AT(1) receptor fused to Galpha(12) was able to induce not only Rho stimulation but also inhibition of PDGF-induced Rac activation and migration. Phospholipase C inhibition did not affect S1P-induced Rho activation, and protein kinase C activation by a phorbol ester did not mimic S1P action, suggesting that S1P inhibition of migration or Rac was not dependent on the phospholipase C pathway.

CONCLUSIONS

These observations together suggest that S1P(2) mediates inhibition of Rac and migration through the coordinated action of G(12/13) and G(q) for Rho activation in VSMCs.

We report conditions for the efficient growth of NIH 3T3 and BALB/c 3T3 cells cultured in a defined medium supplemented with either platelet-derived growth factor (PDGF) or pituitary-derived fibroblast growth factor (FGF). The oncogenes v-mos, v-src, v-sis, and c-H-ras Val 12 can induce morphological transformation of these cells and can release them from the mitogen requirement for growth, while the oncogene v-fos cannot abrogate the PDGF-FGF requirement. The radically different behavior of normal and transformed NIH 3T3 cells in PDGF-FGF-free defined medium can form the basis of a sensitive new fibroblast transformation assay.

Hypoxia results in differential expression of specific genes in certain cell types. In endothelial cells, hypoxia activates several genes that are known to be inducible by transcription factor AP-1, including endothelin-1 and platelet-derived growth factor-B (PDGF-B). In this study we demonstrated that other AP-1-inducible genes are activated by hypoxia in these cells, including collagenase IV and c-jun, and sought to correlate the activation of genes by hypoxia with the activation of transcription factor AP-1. Depending upon the type of cell studied, hypoxic exposure resulted in the induction of AP-1 transcription factor DNA-binding activity with wide variations in levels of binding. The magnitude of activation of transcription factor AP-1 by hypoxia did not always strictly correlate with the level of induction of AP-1-inducible genes. This finding indicates a requirement for additional mechanisms of controlling transcription beyond the simple activation of AP-1 factor DNA-binding activity for the activation of AP-1-inducible genes during hypoxia. Hypoxia has been reported to lower the intracellular redox potential. The effect of redox state changes on AP-1 transcription factor activity and on the activation of AP-1-inducible genes was also studied. PDTC, a potent reducing agent, activated the AP-1 transcription factor in HeLa cells, and also resulted in increased accumulation of c-jun mRNA in these cells. In contrast to PDTC-mediated activation of the AP-1 transcription factor and the subsequent induction of the AP-1-regulated c-jun gene, hypoxic activation of AP-1 transcription factor binding to its cognate DNA sequence did not activate the c-jun gene in HeLa cells, thus documenting distinct differences in signals generated by the reducing intracellular microenvironments created by hypoxia and PDTC. These results demonstrate the induction of AP-1 transcription factor activity by hypoxic environments, but suggest that additional factors or cell-specific signals are involved in the regulation of hypoxia-induced genes.

Hepatocellular carcinoma (HCC) often develops in association with liver cirrhosis, and its high recurrence rate leads to poor patient prognosis. Although recent evidence suggests that peretinoin, a member of the acyclic retinoid family, may be an effective chemopreventive drug for HCC, published data about its effects on hepatic mesenchymal cells, such as stellate cells and endothelial cells, remain limited. Using a mouse model in which platelet-derived growth factor (PDGF)-C is overexpressed (Pdgf-c Tg), resulting in hepatic fibrosis, steatosis, and eventually, HCC development, we show that peretinoin significantly represses the development of hepatic fibrosis and tumors. Peretinoin inhibited the signaling pathways of fibrogenesis, angiogenesis, and Wnt/β-catenin in Pdgf-c transgenic mice. In vitro, peretinoin repressed the expression of PDGF receptors α/β in primary mouse hepatic stellate cells (HSC), hepatoma cells, fibroblasts, and endothelial cells. Peretinoin also inhibited PDGF-C-activated transformation of HSCs into myofibroblasts. Together, our findings show that PDGF signaling is a target of peretinoin in preventing the development of hepatic fibrosis and HCC.

Previously, we have shown that prostaglandins are necessary, but not sufficient, for the stimulation of mitogenesis in BALB/c 3T3 fibroblasts by epidermal growth factor (EGF) (Nolan, R. D., Danilowicz, R. M., and Eling, T. E. (1988) Mol. Pharmacol. 33, 650-656). The purpose of this work was to extend these findings to another potent mitogen, platelet-derived growth factor (PDGF), and to determine if metabolism of arachidonic acid to prostaglandins is necessary for stimulation of expression of the protooncogene c-myc by EGF, which is an early event in the mitogenic cascade. In BALB/c 3T3 cells grown to about 70% confluence and deprived of serum for 16-24 h, PDGF stimulated [3H]thymidine uptake into DNA significantly in a concentration-dependent manner, but did not increase production of prostaglandin E2 (PGE2). The addition of indomethacin, a prostaglandin H synthase inhibitor, or nordihydroguaiaretic acid, a lipoxygenase inhibitor, did not affect PDGF-stimulated thymidine uptake into DNA. In addition, PGE2 enhanced EGF-dependent, but not PDGF-dependent, mitogenesis. Taken together, the data support the hypothesis that prostaglandins are not involved in PDGF-dependent mitogenesis. In contrast, indomethacin (10(-6) M) and nordihydroguaiaretic acid (10(-6) M) inhibited EGF-stimulated thymidine uptake and c-myc expression by approximately 50%. Addition of PGG2 (10(-7) to 10(-5) M) in the presence of indomethacin and EGF restored the ability of EGF to elevate c-myc RNA levels and DNA synthesis. When PGF2 alpha (10(-8) to 10(-5) M) was added in the presence of EGF, c-myc RNA levels and thymidine incorporation were elevated up to 5-6-fold above levels observed with EGF alone. These data support the hypothesis that metabolism of arachidonic acid to prostaglandins is necessary for stimulation of c-myc expression by EGF in BALB/c 3T3 cells.

OBJECTIVE

Connective tissue growth factor (CCN2) is expressed during activation of hepatic stellate cells (HSC) and promotes HSC proliferation, adhesion, and collagen production. The aim of the study was to investigate CCN2 signaling pathways in HSC.

METHODS

Primary HSC were obtained by enzymatic perfusion of rat liver. DNA synthesis was evaluated by [(3)H]thymidine incorporation. Phosphorylation of Elk-1, extracellular signal-regulated kinase (ERK1/2) and focal adhesion kinase (FAK) was evaluated by Western blot. Transcriptional factor binding activity was determined by gel mobility shift assay while c-fos promoter and CCN2 promoter activity was evaluated using luciferase reporters. c-fos mRNA expression was evaluated by Northern blot.

RESULTS

CCN2 stimulated DNA synthesis and phosphorylation of FAK, Elk-1 and ERK1/2, the latter of which was blocked by heparin. The serum response element binding activity and luciferase reporter activity of the c-fos promoter, together with expression of c-fos, were enhanced by CCN2. CCN2-induced c-fos gene activation, expression and cell proliferation were blocked by inhibiting ERK1/2 with PD98059. CCN2 promoter activity was enhanced by TGF-beta1 or PDGF via a Smad7-dependent pathway.

CONCLUSIONS

CCN2-stimulated HSC DNA synthesis is associated with transient induction of c-fos gene activation and expression as well as activation of the ERK1/2 signal pathway.

The receptors involved in the regulation of phospholipase C by hormones, neurotransmitters and other ligands have seven transmembrane-spanning hydrophobic regions (seven-helix motif) and no known enzymatic activity. Furthermore these receptors can be isolated as complexes with guanine nucleotide binding (G) proteins. Guanine nucleotides affect the binding of hormones that stimulate phospholipase C and it has been possible to see activation of GTPase activity in membranes upon addition of these ligands. Further indirect evidence for a Gp (p stands for phospholipase C activation) protein is the finding that in membranes agonist activation of phospholipase C requires the presence of GTP gamma S a non-hydrolyzable analog of GTP. Furthermore, fluoride is able to activate phospholipase C but its inhibition of phosphatidylinositol-4' kinase (PI-4' kinase) can interfere with efforts to demonstrate this in intact cells. There are four major isozymes of phospholipase C that have been cloned and sequenced. Recently it was found that phospholipase C-gamma as well as PI-3'-kinase are substrates for phosphorylation on tyrosine residues by the EGF and PDGF receptors. The PI-3' kinase is able to convert phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3) but the function of this lipid is unknown since it is not a substrate for any known phospholipase C. While much has been learned about the structure and regulation of the phosphoinositide specific kinases and phosphodiesterase enzymes this is a relatively new field in which we can expect many advances during the next few years.

The molecular mechanism involved in the stimulation of hyaluronan synthetase in normal human mesothelial cells was investigated. Exposure of mesothelial cells to platelet-derived growth factor (PDGF)-BB stimulated hyaluronan synthetase activity, measured in isolated membrane preparations, as well as hyaluronan secretion into the medium. The effect on hyaluronan synthetase was maximal after 6 h of treatment. In contrast, the stimulatory effect of transforming growth factor-beta 1 reached a maximum after 24 h. The stimulatory effect of PDGF-BB was inhibited by cycloheximide. The phosphotyrosine phosphatase inhibitor vanadate was found to stimulate hyaluronan synthetase activity, and to potentiate the effect of PDGF-BB. The protein kinase C (PKC) stimulator phorbol 12-myristate 13-acetate (PMA) also stimulated hyaluronan synthetase; furthermore, depletion of PKC by preincubation of the cells with PMA led to an inhibition of the PDGF-BB-induced stimulation of hyaluronan synthetase activity. Thus the PDGF-BB-induced stimulation of hyaluronan synthetase activity is dependent on protein synthesis and involves tyrosine phosphorylation and activation of PKC.

NADPH oxidase is the major source of superoxide production in cardiovascular tissues. We reported previously that PG (prostaglandin) F2alpha caused hypertrophy of vascular smooth muscle cells by induction of NOX1, a catalytic subunit of NADPH oxidase. PGF2alpha-induced NOX1 expression was mediated by transactivation of the EGF (epidermal growth factor) receptor and subsequent activation of ERK (extracellular-signal-regulated kinase) 1/2, PI3K (phosphoinositide 3-kinase) and ATF-1 (activating transcription factor-1), a member of the CREB (cAMP-response-element-binding protein)/ATF family. As the receptor for PGF2alpha is known to activate PKC (protein kinase C), involvement of PKC in up-regulation of NOX1 expression was investigated in A7r5 cells. GF109203x, a non-selective inhibitor of PKC, dose-dependently suppressed the induction of NOX1 mRNA by PGF2alpha. Whereas an inhibitor of the conventional PKC, Gö 6976, and a PKCeta translocation-inhibitor peptide had no effect, an inhibitor of PKCdelta, rottlerin, significantly attenuated the PGF2alpha-induced increase in NOX1 mRNA. Gene silencing of PKCdelta by RNA interference significantly suppressed the PGF2alpha-induced increase in NOX1 mRNA, as well as phosphorylation of the EGF receptor, ERK1/2 and ATF-1. Silencing of the PKCdelta gene also attenuated the PDGF (platelet-derived growth factor)- induced increase in NOX1 mRNA and transactivation of the EGF receptor. Moreover, the augmented synthesis of the protein induced by PGF2alpha or PDGF was abolished by gene silencing of PKCdelta. These results suggest that PKCdelta-mediated transactivation of the EGF receptor is elicited not only by PGF2alpha, but also by PDGF, and that the subsequent activation of ERK1/2 and ATF-1 leads to up-regulation of NOX1 gene expression and ensuing hypertrophy in the vascular cell lineage.

Two approaches have been utilized to investigate the role of individual SH2 domains in growth factor activation of phospholipase C-gamma1 (PLC-gamma1). Surface plasmon resonance analysis indicates that the individual N-SH2 and C-SH2 domains are able to specifically recognize a phosphotyrosine-containing peptide corresponding to Tyr 1021 of the platelet-derived growth factor (PDGF) beta receptor. To assess SH2 function in the context of the full-length PLC-gamma1 molecule as well as within the intact cell, PLC-gamma1 SH2 domain mutants, disabled by site-directed mutagenesis of the N-SH2 and/or C-SH2 domain(s), were expressed in Plcg1(-/-) fibroblasts. Under equilibrium incubation conditions (4 degrees C, 40 min), the N-SH2 domain, but not the C-SH2 domain, was sufficient to mediate significant PLC-gamma1 association with the activated PDGF receptor and PLC-gamma1 tyrosine phosphorylation. When both SH2 domains in PLC-gamma1 were disabled, the double mutant did not associate with activated PDGF receptors and was not tyrosine phosphorylated. However, no single SH2 mutant was able to mediate growth factor activation of Ca2+ mobilization or inositol 1,4,5-trisphosphate (IP3) formation. Subsequent kinetic experiments demonstrated that each single SH2 domain mutant was significantly impaired in its capacity to mediate rapid association with activated PDGF receptors and become tyrosine phosphorylated. Hence, when assayed under physiological conditions necessary to achieve a rapid biological response (Ca2+ mobilization and IP3 formation), both SH2 domains of PLC-gamma1 are essential to growth factor responsiveness.

Stromal cells can dramatically affect the growth and metastatic capability of breast carcinoma cells. Growth factors, considered to be important mediators of this process, act as either mitogenic or mito-inhibitory regulators. We have developed an in vitro coculture system to examine the influence of adipocytes, a dominant mammary stromal cell type, on the growth of a murine mammary carcinoma, SP1. Previously, we have reported that conditioned medium (CM) from 3T3-L1 adipocytes can promote in vitro growth of SP1 cells. We now show that the major mitogenic signal derived from 3T3-L1 adipocyte CM is mediated by hepatocyte growth factor (HGF). Neutralizing antibody against HGF at 15 micrograms/ml completely abrogated mitogenic activity of 3T3-L1 CM. Furthermore, heparin, an inhibitor of biological activity of HGF, inhibited the mitogenic activity of 3T3-L1 CM. Western blot analysis also confirmed the presence of HGF in 3T3-L1 CM. Although basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I) were mitogenic for SP1 cells, neutralizing antibodies against IGF-I, bFGF, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) did not inhibit the mitogenic activity of 3T3-L1 CM. Immunoprecipitation and immunoblotting of HGF receptor/c-met showed that c-met is expressed at high level in SP1 cells, and is phosphorylated following HGF ligation. Together, our present data demonstrate that 3T3-L1 adipocytes secrete HGF, which stimulates SP1 cell growth by a paracrine mechanism. Furthermore, the mitogenic effect of 3T3-L1 CM requires HGF receptor ligation and activation of tyrosine kinase signaling cascades in SP1 cells. These results highlight the importance of stromal-tumor cell interactions and suggest that HGF secreted by adipocytes may be a key regulator of mammary tumor growth.

The mechanism used by the platelet-derived growth factor receptor (PDGFR) to activate the mitogen-activated- protein-kinase (p42/p44 MAPK) pathway was investigated in cultured airway smooth muscle (ASM) cells. We have found that pertussis toxin (PTX, which was used to inactivate the heterotrimeric G-protein Gi) induced an approx. 40-50% decrease in the activation of c-Src and p42/p44 MAPK by PDGF. An essential role for c-Src was confirmed using the c-Src inhibitor, PP1, which abolished p42/p44 MAPK activation (PP1 and PTX were without effect on PDGFR tyrosine phosphorylation). Furthermore, the PTX-dependent decrease in c-Src and p42/p44 MAPK activation appeared correlated. These findings suggest that the PDGFR can utilize the PTX-sensitive G-protein, Gi, to regulate c-Src and subsequent p42/p44 MAPK activation. Phosphoinositide 3-kinase (PI3K) has been shown by others to be involved in p42/p44 MAPK activation. This is confirmed here by experiments which showed that PI3K inhibitors (wortmannin and LY294002) reduced the activation of p42/p44 MAPK by PDGF. PI3K activity was increased in Grb-2 immunoprecipitates from PDGF-stimulated cells and was decreased by pretreating these cells with PTX. These findings show that Gi might also promote Grb-2-PI3K complex formation and that Grb-2 may be a site at which PI3K is integrated into the p42/p44 MAPK cascade. In conclusion, our results demonstrate that Gi enables the PDGFR to signal more efficiently to p42/p44 MAPK, and this appears to be achieved through the regulation of c-Src and Grb-2/PI3K, which are intermediates in the p42/p44 MAPK cascade.

PP1 has previously been described as an inhibitor of the Src-family kinases p56(Lck) and FynT. We have therefore decided to use PP1 to determine the functional role of Src in platelet-derived growth factor (PDGF)-induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed, and the inhibitory activity of PP1/AGL1872 against Src was examined. PP1/AGL1872 potently inhibited recombinant p60(c-src) in vitro and Src-dependent tyrosine phosphorylation in p60(c-srcF572)-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs, as determined in the modified Boyden chamber, as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly, in addition to inhibition of Src kinase, PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells, including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR, PP1/AGL1872 had only a weak effect on vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases, the activities of the epidermal growth factor receptor, fibroblast growth factor receptor-1, and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872, whereas PDGF alpha-receptor was susceptible, albeit to a lesser extent than PDGF beta-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for the Src family of tyrosine kinases. It is also a potent inhibitor of the PDGF beta-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events, creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity, such as vascular remodeling and restenosis.

Protein kinase C (PKC) isoforms exert specific intracellular functions, but the different isoforms display little substrate specificity in vitro. Selective PKC isoform targeting may be a mechanism to achieve specificity. We used a green fluorescent fusion protein (GFP) to test the hypothesis that local changes in [Ca(2+)](i) regulate translocation of PKCalpha and that different modes of Ca(2+) and Ca(2+) release play a role in PKCalpha targeting. We constructed deletion mutants of PKCalpha to analyze the Ca(2+)-sensitive domains and their role in targeting. Confocal microscopy was used and [Ca(2+)](i) was measured by fluo-3. The fusion protein PKCalpha-GFP was expressed in vascular smooth muscle cells and showed a cytosolic distribution similar to the wild-type PKCalpha protein. The Ca(2+) ionophore ionomycin induced a speckled cytosolic PKCalpha-GFP distribution, followed by membrane translocation, while depolarization by KCl induced primarily membrane translocation. Selective voltage-operated Ca(2+) channel opening led to a localized accumulation of PKCalpha-GFP near the plasma membrane. Opening Ca(2+) stores with InsP(3), thapsigargin, or ryanodine induced a specific PKCalpha-GFP targeting to distinct intracellular areas. The G-protein-coupled receptor agonist thrombin induced a rapid translocation of the fusion protein to focal domains. The tyrosine kinase receptor agonist PDGF induced Ca(2+) influx and led to a linear PKCalpha-GFP membrane association. PKCalpha-GFP deletion mutants demonstrated that the CCa(2+)-induced PKCalpha targeting. Targeting was also abolished when the ATP binding site was deleted. We conclude that PKCalpha can rapidly be translocated to distinct intracellular or membrane domains by local increases in [Ca(2+)](i). The targeting mechanism is dependent on the CCa(2+)](i) changes determine the spatial and temporal targeting of PKCalpha.

Inositol lipid turnover was studied in quiescent Swiss mouse 3T3 cells stimulated by platelet-derived growth factor (PDGF). Stimulation of the cells by PDGF for 10 min at 37 degrees C induced the following changes in lipids: in cells prelabelled with [32P]Pi, a 28% decrease in [32P]phosphatidylinositol 4,5-bisphosphate, a 41% decrease in [32P]phosphatidylinositol 4-phosphate and a 1.7-fold increase in the 32P-labelling of phosphatidic acid; in cells prelabelled with [3H8]arachidonic acid, a 17.9-fold increase in [3H]phosphatidic acid, a 20% decrease in [3H]phosphatidylinositol (PtdIns), an 8.6-fold increase in [3H]arachidonic acid released into the medium, a 57-fold increase in [3H]prostaglandin E2 in the medium, and a 5.3-fold increase in [3H]monoacylglycerol released into the medium (the last was identified as the 2-acyl derivative); in cells prelabelled with [2-3H]glycerol, a 1.7-fold increase in [3H]diacylglycerol, a 6.7-fold increase in [3H]phosphatidic acid, a 1.6-fold increase in [3H]lysophosphatidylcholine (lysoPtdCho), a 9% decrease in [3H]PtdIns, and a 1.6-fold increase in [3H]monoacylglycerol released into the medium. PDGF stimulated the formation of inositol tris-, bis- and mono-phosphates in the cells prelabelled with myo-[2-3H]inositol. These results indicate that, in Swiss 3T3 cells stimulated by PDGF, diacylglycerol produced by the hydrolysis of inositol lipids is partly degraded to 2-acylglycerol and partly converted into phosphatidic acid. The increase in lysoPtdCho indicates that a portion of arachidonic acid released from the stimulated cells is formed by the hydrolysis of PtdCho with a phospholipase A2. Different values of half-maximal doses of the partially purified PDGF used in this study were found for the various responses of quiescent Swiss 3T3 cells to PDGF. The values for half-maximal doses suggest that activation of a fraction of the cell-surface receptor for PDGF is sufficient for mitogenesis and for an increase in the cytoplasmic free Ca2+ concentration, and that the PGDF-stimulated lipid metabolism is probably proportional to the number of receptor sites activated by PDGF.

Phosphatidylinositol (PtdIns) 3-kinase is composed of a catalytic p110 subunit and a regulatory p85 subunit. A synthetic phosphopeptide corresponding to the kinase insert of the human PDGF beta subunit receptor and monoclonal antibodies raised against the two described p85 isoforms, p85 alpha and p85 beta were used to isolate PtdIns 3-kinase from human T lymphocytes. We demonstrate that T cells express both p85 alpha and p85 beta proteins. Both isoforms tightly associate with a p110 protein and with PtdIns 3-kinase activity in T cells. Upon triggering of the T cell antigen receptor (TCR)/CD3 complex or activation of protein kinase C (PKC) the p110 protein complexed to p85 alpha becomes rapidly phosphorylated exclusively on serine residues. p85 alpha does not appear to undergo a change in its basal serine phosphorylation during T cell activation. In contrast, stimulation of the TCR/CD3 complex or PKC, results in a marked and rapid increase in phosphorylation of p85 beta on threonine residues. These data show that PtdIns 3-kinase can be a substrate for serine/threonine kinases in T cells. The differential phosphorylation of p85 alpha and p85 beta reveals the potential for divergent regulation and function of these two PtdIns 3-kinase isoforms during T cell activation.

There are four members of the platelet-derived growth factor (PDGF) family; PDGF-A, PDGF-B, PDGF-C and PDGF-D. Their biological effects are mediated via two tyrosine kinase receptors, PDGFR-alpha and PDGFR-beta, and PDGF-mediated signaling is critical for development of many organ systems. Analysis in adult tissues showed that PDGF-C was mainly expressed in kidney, testis, liver, heart and brain. During development, PDGF-C expression was widespread and dynamic, and found in somites and their derivatives, in kidney, lung, brain, and in several other tissues, particularly at sites of developing epidermal openings. PDGF-C may therefore have unique functions during tissue development and maintenance.

We have identified two tyrosine phosphorylation sites, Tyr 1009 and Tyr 1021, in the C-terminal noncatalytic region of the human platelet-derived growth factor (PDGF) receptor beta subunit. Mutant receptors with phenylalanine substitutions at either or both of these tyrosines were expressed in dog epithelial cells. Mutation of Tyr 1021 markedly reduced the PDGF-stimulated binding of phospholipase C (PLC) gamma 1 but had no effect on binding of the GTPase activator protein of Ras or of phosphatidylinositol 3 kinase. Mutation of Tyr 1009 reduced binding of PLC gamma 1 less severely. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, also reduced the PDGF-dependent binding of a transiently expressed fusion protein containing the two Src-homology 2 domains from PLC gamma 1. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, greatly reduced PDGF-stimulated tyrosine phosphorylation of PLC gamma 1 but did not prevent the tyrosine phosphorylation of other cell proteins, including mitogen-activated protein kinase. We conclude that Tyr 1021, and possibly Tyr 1009, is a binding site for PLC gamma 1.

The extracellular signal-regulated kinases 1/2 (ERK1/2) are activated in cardiomyocytes by Gq protein-coupled receptors and are associated with induction of hypertrophy. Here, we demonstrate that, in primary cardiomyocyte cultures, ERK1/2 were also significantly activated by platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or fibroblast growth factor (FGF), but insulin, insulin-like growth factor 1 (IGF-1) and nerve growth factor (NGF) had relatively minor effects. PDGF, EGF or FGF increased cardiomyocyte size via ERK1/2, whereas insulin, IGF-1 or NGF had no effect suggesting minimum thresholds/durations of ERK1/2 signaling are required for the morphological changes associated with hypertrophy. Peptide growth factors are widely accepted to activate phospholipase C gamma1 (PLCgamma1) and protein kinase C (PKC). In cardiomyocytes, only PDGF stimulated tyrosine phosphorylation of PLCgamma1 and nPKCdelta. Furthermore, activation of ERK1/2 by PDGF, but not EGF, required PKC activity. In contrast, EGF substantially increased Ras.GTP with rapid activation of c-Raf, whereas stimulation of Ras.GTP loading by PDGF was minimal and activation of c-Raf was delayed. Our data provide clear evidence for differential coupling of PDGF and EGF receptors to the ERK1/2 cascade, and indicate that a minimum threshold/duration of ERK1/2 signaling is required for the development of cardiomyocyte hypertrophy.

The discovery of a calcium receptor has stimulated interest in the signaling events underlying extracellular calcium ([Ca2+]o)-induced cell-specific responses. In osteoblasts, elevated levels of extracellular calcium mediate both mitogenesis and chemotaxis. Here we provide evidence that [Ca2+]o-stimulated chemotaxis of MCCa2+]o promotes chemotaxis in a concentration-dependent manner. Pertussis toxin blocked almost all of [Ca2+]o-stimulated chemotaxis but had only a small effect on platelet-derived growth factor (PDGF)-stimulated chemotaxis. Consistent with the signaling model for PDGF-mediated chemotaxis, activation of phospholipase C played a critical role in [Ca2+]o-initiated chemotaxis: U-73122, an inhibitor of the activation of phospholipase C, blocked approximately 50% of PDGF-stimulated chemotaxis but blocked nearly all of the [Ca2+]o-stimulated chemotaxis. Down-regulation of protein kinase C also blocked about 50% of PDGF-stimulated chemotaxis but did not block [Ca2+]o-stimulated chemotaxis. Thus, unlike PDGF-mediated chemotaxis, chemotaxis stimulated by [Ca2+]o does not appear to require protein kinase C activation. This finding suggests events downstream of inositol 1,4,5-trisphosphate production rather than diacylglycerol production are critical to [Ca2+]o-promoted chemotaxis of MCPDGF-induced chemotaxis involves the activation of phosphoinositide 3-kinase, as judged by the in vivo production of phosphatidylinositol 3,4-diphosphate and 3,4,5-trisphosphate and the partial sensitivity of chemotaxis to wortmannin, an inhibitor of phosphoinositide 3-kinase. In contrast, [Ca2+]o-stimulated chemotaxis was not blocked by wortmannin and elevations in [Ca2+]o did not increase the production of lipid products of phosphoinositide 3-kinase. Overall, [Ca2+]o-promoted chemotaxis of osteoblasts appears to utilize a unique signaling mechanism via a calcium-sensing receptor.

The effect of tumor promoters and growth factors on the synthesis of urokinase and urokinase mRNA in human carcinoma cells has been investigated. In urokinase-producing human carcinoma cells (A1251), a 20-40-fold increase in urokinase mRNA level is obtained after treatment with 10 nM phorbol myristate acetate (PMA), a smaller effect (two- to fourfold) with 2 ng/ml platelet-derived growth factor (PDGF) and no effect with epidermal growth factor (EGF) (up to 50 nM). After treatment with PMA, urokinase mRNA level increases already at 30 min peaking 2-4 h thereafter. Cell line A431, which has an abnormally high number of EGF receptors, shows the same response to PMA, but also responds to EGF (two- to fourfold increase in mRNA). The kinetics are similar to those of A1251. Nuclear transcription experiments show that the PMA-induced increase in urokinase mRNA is due to increased synthesis. The protein synthesis inhibitor, cycloheximide (10 micrograms/ml), also increases the level of urokinase mRNA. When both cycloheximide and PMA are used, super-induction is observed. This result may indicate that a short-lived protein negatively regulates the level of urokinase. The different efficiency of the effectors (PMA and PDGF better than EGF) and their kinetics, as well as the effect of cycloheximide on urokinase mRNA synthesis, (a) are reminiscent of the effect of PDGF and PMA on competence phase genes (Kelly, K., B.H. Cochran, C.D. Stiles, and P. Leder, 1983, Cell, 35: 603-610), (b) demonstrate that the synthesis of urokinase is part of the early cellular response to these factors, and (c) provide a preliminary insight in the overproduction of urokinase by primary malignant tumors and transformed cells in culture.

IL-13 is a key cytokine involved in airway remodeling in asthma. We previously reported that IL-13 stimulated the mitogenesis of lung fibroblasts via platelet-derived growth factor (PDGF)-AA. In this report, we show that IL-13 increases PDGF-A and PDGF-C mRNA levels through a dual intracellular cascade that requires coactivation of Stat6 and Stat1 to impact transcriptional regulation of the early growth response (Egr)-1 gene, which then drives PDGF expression. Increased levels of PDGF-AA and PDGF-CC protein were observed in vivo in the airways of IL-13 transgenic mice. IL-13 up-regulated PDGF-A and PDGF-C mRNA levels in lung fibroblasts isolated from three different background strains of mice. However, IL-13-induced PDGF-A and PDGF-C mRNA levels were significantly reduced in Stat6-deficient (Stat6(-/-)) fibroblasts as compared with wild-type Stat6(+/+) fibroblasts. In contrast, IL-13-induced PDGF-A and PDGF-C mRNAs were enhanced in Stat1(-/-) fibroblasts as compared with Stat1(+/+) fibroblasts. IL-13 did not up-regulate PDGF-A or PDGF-C mRNA levels in Egr-1(-/-) fibroblasts. Moreover, IL-13 did not increase Egr-1 mRNA and protein levels in Stat6(-/-) fibroblasts and yet enhanced Egr-1 mRNA and protein levels in Stat1(-/-) fibroblasts. Our findings support the hypothesis that Stat6 and Stat1 exert stimulatory and inhibitory effects on Egr-1 and PDGF ligand mRNA transcription, respectively. This novel mechanism could aid in identifying molecular targets for the treatment of chronic airway remodeling and fibrosis in asthma.