Stimulation of the platelet-derived growth factor beta receptor signaling pathway activates protein kinase C-delta.

The murine myeloid progenitor cell line 32D was recently shown to undergo monocytic differentiation when protein kinase C-delta (PKC-delta) was overexpressed and activated by 12-O-tetradecanoylphorbol-13-acetate (TPA) (H. Mischak, J.H. Pierce, J. Goodnight, M.G. Kazanietz, P.M. Blumberg, and J.F. Mushinski, J. Biol. Chem. 268:20110-20115, 1993). Tyrosine phosphorylation of PKC-delta occurred when PKC-delta-transfected 32D cells were stimulated by TPA (W. Li, H. Mischak, J.-C. Yu, L.-M. Wang, J.F. Mushinski, M.A. Heidaran, and J.H. Pierce, J. Biol. Chem. 269:2349-2352, 1994). In order to elucidate the role played by PKC-delta in response to activation of a receptor tyrosine kinase, we transfected platelet-derived growth factor beta receptor (PDGF-beta R) alone (32D/PDGF-beta R) or together with PKC-delta (32D/PDGF-beta R/PKC-delta) into 32D cells. NIH 3T3 cells which endogenously express both PDGF-alpha R and PDGF-beta R were also transfected with PKC-delta (NIH 3T3/PKC-delta). Like TPA treatment, PDGF-BB stimulation caused striking phosphorylation of PKC-delta in vivo and translocation of some PKC-delta from the cytosol fraction to the membrane fraction in both cell systems. Some of the phosphorylation induced by PDGF-BB treatment was found to be on a tyrosine residue(s). Tyrosine-phosphorylated PKC-delta was observed only for the membrane fraction after stimulation with PDGF-BB or TPA. The enzymatic activity of PKC-delta in the membrane fraction also increased after stimulation with TPA or PDGF, providing a positive correlation between PKC-delta tyrosine phosphorylation and its activation. Overnight treatment of 32D/PDGF-beta R/PKC-delta cells with PDGF-BB induced monocytic differentiation as judged by an increase in expression of cell surface macrophage differentiation markers. PDGF-BB had much weaker effects on 32D/PDGF-beta R cell differentiation, suggesting that increased PKC-delta expression enhanced monocytic differentiation. These results indicate that PKC-delta is a downstream molecule in the PDGFR signaling pathway and may play a pivotal role in PDGF-beta R-mediated cell differentiation.     

Integrin-mediated RON growth factor receptor phosphorylation requires tyrosine kinase activity of both the receptor and c-Src

Cooperation between integrins and growth factor receptors plays an important role in the regulation of cell growth, differentiation, and survival. The function of growth factor receptor tyrosine kinases (RTKs) can be regulated by cell adhesion to extracellular matrix (ECM) even in the absence of ligand. We investigated the pathway involved in integrin-mediated RTK activation, using RON, the receptor for macrophage-stimulating protein. Adhesion of RON-expressing epithelial cells to ECM caused phosphorylation of RON, which depended on the kinase activity of both RON itself and c-Src. This conclusion is based on these observations: 1) ECM-induced RON phosphorylation was inhibited in cells expressing kinase-inactive c-Src; 2) active c-Src could phosphorylate immunoprecipitated RON from ECM-stimulated cells but not from unstimulated cells; and 3) ECM did not cause RON phosphorylation in cells expressing kinase-dead RON, nor could active c-Src phosphorylate RON immunoprecipitated from these cells. The data fit a pathway in which ECM-induced integrin aggregation causes both c-Src activation and RON oligomerization followed by RON kinase-dependent autophosphorylation; this results in RON becoming a target for activated c-Src, which phosphorylates additional tyrosines on RON. Integrin-induced epidermal growth factor receptor (EGFR) phosphorylation also depended on both EGFR and c-Src kinase activities. This sequence appears to be a general pathway for integrin-dependent growth factor RTK activation.     

Syndecan-4 regulates platelet-derived growth factor-mediated MAP kinase activation by altering intracellular reactive oxygen species

The cell adhesion receptor, syndecan-4, regulates cellular interactions with both the extracellular matrix and soluble ligands. Accumulating evidence also suggests that cell adhesion is involved in generating reactive oxygen species (ROS). Here, we investigated the role of syndecan-4 in regulating growth factor-induced ROS generation. Rat embryo fibroblasts (REFs) overexpressing syndecan-4 exhibited increased ROS levels compared to control cells. Expression of the non-phagocytic NADH oxidase component Nox1 was increased in syndecan-4-overexpressing REFs and syndecan-4-mediated ROS generation was diminished when levels of Nox1 were knocked-down with small inhibitory RNAs. In addition, syndecan-4 enhanced platelet-derived growth factor (PDGF)-induced MAP kinase activity in parallel with ROS generation. Collectively, these data suggest that syndecan-4 regulates PDGF-induced MAP kinase activation by altering ROS generation.     

Distinct mechanisms of receptor and nonreceptor tyrosine kinase activation by reactive oxygen species in vascular smooth muscle cells: role of metalloprotease and protein kinase C-delta

Reactive oxygen species (ROS) are implicated in cardiovascular diseases. ROS, such as H2O2, act as second messengers to activate diverse signaling pathways. Although H2O2 activates several tyrosine kinases, including the epidermal growth factor (EGF) receptor, JAK2, and PYK2, in vascular smooth muscle cells (VSMCs), the intracellular mechanism by which ROS activate these tyrosine kinases remains unclear. Here, we identified two distinct signaling pathways required for receptor and nonreceptor tyrosine kinase activation by H2O2 involving a metalloprotease-dependent generation of heparin-binding EGF-like growth factor (HB-EGF) and protein kinase C (PKC)-delta activation, respectively. H2O2-induced EGF receptor tyrosine phosphorylation was inhibited by a metalloprotease inhibitor, whereas the inhibitor had no effect on H2O2-induced JAK2 tyrosine phosphorylation. HB-EGF neutralizing antibody inhibited H2O2-induced EGF receptor phosphorylation. In COS-7 cells expressing an HB-EGF construct tagged with alkaline phosphatase, H2O2 stimulates HB-EGF production through metalloprotease activation. By contrast, dominant negative PKC-delta transfection inhibited H2O2-induced JAK2 phosphorylation but not EGF receptor phosphorylation. Dominant negative PYK2 inhibited H2O2-induced JAK2 activation but not EGF receptor activation, whereas dominant negative PKC-delta inhibited PYK2 activation by H2O2. These data demonstrate the presence of distinct tyrosine kinase activation pathways (PKC-delta/PYK2/JAK2 and metalloprotease/HB-EGF/EGF receptor) utilized by H2O2 in VSMCs, thus providing unique therapeutic targets for cardiovascular diseases.     

The role of c-Src in platelet-derived growth factor alpha receptor internalization

We used two approaches to investigate the role of Src family kinases (SFKs) in ligand-stimulated internalization of the platelet-derived growth factor alpha receptor (alphaPDGFR). In cells that normally express SFKs, the internalization rate of the F72/74 mutant alphaPDGFR (which is unable to recruit or activate SFKs) was slower than that of the wild-type (WT) alphaPDGFR. When expressed in cells lacking SFKs (SYF cells), internalization of the WT and mutant receptors was indistinguishable, which indicated that there was not an inherent defect in the mutant receptor's ability to undergo ligand-driven internalization. The internalization difference between the WT and mutant receptors was seen again when c-Src was expressed in the SYF cells. Surprisingly, c-Src slowed the internalization of the mutant receptor but had little effect on WT receptor. We propose the following working hypothesis to explain these findings. In resting cells SFKs suppress internalization of the alphaPDGFR by phosphorylating a hypothetical protein X. This suppression is relieved when cells are exposed to PDGF because SFKs are recruited to the activated WT alphaPDGFR and thereby no longer actively phosphorylate protein X. The internalization of the mutant receptor is slow because it is unable to recruit SFKs and thereby fails to relieve the suppression of internalization. Our findings suggest a role for SFKs in regulating the permissiveness for internalization of the alphaPDGFR.     

Protein kinase B activation by reactive oxygen species is independent of tyrosine kinase receptor phosphorylation and requires SRC activity

Reactive oxygen species (ROS) participate as second messengers in the mitogenic signal transduction. Most of the experimental data supporting the role of ROS as signaling molecules have been obtained by using H2O2. Exposure of cells to H2O2 rapidly increases tyrosine phosphorylation of tyrosine kinase receptors (TKRs) in the absence of growth factor binding, thus inducing the activation of downstream signaling cascades, like that of protein kinase B (AKT). Another molecule able to induce an increase of intracellular ROS levels is diethylmaleate (DEM), which acts by depleting the ROS scavenger reduced glutathione (GSH). A comparison of the effects exerted by H2O2 and DEM shows that the latter induces redox modifications milder than those generated by H2O2. We also demonstrated that DEM-induced redox modifications are not accompanied by platelet-derived growth factor-receptor (PDGF-R) and epidermal growth factor-receptor Tyr phosphorylation, although they are able to activate ERKs and AKT, with kinetics different from those observed following H2O2 treatment. The activation of these two pathways is not blocked by AG1296, a selective inhibitor of PDGF-R Tyr kinase, thus confirming that the effects of DEM are not mediated by the TKR phosphorylation. On the contrary, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazole[3,4-d]pyrimidine), an inhibitor of Src kinase, completely prevents DEM- and H2O2-induced AKT activation but has no effect on the pathway of ERKs. Finally, nitration of Tyr residues in PDGF-R is observed in DEM-treated cells, thus suggesting that ROS-induced modifications different from Tyr phosphorylation can occur at the growth factor-receptor level and can be involved in the regulation of signaling pathways.     

Ligand-independent oncogenic transformation by the EGF receptor requires kinase domain catalytic activity

The retroviral oncogene S3-v-erbB is a transduced, truncated form of the avian EGF (ErbB-1) receptor. Infection of avian fibroblasts with a retroviral vector expressing S3-v-ErbB results in ligand-independent cell transformation, which is accompanied by the assembly of a transformation-specific phosphoprotein signaling complex and anchorage-independent cell growth. It previously had been reported, using lysine-721 mutants (K721), that kinase domain function was required for ErbB-mediated cell transformation. However, since these initial reports, several studies using aspartate-813 mutants (D813) have demonstrated the ability of kinase-impaired ErbB receptors to induce mitogenic signal transduction pathways and cell transformation in a ligand-dependent manner. To determine the necessity of ErbB receptor kinase domain catalytic activity in ligand-independent cell transformation, we created S3-v-ErbB-K(-), a kinase-impaired oncoprotein constructed by replacing aspartate-813 with alanine (D813A). Subcellular routing as well as cell surface membrane and nuclear localization of the S3-v-ErbB-K(-) mutant receptor were unaffected by impairment of kinase activity. In contrast, avian fibroblasts expressing S3-v-ErbB-K(-) do not form the characteristic transformation-specific phosphoprotein complex, or induce soft agar colony growth in vitro. These results suggest that in contrast to ligand-dependent oncogenic signaling, ligand-independent cell transformation by a constitutively activated mutant form of the EGF receptor requires receptor kinase catalytic activity. In addition, these results demonstrate that phosphorylation and assembly of downstream signaling complexes require tyrosine phosphorylation events that are directly mediated by oncogenic forms of the EGF receptor.     

Sphingosine 1-phosphate stimulation of NADPH oxidase activity: relationship with platelet-derived growth factor receptor and c-Src kinase

This study demonstrates for the first time that sphingosine 1-phosphate (S1P) increases H2O2 production in NIH3T3 fibroblasts through NADPH oxidase activation, confirming the involvement of phosphoinositide-3-kinase and protein kinase C in the activation of this enzyme in non-phagocyte mammalian cells. The results demonstrate also that both platelet-derived growth factor (PDGF) and S1P-mediated NADPH oxidase activation and H2O2 production by Gi-protein coupled receptors (GPCRs) and c-Src kinase. Moreover, both PDGF and S1P activate c-Src kinase through GPCRs, indicating that this kinase can constitute a connection factor between PDGF and S1P signaling, confirming the cross-talk previously found between their receptors. Thus, Gi-protein-mediated NADPH oxidase activation with the consequent H2O2 increase constitutes an early event in the PDGF and S1P pathways. However, a different time course of H2O2 production in S1P-stimulated cells compared to that obtained in PDGF-stimulated cells has been observed, and this seems to be related to the different activation behavior of c-Src kinase induced after S1P or PDGF stimulation. Finally, these data demonstrate that S1P-induced H2O2 production is necessary to maximize c-Src kinase activation, confirming that this is a redox regulated kinase. After which, c-Src plays an important role both upstream and downstream from NADPH oxidase activation.     

Preferential inhibition of the platelet-derived growth factor receptor tyrosine kinase by staurosporine

Ligand stimulation of the platelet-derived growth factor receptor (PDGF-R) results in rapid activation of the receptor tyrosine kinase, stimulation of phosphoinositide hydrolysis, an increase in intracellular free Ca2+ concentration ([Ca2+]i), and, ultimately, cellular proliferation. In a previous study, we demonstrated that staurosporine, a known inhibitor of protein kinase C, blocked PDGF-induced [Ca2+]i increases in Swiss mouse 3T3 fibroblasts by a mechanism that appeared unrelated to inhibition of protein kinase activity (Olsen, R., Melder, D., Seewald, M., Abraham, R., and Powis, G. (1990) Biochem. Pharmacol. 39, 968-972). In the present study, we report that staurosporine inhibits ligand-dependent PDGF-R tyrosine kinase activation in cell-free receptor preparations and in intact Swiss 3T3 cells. At the same concentrations (10(-8)-10(-6) M), staurosporine suppressed both the tyrosine phosphorylation of phospholipase C activity and the hydrolysis of phosphoinositides induced by PDGF stimulation of intact cells. In contrast, guanine nucleotide-binding protein-dependent phospholipase C activation induced by bradykinin or fluoroaluminate anion was relatively insensitive to staurosporine. A preferential inhibitory effect of staurosporine on signal generation by the PDGF-R was indicated by findings that epidermal growth factor receptor (EGF-R) tyrosine kinase activity and EGF-dependent phospholipase C in A-431 carcinoma cells were approximately 100-fold less sensitive to this drug. These data indicate that submicromolar concentrations of staurosporine inhibit PDGF-dependent phosphoinositide hydrolysis and Ca2+ mobilization through a proximal inhibitory effect on ligand-induced activation of the PDGF-R tyrosine kinase.     

Stimulation of epidermal growth factor receptor threonine 654 phosphorylation by platelet-derived growth factor in protein kinase C-deficient human fibroblasts

We have tested the hypothesis that the mechanism of platelet-derived growth factor (PDGF) and phorbol diester action to decrease the apparent affinity of the epidermal growth factor (EGF) receptor is the phosphorylation of the EGF receptor at the Ca2+/phospholipid-dependent protein kinase (protein kinase C) phosphorylation site, threonine 654. Protein kinase C-deficient cells were prepared by prolonged incubation of human fibroblasts with phorbol diester. Addition of phorbol diesters to these cells fails to regulate EGF receptor affinity or threonine 654 phosphorylation. In contrast, PDGF treatment of both control and protein kinase C-deficient fibroblasts causes a decrease in the apparent affinity of the EGF receptor and an increase in threonine 654 phosphorylation. Thus, the ability of PDGF or phorbol diester to modulate EGF receptor affinity occurs only when threonine 654 phosphorylation is increased. The stoichiometry of threonine 654 phosphorylation associated with a 50% decrease in the binding of 125I-EGF to high affinity sites was 0.15 versus 0.3 mol of phosphate per mole of EGF receptor when 32P-labeled fibroblasts are treated with PDGF or phorbol diester, respectively. It is concluded that EGF receptor phosphorylation at threonine 654 can be regulated by PDGF independently of protein kinase C, substoichiometric phosphorylation of the total EGF receptor pool at threonine 654 is caused by maximally effective concentrations of PDGF, and different extents of phosphorylation of EGF receptors at threonine 654 are observed for maximally effective concentrations of PDGF and phorbol diester, respectively. The data are consistent with the hypothesis that a specific subpopulation of EGF receptors that exhibit high affinity for EGF are regulated by threonine 654 phosphorylation.     

Epidermal growth factor rescues trophoblast apoptosis induced by reactive oxygen species

Pre-eclampsia and intrauterine growth restriction are associated with increased apoptosis of placental villous trophoblast. This may result from placental hypoperfusion, leading to the generation of reactive oxygen species (ROS). Apoptosis can be induced in villous trophoblast following exposure to oxidative stress. Epidermal growth factor (EGF) reduces trophoblast apoptosis resulting from exposure to hypoxia. We hypothesised that exposure to hydrogen peroxide, a potent generator of ROS, would induce apoptosis in term placental villous explants and that this could be reduced by treatment with EGF. Placental explants were taken from normal term pregnancies and exposed to increasing doses of hydrogen peroxide (0–1,000 μM) or to a combination of increasing doses of hydrogen peroxide and EGF (0–100 ng/ml) for either 6 or 48 h. Apoptosis was assessed by TUNEL, proliferation by Ki-67 immunostaining, necrosis by lactate dehydrogenase activity and trophoblast differentiation by human chorionic gonadotrophin (hCG) secretion in conditioned culture media. Immunoperoxidase staining was performed to identify phosphorylated-AKT (p-AKT) and phosphorylated-PI3 kinase (p-PI3k). Exposure to 1,000 μM hydrogen peroxide for 48 h induced apoptosis in placental explants. The increase in TUNEL positive nuclei predominantly localised to syncytiotrophoblast. The amount of apoptosis was reduced to control levels by treatment with 10 and 100 ng/ml EGF. Proliferation of cytotrophoblasts within villous explants was significantly reduced following exposure to 1,000 μM hydrogen peroxide, this was restored to control levels by simultaneous treatment with 10 or 100 ng/ml EGF. Neither exposure to hydrogen peroxide or EGF altered the amount of necrosis. There was increased immunostaining for pPI3K following treatment with EGF. This study shows that apoptosis may be induced in villous trophoblast following exposure to ROS, and demonstrates the anti-apoptotic effect of EGF in trophoblast, the maintenance of which is essential for normal pregnancy.     

Vibrio vulnificus-induced death of Jurkat T-cells requires activation of p38 mitogen-activated protein kinase by NADPH oxidase-derived reactive oxygen species

Vibrio vulnificus, a pathogenic bacterium causing primary septicemia, exhibited cytotoxicity towards Jurkat cells of T-lymphocytes through intracellular reactive oxygen species (ROS) production. Pretreatment of Jurkat T-cells with diphenyleneiodonium chloride (DPI) abolished V. vulnificus-induced ROS generation and bacterial ability to cause cell death. Jurkat T-cells expressing dominant-negative protein of Rac subunit of NADPH oxidase (NOX) did not show increased ROS production and cell death by V. vulnificus. Vibrio vulnificus also triggered phosphorylation of mitogen-activated protein kinases (MAPKs) including p38 and ERK1/2 in Jurkat T-cells. Experiments using inhibitors or small interfering RNAs for each MAPK showed that both MAPKs are involved in V. vulnificus-induced cell death. DPI only blocked the phosphorylation of p38 MAPK in Jurkat T-cells exposed by V. vulnificus. This study demonstrates that V. vulnificus induces death of Jurkat T-cells via ROS-dependent activation of p38 MAPK, and that NOX plays a major role in ROS generation in V. vulnificus-exposed cells.     

The involvement of the tyrosine kinase c-Src in the regulation of reactive oxygen species generation mediated by NADPH oxidase-1

NADPH oxidase (Nox) family enzymes are one of the main sources of cellular reactive oxygen species (ROS), which have been shown to function as second messenger molecules. To date, seven members of this family have been reported, including Nox1-5 and Duox1 and -2. With the exception of Nox2, the regulation of the Nox enzymes is still poorly understood. Nox1 is highly expressed in the colon, and it requires two cytosolic regulators, NoxO1 and NoxA1, as well as the binding of Rac1 GTPase, for its activity. In this study, we investigate the role of the tyrosine kinase c-Src in the regulation of ROS formation by Nox1. We show that c-Src induces Nox1-mediated ROS generation in the HT29 human colon carcinoma cell line through a Rac-dependent mechanism. Treatment of HT29 cells with the Src inhibitor PP2, expression of a kinase-inactive form of c-Src, and c-Src depletion by small interfering RNA (siRNA) reduce both ROS generation and the levels of active Rac1. This is associated with decreased Src-mediated phosphorylation and activation of the Rac1-guanine nucleotide exchange factor Vav2. Consistent with this, Vav2 siRNA that specifically reduces endogenous Vav2 protein is able to dramatically decrease Nox1-dependent ROS generation and abolish c-Src-induced Nox1 activity. Together, these results establish c-Src as an important regulator of Nox1 activity, and they may provide insight into the mechanisms of tumor formation in colon cancers.     

Differential phosphorylation of the progesterone receptor by insulin, epidermal growth factor, and platelet-derived growth factor receptor tyrosine protein kinases

Purified preparations of insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) receptors were compared for their abilities to phosphorylate purified hen oviduct progesterone receptors. The specific activities of all three peptide hormone-induced receptor kinases were first defined using a synthetic tridecapeptide tyrosine protein kinase substrate. Next, equivalent ligand-activated activities of the three receptor kinases were tested for their abilities to phosphorylate hen oviduct progesterone receptor. Both the insulin and EGF receptors phosphorylated progesterone receptor at high affinity, exclusively at tyrosine residues and with maximal stoichiometries that were near unity. In contrast, the PDGF receptor did not recognize progesterone receptor as a substrate. Insulin decreased the Km of the insulin receptor for progesterone receptor subunits as substrates, but had no significant effect on Vmax values. On the other hand, EGF increased the Vmax of the EGF receptor for progesterone receptor subunits as substrates. Phosphorylation of progesterone receptor by the insulin and EGF receptor kinases differed in two additional ways. 1) EGF-activated receptor phosphorylated the 80- and 105-kDa progesterone receptor subunits to an equal extent, whereas insulin-activated receptor preferentially phosphorylated the 80-kDa subunit. 2) Phosphopeptide fingerprinting analyses revealed that while insulin and EGF receptors phosphorylated one identical major site on both progesterone receptor subunits, they differed in their specificities for other sites.     

Inhibition of platelet-derived growth factor receptor tyrosine kinase and downstream signaling pathways by Compound C

AMP-activated protein kinase (AMPK) has been implicated in anti-proliferative actions in a range of cell systems. Recently, it was observed that Compound C, an inhibitor of AMPK, also reduced the cell viability in human diploid fibroblasts (HDFs). Compound C-induced growth arrest was associated with a decrease in the cell cycle regulatory proteins, such as proliferating cell nuclear antigen, phosphorylated pRB, cyclin-dependent protein kinases (Cdk 2 and 4), cyclins (D and E), and the Cdk inhibitors (p21, p16, and p27). Therefore, the present study examined the molecular mechanism of the antiproliferative effects of Compound C. Although Compound C inhibited serum-induced phosphorylation of Akt and its substrate, glycogen synthase kinase-3β, it did not affect the Akt activity in vitro. Compound C significantly inhibited the receptor tyrosine phosphorylation and the activity of downstream signaling molecules, such as p85 phosphoinositide 3-kinase, phospholipase C-γ1, and extracellular signal-regulated kinase 1/2, induced by platelet-derived growth factor (PDGF) but not by epidermal growth factor- and insulin-like growth factor. In vitro growth factor receptor tyrosine kinase activity profiling revealed the IC₅₀ for PDGF receptor-β (PDGFRβ) to be 5.07 μM, whereas the IC₅₀ for the epidermal growth factor receptor and insulin-like growth factor receptor was ≥ 100 μM. The inhibitory effect of Compound C on PDGFRβ and Akt was also observed in AMPKα₁/α₂-knockout mouse embryonic fibroblasts, indicating that its inhibitory effect is independent of the AMPK activity. The inhibitory effect of Compound C on cell proliferation and PDGFRβ tyrosine phosphorylation was also demonstrated in various PDGFR-expressing cells, including MRC-5, BEAS-2B, rat aortic vascular smooth muscle cells, and A172 glioblastoma cells. These results indicate that Compound C can be used as a potential antiproliferative agent for PDGF- or PDGFR-associated diseases, such as cancer, atherosclerosis, and fibrosis.     

Requirement for reactive oxygen species in serum-induced and platelet-derived growth factor-induced growth of airway smooth muscle.

Reactive oxygen species have been recently identified as important mediators of mitogenic signaling in a number of cell types. We therefore explored their role in mediating mitogenesis of airway smooth muscle. The antioxidants catalase, N-acetylcysteine, and probucol significantly reduced proliferation in primary cultures of rat tracheal smooth muscle stimulated with fetal bovine serum or platelet-derived growth factor, without affecting cell viability or inducing apoptosis. N-Acetylcysteine also significantly reduced serum-stimulated elevation of c-Fos but did not prevent the normal mitogen-induced increase in c-fos mRNA. Fractionation of ribosomes by sucrose density centrifugation and subsequent dot-blot Northern analysis revealed that antioxidants reduced incorporation of c-fos mRNA into the heaviest polyribosomes, suggesting redox regulation of c-fos mRNA translation. Serum treatment of monolayers produced a small but reproducibly significant rise in superoxide dismutase-inhibitable reduction of ferricytochrome c by myocyte monolayers. Serum-induced ferricytochrome c reduction, cellular proliferation, and c-Fos elevation were decreased by the flavoprotein-dependent enzyme inhibitor dipheyleneiodonium. Growth responses to fetal bovine serum and superoxide dismutase-inhibitable reduction of ferricytochrome c were not different between cultured tracheal myocytes from wild-type versus gp91 phagocyte oxidase null mice. These results suggest that mitogen stimulation of airway smooth muscle induces signal transduction of cell proliferation that is in part dependent on generation of partially reduced oxygen species, generated by an NADH or NADPH oxidoreductase that is different from the oxidase in phagocytic cells.     

Role of tyrosine kinase and membrane-spanning domains in signal transduction by the platelet-derived growth factor receptor.

Three types of mutations were introduced into the platelet-derived growth factor (PDGF) receptor to cause a loss of PDGF-stimulated tyrosine kinase activity: (i) a point mutation of the ATP-binding site, (ii) a deletion of the carboxyl-terminal region, and (iii) replacement of the membrane-spanning sequences by analogous transmembrane sequences of other receptors. Transfectants expressing mutated receptors bind, 125I-labeled PDGF with a high affinity but had no PDGF-sensitive tyrosine kinase activity, phosphatidylinositol turnover, increase in the intracellular calcium concentration, change in cellular pH, or stimulation of DNA synthesis. However, PDGF-induced receptor down regulation was normal in the mutant cells. These results indicate that the transmembrane sequence has a specific signal-transducing function other than merely serving as a membrane anchor and that the receptor kinase activity is necessary for most responses to PDGF but is not required for receptor down regulation.     

Ligand-independent activation of vascular endothelial growth factor receptor 1 by low-density lipoprotein

Elevated serum low-density lipoprotein (LDL) is a risk factor for atherosclerotic disorders. However, prominent atherosclerosis, which has been observed in LDL receptor (LDLR)-knockout mice, has diminished the significance of LDLR as a cause of atherosclerosis, while elaborate studies have focused on the receptors for denatured LDL. Here we report that native LDL (nLDL) activates vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1) but not VEGFR2 through LDLR and is as potent as VEGF in macrophage migration. Binding and co-endocytosis of VEGFR1 and LDLR were enhanced by nLDL, which is concomitant with ubiquitination-mediated degradation of VEGFR1. We propose that LDLR-mediated use of VEGFR1 by nLDL could be a potential therapeutic target in atherosclerotic disorders.