SH2/SH3 adaptor proteins are essential components of the signal transduction pathways initiated by tyrosine kinases. Nck is a ubiquitously expressed adaptor protein whose function has been enigmatic. We performed confocal microscopy to localize Nck in NIH3T3 and A431 cells. Surprisingly, Nck was identified in the nucleus as well as the cytoplasm with no visible change in localization due to PDGF or EGF stimulation. Western blot analysis of nuclear and cytosolic fractions confirmed that there was no translocation in response to growth factor and that tyrosine phosphorylation was specific to only cytosolic Nck. Far Western blot analysis with either Nck, the SH2 domain, or the SH3 domains revealed differential binding in nuclear and cytosolic lysates, indicating specific binding partners for each subcellular location. The major target of c-Src during mitosis is SAM68, a RNA-binding protein ordinarily localized to the nucleus. SAM68 was identified as a nuclear specific binding partner of Nck in both nonmitotic and mitotic cells. Several tyrosine kinases can be found in the nucleus but their signal transduction remains undefined. The discovery of an adaptor protein in the nucleus suggests there are signal transduction mechanisms within the nucleus that recapitulate those found in the cytoplasm.

OBJECTIVE

Hepatic stellate cell (HSC) plays a pivotal role in liver fibrosis and is considered as the therapeutic target for the treatment of hepatic fibrosis. Tyrosine protein kinase plays an important role in the proliferation, activation of HSC. The purpose of the study is to investigate the effects of the tyrosine protein kinase inhibitor genistein on the proliferation and activation of cultured rat HSC.

METHODS

Rat HSC were isolated from Wistar rats by in situ perfusion of collagenase and pronase and single-step density Nycodenz gradient. Culture-activated HSC were serum-starved and incubated with 10(-9) to 10(-5) mol/L concentration of genistein for 24, 48 or 72 h. In PDGF-induced HSC proliferation, HSC were stimulated with 10 microg.L(-1) PDGF-BB for 15 min, and then treated with genistein for the same time. Cell proliferation was measured by MTT assay and based on flow cytometric analysis of cell cycle. The a-smooth muscle actin (alpha-SMA) expression in HSC was studied with confocal laser microscopy and flow cytometry. c-fos, c-jun and cyclin D(1) expression in HSC was also detected by flow cytometry.

RESULTS

Genistein inhibited basal and PDGF-induced proliferation of HSC at the concentration of 10(-8) to 10(-5)mol/L, and treatment with 10(-7) mol/L concentration of genistein for 48 h inhibited the HSC proliferation significantly (the inhibition rate was 70.3 %, P<0.05). Immunofluorescence detected by confocal laser microscopy and flow cytometry showed that treatment with 10(-7) mol/L genistein for 48 h suppressed the expression of alpha-SMA significantly in HSC (the specific fluorescence intensity were 60.2+/-21.5 vs 35.3+/-11.6 and 12.8+/-10.4 vs 9.54+/-6.39, respectively, both P<0.05). The intensity of c-fos, c-jun and cyclin D(1) expression of HSCs treated with 10(-7)mol/L genistein for 48 h was also significantly decreased compared with the controls.

CONCLUSIONS

Genistein influences proliferation of HSC, suppresses the expression of alpha-SMA in HSC and t inhibits the intensity of c-fos, c-jun and cyclin D(1) expression of HSCs. Genistein has therapeutic potential against liver fibrosis.

LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1) is a homolog of the hyaluronan receptor CD44, and one of the most widely used markers of lymphatic endothelial cells in normal and tumor tissues. However, the physiologic role of LYVE-1 in the lymphatic system still remains unclear. It is well established that fibroblast growth factor 2 (FGF2) induces lymphangiogenesis. Based on the known interaction between FGF2 and CD44 and based on the structural similarity of CD44 and LYVE-1, we investigated whether FGF2 might interact with LYVE-1. We found that FGF2 is able to bind LYVE-1 using AlphaScreen, or after surface-immobilization or in solution. FGF2 binds to LYVE-1 with a higher affinity than any other known LYVE-1–binding molecules, such as hyaluronan or PDGF-BB. Glycosylation of LYVE-1 is important for FGF2 binding. Furthermore, FGF2 interacts with LYVE-1 when overexpressed in CHO cells. Soluble LYVE-1 and knockdown of LYVE-1 in lymphatic endothelial cells impaired FGF2 signaling and functions. In addition, FGF2 but not VEGF-C-induced in vivo lymphangiogenesis, was also inhibited. Conversely, FGF2 also modulates LYVE-1 expression in cells and ex vivo. Thus, our data demonstrate a functional relationship to the interaction between FGF2 and LYVE-1.

The low molecular weight protein-tyrosine phosphatase (LMW-PTP) is an enzyme that is involved in the early events of platelet-derived growth factor (PDGF) receptor signal transduction. In fact, LMW-PTP is able to specifically bind and dephosphorylate activated PDGF receptor, thus modulating PDGF-induced mitogenesis. In particular, LMW-PTP is involved in pathways that regulate the transcription of the immediately early genes myc and fos in response to growth factor stimulation. Recently, we have found that LMW-PTP exists constitutively in cytosolic and cytoskeleton-associated localization and that, after PDGF stimulation, c-Src is able to bind and phosphorylate LMW-PTP only in the cytoskeleton-associated fraction. As a consequence of its phosphorylation, LMW-PTP increases its catalytic activity about 20-fold. In this study, our interest was to investigate the role of LMW-PTP phosphorylation in cellular response to PDGF stimulation. To address this issue, we have transfected in NIH-3T3 cells a mutant form of LMW-PTP in which the c-Src phosphorylation sites (Tyr(131) and Tyr(132)) were mutated to alanine. We have established that LMW-PTP phosphorylation by c-Src after PDGF treatment strongly influences both cell adhesion and migration. In addition, we have discovered a new LMW-PTP substrate localized in the cytoskeleton that becomes tyrosine-phosphorylated after PDGF treatment: p190Rho-GAP. Hence, LMW-PTP plays multiple roles in PDGF receptor-mediated mitogenesis, since it can bind and dephosphorylate PDGF receptor, and, at the same time, the cytoskeleton-associated LMW-PTP, through the regulation of the p190Rho-GAP phosphorylation state, controls the cytoskeleton rearrangement in response to PDGF stimulation.

Profibrogeneic cytokines contribute to the accumulation of myofibroblasts in the lung interstitium in idiopathic pulmonary fibrosis (IPF). Imatinib mesylate, a tyrosine kinase inhibitor specific for Abl, platelet-derived growth factor receptor (PDGFR) and c-Kit tyrosine kinases, has been shown to inhibit fibrosis and profibrotic signaling in mouse models of inflammation-mediated lung reactions. The authors tested imatinib mesylate in vivo in a mouse model of crocidolite asbestos-induced progressive fibrosis. The ability of imatinib mesylate to inhibit profibrogeneic cytokine-induced human pulmonary fibroblast migration was tested in vitro and the expression of its target protein tyrosine kinases was assessed with immunofluorescence. In vivo, 10 mg/kg/day imatinib mesylate inhibited histological parenchymal fibrosis and led to a decrease in collagen deposition, but had no significant effect on asbestos-induced neutrophilia. However, 50 mg/kg/day imatinib mesylate did not inhibit collagen deposition. In vitro, IPF fibroblasts expressed Abl, PDGFR-alpha, PDGF-beta, but not c-Kit, and 1 microM imatinib mesylate inhibited profibrogeneic cytokine-induced IPF fibroblast migration. These results suggest that imatinib mesylate is a potential and specific inhibitor of fibroblast accumulation in asbestos-induced pulmonary fibrosis.

NMDA receptor function is modulated by both G-protein-coupled receptors and receptor tyrosine kinases. In acutely isolated rat hippocampal neurons, direct activation of the platelet-derived growth factor (PDGF) receptor or transactivation of the PDGF receptor by D4 dopamine receptors inhibits NMDA-evoked currents in a phospholipase C (PLC)-dependent manner. We have investigated further the ability of D2-class dopamine receptors to modulate NMDA-evoked currents in isolated rat prefrontal cortex (PFC). We have demonstrated that, similar to isolated hippocampal neurons, the application of PDGF-BB or quinpirole to isolated PFC neurons induces a slow-onset and long-lasting inhibition of NMDA-evoked currents. However, in contrast to hippocampal neurons, the inhibition of NMDA-evoked currents by quinpirole in PFC neurons is dependent upon D2/3, rather than D4, dopamine receptors. In PFC slices, application of both PDGF-BB and quinpirole induced a phosphorylation of the PDGF receptor at the PLCgamma binding and activation site, Tyr1021. The PDGF receptor kinase inhibitor, tyrphostin A9, and the D2/3 dopamine receptor antagonist, raclopride, inhibited quinpirole-induced Tyr1021 phosphorylation. These finding suggest that quinpirole treatment inhibits NMDAR signaling via PDGF receptor transactivation in both the hippocampus and the PFC, and that the effects of quinpirole in these regions are mediated by D4 and D2/3 dopamine receptors, respectively.

Glioblastoma (GBM), the most frequent and aggressive brain tumor, is characterized by marked angiogenesis directly related to invasiveness and poor prognosis. Hypoxia is considered to be an important stimulus for angiogenesis by inducing hypoxia-inducible factor 1-alpha (HIF-1α) overexpression that activates platelet-derived growth factor (PDGF) and VEGF. The aim of this study is to analyze the expression of PDGF-C, VEGF in endothelial and tumor cells of GBM and their relation to HIF-1α expression. Two hundred and eight GBM cases were studied by tissue microarray immunohistochemical preparation. Expression of HIF-1α, VEGF and PDGF-C was observed in 184 (88.5%), 131 (63%) and 160 (76.9%) tumor cases, respectively. The numbers of vessels were quantified by CD34, PDGF-C, VEGF and CD105 staining, and were in median 20, 16, 5 and 6, respectively. The GBMs that showed positive or negative expression for HIF-1α showed a median vascular density of 30 and 14, respectively, for CD34 (P < 0.015). Positive expression for HIF-1α was correlated with VEGF and PDGF-C expression in tumors (P < 0.001). There was a significant correlation between VEGF and PDGF-C expression in the cytoplasm of GBM tumor cells (P < 0.0001). We showed that VEGF expression in tumor cells was correlated with its expression in blood vessels (P < 0.0001). Endothelial cells with PDGF-C and VEGF positive expression were also positive for CD105 and their nuclei for Ki-67, confirming the neoangiogenic and proliferative influence of VEGF and PDGF-C. VEGF nuclear staining in tumor cells (P = 0.002) as well as nuclear staining for HIF-1α and VEGF (P = 0.005) correlated with survival. In summary, our present findings of the concomitant upregulation of PDGF-C with VEGF in GBM tumor cells and vessels further reinforce the benefit of using combined anti-angiogenic approaches to potentially improve the therapeutic response for GBM.

BACKGROUND

Formation of haemorrhagic neovessels in the intima of developing atherosclerotic plaques is thought to significantly contribute to plaque instability resulting in thrombosis. C-reactive protein (CRP) is an acute phase reactant whose expression in the vascular wall, in particular, in reactive plaque regions, and circulating levels increase in patients at high risk of cardiovascular events. Although CRP is known to induce a pro-inflammatory phenotype in endothelial cells (EC) a direct role on modulation of angiogenesis has not been established.

RESULTS

Here, we show that CRP is a powerful inducer of angiogenesis in bovine aortic EC (BAEC) and human coronary artery EC (HCAEC). CRP, at concentrations corresponding to moderate/high risk (1-5 microg/ml), induced a significant increase in proliferation, migration and tube-like structure formation in vitro and stimulated blood vessel formation in the chick chorioallantoic membrane assay (CAM). CRP treated with detoxi-gel columns retained such effects. Western blotting showed that CRP increased activation of early response kinase-1/2 (ERK1/2), a key protein involved in EC mitogenesis. Furthermore, using TaqMan Low-density Arrays we identified key pro-angiogenic genes induced by CRP among them were vascular endothelial cell growth factor receptor-2 (VEGFR2/KDR), platelet-derived growth factor (PDGF-BB), notch family transcription factors (Notch1 and Notch3), cysteine-rich angiogenic inducer 61 (CYR61/CCN1) and inhibitor of DNA binding/differentiation-1 (ID1).

CONCLUSIONS

This data suggests a role for CRP in direct stimulation of angiogenesis and therefore may be a mediator of neovessel formation in the intima of vulnerable plaques.

Liver fibrosis is a wound-healing response which engages a variety of cell types to encapsulate injury. Telocyte (TC), a novel type of interstitial cell, has been identified in a variety of tissues and organs including liver. TCs have been reported to be reduced in fibrotic areas after myocardial infarction, human interstitial wall's fibrotic remodelling caused either by ulcerative colitis or Crohn's disease, and skin of systemic sclerosis. However, the role of TCs in human liver fibrosis remains unclear. Liver samples from human liver biopsy were collected. All samples were stained with Masson's trichrome to determine fibrosis. TCs were identified by several immunofluorescence stainings including double labelling for CD34 and c-kit/CD117, or vimentin, or PDGF Receptor-α, or β. We found that hepatic TCs were significantly decreased by 27%-60% in human liver fibrosis, suggesting that loss of TCs might lead to the altered organization of extracellular matrix and loss the control of fibroblast/myofibroblast activity and favour the genesis of fibrosis. Adding TCs might help to develop effective and targeted antifibrotic therapies for human liver fibrosis.

Current treatments for tendon injuries often fail to fully restore joint biomechanics leading to the recurrence of symptoms, and thus resulting in a significant health problem with a relevant social impact worldwide. Cell-based approaches involving the use of stem cells might enable tailoring a successful tendon regeneration outcome. As growth factors (GFs) powerfully regulate the cell biological response, their exogenous addition can further stimulate stem cells into the tenogenic lineage, which might eventually depend on stem cells source. In the present study we investigate the tenogenic differentiation potential of human- amniotic fluid stem cells (hAFSCs) and adipose-derived stem cells (hASCs) with several GFs associated to tendon development and healing; namely, EGF, bFGF, PDGF-BB and TGF-β1. Stem cells response to biochemical stimuli was studied by screening of tendon-related genes (collagen type I, III, decorin, tenascin C and scleraxis) and proteins found in tendon extracellular matrix (ECM) (Collagen I, III, and Tenascin C). Despite the fact that GFs did not seem to influence the synthesis of tendon ECM proteins, EGF and bFGF influenced the expression of tendon-related genes in hAFSCs, while EGF and PDGF-BB stimulated the genetic expression in hASCs. Overall results on cellular alignment morphology, immunolocalization and PCR analysis indicated that both stem cell source can be biochemically induced towards tenogenic commitment, validating the potential of hASCs and hAFSCs for tendon regeneration strategies.

Several growth factors activate signal transducers and activators of transcription (Stats) but the mechanism of Stat activation in receptor tyrosine kinase signalling has remained elusive. In the present study we have analysed the roles of different platelet-derived growth factor (PDGF)-induced tyrosine kinases in the activation of Stat5. Co-expression experiments in insect and mammalian cells demonstrated that both PDGF beta-receptor (PDGF beta-R) and Jak1, but not c-Src, induced the activation of Stat5. Furthermore, immune-complex-purified PDGF beta-R was able to phosphorylate Stat5 directly. The role of the cytoplasmic tyrosine kinases in the PDGF-induced activation of Stat5 was further investigated by overexpressing kinase-negative (KN) and wild-type Jak and c-Src kinases. Jak1-KN or Jak2-KN had no effect but both Src-KN and wild-type c-Src similarly decreased the PDGF-beta-R-induced activation of Stat5. The activation of both Src and Stat5 is dependent on the same tyrosine residues Tyr(579) and Tyr(581) in PDGF beta-R; thus the observed inhibition by Src might result from competition for binding of Stat5 to the receptor. Finally, fibroblasts derived from Src(-/-) and Fyn(-/-) mice showed normal pattern of PDGF-induced tyrosine phosphorylation of Stat5. Taken together, these results indicate that Stat5 is a direct substrate for PDGF beta-R and that the activation does not require Jak1, Jak2, c-Src or Fyn tyrosine kinases.

Imatinib mesylate, also known as STI571 or CGP57148, is a competitive inhibitor of a few tyrosine kinases, including BCR-ABL, ABL, KIT, and the platelet-derived growth factor receptors (PDGF-R). It binds to the ATP-binding site of the target kinase and prevents the transfer of phosphate from ATP to the tyrosine residues of various substrates. At oral doses of 300 mg or greater, the vast majority of patients with chronic myeloid leukaemia achieve a haematological response and this is usually associated with limited toxicity. Imatinib also has substantial activity in Philadelphia chromosome-positive acute lymphoblastic leukaemia expressing the BCR-ABL fusion protein. Gastrointestinal stromal tumours (GISTs) have also been evaluated for clinical activity of imatinib. About 90% of malignant GISTs harbour a mutation in c-kit leading to KIT receptor autophosphorylation and ligand-independent activation. According to initial clinical studies, more than 50% of GISTs respond to therapy within a few months, and only about 10-15% progress. The potential for cure and the optimal length of treatment are currently not known. Several other human cancers may over-express KIT or PDGF-R, and clinical trials to evaluate the role of imatinib in the treatment of such cancers are currently ongoing. Imatinib is an example of a specifically designed, highly targeted cancer therapy, which poses novel requirements for both pathology laboratories and clinicians in terms of identifying the major molecular mechanisms involved in tumour growth.

Platelet-derived growth factor AA (PDGF AA), in contrast to PDGF AB and BB, is a poor mitogen for smooth muscle cells (SMC). However, together with basic fibroblast growth factor (bFGF) it acts synergistically on DNA synthesis of these cells. Northern blot analysis revealed that bFGF selectively increases the PDGF-receptor alpha subtype (PDGF-R alpha) mRNA level without a significant effect on the PDGF-R beta mRNA level. The amount of PDGF-R alpha protein is also selectively increased after stimulating SMC with bFGF as shown by immunoprecipitation of lysates from SMC with anti-PDGF-R alpha antibodies. The number of binding sites for 125I-PDGF AA is more than doubled after bFGF-treatment, whereas the specific binding for PDGF AB and BB increased only by approximately 30 and 20%, respectively. The increase in the number of PDGF-R alpha renders the SMC responsive for PDGF AA as demonstrated by the induction of the proto-oncogene c-fos as well as by an increased cell proliferation. The enhanced PDGF binding after bFGF treatment may in fact explain the observed synergistic behavior. These data are discussed with regard to a possible role of growth factor-induced transmodulation of receptor expression during atherogenesis.

Agrin, a molecule produced by motoneurons that induces the aggregation of nicotinic acetylcholine receptors (nAChRs), has recently been structurally characterized. Agrin-related proteins (ARPs) that arise from differential splicing are synthesized by neurons and muscle. The C-terminal region of agrin that instructs muscle to aggregate nAChRs contains three laminin A modules separated by epidermal growth factor-like modules. Alternative splicing in the laminin A modules leads to the formation of at least three ARPs that are devoid of nAChR-aggregating activity. In their N-terminal regions, both agrin and ARPs contain nine follistatin-related modules that, like those in follistatin and in another related protein, osteonectin, may have the capability to bind members of the transforming growth factor beta (TGF-beta) or platelet-derived growth factor (PDGF) families. This review proposes that these follistatin-like regions of agrin and ARPs might bind and localize growth factors, and thus provide a matrix-bound concentration of them. Beyond agrin's role in inducing AChR aggregation, the function of agrin and ARPs to provide a localized reservoir of growth factors could contribute to the formation and maintenance of the long-lasting synaptic architecture by specifying and limiting the area of influence of these molecules.

Transcriptional up-regulation of the c-sis/platelet-derived growth factor-B (PDGF-B) proto-oncogene by the Tax protein of human T-cell leukemia virus type 1 has been implicated as one possible mechanism of cellular transformation by human T-cell leukemia virus type 1. In previous work, we identified an essential site in the c-sis/PDGF-B promoter, Tax-responsive element 1 (TRE1), necessary for transactivation by Tax. We also identified Sp1, Sp3, and NGFI-A/Egr-1 as the primary nuclear transcription factors binding to TRE1 which mediate Tax responsiveness. In the present work, we have investigated the mechanism(s) whereby Tax transactivates the c-sis/PDGF-B proto-oncogene. In vitro transcription assays showed that Tax was able to significantly increase the transcriptional activity of a template containing the -257 to +74 region of the c-sis/PDGF-B promoter. Electrophoretic mobility shift assay analysis showed that Tax increased the DNA binding activity of both Sp1 and NGFI-A/Egr-1 using a TRE1 probe. Analysis of Tax mutants showed that two mutants, IEXC29S and IEXL320G, were unable to significantly transactivate the c-sis/PDGF-B promoter. Finally, co-immunoprecipitation analysis revealed that Tax is able to stably bind to both Sp1 and NGFI-A/Egr-1. Interestingly, co-immunoprecipitation analysis also revealed that Tax mutant IEXC29S is unable to interact with NGFI-A/Egr-1, whereas Tax mutant IEXL320G is able to interact with NGFI-A/Egr-1.

Adherence is an important initial step in the transition of a circulating monocyte to a tissue macrophage. This differentiation is accompanied by an augmented capacity to generate growth factors. We hypothesized that adherence itself might be an important trigger for a sequence of gene activation culminating in cells with increased mRNA encoding profibrotic growth factors such as platelet-derived growth factor B subunit (PDGF[B]) and transforming growth factor-beta (TGF-beta). After in vitro adherence, human monocytes had a biphasic increase in PDGF(B) mRNA with peaks at 6 h and 13 d. No increase in TGF-beta mRNA was observed. The 6-h increase in PDGF(B) mRNA was adherence dependent, and in addition, was abrogated when the cytoskeletal integrity was compromised by cytochalasin D. The 6-h increase in PDGF(B) mRNA was unaltered by adherence in the presence of the monocyte stimulus lipopolysaccharide. Adherence to either fibronectin or collagen-coated plastic had little consistent effect on PDGF(B) mRNA accumulation. The increased PDGF(B) mRNA observed in adherent monocytes was accompanied by increases in mRNAs of the early growth response genes c-fos (maximal at 20 min), c-jun, and EGR2 (maximal at 6-24 h). The increase in c-jun and EGR2, but not c-fos, mRNA was also abrogated by cytochalasin D. These observations suggest that adherence results in increases of c-fos, c-jun, EGR2, and PDGF(B) mRNA. In addition, the increases in c-jun, EGR2, and PDGF(B) may depend on cytoskeletal rearrangement. Modulation of these events at the time of adherence offers a mechanism by which differential priming of the cells may be accomplished.

BACKGROUND

Platelet derived growth factors (PDGFs) are mitogens for fibroblasts and smooth muscle cells. This growth factor family contains four members PDGF-A, PDGF-B, PDGF-C and PDGF-D. Biology of recently discovered PDGF-C and PDGF-D is not well-established. Here we studied the expression of PDGF-C and PDGF-D and their receptors PDGFR-alpha and PDGFR-beta in normal and atherosclerotic human arteries.

METHODS

Human arterial samples from amputations and autopsies were classified according to the atherosclerotic stage and the expression of PDGF-C and PDGF-D proteins and their receptors was studied by immunohistochemistry. In situ hybridization and reverse transcriptase-PCR were used to study mRNA expression.

RESULTS

Both growth factors were expressed in medial smooth muscle cells (SMCs) in normal arteries and atherosclerotic lesions. However, clear differences were found in the expression profiles in endothelium: PDGF-C was strongly expressed in endothelial cells in both normal arteries and lesions whereas PDGF-D was only weakly expressed in endothelium. PDGF-C expression was very prominent in lesion macrophages. PDGF-D was expressed throughout the artery wall in lesions. PDGFR-alpha expression was strong in endothelium and in lesion macrophage-rich areas, whereas PDGFR-beta was mostly expressed in SMCs.

CONCLUSIONS

Our results suggest that PDGF-C may play an important role in endothelium in normal and atherosclerotic arteries and in macrophages in lesions. PDGF-D was expressed in all types of lesions with the same intensity and thus differs from the expression of PDGF-C.

In rat type I astrocytes and CPDGF) and endothelin. In CC, an inhibitor of protein kinase C (PKC), or pertussis toxin (PTX), and completely inhibited by the combination of these agents. Essentially, the same inhibitory pattern by these agents has been observed for S1P-induced extracellular signal-regulated kinase (ERK) activation. The S1P-induced expression of Egr-1 was also completely inhibited in association with complete inhibition of ERK by PD 98059, an ERK kinase inhibitor. Thus, the S1P-induced activation of the Egr-1/FGF-2 system may be mediated through ERK activation, which may involve at least two signaling pathways, i.e., a PTX-sensitive G-protein-dependent pathway and a PKC-dependent pathway.

Meningioma is a generally benign tumor derived from arachnoid tissue. We have investigated the presence of functionally active PDGF-receptors on human meningioma cells in culture. Tumor samples were obtained from 3 surgically removed benign meningiomas and normal arachnoid tissue from an autopsy case. Binding studies were performed by using 125I-labelled recombinant PDGF-AA and PDGF-BB. Only 125I-PDGF-BB showed specific binding to all tumor-cell cultures after incubation of cells for 2 hr at 4 degrees C. Effects of PDGF-AA and PDGF-BB on DNA synthesis were measured as 3H-thymidine incorporation during 48 hr of labelling cells maintained in Eagle's minimum essential medium 0.5% fetal calf serum. PDGF-BB but not PDGF-AA stimulated DNA synthesis in all 3 tumor-cell cultures. Total cellular RNA was analyzed by Northern blotting and hybridization with a 32P-labelled human PDGF beta-receptor probe, and PDGF beta-receptor mRNA was found in both tumor and arachnoid cell cultures. Furthermore, PDGF beta-receptor mRNA was shown to be present in 2 meningioma biopsies and immunohistochemical staining revealed that PDGF beta-receptors are present in meningioma and arachnoid tissues in vivo. It appears that a possible way of maintaining human meningioma cell growth in vivo is through activation of PDGF beta-receptors.

Proliferation of vascular smooth muscle cells (SMC) contributes to formation of the complicated human atherosclerotic plaque. These lesions also contain macrophages, known to secrete SMC mitogens, and T lymphocytes. Many of the SMC in the lesions express class II major histocompatibility antigens, an indication that activated T cells secrete immune IFN-gamma locally in the plaque. We therefore studied the effect of IFN-gamma on the proliferation of cultured SMC derived from adult human blood vessels. IFN-gamma (1,000 U/ml) reduced [3H]thymidine (TdR) incorporation into DNA by SMC stimulated with the well-defined mitogens IL 1 (from 15.3 +/- 0.7 to 6.2 +/- 0.7 dpm X 10(-3)/24 h) or platelet-derived growth factor (PDGF) (from 18.5 +/- 1.0 to 7.3 +/- 0.7 dpm X 10(-3)/24 h). Kinetic and nuclear labeling studies indicated that this effect of IFN-gamma was not due to altered thymidine transport or specific radioactivity of TdR in the cell. In longer term experiments (4-16 d) IFN-gamma prevented net DNA accumulation by SMC cultures stimulated by PDGF. IFN-gamma also delayed (from 30 to 60 min) the time to peak level of c-fos RNA in IL 1-treated SMC. It is unlikely that cytotoxicity caused these effects of IFN-gamma, as the inhibition of growth was reversible and we detected no cell death in SMC cultures exposed to this cytokine. Activation of 2'-5' oligoadenylate synthetase gene expression may mediate certain antiproliferative and antiviral effects of interferons. Both IFN-gamma and type I IFNs (IFN-alpha or IFN-beta) induced 2'-5' oligoadenylate synthetase mRNA and enzyme activity in SMC cultures, but with concentration dependence and time course that may not account for all of IFN-gamma's cytostatic effect on SMC. The accumulation of SMC in human atherosclerotic lesions is a long-term process that must involve altered balance between growth stimulatory and inhibitory factors. The cytostatic effect of IFN-gamma on human SMC demonstrated here may influence this balance during human atherogenesis, because T cells present in the complicated atherosclerotic plaque likely produce this cytokine.

Phorbol esters induce the differentiation of human myeloid leukemia cells HL-60 and U-937 along the monocytic-macrophage lineage. This process has been associated with the induction of several cellular protooncogenes, including the c-fos and c-fms genes. We now report that phorbol ester-induced differentiation of the HL-60 and U-937 cells results in the induction of the expression of the c-sis platelet-derived growth factor 2 (PDGF-2) protooncogene. sis mRNA transcripts were not detectable in the uninduced cells but were detectable within 12 hr of phorbol ester induction. Concomitantly, the induced cells were shown to synthesize and secrete biologically active PDGF-like proteins, identified in the conditioned medium of the phorbol ester-treated cells by direct immunoprecipitation with PDGF antiserum. Addition of cycloheximide to phorbol ester-treated HL-60 cells superinduced sis mRNA transcripts. c-sis gene transcripts were also detected in freshly isolated human monocytes but not in human granulocytes or in HL-60 cells induced to differentiate along the granulocytic lineage. Activation of the c-sis/PDGF-2 gene in human hematopoietic cells during monocytic differentiation may serve in the mediation of physiologic functions of the differentiated cells by means of the secretion of potent PDGF-like mitogen.

BACKGROUND

To explore the role of intracellular oxidative stress in high glucose-induced atherogenesis, we examined the effect of probucol and/or alpha-tocopherol on the migration and growth characteristics of cultured rabbit coronary vascular smooth muscle cells (VSMCs).

RESULTS

Chronic high-glucose-medium (22. 2 mmol/L) treatment increased platelet-derived growth factor (PDGF)-BB-mediated VSMC migration, [3H]thymidine incorporation, and cell number compared with VSMCs treated with normal-glucose medium (5.6 mmol/L+16.6 mmol/L mannose). Probucol and alpha-tocopherol significantly suppressed high glucose-induced increase in VSMC migration, cell number, and [3H]thymidine incorporation. Probucol and alpha-tocopherol suppressed high glucose-induced elevation of the cytosolic ratio of NADH/NAD+, phospholipase D, and membrane-bound protein kinase C activation. Probucol, alpha-tocopherol, and calphostin C improved the high glucose-induced suppression of insulin-mediated [3H]deoxyglucose uptake. Chronic high-glucose treatment increased the oxidative stress, which was significantly suppressed by probucol, alpha-tocopherol, suramin, and calphostin C.

CONCLUSIONS

These findings suggest that probucol and alpha-tocopherol may suppress high glucose-induced VSMC migration and proliferation via suppression of increases in the cytosolic ratio of free NADH/NAD+, phospholipase D, and protein kinase C activation induced by high glucose, which result in reduction in intracellular oxidative stress.

Using site-directed mutagenesis of a PDGF-A cDNA clone, we identify two domains that are required to generate stable, mitogenically active PDGF-AA homodimers. Alteration of the tetra-basic amino acid sequence (Arg84-Arg-Lys-Arg to Arg-Ser-Asn-Gly) results in the formation of stable pro-PDGF-A homodimers that lack mitogenic activity. Substitution of serine for Cys129 destabilizes PDGF-A subunits within the cell. Genes incorporating either the processing lesion or the cysteine substitution suppress wild-type PDGF-A gene expression in a trans-dominant fashion. Suppression occurs because the mutant PDGF subunits dimerize with wild-type subunits to form inactive or unstable heterodimers. Suppression is exerted across phylogenetic boundaries; thus, the mouse PDGF-A chain mutants inhibit the activity of the wild-type Xenopus PDGF-A. The cysteine mutant gene suppresses expression of PDGF-B (c-sis), as well as PDGF-A. The processing mutant gene, however, suppresses only PDGF-A. Dominant-negative mutations of PDGF and other growth factors which, like PDGF, function as dimers may prove useful for creating animals models of growth factor deficiency disease states and for revealing the function of growth factors during early embryonic development.