OBJECTIVE

The protein kinase C (PKC), platelet-derived growth factor (PDGF) and polyol pathway play important parts in the hyperproliferation of smooth muscle cells, a characteristic feature of diabetic macroangiopathy. The precise mechanism, however, remains unclear. This study investigated the relation between polyol pathway, protein kinase C and platelet-derived growth factor in the development of diabetic macroangiopathy.

METHODS

Smooth muscle cells were cultured with 5.5 or 20 mmol/l glucose with or without an aldose reductase inhibitor, epalrestat, or a PKC-beta specific inhibitor, LY333531. Protein kinase C activities, the expression of PKC-beta II isoform and PDGF-beta receptor protein, free cytosolic NAD+:NADH ratio, the contents of reduced glutathione, and proliferation activities were measured.

RESULTS

Smooth muscle cells cultured with 20 mmol/l glucose showed statistically significant increases in protein kinase C activities, the expression of PKC-beta II isoform and PDGF-beta receptor protein, and proliferation activities, compared with smooth muscle cells cultured with 5.5 mmol/l glucose. Although epalrestat and LY333531 inhibited protein kinase C activation induced by glucose to the same degree, the effects of epalrestat on proliferation activities and expression of the PDGF-beta receptor were more prominent than those of LY333531. Epalrestat improved the glucose-induced decrease in free cytosolic NAD+:NADH ratio and reduced glutathione content, but LY333531 did not. The increased expression of membranous PKC-beta II isoform was normalized by epalrestat.

CONCLUSIONS

These observations suggest that polyol pathway hyperactivity contributes to the development of diabetic macroangiopathy through protein kinase C, PDGF-beta receptor, and oxidative stress, and that an aldose reductase inhibitor has a therapeutic value for this complication.

Malignant solitary fibrous tumor (MSFT) of the pleura is a rare neoplasm, with unpredictable biologic behavior and a low sensitivity to chemotherapy. To the authors' knowledge, no other effective medical treatment is available for this disease. Imatinib mesylate is a tyrosine kinase inhibitor targeting the platelet-derived growth factor (PDGFR-alpha and PDGFR-beta), the BCR-ABL, and c-KIT receptors. We report the first evidence of the activity of imatinib in a symptomatic patient with a chemo- and radio-resistant advanced MSFT, who obtained a 21-months lasting major clinical benefit with a consistent reduction in tumor metabolism. Immunostaining of tumor cells demonstrated the positivity for PDGFR-alpha and PDGFR-beta and the absence of c-KIT over-expression, in the absence of c-KIT and PDGRFR mutations; all the cells strongly and diffusely expressed the ligand PDGF A in the cytoplasm. This profile suggests that the observed tumor response was mediated through the inhibition of the tyrosine kinase activity of PDGFR. Treatment with imatinib should be considered for patients with recurrent or unresectable MSFTs with PDGFR expression.

Platelet-derived growth factor (PDGF) is a multifunctional protein that plays important roles in many tissues, including the mammalian central nervous system. PDGF and PDGF receptors (PDGFRs) are expressed in virtually every region of the central nervous system where they are involved in the development, survival, growth, and differentiation of both neuronal and glial cells. We now report that a brief activation of PDGFRs produced a long-lasting inhibition of N-methyl-D-aspartate (NMDA)-dependent excitatory postsynaptic currents in CA1 pyramidal neurons in rat hippocampal slices. PDGF also inhibited NMDA receptors (NMDA-Rs) in cultured hippocampal neurons by a mechanism that involves a decrease in single channel open probability. Non-NMDA receptor function was not affected by PDGF in hippocampal neurons. Experiments with mutant PDGFRs and chelation of intracellular Ca2+ in Xenopus oocytes indicate that this inhibition depends on a phospholipase C-gamma-induced elevation of intracellular Ca2+ levels. The PDGF-induced inhibition of NMDA-Rs is produced by a mechanism different than the well characterized phenomenon of Ca2+-dependent NMDA-R run down because the effect of PDGF was blocked by the phosphatase inhibitor, calyculin A, and was not affected by the microtubule polymerizing agent, phalloidin. Because elevations of PDGF levels are associated with neurological trauma or disease, we propose that PDGF can exert neuroprotective effects by inhibiting NMDA-R-dependent excitotoxicity.

BACKGROUND

All-trans retinoic acid (ATRA) has antiproliferative and anti-inflammatory effects and is currently used in the treatment of leukemia and dermatologic diseases. We tested the therapeutic potential of ATRA on anti-glomerular basement membrane (GBM) glomerulonephritis rats.

METHODS

Glomerulonephritis was induced in male Wistar-Kyoto rats on day 0 by an intravenous injection of antirat GBM antibody. On day 14 after the induction of anti-GBM glomerulonephritis, some rats were sacrificed (N = 5). Another 10 rats were divided into two groups: the vehicle group (N = 5) and the ATRA treated group (N = 5). ATRA was orally administrated from day 14 to day 27 after disease induction. Blood pressure, body weight, urinary protein excretion, and blood chemistry was determined on days 1, 14, 21, and 27. Kidney samples were obtained on day 28. The kidneys were examined with periodic acid-Schiff staining (PAS) and immunohistochemistry using antibodies against the proliferative cell nuclear antigen (PCNA), rat monocyte and macrophage (ED-1), and alpha-smooth muscle actin (alpha-SMA). Glomerular RNA was extracted from isolated glomeruli, and reverse transcription (RT) followed by polymerase chain reaction (PCR) was performed.

RESULTS

ATRA administration produced a 55% reduction of proteinuria in glomerulonephritis rats. Light microscopic analysis revealed severe necrosis/crescent formation (>50% of the glomerulus) affecting 34% of glomeruli in vehicle rats, whereas ATRA treatment reduced the glomeruli showing severe change to 14%. ATRA also significantly reduced PCNA-positive cells, ED-1-positive cells and alpha-SMA-positive area in the glomeruli. RT-PCR analyses revealed that a wide variety of genes including inflammation related [tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and CCAAT enhancer-binding protein delta (C/EBPdelta)], cell proliferation-related [platelet-derived growth factor (PDGF)] and fibrosis-related [transforming growth factor-beta1 (TGF-beta1), type I collagen, and alpha-SMA) genes were suppressed in the glomeruli of ATRA-treated rats.

CONCLUSIONS

ATRA administration significantly reduced severe necrosis/crescent formation and urinary protein excretion in glomerulonephritis rats. Suppression of a wide variety of gene expression may partly explain the mechanism of ATRA's antiproliferative and anti-inflammatory effects. These data suggest a novel therapeutic application of ATRA toward glomerulonephritis.

BALB/c 3T3 mouse cells exposed briefly to platelet-derived growth factor (PDGF) become "competent" to replicate their DNA and divide. When cells are treated with PDGF and then fused to untreated cells, the resulting heterokaryons become competent to replicate their DNA. Cytoplasts derived from PDGF-treated cells are also able to transfer the growth response to untreated cells. After cytoplasmic transfer to another cell, the strength of the PDGF-induced mitogenic signal is attenuated by a factor roughly proportional to the increase in total cytoplasmic volume. When RNA synthesis is blocked during PDGF treatment, cells do not acquire the capacity to transfer the PDGF growth signal to untreated cells. By contrast, exposure to cycloheximide during PDGF treatment has no effect. These observations suggest that cytoplasmic transfer of the growth response to PDGF (competence) is mediated by a PDGF-induced stable RNA rather than by PDGF itself or a PDGF--receptor complex. The onset of DNA synthesis in PDGF--control heterokaryons occurs a minimum of 11 hr after cell fusion. Thus the substance that is transferred in these cell fusions is not directly involved in DNA synthesis; rather, it seems to trigger a sequence of events culminating in DNA synthesis.

The mechanism of Raf-1 activation by platelet-derived growth factor (PDGF) is poorly defined. We previously reported that, in Rat-1 fibroblasts, PDGF activates a phosphatidylcholine-specific phospholipase C (PC-PLC) and that the product, diacylglycerol, somehow activates protein kinase C-zeta (PKC-zeta). Both PC-PLC and PKC-zeta activities were required for PDGF activation of mitogen-activated protein kinase (MAPK). Now we report that MAPK activation by exogenous PC-PLC depends on Raf-1 activation. PKC-zeta co-immunoprecipitates with, phoshorylates and activates Raf-1, suggesting that in the PDGF- and PC-PLC-activated MAPK pathway, PKC-zeta operates in a signalling complex as a direct activator of Raf-1.

Five randomly identified cosmids have been mapped proximal to the Leh66D locus on mouse chromosome 17. Two of these cosmids, Au10 and Au119, map near the neurological mutation quaking. Au119 is deleted in qkviable/qkviable DNA, whereas Au10 is not. Au76 maps to a gene-rich region near the Time locus. The Au76 locus encodes a member of a low copy gene family expressed in embryos, the adult central nervous system and testis. A second member of this family has been mapped to chromosome 15 near c-sis (PDGF-B). At the centromeric end of chromosome 17, Au116 maps near the Tu1 locus, and along with Au217rs identifies a region of unusually high recombinational activity between t-haplotypes and wild-type chromosomes. Au217I and II map to the large inverted repeats found at the proximal end of the wild-type chromosome. In addition, the Au217I and/or II loci encode testis transcripts not expressed from t-haplotypes.

We examined whether PDGF may directly stimulate the expression of VEGF by retinal pigment epithelial (RPE) cells in vitro, and the involvement of three signal transduction pathways in the regulation of PDGF-evoked cell proliferation, migration, and production of VEGF-A was investigated. PDGF stimulated the gene and protein expression of VEGF-A by RPE cells, and increased cell proliferation and chemotaxis. PDGF activated all signaling pathways investigated, as determined by increased phosphorylation levels of ERK1/2, p38, and Akt proteins. The three signaling pathways were involved in the mediation of PDGF-evoked cell proliferation, while p38 and PI3K mediated cell migration, and PI3K mediated secretion of VEGF-A. In addition to VEGF-A, the cells expressed mRNAs for various members of the VEGF family and for their receptors, including VEGF-B, -C, -D, flt-1, and KDR. The data indicate that PDGF selectively stimulates the expression of VEGF-A in RPE cells. PDGF evokes at least three signal transduction pathways which are differentially involved in various cellular responses.

We studied the expression of the genes encoding the A and B chains of platelet-derived growth factor (PDGF) in a number of human leukemia cell lines. Steady-state expression of the A-chain RNA was seen only in the promonocytic leukemia cell line U937 and in the T-cell leukemia cell line MOLT-4. It has previously been reported that both PDGF A and PDGF B genes are induced during megakaryoblastic differentiation of the K562 erythroleukemia cells and transiently during monocytic differentiation of the promyelocytic leukemia cell line HL-60 and U937 cells. In this study we show that PDGF A RNA expression was induced in HL-60 and Jurkat T-cell leukemia cells and increased in U937 and MOLT-4 cells after a 1- to 2-h stimulation with an 8 pM concentration of transforming growth factor beta (TGF-beta). PDGF A RNA remained at a constant, elevated level for at least 24 h in U937 cells, but returned to undetectable levels within 12 h in HL-60 cells. No PDGF A expression was induced by TGF-beta in K562 cells or in lung carcinoma cells (A549). Interestingly, essentially no PDGF B-chain (c-sis proto-oncogene) RNA was expressed simultaneously with PDGF A. In the presence of TGF-beta and protein synthesis inhibitors, PDGF A RNA was superinduced at least 20-fold in the U937 and HL-60 cells. PDGF A expression was accompanied by secretion of immunoprecipitable PDGF to the culture medium of HL-60 and U937 cells. The phorbol ester tumor promoter tetradecanoyl phorbol acetate also increased PDGF A expression with similar kinetics, but with a mechanism distinct from that of TGF-beta. These results suggest a role for TGF-beta in the differential regulation of expression of the PDGF genes.

The G0/G1 to S transition in quiescent BALB/c 3T3 cells stimulated by serum growth factors can be specifically blocked by the administration of interferon (IFN) to the system. In the present communication, we studied whether IFN inhibits the early events in the G0/G1 phase that are initiated by the platelet-derived growth factor (PDGF). The results show that IFN inhibits most of the PDGF-mediated increase of c-myc, ornithine decarboxylase, and beta-actin mRNAs measured 3 hr after stimulation. c-fos mRNA levels are reduced by IFN as early as 20 min after exposure of the quiescent cells to PDGF. The expression of several genes that belong to the competence gene family is, therefore, inhibited by IFN and this could account for the failure of the IFN-treated cells to enter into the S phase when growth factors present in the platelet-poor plasma are added. We also report that the PDGF-mediated increase in the uptake of deoxyglucose is not impaired by IFN, thus suggesting that the early effects of IFN on gene expression do not result from inhibition of binding of PDGF to its cell-surface receptors. Unlike the direct stimulatory effect of PDGF, which is not sensitive to cycloheximide, the inhibitory effect of IFN on c-myc mRNA levels depends in part on protein synthesis. We propose that a putative product of one of the IFN-induced genes could mediate the decrease in expression of the PDGF-regulated gene family.

Diverse signals have the potential to modulate gene transcription through the Ca2+ and cAMP response element binding protein (CREB) in vascular smooth muscle cells (VSMCs). A key step in the transmission of these signals is import into the nucleus. Here, we provide evidence that the Ran GTPase, which regulates nuclear import, exerts different regulation over PDGF-BB, Ca2+, and cAMP signaling to CREB in VSMCs. PDGF-BB, membrane depolarization, and forskolin increased levels of activated CREB (P-CREB) and c-fos in VSMCs and intact aorta. The calcium channel antagonist nimodipine reduced the level of P-CREB stimulated by membrane depolarization, but not by PDGF-BB or forskolin. Block of Ran-mediated nuclear import, by wheat germ agglutinin or an inactivating Ran mutant (T24N Ran), significantly reduced nuclear P-CREB in response to PDGF-BB or membrane depolarization, but enhanced levels of P-CREB in response to forskolin. Contrary to expectation, block of nuclear import led to the appearance of P-CREB in the cytoplasm after depolarization. Furthermore, blocking nuclear export with leptomycin B reduced P-CREB stimulation by both depolarization and PDGF-BB. These results suggest that translocation of CREB between the nucleus and the cytoplasm provides an important role in CREB activating pathways in VSMCs.

Thrombospondin-1 (TSP1), a multifunctional extracellular matrix glycoprotein, has been shown to suppress the angiogenic response in vivo and in vitro. We hypothesized that TSP1 might play a role in the inhibition of capillary morphogenesis during the endometrial cycle and examined its expression in 46 human endometrial specimens. Our results show that the expression of TSP1 in the endometrium is (a) cycle-dependent, (b) associated with periods of low capillary growth, and (c) regulated by progesterone. TSP1 protein was identified in the basement membrane of capillaries of the functional endometrium during the secretory phase. Abundant expression of TSP1 mRNA in the secretory phase was also detected by in situ hybridization, in contrast to the low levels seen in the proliferative phase. These findings were confirmed by Northern analysis of proliferative and secretory endometrium. Transcripts for TSP1 were observed predominantly in stromal cells, but signal was also detected in some endothelial and smooth muscle cells. Since the proliferation of endometrial tissue is regulated by steroid hormones, we tested the effects of estrogen and progesterone on TSP1 expression by stromal cells isolated from human endometrium. We found that levels of TSP1 mRNA and protein were increased after incubation with progesterone. Maximal stimulation of mRNA was observed after 8 h of treatment with 10-50 microM progesterone, and the effect was suppressed by the progesterone antagonist RU-486. Induction by progesterone was cell-specific and equivalent to the stimulation mediated by PDGF. Finally, the levels of TSP1 present in progesterone-stimulated cultures were sufficient to inhibit the migration of endothelial cells in vitro; this effect was nullified by anti-TSP antibodies. We therefore propose that the production of TSP1 at later stages of the endometrial cycle is linked to the inhibition of vessel formation and that TSP1 expression is progesterone-dependent in this tissue.

The receptor for angiotensin II (Ang II) has recently been cloned; it is a receptor with seven transmembrane spanning domains that stimulates phosphoinositide hydrolysis upon ligand binding. The physiologic effects of Ang II are important in the regulation of vascular function. In this study, we examined the ability of Ang II to regulate the enzymatic activity of phospholipase C (PLC) in rat aortic vascular smooth muscle cells (VSMC). In cultured VSMC, PLC-gamma 1 and PLC-delta 1 isozymes, but not PLC-beta 1, were identified by Western analysis. Ang II (10(-7) M)-stimulated PLC-gamma 1 phosphotyrosine phosphorylation with a maximum increase of 4.5-fold at 0.5 min. This followed the same time course as the Ang II-stimulated increase in inositol 1,4,5-trisphosphate (1,4,5-IP3) levels. 1,4,5-IP3 formation was inhibited 75% by the tyrosine kinase inhibitor genistein (120 microM). Several growth factor receptors, such as the platelet-derived growth factor (PDGF) receptor are themselves tyrosine kinases and have been shown to phosphorylate PLC-gamma 1 and increase intracellular Ca2+ concentrations. The time course for PLC-gamma 1 phosphorylation, IP3 formation, and Ca2+ mobilization by PDGF differed from Ang II in VSMC. The kinetics of the PDGF effects were slower in onset and more prolonged than those of Ang II. In summary, these findings show that Ang II stimulates VSMC phosphoinositide hydrolysis in association with tyrosine phosphorylation of PLC-gamma 1 and support the concept that Ang II-stimulated tyrosine phosphorylation is responsible for early signal transduction events.

The raf genes encode for a family of cytoplasmic proteins (A-raf, B-raf and c-raf-1) with associated serine/threonine kinase activities. Raf-1 is an important mediator of signals involving cell growth, transformation and differentiation. It is activated in response to a wide variety of extracellular stimuli such as insulin, nerve growth factor (NGF), platelet derived-growth factor (PDGF), and in response to expression of oncogenes, v-src and v-ras, in a cell-specific manner. Recently, the first physiological substrate for Raf-1 protein kinase was identified. Raf-1 was found to phosphorylate and activate Mitogen-Activated Protein Kinase Kinase (MEK), an activator of MAP kinase, thus linking the Raf-1 signaling pathway with that of MAP kinase. Cell specific differences in signalling pathways involving Raf-1 and MAP kinase have also been discovered. Accumulating evidence indicates that membrane tyrosine kinases, ras, Raf-1, MEK and MAP kinase are interconnected via a complex network rather than via a linear pathway involving multiple substrates and feedback loops.

nef is a human immunodeficiency virus (HIV) gene encoding a 27-kDa myristoylated protein with structural features of a signal transducing molecule, but whose functions are largely unknown. We studied the interactions of Nef with the signal transduction pathways triggered by the platelet-derived growth factor (PDGF) receptor. The association of phosphatidylinositol (PI) 3-kinase with the activated receptor was severely impaired by nef expression. Conversely, PDGF-induced receptor tyrosine phosphorylation, binding to phospholipase C-gamma and to Ras-GAP were not modified. Microtubule-associated protein kinase activation and intracellular calcium influx in response to PDGF were either unaffected or only slightly enhanced. Nef significantly reduced the proliferative response to the growth factor, while the chemotactic response was unchanged. These data show that Nef affects selectively the PI 3-kinase signaling pathway and suggest that this interference results in some of the HIV adverse effects on host cell functions.

Platelet-derived growth factor (PDGF) plays an important role in the process of atherosclerosis which is characterized by the presence of macrophage-derived foam cells. In the present study, the induction of the mRNA of PDGF-beta receptor was demonstrated during cell differentiation of human monocyte-macrophages, whereas no mRNA was detected in the cells during the early days of culture. Flow cytometry analysis using antibodies specific for PDGF-beta receptor and CD14 showed the presence of both PDGF-beta receptor and CD14 on human monocyte-derived macrophages, whereas no PDGF-beta receptor was detected on human monocytes 4 h after cell adhesion to a culture dish. In the binding assay of PDGF-BB on human monocyte-derived macrophages, a saturable and high affinity binding site with Kd of 27.5 pM and Bmax of 23.3 fmol/mg of cell protein was demonstrated. When human monocytes were cultured in the presence of the protein kinase C inhibitor staurosporine, PDGF-beta receptor induction was inhibited, and tetradecanoylphorbol acetate enhanced PDGF-beta receptor expression in human monocyte-derived macrophages, indicating that PDGF-beta receptor expression is associated with maturation and differentiation of monocyte-macrophages through the activation of protein kinase C. In response to PDGF-BB homodimer, PDGF-beta receptor was phosphorylated, and thymidine uptake and inositol trisphosphate production were stimulated in monocyte-derived macrophages. Furthermore, PDGF-BB suppressed the production of macrophages colony-stimulating factor in macrophages. The expression of PDGF-beta receptor on human monocyte-derived macrophages suggests that PDGF influences the process of atherosclerosis by regulating the function of macrophages as well as smooth muscle cells in the vascular wall.

c-Fms, a member of the Platelet-derived Growth Factor (PDGF) receptor family of receptor tyrosine kinases (RTKs), is the receptor for macrophage colony stimulating factor (CSF-1) that regulates proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. Abnormal expression of c-fms proto-oncogene is associated with a significant number of human pathologies, including a variety of cancers and rheumatoid arthritis. Accordingly, c-Fms represents an attractive therapeutic target. To further understand the regulation of c-Fms, we determined the 2.7 A resolution crystal structure of the cytosolic domain of c-Fms that comprised the kinase domain and the juxtamembrane domain. The structure reveals the crucial inhibitory role of the juxtamembrane domain (JM) that binds to a hydrophobic site immediately adjacent to the ATP binding pocket. This interaction prevents the activation loop from adopting an active conformation thereby locking the c-Fms kinase into an autoinhibited state. As observed for other members of the PDGF receptor family, namely c-Kit and Flt3, three JM-derived tyrosine residues primarily drive the mechanism for autoinhibition in c-Fms, therefore defining a common autoinhibitory mechanism within this family. Moreover the structure provides an understanding of c-Fms inhibition by Gleevec as well as providing a platform for the development of more selective inhibitors that target the inactive conformation of c-Fms kinase.

Platelet-derived growth factor (PDGF) stimulates protein kinase D (PKD) in a time- and dose-dependent manner. We have used a series of PDGF receptor mutants that display a selective impairment of the binding of SH2-containing proteins (GTPase-activating protein, SHP-2, phospholipase Cgamma (PLCgamma), or phosphatidylinositol 3'-kinase (PI3K)) to show that Tyr-1021, the PLCgamma-binding site, is essential for PKD stimulation by PDGF in A431 cells. We next investigated whether any one of these four binding sites could mediate PKD activation in the absence of the other three sites. F5, a receptor mutant that lacks all four binding sites for GTPase-activating protein, PLCgamma, PI3K, and SHP-2, fails to activate PKD. A panel of single add-back mutants was used to investigate if any one of these four sites could restore signaling to PKD. Of the four sites, only the PLCgamma+ single add-back receptor restored PDGF-mediated activation of PKD, and only this add-back receptor produced diacylglycerol (DAG) in a PDGF-dependent manner. 1,2-Dioctanoyl-sn-glycerol, a membrane-permeant DAG analog, was found to be sufficient for activation of PKD. Taken together, these data indicate that PLCgamma activation is not only necessary, but also sufficient to mediate PDGF-induced PKD activation. Although the presence of a pleckstrin homology domain makes PKD a potential PI3K target, PKD was not stimulated by selective PI3K activation, and wortmannin, an inhibitor of PI3K, did not inhibit PDGF signaling to PKD. The activation of PKD by DAG or by the wild-type and PLCgamma+ add-back PDGF receptors was inhibited by GF109203X, suggesting a role for protein kinase C in the stimulation of PKD by PDGF. PDGF induced a time-dependent phosphorylation of PKD that closely correlated with activation. The PDGF-induced activation and phosphorylation of PKD were reversed by in vitro incubation of PKD with protein phosphatase 1 or 2A, indicating that PDGF signaling to PKD involves the Ser/Thr phosphorylation of PKD. Taken together, these results conclusively show that PDGF activates PKD through a pathway that involves activation of PLCgamma and, subsequently, protein kinase C.

The proliferation of cells in vivo and in culture is regulated by polypeptide growth factors, such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Growth factors initiate their action by binding to specific cell surface receptors. Receptor occupancy triggers a cascade of physiological changes in the target cell which ultimately lead to DNA synthesis and cell division. Immediate consequences of receptor activation include tyrosine-specific protein phosphorylations, a sustained increase in cytoplasmic pH (pHi) and a transient rise in free Ca2+. The rise in pHi has a permissive effect on DNA synthesis and is mediated by an otherwise quiescent Na+/H+ exchange mechanism in the plasma membrane, which is turned on by protein kinase C, the cellular receptor for phorbol esters. The rapid Ca2+ signal is due to either release from internal stores (PDGF) or net entry via a voltage-independent channel in the plasma membrane (EGF). Phorbol esters, acting via kinase C, inhibit the growth factor-induced Ca2+ signals without affecting resting Ca2+ levels. Monoclonal antibodies against the human EGF receptor can act as partial agonists in that they activate the tyrosine-specific protein kinase without inducing any of the ionic signals. These antibodies fail to induce DNA synthesis when added to quiescent fibroblasts, indicating that the Ca2+ and pHi signals can be dissociated from tyrosine kinase activity and suggesting that these signals are indispensable for the stimulation of cell proliferation.

Simultaneous discovery of members of the annexin family of calcium and phospholipid binding proteins by several groups is intimately linked to the possibility that these proteins may be controlled by phosphorylation. Indeed, annexin I and annexin II have been identified as major substrates for the tyrosine kinase activity associated with epidermal growth factor receptor (EGF-R) and for the retrovirus encoded protein tyrosine kinase pp60v-arc. Both annexins are also in vitro and/or in situ substrates for platelet derived growth factor (PDGF), insulin and hepatocyte growth factor/scatter factor (HGF/SF) receptor tyrosine kinases. In addition, to serve as substrates for tyrosine protein kinases some annexins are cellular targets for serine threonine protein kinases such as protein kinase C (PKC) and cAMP-dependent protein kinase A (PKA). Although the role of annexin phosphorylation has not been studied in detail, it is thought to influence their vesicle aggregation and phospholipid binding properties. Some annexins are also potent inhibitors of various serine/threonine and tyrosine kinases. The physiological functions of the annexins have still not been clearly defined. Therefore the identification of the ability of these proteins to undergo phosphorylation may be helpful in assigning them a precise biological role.

Cultured endothelial cells (EC) produce a platelet-derived growth factor-like protein (PDGF-c) that stimulates the growth of cultured cells of mesenchymal origin. We have examined the effect of native plasma low density lipoprotein (LDL) and chemically modified LDL on production of PDGF-c by EC. Acetyl-LDL, but not native LDL, suppressed the production of PDGF-c by bovine aortic EC. Half-maximal inhibition was observed at a concentration of 25-75 micrograms of cholesterol per ml, and maximal inhibition (0-25% of control) at 150 micrograms of cholesterol per ml. EC treated with acetyl-LDL showed no morphological damage, there was no change in cell number, and the effect on production of PDGF-c was substantially reversed upon removal of the acetyl-LDL. The observed inhibition of PDGF-c production was specific, since total cellular and secreted protein synthesis were unaffected by acetyl-LDL. Acetyl-LDL suppressed PDGF-c production in both bovine aortic and human umbilical vein EC, but not in rat heart EC. This cell specificity correlated with the presence of scavenger receptors as measured by degradation of 125I-labeled acetyl-LDL and uptake of fluorescently labeled acetyl-LDL. Dimethylpropanediamine-LDL, a cationic modified lipoprotein, also inhibited PDGF-c production. The inhibition by both types of modified LDL was accompanied by significant intracellular cholesterol accumulation, suggesting a role for EC lipid composition in the regulation of production of PDGF-c.

The neuropathological abnormalities of human immunodeficiency virus (HIV)-1 patients abusing illicit drugs suggest extensive interactions between the two agents, thereby leading to increased rate of progression to neurodegeneration. The role of HIV-1 transactivating protein, Tat has been elucidated in mediating neuronal damage via apoptosis, a hallmark of HIV-associated dementia (HAD), however the underlying mechanisms involved in enhanced neurodegeneration by illicit drugs remain elusive. In this study, we demonstrated that morphine enhances HIV-Tat induced toxicity in human neurons and neuroblastoma cells. Enhanced toxicity by Tat and morphine was accompanied by increased numbers of TUNEL positive apoptotic neurons, elevated caspase-3 levels and decreased ratio of anti- and pro-apoptotic proteins, Bcl2/Bax. Tat and morphine together elicited high levels of reactive oxygen species that were NADPH dependent. Significant alterations in mitochondrial membrane homeostasis were also observed with co-exposure of these agents. Extensive studies of mitogen activated protein kinase (MAPK) signaling pathways revealed the involvement of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase-1/2 (ERK1/2) pathways in enhanced toxicity of Tat and morphine. In addition to this, we found that pre-treatment of cells with platelet derived growth factor (PDGF-BB) protected neurons from HIV-Tat and morphine induced damage. PDGF-BB alleviated ROS production, maintained mitochondrial membrane potential, decreased caspase-3 activation and hence protected the cells from undergoing apoptosis. PDGF-BB mediated protection against Tat and morphine involved the phosphatidylinositol-3 kinase (PI3K) pathway, as specific inhibitor of PI3K abrogated the protection conferred by PDGF-BB. This study demonstrates the mechanism of enhanced toxicity in human neurons subjected to co-exposure of HIV protein Tat and morphine, thus implying its importance in HIV positive drug abusers, where damage to the brain is reported to be more severe than non-drug abusers. We have also showed for the first time that PDGF-BB can protect against simultaneous exposure of Tat and morphine, strengthening its role as a neuroprotective agent that could be considered for therapeutic intervention.

Growth arrest-specific (Gas2) protein has been shown to be a component of the microfilament system, that is highly expressed in growth arrested mouse and human fibroblasts and is hyperphosphorylated upon serum stimulation of quiescent cells. (Brancolini, C., S. Bottega, and C. Schneider. 1992. J. Cell Biol. 117:1251-1261). In this study we demonstrate that the kinetics of Gas2 phosphorylation, during Go->>G1 transition, as induced by addition of 20% FCS to serum starved NIH 3T3 cells, is temporally coupled to the reorganization of actin cytoskeleton. To better dissect the relationship between Gas2 phosphorylation and the modification of the microfilament architecture we used specific stimuli for both membrane ruffling (PDGF and PMA) and stress fiber formation (L-alpha-lysophosphatidic acid LPA) (Ridley, A. J., and A. Hall. 1992. Cell. 70:389-399). All of them, similarly to 20% FCS, are able to downregulate Gas2 biosynthesis. PDGF and PMA induce Gas2 hyperphosphorylation that is temporally coupled with the appearance of membrane ruffling where Gas2 localizes. On the other hand LPA, a specific stimulus for stress fiber formation, fails to induce a detectable Gas2 hyperphosphorylation. Thus, Gas2 hyperphosphorylation is specifically correlated with the formation of membrane ruffling possibly implying a role of Gas2 in this process.

The aim of this study was to evaluate the effects of advanced glycation end-products (AGEs) on the proliferative activity and fibronectin production of smooth muscle cells (SMCs). AGE-bovine serum albumin (AGE-BSA) was prepared by incubation with D-glucose at 37 degrees C for 60 days. Cultured SMCs were obtained from explants isolated from porcine abdominal aorta and used between passages 3 and 10. The proliferative activity of SMCs was examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and by incorporation of 3H-thymidine into DNA. Fibronectin production was assessed by competitive ELISA assay for both fibronectin secreted into the culture medium (M-FN) and cell-associated fibronectin (C-FN), i.e., both intra- and peri-cellular fibronectin. Theassay revealed that AGE-BSA did not produce any change in optical density (A570) of SMCs at concentrations of up to 20 microg/ml, but decreased that of SMCs at a concentration of 40 microg/ml. The addition of PDGF (5 ng/ml) induced an increase in 3H-thymidine incorporation into DNA of quiescent SMCs, while the addition of AGE-BSA (20 microg/ml) had no effect. In contrast, AGE-BSA significantly increased C-FN of SMCs (30.8+/-8.58 ng/microg TP), compared to unmodified BSA (16.5+/-4.19 ng/microg TP). However, no difference in M-FN levels was observed between cells treated with AGE-BSA and unmodified BSA. The addition of anti-transforming growth factor (TGF)-beta antibody restored the levels of C-FN in SMCs cultured in 20 microg/ml of AGE-BSA, suggesting that TGF-beta might act as an intermediate factor in AGE-induced fibronectin production by SMCs. Our results suggest that interaction of AGE-modified proteins with SMCs may play a role in the development of atherosclerosis in diabetic and non-diabetic patients.