Regulation of uridine kinase activity in BALB/C-3T3 cells by serum components

After the stimulation of quiescent density-inhibited BALB/c-3T3 cells with fresh bovine calf serum, uridine kinase activity measured in cellular extracts increased between hours 3 and 6 of incubation and remained elevated through 12 h after stimulation. The addition of either partially purified platelet-derived growth factor (PDGF) or platelet-poor plasma (PPP) also caused increased uridine kinase activity by 6 h, but the increased activity was not maintained and the activity returned to the prestimulated level by 12 h. However, when PDGF and PPP were added in combination an increased level of uridine kinase activity was maintained in a manner similar to that seen after the addition of serum. The components of PPP eluted in the void volume from Sephadex G-50 chromatography did not induce uridine kinase activity when present alone, although they did act synergistically with PDGF to allow the maintenance of elevated levels or uridine kinase activity over the period from 6 to 12 h after stimulation. Thymidine kinase activity was not induced by the addition of either PDGF or PPP alone, although either serum or the combination of PDGF and PPP did produce and induction of thymidine kinase activity in late G1.     

Effects of mitogens on ornithine decarboxylase activity and messenger RNA levels in normal and protein kinase C-deficient NIH-3T3 fibroblasts

Ornithine decarboxylase activity was assessed in serum-deprived quiescent NIH-3T3 murine fibroblasts after exposure to a variety of growth-promoting factors. Ornithine decarboxylase activity increased after treatment with phorbol 12-myristate 13-acetate (PMA), fetal calf serum, bovine pituitary fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and the synthetic diacyglycerol sn-1,2-dioctanolyglycerol but not after treatment with epidermal growth factor, insulin, 4 alpha-phorbol 12,13-didecanoate, sn-1,2-dibutyrylglycerol, or the calcium ionophore A23187. Activity peaked at 3-4 h and returned to basal levels after 8 h. To determine the importance of protein kinase C in this increase, cells were pretreated with PMA for 16 h to make the cells effectively deficient in protein kinase C; this deficiency was documented by direct measurement of enzyme activity and immunoreactivity. The ornithine decarboxylase response to each mitogen was then compared in cells pretreated with PMA or control conditions. PMA pretreatment abolished the increase in ornithine decarboxylase activity due to additional PMA and decreased but did not eliminate the ability of serum, FGF, and PDGF to cause increases in ornithine decarboxylase activity. Similarly, pretreatment with PMA abolished the ability of additional PMA to increase ornithine decarboxylase mRNA levels but did not prevent the increases in these mRNA levels caused by FGF or serum. These data suggest that the increases in ornithine decarboxylase activity and mRNA levels that occur in quiescent fibroblasts in response to serum, FGF, or PDGF are due to activation of at least two separate pathways, one involving protein kinase C and the other independent of protein kinase C.     

Platelet-derived growth factor-induced alterations in vinculin and actin distribution in BALB/c-3T3 cells

Exposure of BALB/c-3T3 cells (clone A31) to platelet-derived growth factor (PDGF) results in a rapid time- and dose-dependent alteration in the distribution of vinculin and actin. PDGF treatment (6-50 ng/ml) causes vinculin to disappear from adhesion plaques (within 2.5 min after PDGF exposure) and is followed by an accumulation of vinculin in punctate spots in the perinuclear region of the cell. This alteration in vinculin distribution is followed by a disruption of actin-containing stress fibers (within 5 to 10 min after PDGF exposure). Vinculin reappears in adhesion plaques by 60 min after PDGF addition while stress fiber staining is nondetectable at this time. PDGF treatment had no effect on talin, vimentin, or microtubule distribution in BALB/c-3T3 cells; in addition, exposure of cells to 5% platelet-poor plasma (PPP), 0.1% PPP, 30 ng/ml epidermal growth factor (EGF), 30 ng/ml somatomedin C, or 10 microM insulin also had no effect on vinculin or actin distribution. Other competence-inducing factors (fibroblast growth factor, calcium phosphate, and choleragen) and tumor growth factor produced similar alterations in vinculin and actin distribution as did PDGF, though not to the same extent. PDGF treatment of cells for 60 min followed by exposure to EGF (0.1-30 ng/ml for as long as 8 h after PDGF removal), or 5% PPP resulted in the nontransient disappearance of vinculin staining within 10 min after EGF or PPP additions; PDGF followed by 0.1% PPP or 10 microM insulin had no effect. Treatment of cells with low doses of PDGF (3.25 ng/ml), which did not affect vinculin or actin organization in cells, followed by EGF (10 ng/ml), resulted in the disappearance of vinculin staining in adhesion plaques, thus demonstrating the synergistic nature of PDGF and EGF. These data suggest that PDGF-induced competence and stimulation of cell growth in quiescent fibroblasts are associated with specific rapid alterations in the cellular organization of vinculin and actin.     

Thymidine transport in phytohaemagglutinin-stimulated pig lymphocytes.

Thymidine and uridine transporters in peripheral pig lymphocytes have structural features in common, but are not identical. Accelerated entry of [3H]thymidine begins 12h after the addition of phytohaemagglutinin. The increased thymidine uptake into the cells is characterized by an increase in Vmax. Without alteration of the apparent Km(0.6+/-0.08muM). Thymidine kinase activity is increased 12h after stimulation. Both the increased thymidine uptake and the increased thymidine kinase activity are inhibited in cultures incubated with puromycin: rates of degradation of the two systems are unchanged after phytohaemagglutinin addition, and indicate similar half-lives of about 2h. Thymidine kinase is rate-limiting for thymidine entry up to 18h after phytohaemagglutinin addition; increase in its synthesis is detectable about 6h before net incorporation of thymidine into DNA is significantly promoted.     

Regulation of phosphoinositide phosphorylation in Swiss 3T3 cells stimulated by platelet-derived growth factor

The regulation of phosphoinositide phosphorylation was studied in Swiss 3T3 cells that were stimulated by platelet-derived growth factor (PDGF). Studies with intact cells showed that the mitogen increased the incorporation of 32P into phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns-P), and phosphatidylinositol 4,5-bisphosphate (PtdIns-P2) during the cell cycle, with distinct peaks of incorporation for all three phosphoinositides after 1 h, and for PtdIns and PtdIns-P2 after 20 h. Direct measurements of the activities of PtdIns kinase and PtdIns-P kinase in freeze-thawed cells revealed that the activity of PtdIns kinase was rate-limiting for the synthesis of PtdIns-P2. Maximal activities of PtdIns kinase and PtdIns-P kinase, with exogenous substrates, were unchanged during the 1st h of PDGF treatment, but doubled during the next 24 h. The increase in PtdIns kinase activity began within 2-4 h, exceeded the increase in cell protein, and was abolished by cycloheximide, which suggests that the enzyme was induced specifically in response to PDGF. The increase in activity of PtdIns-P kinase paralleled the increase in cell protein. Dose-response curves for PDGF showed that the activities of PtdIns kinase and PtdIns-P kinase at 24 h increased in proportion to the extent of mitogenic stimulation of the cells. Our results support the conclusion that the activities of PtdIns kinase and PtdIns-P kinase increase in response to PDGF, but only after several hours of cell cycle traverse.     

MicroRNA‐125b induces tau hyperphosphorylation and cognitive deficits in Alzheimer's disease

Sporadic Alzheimer's disease (AD) is the most prevalent form of dementia, but no clear disease‐initiating mechanism is known. Aβ deposits and neuronal tangles composed of hyperphosphorylated tau are characteristic for AD. Here, we analyze the contribution of microRNA‐125b (miR‐125b), which is elevated in AD. In primary neurons, overexpression of miR‐125b causes tau hyperphosphorylation and an upregulation of p35, cdk5, and p44/42‐MAPK signaling. In parallel, the phosphatases DUSP6 and PPP1CA and the anti‐apoptotic factor Bcl‐W are downregulated as direct targets of miR‐125b. Knockdown of these phosphatases induces tau hyperphosphorylation, and overexpression of PPP1CA and Bcl‐W prevents miR‐125b‐induced tau phosphorylation, suggesting that they mediate the effects of miR‐125b on tau. Conversely, suppression of miR‐125b in neurons by tough decoys reduces tau phosphorylation and kinase expression/activity. Injecting miR‐125b into the hippocampus of mice impairs associative learning and is accompanied by downregulation of Bcl‐W, DUSP6, and PPP1CA, resulting in increased tau phosphorylation in vivo. Importantly, DUSP6 and PPP1CA are also reduced in AD brains. These data implicate miR‐125b in the pathogenesis of AD by promoting pathological tau phosphorylation.     

The beta-PDGF receptor induces neuronal differentiation of PC12 cells.

Expression of the mouse beta-PDGF receptor by gene transfer confers PDGF-dependent and reversible neuronal differentiation of PC12 pheochromocytoma cells similar to that observed in response to NGF and basic FGF. A common property of the PDGF, NGF, and basic FGF-induced differentiation response is the requirement for constant exposure of cells to the growth factor. To test the hypothesis that a persistent level of growth factor receptor signaling is required for the maintenance of the neuronal phenotype, we examined the regulation of the serine/threonine-specific MAP kinases after either short- (10 min) or long-term (24 h) stimulation with growth factors. Mono Q FPLC resolved two peaks of growth factor-stimulated MAP kinase activity that coeluted with tyrosine phosphorylated 41- and 43-kDa polypeptides. MAP kinase activity was markedly stimulated (approximately 30-fold) within 5 min of exposure to several growth factors (PDGF, NGF, basic FGF, EGF, and IGF-I), but was persistently maintained at 10-fold above basal activity after 24 h only by the growth factors that also induce PC12 cell differentiation (PDGF, NGF, and basic FGF). Thus the beta-PDGF receptor is in a subset of tyrosine kinase-encoded growth factor receptors that are capable of maintaining continuous signals required for differentiation of PC12 cells. These signals include the constitutive activation of cytoplasmic serine/threonine protein kinases.     

Stimulation of phosphatidylcholine breakdown and diacylglycerol production by growth factors in Swiss-3T3 cells.

The effect of a number of growth factors on phosphatidylcholine (PtdCho) turnover in Swiss-3T3 cells was studied. Phorbol 12-myristate 13-acetate (PMA), bombesin, platelet-derived growth factor (PDGF) and vasopressin rapidly stimulated PtdCho hydrolysis, diacylglycerol (DAG) production, and PtdCho synthesis. Insulin and prostaglandin F2 alpha (PGF2 alpha) stimulated PtdCho synthesis, but not its breakdown, whereas epidermal growth factor (EGF) and bradykinin were without effect. Stimulation of PtdCho hydrolysis by the above ligands resulted in increased production of phosphocholine and DAG (due to phospholipase C activity) and significant amounts of choline, suggesting activation of a phospholipase D as well. CDP-choline and glycerophosphocholine levels were unchanged. Down-regulation of protein kinase C with PMA (400 nM, 40 h) abolished the stimulation of PtdCho hydrolysis and PtdCho synthesis by PMA, bombesin, PDGF and vasopressin, but not the stimulation of PtdCho synthesis by insulin and PGF2 alpha. PtdCho hydrolysis therefore occurs predominantly by activation of protein kinase C (either by PMA or PtdIns hydrolysis) leading to elevation of DAG levels derived from non-PtdIns(4,5)P2 sources. PtdCho synthesis occurs by both a protein kinase C-dependent pathway (stimulated by PMA, PDGF, bombesin and vasopressin) and a protein kinase C-independent pathway (stimulated by insulin and PGF2 alpha). DAG production from PtdCho hydrolysis is not the primary signal to activate protein kinase C, but may contribute to long-term activation of this kinase.     

Two signal transduction pathways mediate interleukin-1 receptor expression in Balb/c3T3 fibroblasts

Our previous studies showed that platelet-derived growth factor (PDGF) modulated interleukin-1 (IL-1) activity and IL-1 binding to Balb/c3T3 fibroblasts (Bonin, P. D., and Singh, J. P. (1988) J. Biol. Chem. 263, 11052-11055). Subsequent studies have demonstrated an action of PDGF at the level of IL-1 receptor (IL-1R) gene expression. PDGF treatment of Balb/c3T3 cells produces a 10-20-fold stimulation of mRNA for IL-1 receptor. Investigation of the signal transduction pathways shows that activation of either the protein kinase C pathway or the cAMP-mediated pathway leads to the stimulation of IL-1 receptor expression in Balb/c3T3 cells. Treatment of Balb/c3T3 cells with phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, produced an increased 125I-IL-1 binding to cells and stimulation of IL-1R mRNA. Staurosporine, an inhibitor of protein kinase C, blocked the induction of IL-1 binding by PDGF or PMA. Down-regulation of protein kinase C by pretreatment with PMA reduced the subsequent stimulation by PDGF. Chronic treatment with PMA, however, did not produce a complete inhibition of PDGF effect on IL-1R. Further studies showed that the agents that stimulate cAMP accumulation (isobutyl methylxanthine, dibutyryl), directly stimulate adenylate cyclase (forskolin), or activate G protein (choleragen) stimulated 125I-IL-1 binding and IL-1R mRNA accumulation in Balb/c3T3 cells. These studies suggest that potentially two signal transduction pathways mediate IL-1 receptor expression in Balb/c3T3 fibroblasts. Evidence is presented that suggests that stimulation of IL-1R through these two pathways (PMA/PDGF-stimulated and cAMP-stimulated) occurs independent of each other.     

Bombesin and platelet-derived growth factor stimulate formation of inositol phosphates and Ca2+ mobilization in Swiss 3T3 cells by different mechanisms.

Highly purified platelet-derived growth factor (PDGF) or recombinant PDGF stimulate DNA synthesis in quiescent Swiss 3T3 cells. The dose-response curves for the natural and recombinant factors were similar, with half-maximal responses at 2-3 ng/ml and maximal responses at approx. 10 ng/ml. Over this dose range, both natural and recombinant PDGF stimulated a pronounced accumulation of [3H]inositol phosphates in cells labelled for 72 h with [3H]inositol. In addition, mitogenic concentrations of PDGF stimulated the release of 45Ca2+ from cells prelabelled with the radioisotope. However, in comparison with the response to the peptide mitogens bombesin and vasopressin, a pronounced lag was evident in both the generation of inositol phosphates and the stimulation of 45Ca2+ efflux in response to PDGF. Furthermore, although the bombesin-stimulated efflux of 45Ca2+ was independent of extracellular Ca2+, the PDGF-stimulated efflux was markedly inhibited by chelation of external Ca2+ by using EGTA. Neither the stimulation of formation of inositol phosphates nor the stimulation of 45Ca2+ efflux in response to PDGF were affected by tumour-promoting phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (TPA). In contrast, TPA inhibited phosphoinositide hydrolysis and 45Ca2+ efflux stimulated by either bombesin or vasopressin. Furthermore, whereas formation of inositol phosphates in response to both vasopressin and bombesin was increased in cells in which protein kinase C had been down-modulated by prolonged exposure to phorbol esters, the response to PDGF was decreased in these cells. These results suggest that, in Swiss 3T3 cells, PDGF receptors are coupled to phosphoinositidase activation by a mechanism that does not exhibit protein kinase C-mediated negative-feedback control and which appears to be fundamentally different from the coupling mechanism utilized by the receptors for bombesin and vasopressin.     

Somatomedin-C and platelet-derived growth factor stimulate human fibroblast replication

Previous studies have demonstrated that serum contains mitogens, such as platelet-derived growth factor (PDGF), which may alter fibroblast responsiveness to growth factors contained in plasma. Somatomedin-C (SM-C) has been identified as one of the plasma growth factors required for mouse Balb/c 3T3 fibroblasts to initiate DNA synthesis. The present experiments were undertaken to explore the interaction between PDGF, human growth hormone (hGH), SM-C, and other growth-promoting agents in stimulating the growth of human fibroblasts. Proliferation of human dermal fibroblasts plated at low density (3,000 cells/cm²) was found to be equally stimulated by continuous exposure to either normal or somatomedin-C-deficient serum. In contrast, when confluent monolayers were sequentially exposed to PDGF, followed either by normal platelet poor plasma (PPP) or hypopituitary PPP, the cells exposed to normal PPP entered the “S” phase of the cell cycle 50% faster. This difference could be abolished by a 6-hour incubation with growth hormone (10 ng/ml) or somatomedin-C (5 ng/ml) preceding the addition of plasma. When medium containing either hGH or Sm-C was changed frequently so as to remove factors secreted by fibroblasts, only those cells exposed to exogenous somatomedin-C entered DNA synthesis. This finding is in agreement with previous findings that human fibroblasts are capable of making Sm-C in response to hGH. These findings support the hypothesis that somatomedin is required for fibroblast replication in vitro, and that growth hormone appears to stimulate replication indirectly through somatomedin production.     

Uridine transport and phosphorylation in 3T3 and CHO cells with induced cell proliferation

In the serum-deficient medium, the cultured Swiss 3T3 and CHO-K1 cells transit to the resting state. The rates of uridine phosphorylation and RNA synthesis in these cells are lowered. After the addition of fresh medium containing 10% serum, cell proliferation is induced. At the early stage of cell entrance into the cell cycle uridine transport through the cell plasma membrane remains unchanged in both cultures. During the 1st hour after serum addition the rate of uridine phosphorylation increases in 3T3 cells to remain practically unchanged in CHO-K1 cells. At this time, RNA synthesis in cells increases almost twofold in both cultures. A correlation has been revealed between the initial level of uridine phosphorylation in 3T3 cells and the percentage of its maximal elevation after serum addition. No such a correlation was observed for CHO-K1 cells. The rate of uridine phosphorylation in arrested CHO-K1 cells is higher than that in 3T3 cells. It has been included that the initial increase of uridine phosphorylation during serum stimulation may be not obligatory for all cell types, but depends on the level of uridine kinase activity before serum addition to the cells.     

Vasopressin and norepinephrine stimulation of inositol phosphate accumulation in rat hepatocytes are modified differently by protein f1nase C and protein kinase A

Rat hepatocytes were maintained in primary monolayer culture for 24 h in the presence of serum. Treatment of hepatocytes with 1 microM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) for 5-15 min increased membrane-associated protein kinase C activity and concomitantly decreased soluble activity. Membrane protein kinase C activity returned to basal values within 1 h then decreased by more than 50% within 2 h. Prolonged (2-18 h) incubation with PMA did not further decrease protein kinase C activity. Pretreatment of hepatocytes with PMA for 5-15 min had little effect on the subsequent actions of 100 nM vasopressin but abolished the stimulation of inositol phosphate accumulation by 3 nM vasopressin and 20 microM norepinephrine. Long-term exposure (2-18 h) of hepatocytes to 1 microM PMA actually enhanced the effects of vasopressin and 20 microM norepinephrine. The stimulation by norepinephrine (20 microM) of inositol phosphate accumulation was abolished by the alpha 1-adrenergic antagonist prazosin (1 microM), whereas the beta-adrenergic antagonist propranolol (30 microM) had little effect. Addition of 8Br-cAMP (100 microM) or glucagon (10 nM) for 5 min or 8 h had no significant effect alone, but enhanced the subsequent vasopressin stimulation of inositol phosphate accumulation. There was no effect of 8Br-cAMP or glucagon on norepinephrine stimulation of phosphoinositide breakdown. These data indicate that the stimulation of phospholipase C activity in rat hepatocytes by 3 nM vasopressin is enhanced by cyclic AMP-dependent kinase but inhibited by protein kinase C. In contrast, down regulation of protein kinase C markedly enhanced the maximal phosphoinositide response due to both vasopressin and norepinephrine.     

Tyrosine mutations within the alpha platelet-derived growth factor receptor kinase insert domain abrogate receptor-associated phosphatidylinositol-3 kinase activity without affecting mitogenic or chemotactic signal transduction.

A phosphatidylinositol-3 (PI-3) kinase activity of unknown biological function associates with tyrosine kinase-containing proteins, including a number of growth factor receptors after ligand stimulation. In the beta platelet-derived growth factor (beta PDGF) receptor, phosphorylation of a specific tyrosine residue within the kinase insert domain was required for its interaction with this enzyme. We show that substitutions of phenylalanine for tyrosine residue 731 or 742 within the kinase insert domain of the alpha PDGF receptor do not impair PDGF-induced tyrosine phosphorylation of the receptor or of an in vivo substrate, phospholipase C-gamma. Moreover, phosphatidylinositol turnover in response to ligand stimulation is unaffected. However, both lesions markedly impair receptor association with PI-3 kinase. Antiphosphotyrosine antibody-recoverable PI-3 kinase was also dramatically reduced in PDGF-stimulated cells expressing either mutant receptor. Since neither mutation abolished PDGF-induced mitogenesis or chemotaxis, we conclude that alpha PDGF receptor-associated PI-3 kinase activity is not required for either of these major PDGF signalling functions.     

Platelet-derived growth factor induces rapid and sustained tyrosine phosphorylation of phospholipase C-gamma in quiescent BALB/c 3T3 cells.

Platelet-derived growth factor (PDGF) stimulates the proliferation of quiescent fibroblasts through a series of events initiated by activation of tyrosine kinase activity of the PDGF receptor at the cell surface. Physiologically significant substrates for this or other growth factor receptor or oncogene tyrosine kinases have been difficult to identify. Phospholipase C (PLC), a key enzyme of the phosphoinositide pathway, is believed to be an important site for hormonal regulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate, which produces the intracellular second-messenger molecules inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Treatment of BALB/c 3T3 cells with PDGF led to a rapid (within 1 min) and significant (greater than 50-fold) increase in PLC activity, as detected in eluates of proteins from a phosphotyrosine immunoaffinity matrix. This PDGF-stimulated increase in phosphotyrosine-immunopurified PLC activity occurred for up to 12 h after addition of growth factor to quiescent cells. Interestingly, the PDGF stimulation occurred at 3 as well as 37 degrees C and in the absence or presence of extracellular Ca2+. Immunoprecipitation of cellular proteins with monoclonal antibodies specific for three distinct cytosolic PLC isozymes demonstrated the presence of a 145-kilodalton isozyme, PLC-gamma (formerly PLC-II), in BALB/c 3T3 cells. Furthermore, these immunoprecipitation studies showed that PLC-gamma is rapidly phosphorylated on tyrosine residues after PDGF stimulation. The results suggest that mitogenic signaling by PDGF is coincident with tyrosine phosphorylation of PLC-gamma.     

Effect of the tyrosine kinase inhibitor, genistein, on interleukin-1 stimulated PGE2 production in mesangial cells

Prostaglandin production and cAMP formation are two signaling pathways identified for IL-1, though neither adequately account for the multitude of effects of IL-1. To investigate the role of tyrosine phosphorylation in IL-1 signaling, we used the tyrosine kinase inhibitor, genistein. At 10-30 micrograms/ml, genistein blocked IL-1 stimulated prostaglandin production and induction of prostaglandin endoperoxide synthase (PES) in glomerular mesangial cells maintained in 10% serum. Addition of genistein hours after IL-1 addition also halted further PGE2 synthesis. Genistein failed to block PES activity in vitro, indicating it was not acting as a PES inhibitor. Overall these data suggest that tyrosine phosphorylation may be a required event for IL-1 stimulation of PGE2 and PES activity, either directly as part of IL-1 signaling, or indirectly as part of a serum/PDGF competence effect on mesangial cells.     

Platelet-derived growth factor rapidly increases activity and cell surface expression of the EAAC1 subtype of glutamate transporter through activation of phosphatidylinositol 3-kinase

Na(+)-dependent glutamate transporters are the primary mechanism for removal of excitatory amino acids (EAAs) from the extracellular space of the central nervous system and influence both physiologic and pathologic effects of these compounds. Recent evidence suggests that the activity and cell surface expression of a neuronal subtype of glutamate transporter, EAAC1, are rapidly increased by direct activation of protein kinase C and are decreased by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K). We hypothesized that this regulation could be analogous to insulin-induced stimulation of the GLUT4 subtype of glucose transporter, which is dependent upon activation of PI3-K. Using C6 glioma, a cell line that endogenously and selectively expresses EAAC1, we report that platelet-derived growth factor (PDGF) increased Na(+)-dependent L-[(3)H]-glutamate transport activity within 30 min. This effect of PDGF was not due to a change in total cellular EAAC1 immunoreactivity but was instead correlated with an increase cell surface expression of EAAC1, as measured using a membrane impermeant biotinylation reagent combined with Western blotting. A decrease in nonbiotinylated intracellular EAAC1 was also observed. These studies suggest that PDGF causes a redistribution of EAAC1 from an intracellular compartment to the cell surface. These effects of PDGF were accompanied by a 35-fold increase in PI3-K activity and were blocked by the PI3-K inhibitors, wortmannin and LY 294002, but not by an inhibitor of protein kinase C. Other growth factors, including insulin, nerve growth factor, and epidermal growth factor had no effect on glutamate transport nor did they increase PI3-K activity. These studies suggest that, as is observed for insulin-mediated translocation of GLUT4, EAAC1 cell surface expression can be rapidly increased by PDGF through activation of PI3-K. It is possible that this PDGF-mediated increase in EAAC1 activity may contribute to the previously demonstrated neuroprotective effects of PDGF.     

Platelet-derived growth factor-induced c-myc RNA expression. Analysis of an inducible pathway independent of protein kinase C

Platelet-derived growth factor (PDGF) is generally considered to stimulate phosphoinositide turnover resulting in activation of protein kinase C and increased cytoplasmic [Ca2+]. We have examined the role of these secondary effects in regulation of c-myc mRNA accumulation in the MG-63 human osteogenic sarcoma line. Treatment of quiescent cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA) to down-regulate protein kinase C inhibited TPA-stimulated c-myc expression but did not affect the PDGF-modulated process. When cytoplasmic [Ca2+] was increased by addition of a Ca2+ ionophore (A23187 or ionomycin), no stimulation of c-myc RNA was seen; furthermore, these agents did not enhance the PDGF-modulated c-myc expression. Addition of EGTA to cultures treated with both PDGF and a Ca2+ ionophore did not inhibit c-myc induction but rather caused a superinduction of c-myc RNA accumulation. Superinduction occurred only if the [EGTA] was greater than [Ca2+] in the medium. This superinduction was distinct from the increased induction caused by inhibition of protein synthesis. Because PDGF-induced c-myc expression is independent of protein kinase C and increased cytoplasmic [Ca2+], the evidence suggests that PDGF modulates c-myc RNA accumulation in MG-63 cells via a novel pathway, seemingly uncoupled from the classic action of increased phosphoinositide metabolism.     

CSF-1 stimulates nucleoside transport in S1 macrophages

We have examined nucleoside transport (NT) in a cell line derived from primary day 7 murine bone marrow macrophages (S1 macrophages) in response to the macrophage growth factor, colony-stimulating factor 1 (CSF-1). Adenosine and uridine transport in quiescent S1 macrophages occurred primarily by two facilitated diffusional routes, one that was sensitive and one that was relatively resistant to the inhibitor nitrobenzylthioinosine (NBMPR). Addition of CSF-1 to quiescent cultures resulted in increased adenosine and uridine transport with biphasic kinetics with respect to the cell cycle. Basal NT activity was elevated (about twofold) within 15 min of CSF-1 addition, returned to near basal levels by 1 h, and then increased again (three- to fourfold) 8–12 h later, returning again to basal levels by 48 h post CSF-1 stimulation. We propose that the large increase in NT activity at 8–12 h corresponded with the time when cultures synchronously began to enter the S phase of the cell cycle. In addition to these changes in the absolute rates, the proportions of NBMPR-sensitive and NBMPR-insensitive transport also change after CSF-1 addition. Quiescent cultures exhibited primarily NBMPR-insensitve transport while logrithmically growing cultures exhibited primarily NBMPR-sensitive nucleoside transport activity. The increase in the NBMPR-sensitive component of the transport process paralleled a similar increase in the number of high-affinity NBMPR binding sites, suggesting that the mechanism for upregulating NBMPR-sensitive NT activity involves increases in the number of NBMPR-sensitive transporter sites. Interestingly, we were unable to detect Na+-dependent concentrative uptake of adenosine, uridine, or formycin-B either in the S1 macrophage cell line or in primary (day 7) murine macrophages. Thus these bone marrow derived macrophages did not display the characteristically large Na+-dependent transport systems observed by others in peritoneal macrophages, implying that these two populations of macrophages are, indeed, functionally distinct. © 1993 Wiley-Liss, Inc.