Effects of homo- and heterodimeric isoforms of PDGF on signalling events in rat renal mesangial cells.

Platelet-derived growth factor (PDGF) exists in three dimeric isoforms, AA, BB and AB. Mesangial cells exclusively bound the BB homodimer and responded only to the BB isoform in terms of DNA synthesis and phosphoinositide hydrolysis. PDGF-BB stimulated a dose-dependent formation of inositol trisphosphate (InsP3). Neither pertussis toxin nor short-term (10 min) treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited the PDGF-BB-evoked production of InsP3. In contrast, the response to PDGF-BB was attenuated in cells in which protein kinase C has been down-regulated by long-term (24 h) treatment with TPA. In parallel to the generation of InsP3, there was a biphasic increase in 1,2-diacylglycerol (DAG). The second peak of DAG generation was associated with a concomitant 2-fold increase in choline formation. In addition, PDGF-BB stimulated the accumulation of phosphatidylpropanol, produced by phospholipase D phosphatidyl transferase activity, when 1-propanol was added to mesangial cells. Stimulation of mesangial cells with PDGF-BB caused a dose-dependent formation of prostaglandin E2. Furthermore, mesangial cells secreted PDGF-AA into the culture supernatant.     

Different sensitivity to phorbol esters and pertussis toxin of bombesin- and platelet-derived growth factor-induced, phospholipase C-mediated hydrolysis of phosphoinositides in NIH/3T3 cells

Incubation of the serum-deprived cultures of NIH/3T3 cells with bombesin or platelet-derived growth factor (PDGF) induced the phospholipase C-mediated hydrolysis of phosphoinositides. Protein kinase C-activating 12-O-tetradecanoylphorbol 13-acetate (TPA) and pertussis toxin inhibited the bombesin-induced phospholipase C reactions. AlF4-, a direct activator of GTP-binding proteins (G proteins), also induced the phospholipase C reactions and TPA inhibited the AlF4- -induced reactions. These results suggest that a pertussis toxin-sensitive G protein is involved in the coupling of the bombesin receptor to the phospholipase C and that the coupling of the G protein to the phospholipase C is inhibited by protein kinase C. In contrast, neither TPA nor pertussis toxin inhibited the PDGF-induced phospholipase C reactions, indicating that a pertussis toxin-sensitive G protein is not involved in the coupling of the PDGF receptor to the phospholipase C and that this coupling is insensitive to protein kinase C. These results suggest that the regulatory mechanism of the PDGF receptor for the phospholipase C activation is different from that of the bombesin receptor.     

Gonadotropin-releasing hormone activates phospholipase D in ovarian granulosa cells. Possible role in signal transduction

We have investigated the stimulation of phospholipase D activity by the gonadotropin-releasing hormone receptor agonist [D-Ala6, des-Gly10]GnRH N-ethylamide (GnRH-A) in preovulatory, cultured granulosa cells. GnRH-A stimulated up to 10-fold accumulation of phosphatidylethanol, produced by phospholipase D phosphatidyl transferase activity when ethanol acts as the phosphatidyl group acceptor. The effect of GnRH-A was concentration dependent (EC50 = 1 nM) and was inhibited by a specific GnRH receptor antagonist. Low GnRH-A concentrations (less than 10 nM) stimulated also accumulation of phosphatidic acid, but at higher concentrations this response was attenuated. Propranolol, which inhibits phosphatidic acid phosphohydrolase, increased both basal and GnRH-A-stimulated production of phosphatidic acid. A protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM), increased up to 30-fold phosphatidylethanol levels. The effects of supramaximal concentrations of GnRH-A (50 nM) and TPA (1 microM) on the accumulation of phosphatidylethanol were additive, suggesting that the two agents may not act via the same mechanism. This is supported by the fact that 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, a protein kinase C inhibitor, inhibited the effect of TPA 50%, but not that of GnRH-A. However, 24 h pretreatment with TPA abolished cellular response to subsequent treatment with either TPA or GnRH-A. The stimulatory action of GnRH on steroidogenesis could be mimicked by elevating endogenous phosphatidic acid levels in granulosa cells. Exogenous phospholipase D (from Streptomyces chromofuscus, 10 IU/ml) significantly increased (2.7-fold) progesterone production by the cells; under the same conditions, GnRH-A and FSH stimulated progesterone production 3- and 2.6-fold, respectively. Similarly, propranolol stimulated progesterone production 2.2-fold. These results suggest that, in granulosa cells, GnRH receptors are coupled to a phospholipase D whose activation may participate in transducing the GnRH signal for accelerated steroidogenesis. Phospholipase D activity can be independently regulated also by protein kinase C. The possible interrelationships between phospholipase D and other phospholipases which may be activated by GnRH in these ovarian cells are discussed.     

Phospholipase D in cultured rat vascular smooth muscle cells and its activation by phorbol ester

We determined the phospholipase D (PLD) activity in rat vascular smooth muscle cells by the formation of phosphatidylethanol in cells prelabeled with [3H] myristic acid. The enzyme was markedly activated by a phorbol ester (TPA). Down regulation of protein kinase C (PKC) resulted in almost complete inhibition indicating PKC-dependent mechanism of its activation. Depletion of calcium by EGTA and TMB-8 caused 53% inhibition. Chelator-stable association of PKC to membrane by TPA was observed in the absence of extracellular Ca2+. The mitogenic peptide PDGF also caused a marked stimulation of PLD. These results indicate that PLD in vascular smooth muscle cells is stimulated by TPA through the activation of PKC both by calcium-dependent and independent mechanisms.     

Delayed activation of phospholipase D by gonadotropin-releasing hormone in a clonal pituitary gonadotrope cell line (alpha T3-1).

Stimulation of cultured pituitary cells from a gonadotrope lineage (alpha T3-1) by the gonadotropin-releasing hormone agonist analog [D-Trp6]GnRH (GnRH-A) resulted in a manifold increase in accumulation of phosphatidylethanol, a specific product of phospholipase D phosphatidyl transferase activity when ethanol is the phosphatidyl group acceptor. Levels of the natural lipid product of phospholipase D, phosphatidic acid, were increased 2-3-fold. Activation of phospholipase D by GnRH-A was dose- and time-dependent and was blocked by a GnRH receptor antagonist [D-pClPhe2,D-Trp3.6]GnRH. GnRH-A stimulated phospholipase D activity after a lag of 1-2 min. We conclude that in alpha T3-1 gonadotropes GnRH receptor occupancy results in delayed activation of phospholipase D which could participate in late phases of gonadotrope regulation by the neurohormone.     

Phosphatidylethanol biosynthesis in ethanol-exposed NG108-15 neuroblastoma X glioma hybrid cells. Evidence for activation of a phospholipase D phosphatidyl transferase activity by protein kinase C

12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulates the release of free choline from intact NG108-15 cells into the medium, without affecting the release of phosphocholine (Liscovitch, M., Blusztajn, J.K., Freese, A., and Wurtman, R.J. (1987) Biochem. J. 241, 81-86). To test the hypothesis that this response reflects activation of cellular phospholipase D, via protein kinase C (Ca2+/phospholipid-dependent enzyme), I examined in NG108-15 cells the biosynthesis of the abnormal phospholipid phosphatidylethanol, produced by phospholipase D in the presence of ethanol by transphosphatidylation. Phosphatidylethanol production was quantitated by measuring the incorporation of phosphatidyl moieties (prelabeled metabolically with [3H]oleic acid) into phosphatidylethanol. The production of phosphatidylethanol in NG108-15 cells was virtually dependent on stimulation by TPA, in a time- and concentration-dependent manner (EC50 = 18 nM). The rate of 3H-phosphatidylethanol formation reached a peak after 10 min of incubation with TPA and declined gradually thereafter. The levels of 3H-phosphatidylethanol in TPA-treated cells were directly related to ethanol concentration in the physiologically attainable range (20-80 mM). Phosphatidylethanol production was activated only by phorbol derivatives that are activators of protein kinase C (i.e. TPA, 4 beta-phorbol-12,13-dibutyrate, and 4 beta-phorbol-12,13-didecanoate) and could be mimicked by a cell-permeant diacylglycerol, 1,2-dioctanoyl-sn-glycerol, in a nonadditive manner. The effect of TPA was inhibited by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (0.1 mM) by 70% but not by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide. Phosphatidylethanol formation was completely abolished in cells in which protein kinase C was down-regulated by pretreatment of the cells with TPA. These results indicate that phosphatidylethanol biosynthesis in NG108-15 cells depends largely on activation of protein kinase C. In contrast to its effects on the release of free choline and on the accumulation of phosphatidylethanol, TPA did not affect the levels of phosphatidic acid in NG108-15 cells. It is therefore proposed that protein kinase C selectively activates the phosphatidyl transferase activity of phospholipase D, reflecting a signal termination mechanism which may be operative in phospholipase D-mediated signal transduction cascades.     

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.     

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.     

Protein kinase C mediates platelet-derived growth factor-induced tyrosine phosphorylation of p42

One of the early events after stimulation of Swiss 3T3 cells with either platelet-derived growth factor (PDGF), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), diacylglycerol, or several other mitogens is the near stoichiometric phosphorylation at tyrosine and serine of a scarce cytoplasmic protein (p42). TPA and diacylglycerol are known to directly stimulate the activity of a protein-serine/threonine kinase, protein kinase C (PKC). PDGF and several other mitogens stimulate tyrosine kinases directly and PKC indirectly. We have therefore examined the involvement of PKC in p42 tyrosine phosphorylation in Swiss 3T3 cells. Firstly, six agents which stimulated phosphorylation of p42 also stimulated phosphorylation of a known PKC substrate, an 80,000-Mr protein (p80). Secondly, in PKC-deficient cells (cells in which PKC activity was reduced to undetectable levels by prolonged exposure to TPA), PDGF-induced p42 phosphorylation was reduced three- to fourfold. Phosphoamino acid analysis of phosphorylated p42 from PDGF-stimulated PKC-deficient cells revealed primarily phosphoserine and only a trace of phosphotyrosine, suggesting that the reduction in PDGF-stimulated tyrosine phosphorylation of p42 resulting from PKC deficiency is greater than three- to fourfold. Finally, comparison of antiphosphotyrosine immunoprecipitates of PKC-deficient versus naive cells revealed that most other PDGF-induced tyrosine phosphorylation events were quite similar. These data suggest that mitogens such as PDGF, which directly stimulate phosphorylation of some proteins at tyrosine, induce p42 tyrosine phosphorylation via a cascade of events involving PKC.     

Induction of tumor promotor-inducible genes in murine 3T3 cell lines and tetradecanoyl phorbol acetate-nonproliferative 3T3 variants can occur through protein kinase C-dependent and -independent pathways.

We isolated a group of genes that are rapidly and transiently induced in 3T3 cells by tetradecanoyl phorbol acetate (TPA). These genes are called TIS genes (for TPA-inducible sequences). Epidermal growth factor (EGF), fibroblast growth factor (FGF), and TPA activated TIS gene expression with similar induction kinetics. TPA pretreatment to deplete protein kinase C activity did not abolish the subsequent induction of TIS gene expression by epidermal growth factor or fibroblast growth factor; both peptide mitogens can activate TIS genes through a protein kinase C-independent pathway(s). We also analyzed TIS gene expression in three TPA-nonproliferative variants (3T3-TNR2, 3T3-TNR9, and A31T6E12A). The results indicate that (i) modulation of a TPA-responsive sodium-potassium-chloride transport system is not necessary for TIS gene induction either by TPA or by other mitogens and (ii) TIS gene induction is not sufficient to guarantee a proliferative response to mitogenic stimulation.     

Platelet-Derived growth factor activates phospholipase D and chemotactic responses in vascular smooth muscle cells

Summary Platelet-derived growth factor (BB dimer; PDGF-BB) stimulates a mitogenic response in A-10 vascular smooth muscle cells. In addition, PDGF-BB stimulates phospholipase D activity against phosphatidylcholine in A-10 cells. This response was observed as a rapid metabolism of phosphatidylcholine to phosphatidate and choline; a subsequent metabolism generates sustained levels of diacylglycerol. The accumulation of phosphatidylethanol, a transphosphatidylation product of phospholipase D, was obvious in PDGF-treated cells. PDGF-BB also stimulates a chemotactic response in A-10 cells. The concentrations of PDGF-BB required to stimulate mitogenesis, phospholipase D activity and chemotaxis are similar. This finding shows that PDGF induces a variety of cellular responses and suggests that these responses may share common metabolic pathways. That conception was tested by investigating the activity of the different PDGF dimers. PDGF-AA had little or no activity in A-10 cells for any of the responses measured. PDGF-AB and PDGF-BB were equally potent in stimulating mitogenic responses. However, the AB heterodimer was only half as active as PDGF-BB with respect to activation of phospholipase D and chemotactic responses. These results demonstrate that PDGF stimulates phospholipase D in vascular smooth muscle cells. In addition, the data indicate that different PDGF dimers can transduce varying signals and suggest a link between the mechanisms by which PDGF-BB activates phospholipase D and the chemotactic response. Partial support for this project was obtained through a grant to C. J. W. from the American Heart Association (#88-034G) and from the W. Alton Jones Foundation.     

Mitogen-mediated protein phosphorylation in Werner's syndrome fibroblasts

DNA synthesis of WF-1 fibroblasts derived from a patient with Werner's syndrome was stimulated by fetal calf serum and adult human serum but not by various mitogens including epidermal growth factor, platelet-derived growth factor (PDGF), fibroblast growth factor, insulin and 12-O-tetradecanoylphorbol-13-acetate (TPA). To clarify the cause of nonresponsiveness to these mitogens, we compared the rate of protein phosphorylation in normal fibroblasts HF-O and Werner's WF-1 cells. PDGF and TPA enhanced the phosphorylation of a Mr 80 K protein, which is known to be a substrate for protein kinase C, both in HF-O and WF-1 cells. This indicates that the pathway involving PDGF receptor, phosphatidylinositol turnover and protein kinase C activation is operational in WF-1 cells. Several species of phosphoproteins of Mr 250 K, 135 K, 110 K, 78 K and 42 K were detected in normal HF-O cells by immunoprecipitation using an anti-phosphotyrosine antibody. The same species of phosphoproteins were detected in Werner's WF-1 cells at passage 6, but only when treated with various mitogens and were not detected in WF-1 cells at passage 10 even after the PDGF- or TPA-treatment. These results suggest that the reduction of phosphorylation of these target proteins may be in part responsible for the diminished mitogenic responsiveness of Werner's fibroblasts.     

Relationship of cytosolic ion fluxes and protein kinase C activation to platelet-derived growth factor induced competence and growth in BALB/c-3T3 cells

Platelet-derived growth factor (PDGF) and other agents that activate protein kinase C (PKC) rapidly alter cytosolic pH (pHi) and intracellular free calcium ([Ca++]i) in BALB/c-3T3 fibroblasts. To define whether changes in pHi or [Ca++]i are linked to PDGF-stimulated mitogenesis, these parameters were assessed in control and PKC depleted fibroblasts. PDGF addition to BALB/c-3T3 fibroblasts resulted in transient acidification of the cytoplasm followed by prolonged cytosolic alkalinization. Exposure of cells to 12-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester that activates PKC, resulted in cytosolic alkalinization without prior acidification. Overnight incubation with 600 nM TPA decreased the total cell PKC histone phosphorylating activity in BALB/c-3T3 fibroblasts by greater than 90%. In PKC-deficient fibroblasts, TPA, and PDGF-induced alkalinization was abolished. In addition, the transient drop in pHi seen initially in control cells treated with PDGF is sustained to the point where pHi is fully 0.6-0.7 pH units below control cell values for up to 30 minutes. PDGF increased [Ca++]i threefold; this transient rise in [Ca++]i was only minimally affected (less than 15%) by lowering of the extracellular calcium level with ethylene glycol bis(b-aminoethyl ether)0 N,N,N' tetraacetic acid (EGTA) or blocking calcium influx with CoCl2. In contrast, 8-(diethylamine)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an agent thought to inhibit calcium release from intracellular stores, substantially inhibited the rise in [Ca++]i caused by PDGF. TPA and 1-oleoyl-2-acetylglycerol (OAG) increased [Ca++]i but in contrast to PDGF this effect was blocked by pretreatment of cells with EGTA or CoCl2. In PKC-deficient fibroblasts, PDGF still increased [Ca++]i and stimulated DNA synthesis as effectively as in controls. TPA and OAG however, no longer increased [Ca++]i. The continued ability of PDGF to stimulate DNA synthesis in the face of sustained acidification and the absence of PKC activity suggests that cytosolic alkalinization and PKC activation are not essential for PDGF-induced competence in BALB/c-3T3 fibroblasts.     

Na+/K+/Cl- cotransporter activates MAP-kinase cascade downstream to protein kinase C, and upstream to MEK

In this study, we demonstrated that the specific inhibitors of the Na+/K+/Cl- cotransporter (NKCC1), bumetanide and furosemide, inhibited extracellular regulated kinase (ERK) phosphorylation in Balb/c 3T3 fibroblasts, stimulated with a variety of mitogens. In addition to fibroblast growth factor (FGF) shown before, the various mitogens tested in the present study (endothelial growth factor (EGF), platelet-derived growth factor (PDGF), insulin, thrombin, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA)). Enter, the Ras/Raf/MEK/ERK cascade via different growth factors receptors and through one of the two main routes. The results of the present study provide evidence that have led us to conclude that the target protein which is controlled by the Na+/K+/Cl- cotransporter, is downstream of tyrosine kinase receptors, as well as of the G-protein-coupled receptor (GPCR). Several additional lines of evidence supported the above conclusion: (i) furosemide inhibits phosphorylation of MAPK kinase (MEK) induced by receptor tyrosine kinase (RTK) ligands, such as PDGF, FGF, and EGF. (ii) Furosemide also inhibited ERK phosphorylation, induced by thrombin, a GPCR. (iii) Furosemide inhibited MEK and ERK phosphorylation even when ERK phosphorylation was induced by direct activation of protein kinase C (PKC) by TPA, which bypasses early steps of the mitogenic cascade. In addition, we found that furosemide did not affect PKC phosphorylation induced directly by TPA. Taken together, the results of the present study indicate that the signal transduction protein, controlled by the Na+/K+/Cl- cotransporter, must be downstream of the PKC, and at/or upstream to MEK in the Ras/Raf/MEK/ERK cascade.     

Bradykinin Activates a Phospholipase D that Hydrolyzes Phosphatidylcholine in PC12 Cells

In PC12 pheochromocytoma cells whose phospholipids had been prelabelled with [3H]palmitic acid, bradykinin increased the production of [3H]phosphatidic acid. The increase in [3H]phosphatidic acid occurred within 1-2 min. before the majority of the increase in [3H]diacylglycerol. When the phospholipids were prelabeled with [3H]choline, bradykinin increased the intracellular release of [3H]choline. The production of phosphatidic acid and choline suggests that bradykinin was increasing the activity of phospholipase D. Transphosphatidylation is a unique property of phospholipase D. In cells labeled with [3H]palmitic acid, bradykinin stimulated the transfer of phosphatidyl groups to both ethanol and propanol to form [3H]phosphatidylethanol and [3H]phosphatidylpropanol, respectively. The effect of bradykinin on [3H]phosphatidic acid and [3H]phosphatidylethanol formation was partially dependent on extracellular Ca2+. In cells treated with nerve growth factor, carbachol also increased [3H]phosphatidylethanol formation. To investigate the substrate specificity of phospholipase D, cells were labeled with [14C]stearic acid and [3H]palmitic acid, and then incubated with ethanol in the absence or presence of bradykinin. The 14C/3H ratio of the phosphatidylethanol that accumulated in response to bradykinin was almost identical to the 14C/3H ratio of phosphatidylcholine. The 14C/3H ratio in phosphatidic acid and diacylglycerol was higher than the ratio in phosphatidylcholine. These data provide additional support for the idea that bradykinin activates a phospholipase D that is active against phosphatidylcholine. The hydrolysis of phosphatidylcholine by phospholipase D accounts for only a portion of the phosphatidic acid and diacylglycerol that accumulates in bradykinin-stimulated cells: bradykinin evidently stimulates several pathways of phospholipid metabolism in PC12 cells.     

Differential activation of phospholipases by mitogenic EGF and neurogenic PDGF in immortalized hippocampal stem cell lines

In several neuronal systems, nerve growth factor (NGF) and platelet-derived growth factor (PDGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogenic agent. Hippocampal stem cell lines (HiB5) immortalized by the expression of a temperature-sensitive SV40 large T antigen also respond differentially to EGF and PDGF. While EGF treatment at the permissive temperature induces proliferation, the addition of PDGF induces differentiation at the non-permissive temperature. However, the mechanism responsible for these different cellular fates has not been clearly elucidated. In order to clarify possible critical signaling events leading to these distinct cellular outcomes, we examined whether either EGF or PDGF differentially induces the activation of phospholipases, such as phospholipase A(2) (PLA(2)), C (PLC), or D (PLD). Although EGF stimulation did not induce phospholipases, PDGF caused a rapid and transient activation of PLC and PLD, but not PLA(2). When the activation of PLC or PLD was blocked, the neurite outgrowth induced by PDGF was significantly inhibited. Although the activation of PLD occurred faster than PLC, blocking of PLD activity by transient expression of lipase-inactive mutants did not inhibit the induction of PLC activity by PDGF. These results suggest that the differential activation of phospholipases may play an important role in signal transduction by mitogenic EGF and neurotrophic PDGF in HiB5 neuronal hippocampal stem cells. In particular, the activation of phospholipase C and D may contribute to neuronal differentiation by neurogenic PDGF in the HiB5 cells.     

Stimulation of Phospholipase D Activity by Phorbol Esters in Cultured Astrocytes

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) was found to stimulate phospholipase D activity in cultured primary astrocytes. Both the hydrolysis and the transphosphatidylation reaction catalyzed by phospholipase D were studied in cells labeled with [3H]glycerol. Phosphatidic acid (PA) synthesis was increased after addition of 100 nM TPA. When ethanol was present in the cell culture medium, phosphatidylethanol (Peth), a product of phospholipase D-catalyzed transphosphatidylation, was formed. The half-maximum effective concentrations (EC50) of TPA were 25 nM for PA increase as well as for Peth formation. The formation of Peth in ethanol-treated cells was accompanied by an inhibition of the TPA-induced increase in labeled PA. Increasing ethanol concentrations led to an increase in [3H]Peth and a decrease in [3H]PA. A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), inhibited both the synthesis of PA and the formation of Peth observed after TPA addition to the astrocytes. Dioctanoyl-glycerol (100 microM) stimulated the formation of Peth in the presence of ethanol. In addition to the induction of Peth formation in astrocytes, TPA induced Peth formation in ethanol-treated neurons. The present results indicate that phospholipase D activity is stimulated by TPA in cultured primary brain cells. Modulation of phospholipase D activity by protein kinase C is a mechanism that may be important in signal transduction cascades.     

Different regulation of phospholipase D activity in glioma C6 cells by sphingosine, propranolol, imipramine and phorbol ester

In has been found that sphingosine, propranolol, imipramine and phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) have a stimulatory effect on phospholipase D activity in glioma C6 cells. The cells were prelabelled with [1-(14)C]palmitic acid and phospholipase D-mediated synthesis of [(14)C]phosphatidylethanol was measured. The enhancing effect of TPA was almost completely blocked by a specific protein kinase C inhibitor, GF 109203X. In contrast, GF 109203X failed to inhibit the sphingosine, imipramine and propranolol stimulatory effects, indicating that their stimulation was independent of protein kinase C. The effect of TPA on phospholipase D was also blocked by imipramine and propranolol, whereas sphingosine additively potentiated TPA-mediated phospholipase D activity, both at shorter and longer (2-60 min) times of incubation. These results suggest that in glioma C6 cells, sphingosine is not only involved in a different phospholipase D activation than the TPA regulatory system, but also that it operates in a different compartment of the cell.     

Heterologous regulation of EGF receptors in fibroblastic cells

Platelet-derived growth factor (PDGF) increases the mitogenic activity of epidermal growth factor (EGF) in several cells lines, including BALB/C-3T3. PDGF-treated BALB/C-3T3 cells manifest a reduced capacity to bind 125I-labeled EGF due to a loss of high affinity EGF receptors. Cholera toxin potentiates the ability of PDGF to both decrease EGF binding and initiate mitogenesis. Whether PDGF increases EGF sensitivity via its effects on EGF receptors is not known and requires a more complete understanding of the mechanism by which PDGF decreases EGF binding. 12-O-tetradecanoylphorbol 13-acetate (TPA) also reduces EGF binding in BALB/C-3T3 and other cells, presumably by activating protein kinase C and, consequently, inducing the phosphorylation of EGF receptors at threonine-654. PDGF indirectly activates protein kinase C, and EGF receptors in PDGF-treated WI-38 cells are phosphorylated at threonine-654. Thus, the effects of PDGF on EGF binding may also be mediated by protein kinase C. We investigated this hypothesis by comparing the actions of PDGF and TPA on EGF binding in density-arrested BALB/C-3T3 cells. Both PDGF and TPA caused a rapid, transient, cycloheximide-independent loss of 125I-EGF binding capacity. The actions of both agents were potentiated by cholera toxin. However, whereas TPA allowed EGF binding to recover, PDGF induced a secondary and cycloheximide-dependent loss of binding capacity. Most importantly, PDGF effectively reduced binding in cells refractory to TPA and devoid of detectable protein kinase C activity. These findings indicate that PDGF decreases EGF binding by a mechanism that involves protein synthesis and is distinct from that of TPA.