Effects of platelet-derived growth factor on Ca in 3T3 cells

The effect of platelet-derived growth factor (PDGF) on cellular Ca²⁺ was examined in BALB/c-3T3 cells. PDGF induced:

1. 1. A decrease in cell ⁴⁵Ca²⁺ content.

2. 2. An apparent increased rate of efflux of preloaded ⁴⁵Ca²⁺.

3. 3. A decrease in residual intracellular ⁴⁵Ca²⁺ remaining after rapid efflux.

4. 4. When added after the rapid phase of efflux of ⁴⁵Ca²⁺ had occurred, an immediate decrease in post-efflux residual intracellular ⁴⁵Ca²⁺.

All of the observed changes in ⁴⁵Ca²⁺ induced by PDGF are consistent with a rapid release of Ca²⁺ from an intracellular Ca²⁺ pool that has the slowest efflux and is relatively inaccessible to extracellular EDTA. When incubated with chlortetracycline (CTC), a fluorescent Ca²⁺ probe, 3T3 cell mitochondria became intensely fluorescent. Addition of PDGF resulted in a rapid decrease in CTC fluorescence intensity in both adherent and suspended 3T3 cells. The effects of PDGF on 3T3 cell Ca²⁺ stores and CTC fluorescence intensity were identical with the effects of the Ca²⁺ ionophore A23187 and of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone. Serum, which contains PDGF, also altered intracellular Ca²⁺ stores, but platelet-poor plasma, which does not contain PDGF, had no effect. EGF, insulin, and tetradecanoyl phorbol acetate (TPA), other factors which stimulate 3T3 cell growth, did not alter 3T3 cell Ca²⁺ stores. Release of Ca²⁺ from intracellular sequestration sites may be a mechanism by which PDGF Stimulates Cell growth.     

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.     

Platelet-derived growth factor stimulates non-mitochondrial Ca2+ uptake and inhibits mitogen-induced Ca2+ signaling in Swiss 3T3 fibroblasts

Changes in intracellular free Ca2+ concentration [( Ca2+]i) were used to study the interaction between mitogens in Swiss 3T3 fibroblasts. Platelet-derived growth factor (PDGF) produced an increase in [Ca2+]i and markedly decreased the increases in [Ca2+]i caused by subsequent addition of bradykinin and vasopressin. If the order of the additions was reversed the [Ca2+]i response to PDGF was not inhibited by bradykinin or vasopressin. Inhibition of protein kinase C by staurosporine or chronic treatment of the cells with phorbol 12-myristate 13-acetate prevented the inhibitory effect of PDGF on the [Ca2+]i response to vasopressin but not bradykinin. PDGF did not decrease the receptor binding of bradykinin and produced only a small decrease in the receptor binding of vasopressin. PDGF decreased the rise in [Ca2+]i caused by the Ca2+ ionophores 4-bromo-A23187 and ionomycin and by a membrane perturbing ether lipid, 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine, both in the presence and absence of external Ca2+. There was no change in cell 45Ca2+ influx caused by PDGF, vasopressin, or bradykinin. 45Ca2+ efflux from cells exposed to PDGF and vasopressin mirrored the changes in [Ca2+]i caused by the agents, that is, PDGF added after vasopressin produced a further increase in 45Ca2+ efflux but vasopressin did not increase 45Ca2+ efflux after PDGF. PDGF but not vasopressin caused an increase in the uptake of 45Ca2+ into an inositol 1,4,5-trisphosphate-insensitive non-mitochondrial store in permeabilized cells. The results suggest that the decreased [Ca2+]i response to mitogens after PDGF represents an action of PDGF at a point beyond the release of intracellular Ca2+ and the influx of external Ca2+, caused by an increase in the rate of removal of cytoplasmic free Ca2+. This increased removal of cytoplasmic Ca2+ by PDGF is not due to the increased export of Ca2+ from the cell but results from increased Ca2+ uptake into non-mitochondrial stores.     

Temporal relationship between inositol polyphosphate formation and increases in cytosolic Ca2+ in quiescent 3T3 cells stimulated by platelet-derived growth factor, bombesin and vasopressin.

We determined the temporal relationship between the formation of inositol phosphates and increase in cytosolic [Ca2+] elicited by bombesin, vasopressin and platelet-derived growth factor (PDGF) in quiescent Swiss 3T3 cells. These responses were measured under identical conditions. Bombesin caused a rapid increase in inositol 1,4,5-trisphosphate which coincided with the increase in cytosolic [Ca2+]. This was followed by a slower but marked increase in inositol 1,3,4-trisphosphate and inositol-bisphosphate. Vasopressin elicited a similar sequence of events. In sharp contrast, highly purified porcine PDGF induced increases in cytosolic [Ca2+] and inositol 1,4,5-trisphosphate that were temporally uncoupled: detectable inositol polyphosphate formation occurred after Ca2+ mobilization from intracellular stores. The same temporal dissociation was observed when a recombinant v-sis product was used instead of porcine PDGF. However, PDGF was as effective as bombesin in stimulating the formation of inositol phosphates after 5-10 min of incubation. The data suggest that PDGF increases cytosolic [Ca2+] via a different signal transduction pathway from that utilized by bombesin and vasopressin. These findings have important implications for understanding the signal transduction pathway activated by PDGF.     

Retinyl acetate inhibits platelet-derived growth factor-induced Ca2+ signals in C3H 10T1/2 fibroblasts

Mitogenic stimulation of density-arrested C3H 10T1/2 mouse fibroblasts by serum or purified platelet-derived growth factor (PDGF) was potently inhibited by retinyl acetate (RAc; IC50 = 0.1 microgram/ml, 0.3 x 10(-6) M) when administered during the first 2 hours of mitogen exposure. This inhibitory effect of RAc coincided with a period early in the cell growth-division cycle when density-arrested C3H 10T1/2 cells stimulated by PDGF were found to require physiological levels of extracellular Ca2+ for the transition from G0 to G1 of the cell cycle. To determine if the inhibitory effect of RAc was mediated through alterations in the Ca2+ signaling pathway induced by mitogens, we examined Fura-2-loaded fibroblasts for changes in the Ca2+ response elicited by PDGF. Addition of PDGF (5 ng/ml) induced a transient increase in the [Ca2+]i that was not significantly effected by the extracellular Ca2+ concentration. Treatment of cells with RAc caused a concentration- and time-dependent inhibition of this PDGF-stimulated Ca2+ flux (IC50 = 0.45 microgram/ml or 1.5 x 10(-6) M; t1/2 = 15 min), whereas release of intracellularly stored Ca2+ by thrombin was unaffected by RAc (1.2 micrograms/ml, 4 x 10(-6) M). Treatment with RAc did not significantly affect PDGF binding to cell surface receptors or the generation of inositol phosphates. These results suggest that the mechanism by which RAc inhibits PDGF- or serum-induced mitogenesis is through modulation of the Ca2+ signal stimulated by PDGF, and thereby depriving the cell of a rise in intracellular Ca2+ necessary for progression through the cell cycle.     

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.     

Ionic responses rapidly elicited by porcine platelet-derived growth factor in Swiss 3T3 cells.

Addition of porcine platelet-derived growth factor (PDGF) to quiescent cultures of Swiss 3T3 cells caused a marked, dose-dependent stimulation of Na+ influx and Na-K pump-mediated 86Rb+ uptake. Porcine PDGF (a single component in SDS polyacrylamide gels) stimulated ion fluxes to the same maximal extent as partially purified preparations, and exhibited half-maximal effect at 6 ng/ml (2 X 10(-10) M). Maximal effect was achieved at 30 ng/ml (10(-9) M). In the presence of insulin, PDGF elicited mitogenesis at comparable concentrations. PDGF stimulated ion uptake in a time-dependent fashion; maximal effect was obtained after 5 min of exposure to the growth factor. PDGF stimulates Na+ influx via an amiloride-sensitive pathway, suggesting that PDGF enhances the activity of a Na+/H+ antiport system. In accordance with this possibility, the mitogen caused an increase of intracellular pH by 0.15 pH units, as judged by the steady-state distribution of labelled 5,5-dimethyloxazolidine-2,4-dione (DMO). Porcine PDGF stimulated E-type prostaglandin synthesis and cAMP accumulation but these events could be dissociated from the stimulation of the ionic fluxes, which was detected within minutes and was not blocked by indomethacin. It is suggested that PDGF elicits multiple signals to stimulate cell proliferation in 3T3 cells.     

Fibroblast growth factor stimulates protein kinase C in quiescent 3T3 cells without Ca2+ mobilization or inositol phosphate accumulation

To elucidate the transmembrane signalling processes initiated by fibroblast growth factor (FGF), we have studied the effect of recombinant basic FGF (bFGF) on various early events associated with mitogenesis in Swiss 3T3 fibroblasts. bFGF, at mitogenic concentrations, neither induced Ca2+ mobilization from intracellular stores nor increased the accumulation of inositol phosphates. In contrast, bFGF stimulated the phosphorylation of the Mr 80,000 (80K) cellular protein which is a major substrate of protein kinase C. This effect was potentiated by the diacylglycerol kinase inhibitor R59022. Two-dimensional polyacrylamide gel electrophoresis and phosphopeptide mapping showed that the 80K phosphoproteins generated in response to bFGF, bombesin, and phorbol 12,13-dibutyrate were indistinguishable. Down-regulation of protein kinase C prevented bFGF stimulation of 80K phosphorylation. Other protein kinase C-dependent early events such as transmodulation of the epidermal growth factor receptor, cytoplasmic alkalinization, inhibition of vasopressin induced increase in cytosolic [Ca2+], and enhancement of cAMP accumulation in response to forskolin were also induced by bFGF. Similar results were obtained when bFGF was added to quiescent cultures of tertiary mouse embryo fibroblasts. We conclude that bFGF stimulates protein kinase C through a signal transduction pathway distinct from inositol phospholipid turnover and Ca2+ mobilization.     

Inhibition of early platelet-derived growth factor responses in BALB/c-3T3 cells by interferon

Stimulation of total inositol phosphate production, alteration of cytosolic free calcium [( Ca++]i), vinculin disruption from adhesion plaques, and DNA synthesis caused by PDGF were examined in normal and INF pretreated density arrested BALB/c-3T3 fibroblasts. In normal cells, PDGF caused an increase in total inositol phosphates, a rapid, transient increase in [Ca++]i, disappearance of vinculin from adhesion plaques, and stimulation of DNA synthesis. Pretreatment of cells with INF inhibited PDGF-stimulated increases in [Ca++]i, vinculin disruption from adhesion plaques, and DNA synthesis, but had no effect on PDGF-induced increase in total inositol phosphate levels. These findings suggest that INF prevents entry of quiescent BALB/c-3T3 cells into G1 by inhibiting PDGF-induced release of Ca++ from intracellular stores.     

Epidermal growth factor induces intracellular Ca2+ oscillations in microvascular endothelial cells

An increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) may play a role in the proliferative effect of several growth factors. In this study, the changes in [Ca(2+)](i) elicited by epidermal growth factor (EGF) in rat cardiac microvascular endothelial cells (CMEC) have been investigated by using fura-2 conventional and confocal microscopy. A large heterogeneity in the latency and in the pattern of the Ca(2+) response was found at each dose of EGF (2.5-100 ng/ml), whereas some cells displayed a non-oscillatory behavior and others exhibited a variable number of Ca(2+) oscillations. On average, the fraction of responsive cells, the total number of oscillations and the duration of the Ca(2+) signal were higher at around 10 ng/ml EGF, while there was no dose-dependence in the lag time and in the amplitude of the [Ca(2+)](i) increase. EGF-induced Ca(2+) spikes were abolished by the tyrosine kinase inhibitor genistein, but not by its inactive analogue daidzein, and by the phospholipase C blocker NCDC. Only 1-2 transients could be elicited in Ca(2+)-free solution, while re-addition of extracellular Ca(2+) recovered the spiking activity. The oscillatory signal was prevented by the SERCA inhibitor thapsigargin and abolished by the calcium entry blockers Ni(2+) and La(3+). Moreover, EGF-induced Ca(2+) transients were abolished by the InsP(3) receptor blocker caffeine, while ryanodine was without effect. Confocal imaging microscopy showed that the Ca(2+) response to EGF was localized both in the cytoplasm and in the nucleus. We suggest that EGF-induced [Ca(2+)](i) increase may play a role in the proliferative action of EGF on endothelial cells.     

Inositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet-derived growth factor.

Swiss 3T3 cells incubated for 60 h with [3H]inositol incorporated radioactivity into phosphatidylinositol (PI) and the two polyphosphoinositides phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2). On stimulation with platelet-derived growth factor (PDGF) there were significant increases in the levels of inositol 1-phosphate (IP1), inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). The effect of PDGF and IP3 on Ca2+ mobilization was studied in both intact cells and in 'leaky' cells that had been permeabilized with saponin. In intact cells, PDGF stimulated the efflux of 45Ca2+, whereas IP3 had no effect. Conversely, IP3 stimulated 45Ca2+ efflux from 'leaky' cells, which were insensitive to PDGF. 'Leaky' cells, which accumulated 45Ca2+ to a steady state within 20 min, were found to release approx. 40% of the label within 1 min after addition of 10 microM-IP3. This stimulation of 45Ca2+ release by IP3 was reversible and was also dose-dependent, with a half-maximal effect at approx. 0.3 microM. It seems likely that an important action of PDGF on Swiss 3T3 cells is to stimulate the hydrolysis of PIP2 to form IP3 and diacylglycerol, both of which may function as second messengers. Our results indicate that IP3 mobilizes intracellular Ca2+, and we propose that diacylglycerol may act through C-kinase to activate the Na+/H+ antiport. By generating two second messengers, PDGF can simultaneously elevate the intracellular level of Ca2+ and alkalinize the cytoplasm by lowering the level of H+.     

Distribution of intracellular free calcium in quiescent BALB/c 3T3 cells stimulated by platelet-derived growth factor

A digital imaging microscope and fluorescent Ca(2+)-sensitive probe (Fura 2) were used to study the spatial location and time course of increases in free intracellular calcium (Cai) induced by platelet-derived growth factor (PDGF). Microinjection of Fura 2 acid avoided problems of incomplete deesterification of Fura 2-acetoxymethyl ester (Fura 2/AM) and dye localization in cellular organelles. PDGF stimulated a rapid increase in Cai (up to 8-fold increase) in both the nucleus and the cytoplasm in approximately half of the quiescent BALB/c 3T3 cells. Cai changes were both spatially and temporally heterogeneous, the latter including both transient (1-2 min) and prolonged increases (greater than 5 min) in the same cell. PDGF stimulated mitogenesis and Cai increases in approximately the same percentage of cells. Moreover, large intracellular concentrations of a Ca2+ buffer (Quin 2) inhibited both Cai increases and mitogenesis stimulated by PDGF. Thus, Ca2+ increases in the nuclear and/or cytosolic compartments appear to be required for the stimulation of mitogenesis by polypeptide growth factors such as PDGF.     

Plasma membrane hyperpolarization and [Ca2+]i increase induced by fibroblast growth factor in NIH-3T3 fibroblasts: resemblance to early signals generated by platelet-derived growth factor

The effects of different substances on [Ca2+]i and membrane potential (measured by fura-2 and bis-oxonol fluorescence techniques, respectively) were studied in wild-type and NIH-3T3 fibroblasts transfected with the cDNA encoding the human epidermal growth factor receptor. Application of partially purified PDGF or FGF induced, after a lag (0.5-1 min), a [Ca2+]i increase composed by an initial, slow peak, sustained primarily by intracellular Ca2+ release followed by a plateau, sustained by Ca2+ influx from the medium. The [Ca2+]i changes were paralleled by plasma membrane hyperpolarization mainly due to the activation of a K+ efflux, since raising the extracellular K+ concentration progressively reversed the effect of both growth factors. These responses were much slower than those induced by other agents (bradykinin, extracellular ATP, and EGF). The close resemblance between PDGF- and FGF-induced early signals (time-course and insensitivity to phorbol esters) suggests similar transmembrane signalling mechanisms at the cognate receptor.     

Effects of platelet-derived growth factor on bone formation in vitro

Platelet-derived growth factor (PDGF) is a polypeptide found in a variety of tissues, including bone, where it could act as an autologous regulator of skeletal remodeling. Therefore, a recombinant B chain homodimer of human PDGF was studied for its effects on bone formation in cultured rat calvariae. PDGF at 10-100 ng/ml stimulated [3H]thymidine incorporation into DNA by up to sixfold and increased the DNA content and the number of colcemid-induced metaphase arrested cells. This effect was observed in the fibroblast and precursor cell-rich periosteum. As a result of its mitogenic actions, PDGF enhanced [3H]proline incorporation into collagen, an effect that was observed primarily in the osteoblast-rich central bone. The effect of PDGF was not specific for collagen since it also increased noncollagen protein synthesis. In addition, PDGF increased bone collagen degradation. PDGF and insulin-like growth factor (IGF) I had additive effects on calvarial DNA synthesis, but PDGF opposed the stimulatory effect of IGF I on collagen synthesis and IGF I prevented the PDGF effect on collagen degradation. In conclusion, PDGF stimulates calvarial DNA synthesis which causes an increased number of collagen-synthesizing cells, but PDGF also enhances bone collagen degradation.     

Post-transcriptional control of protein synthesis in Balb/c-3T3 cells by platelet-derived growth factor and platelet-poor plasma

Platelet-derived growth factor (PDGF) and platelet-poor plasma, which lacks PDGF, both induce a rapid increase in the rate of total protein synthesis within quiescent, density-arrested Balb/c-3T3 cells. This stimulation of protein synthesis is associated with an increased aggregation of ribosomes into polyribosomes. Nuclear functions are not required for this response, as demonstrated by the observation that this stimulation of protein synthesis occurs in cells pretreated with actinomycin D and in enucleated cells (cytoplasts). The response to PDGF persists even after PDGF has been removed from the culture medium, but in contrast, when plasma is removed from the medium, polysomes disaggregate and protein synthesis declines. PDGF and plasma do not function synergistically to increase protein synthesis, whereas they do to induce optimum DNA synthesis. Thus stimulation of the translational apparatus may be necessary for the mitogenic response of Balb/c-3T3 cells to growth factors, but it is not by itself sufficient.     

Repression of p27kip1 synthesis by platelet-derived growth factor in BALB/c 3T3 cells.

We have investigated the regulation of p27kip1, a cyclin-dependent kinase inhibitor, in BALB/c 3T3 cells during growth factor-stimulated transition from quiescence (G0) to a proliferative (G1) state. The level of p27kip1 protein falls dramatically after mitogenic stimulation and is accompanied by a decrease in cyclin E associated p27kip1, as well as a transient increase in cyclin D1-associated p27kip1 that later declines concomitantly with the loss of total p27kip1. Analysis of metabolically labelled cells revealed that cyclin D2, cyclin D3, and cdk4 were also partnered with p27kip1 in quiescent BALB/c 3T3 cells and that this association decreased after platelet-derived growth factor (PDGF) treatment. Furthermore, the decline in p27kip1 and reduced association with cyclin D3, initiated by the addition of PDGF but not plasma-derived factors, suggested that these changes are involved in competence, the first step in the exit from G0. Synthesis of p27kip1 as determined by incorporation of [35S]methionine was repressed upon mitogenic stimulation, and PDGF was sufficient to elicit this repression within 2 to 3 h. Pulse-chase experiments demonstrated the reduced rate of synthesis was not the result of an increased rate of degradation. Full repression of p27kip1 synthesis required the continued presence of PDGF and failed to occur in the presence of the RNA polymerase inhibitor 5,6-dichlorobenzimidazole riboside. These characteristics demonstrate that repression was a late effect of PDGF and was consistent with our finding that conditional expression of activated H-ras did not affect synthesis of p27kip1. Northern (RNA) analysis of p27kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27kip1 mRNA, suggesting that the PDGF-regulated decrease in p27kip1 expression occurred through a translational mechanism.