studies on the mechanisms of signalling and inhibition by pertussis toxin on fibroblast growth...

Cellular Signalling Vol. 3, No. 3, pp. 215-223, 1991. 0898-6568/91 $3.00+.00 Printed in Great Britain. © 1991 Pergamon Press plc

S T U D I E S O N T H E M E C H A N I S M S O F S I G N A L L I N G A N D I N H I B I T I O N

B Y P E R T U S S I S T O X I N O F F I B R O B L A S T G R O W T H F A C T O R -

S T I M U L A T E D M I T O G E N E S I S I N B A L B / C 3T3 C E L L S

ANN LOGAN* and STEPHEN D. LOGAN

Department of Physiology, The Medical School, University of Birmingham, Birmingham B! 5 2TJ, U.K.

(Received 15 October 1990; and accepted 14 January 1991)

Abstract--Basic fibroblast growth factor (bFGF) stimulates mitogenesis of Balb/c 3T3 fibroblast cells. This stimulation may be mediated by multiple signal pathways as it is accompanied by the formation of inositol phosphates, activation of PKC (protein kinase C) and a decrease in intracellular cAMP levels. The multiple positive and negative pathways implicated for FGF-induced mitogenesis may interact and each may contribute in varying degrees to the final cellular response. At least two types of G-proteins may be involved in the intracellular signalling pathways of FGF. Pertussis toxin blocks FGF and TPA (12-O-tetradecanoylphorbol- 13-acetate) induced, PKC-mediated mitogenesis and also the associated fall in intracellular cAMP levels. However, pertussis toxin has no effect upon FGF-induced inositol phosphates formation. Thus, inhibition of mitogenesis by pertussis toxin may involve pertussis toxin sensitive G-proteins which may affect at least two separate putative signal pathways involving adenylate cyclase and protein kinase C. Pertussis toxin insensitive G-proteins may also be involved in coupling the FGF receptor to phosphoinositidase C.

Key words: bFGF, inositol phosphates, protein kinase C, cAMP, mitogenesis, 3T3 cells, pertussis toxin.

INTRODUCTION

BASIC fibroblast growth factor (bFGF) is a mitogen which was originally isolated from bovine pituitary as a factor that stimulates the division of Balb/c 3T3 fibroblast cells [1]. This growth factor is known to exert its activity by interaction with specific, high affinity, cell- surface FGF receptors which may be of different species [2, 3]. At least one of these, the fig receptor, has been cloned and sequenced and shown to belong to the tyrosine kinase family of growth factor receptors [4]. Many receptors require interaction with guanine

*Author to whom correspondence should be addressed at: Department of Clinical Chemistry, Wolfson Labora- tories, Queen Elizabeth Medical Centre, Edgbaston, Birmingham B 15 2TH.

Abbreviations: bFGF, basic fibroblast growth factor; cAMP, cyclic Y,5'-adenosine monophosphate; DMEM, Dulbeeeo's modified Eagle's medium; fcs, foetal calf serum; OAG, 1-oleoyl-2-acetylglycerol; PCA, pcrehloric acid; PDBu, phorbol-12,13-dibutyrate; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol- 13-acetate.

215

nucleotide binding proteins (G-proteins) for transduction of the agonist signal to occur. Whether G-proteins are required by FGF receptors for transduction and which this G-protein might be have yet to be established.

The intracellular signalling pathways activated following interaction of bFGF and its receptor(s) have yet to be rigorously defined. A number of alternative pathways are possible including those involving phosphoinositidase C, adenylate cyclase, kinases such as the tyrosine kinases and additionally a direct modulation of transcription following translocation of bFGF to the nucleus.

We have previously demonstrated that stimulation of DNA synthesis in Balb/c 3T3 cells is accompanied by hydrolysis of inositol lipids and the production of inositol phosphates, presumably by activation of a phosphoinositidase [5, 6]. The adenylate cyclase system has been implicated in the mitogenic signalling pathways of some growth factors [7, 8]. The relevance of these systems to the mitogenic activity of bFGF

216 A. LOGAN and S. D. LOGAN

remains to be established. Therefore in this study we have further investigated the putative signalling pathways for b F G F including hydrolysis of polyphosphoinositides and cAMP production.

Furthermore, we have investigated the mechanism of the inhibition by pertussis toxin of bFGF-induced mitogenesis. In addition to its well established effects on ribosylation of Gi, the G-protein that couples receptors to the inhibition of adenylate cyclase [9], pertussis toxin has the ability to inhibit agonist-induced stimulation of phosphoinositidase C in some cells [10]. This may also occur by ribosylation of a G-protein associated with this enzyme [11, 12]. Reports suggest that although pertussis toxin does inhibit some of the responses of Swiss 3T3 cells to FGF, this inhibition is not mediated by the modulation of phosphoinositide hydrolysis since inositol phosphates production was unaffected [13, 14]. In this study, we have further characterized the inhibition by pertussis toxin of FGF-stimulated D N A synthesis and in addition have investigated this toxin's effects on cAMP production, inositol phosphates production and protein kinase C activation in Balb/c 3T3 cells.

MATERIALS AND M E T H O D S

Materials

Tissue culture medium and supplements were obtained from Flow Laboratories. Methyl- [3H]-thymidine (2 Ci/mmol) and myo[2-3H]-inositol (10-20 Ci/mmol) and the cyclic AMP assay kit were from Amersham, U.K. Salts were from Fluka and BDH. Pertussis toxin was a gift from Dr F. S. Walsh, Institute of Neurology, London. Bovine pituitary basic FGF were purchased from Collaborative Research, Inc. 12-O-tetradeeanoylphorbol- 13-acetate (TPA), phorbol-12,13-dibutyrate (PDBu) and l-oleoyl-2-acetylglycerol (OAG) were purchased from Sigma.

Cell culture

Low passage stock cultures of Balb/c 3T3 fibroblast cells were maintained in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% foetal calf serum (fcs), 100#/ml penicillin and 100 #g/ml streptomycin. For the inositol phosphates

assays, cells were cultured in inositoi-free DMEM (Flow Laboratories). All agonists were dissolved in less than 20 #1 of double distilled water and added directly to the growth medium.

Assay of DNA synthesis

DNA synthesis was assessed in cultures of growth arrested 3T3 cells in low serum medium (0.25% fcs) stimulated with bovine pituitary bFGF or other agonists. This was done by measuring incorporation of methyl[3H]-thymidine into newly synthesized DNA as described previously [15]. DNA synthesis in cultures was expressed as dpm/well.

Stimulation of inositol phosphates accumulation

3T3 cells were seeded into 12-well microtitre plates in 1 ml of supplemented inositol-free DMEM and allowed to grow to confluence over 3-5 days by which time they became growth arrested. Medium was replaced and 3/~Ci/ml [3H]myo-inositol was added to each culture for 24 h. Prior to stimulation, the cells were washed twice and pre-incubated for 30 rain with 1 ml ofinositol-free DMEM plus 10 mM LiCI and 0.1% glucose. Bovine pituitary bFGF was added to each culture and after a 60 rain stimulation period reactions were terminated with 200/zl of 10% ice-cold perchloric acid (PCA).

After 15 min on ice, water soluble inositol phosphates were extracted from the cells by the method of Sharps and McCarl [16]. The PCA extract was separated from the cells by centrifugation at 2000 g for 5 min before neutralization with 0.25 ml octyla- mine-Freon (1 : 1). Sodium maleate (pH 7) was also added to give a final concentration of 75 mM to minimize losses of the inositol trisphosphates [17]. The uppermost aqueous layer was separated from the organic layer by centrifugation at 2000g for 5 min. Samples were stored at -20°C for not more than 48 h before assaying.

Estimation of [3H]-inositol phosphates accumulation

Samples were applied to 1 ml Dowex 1 × 10, 200--400 mesh, formate form, anion exchange columns in 10ml 5mM Na tetraborate/0.5mM EDTA. The columns were washed with 20 ml of distilled water and 15 ml of 50 mM ammonium formate to remove [3H]-inositol and glycerophos- phoinositides respectively. Elution of the [3H]-inositol phosphates was effccted with 18 ml of 1.7M ammonium formate/0.1 M formic acid (see Ref. 18). Following the addition of Optiphase 'X' scintillant (LKB), radioactivity was measured in samples by scintillation counting and expressed as dpm/culture well.

Mechanisms of signalling FGF-stimulated mitogenesis 217

c A M P assay

Levels of cAMP in 3T3 cells were measured using the cyclic AMP assay kit supplied by Amersham, U.K. which is based on the method of Tovey et al. [19]. Cultures of growth arrested 3T3 cells were treated with test agents in fresh unsupplemented DMEM for 15min at 37°C. After treatment, medium was removed and cultures were washed twice with cold phosphate buffered saline and harvested. After removal of aliquots of known volume for protein assay [20] and cell counts, samples were lysed with 0.1 M hydrochloric acid and boiled for 2 min. After centrifugation to remove the precipitate, the supernatant was frozen for later cAMP assay. After assay as described, cAMP levels were expressed as pmols cAMP//~g protein. Signifi- cance values were obtained using Student's unpaired t-test.

RESULTS AND DISCUSSION

We have previously shown [6] that addition of b F G F to quiescent cultures of Balb/c 3T3 fibroblast cells stimulates DNA synthesis in a concentration dependent manner over the range 1-100 ng/ml. The stimulation of cell division is accompanied by a similar concentration dependent stimulation of inositol phosphates production. This presumably occurs by activation of phosphoinositidase C as measured by the increase in total inositol phosphates accumu- lated in lithium-treated cells following treatment with bFGF. Although there has been some controversy surrounding the ability of the activated F G F receptor to hydrolyse inositol

lipids [4, 14, 21-23] our laboratory has demonstrated a clear association of mitogenesis and inositol phosphates production [5, 6].

The proposed association of the b F G F receptor(s) with phosphoinositidase C is further supported by the demonstration that levels of diacylglycerol rise rapidly in FGF-stimulated fibroblast cells [24]. The diacylglyceroi production was also linked to a subsequent activation of protein kinase C (PKC). Our results also implicate PKC in the post-receptor mi togenic signalling pathway of F G F in fibroblasts. It has been shown in a number of cell types that prolonged treatment with a high dose of phorbol ester results in down-regulation of protein kinase C activity [25,26], due to an increased degradation of the enzyme [27]. Quiescent cultures of 3T3 cells were pre-treated with 200 nM PDBu for 24 h under conditions known to down-regulate PKC in these cells [23]. Under these conditions the magnitude of the FGF-induced increase in DNA synthesis was reduced by approximately one half (Table 1). These results imply that PKC contri- butes to the intracellular signalling pathways used by b F G F in 3T3 fibroblasts and furthermore that it may be directly or indirectly relevant to mitogenic signalling by bFGF.

The link between PKC activation and cell division in 3T3 fibroblasts is also suggested by the observation that TPA, one of the most potent tumour promoting phorbol esters, is also able to stimulate [3H]-thymidine incorporation

TABLE 1. E ~ C ~ OF PDBu PRE-TREATMENT ON F G F - S ~ L A ~ D DNA SYNOPSIS

PDBu [aH]-thymidine incorporation Treatment Pre-treatment (dpm/well_+ S.E.M.)

Unstimulated - 1041 _+ 62 Unstimulated + 1155 _+ 132 50 ng/ml FGF - 5024 + 59 50 ng/ml FGF + 2756_+ 92 200 nM PDBu - 3253 _+ 73 200 nM PDBu + 2202__ 51

3T3 cells were pre-incubated for 24 h in the absence and presence of 200 nM PDBu. Cells were stimulated with 50ng/ml FGF or 200nM PDBu and [3H]-thymidine incorporation into cultures was measured as described in the Materials and Methods section. Results are the m e a n s + S . E . M , o f 6 replicates. Repeat experiments gave similar results.

218 A.l.~An and S. D. LoG^lq

o - Pertus,.Is toxin A.P~'tuula toxin

"~ 2 0 0 0 -

"o

1000-

8 ~r

-1- / / I t i I .I t~

0 10 -12 10-11 10-10 10-9 10 -8

M TPA

FIG. 1. Effects of pertussis toxin on TPA-induced DNA synthesis. 3T3 cells were treated with increasing concentrations of TPA between 10 -s M a n d l0 -12 M in the presence and absence of 10 ng/ml pertussis toxin. [3H]-thymidine incorporation was measured as described in the Materials and Methods section. Results are the means +_ S.E.M. of 6 replicate determinations. Repeat experiments gave similar results.

into these cells (Fig. 1). Phorbol ester tumour promoters such as TPA have been shown to bind to and activate PKC by acting as ana- logues of diacylglycerol [28].

In addition to stimulating the hydrolysis of inositol lipids, releasing inositol phosphates and stimulating PKC, b F G F also apparently inhibits adenylate cyclase activity in 3T3 cells as evidenced by the fall in intracellular cAMP levels from 3.37___0.2 to 2 .73_0.23pmol /#g protein in bFGF-stimulated cells (Table 2). This fall of cAMP levels to approximately 80% of the levels seen in unstimulated cells is statisti- cally significant (P < 0.05 vs control) and is similar to the changes observed in fibroblasts stimulated with other factors. For example, thrombin inhibits adenylate cyclase and causes a fall to approximately 80% of cAMP levels in control fibroblast cells [29].

These results suggest that the activated b F G F receptor(s) may be associated simul-

T A B L E 2. EF F ECTS OF F O R S K O L I N A N D PERTUSSIS TOXIN ON FGF-INDUCED CH A N G E S IN cAMP A N D DNA SYNTHESIS

[3H]-thymidine incorporation pmoles cAMP/pg protein

Treatment (dpm/well +_ S.E.M.) _+ S.E.M.

Experiment 1 Control 112 +_ 13 3.37 + 0.21 50 ng/ml FGF 1022+24 2.73+0.23 20~tM forskolin 156+ 15 8.12+0.85 10 ng/ml pertussis toxin 172 _+ 9 3.27 + 0.34 50 ng/ml FGF and 20/zM forskolin 221 + 21 6.43 _+ 1.45 50 ng/ml FGF and 10 ng/ml pertussis toxin 472 + 16 3.42 _+ 0.36

Experiment 2 Control 243 + 29 4.54 + 0.47 50 ng/ml FGF 2342 + 104 3.25 + 0.29 10#M forskolin 375+ 55 12.44+0.72 1.0/zM forskolin 343 _+ 35 11.52 _+ 0.89 0.1 gM forskolin 259 + 20 6.77 + 0.66 0.01/zM forskolin 221 _+ 17 4.81 +_0.21 50 ng/ml FGF and 10/~M forskolin 458 +_ 53 11.25 +_ 0.42 50 ng/ml FGF and 1.0/tM forskolin 543 +_ 38 10.14 +_ 0.95 50 ng/ml FGF and 0.1/tM forskolin 546+27 5.12+_0.52 50 ng/ml FGF and 0.01/tM forskolin 1824+_69 3.93 +_0.47

3T3 cells were stimulated with FGF in the prelam~ or absence of forskolin and pertussis toxin. [3H]-thymidine incorporation and cAMP formation was measured as described in the Materials and Methods section. Results are the means+S.E.M, of 6 and 3 replicates respectively.


taneously with a number of putative signalling pathways which include those utilizing inositol phosphates, PKC and cAMP. Clearly, the modulation of these various signal pathways may be due to a direct coupling to the bFGF receptor(s) or the effects may be indirect via cross-talk of intracellular signalling molecules. Additionally, there are reports of other possible signalling pathways associated with bFGF activity including translocation of FGF to the nucleus where it can directly affect ribosomal RNA transcription [30] and PKC-independent phosphorylation by kinases which includes a tyrosine kinase [4, 31-33].

In view of the increasing evidence that bFGF is a multifunctional regulatory factor in target cells [34] it is important to define the relative importance of each signal pathway to individual cellular responses. These experiments do provide some evidence concerning their relative contributions to the regulation of cell proliferation.

Elevation of intracellular cAMP levels in 3T3 cells by forskolin treatment in the presence of bFGF markedly reduced, but did not com- pletely abolish, FGF-stimulated [3H]-thymidine incorporation (Table 2). A small stimulation of DNA synthesis by bFGF was still apparent even though bFGF was unable to overcome, in any marked degree, the stimulatory influence of

forskolin upon cAMP levels. In experiment 2 (Table 2) it can be seen that when forskolin is titrated so that on bFGF addition the cAMP levels are increased back to approximate control values (0.1/~M forskolin), the mitogenic stimulation by bFGF was still significantly reduced. This experiment provides some evidence of the relative importance of bFGF- induced modulation of the adenylate cyclase pathway to this growth factor's mitogenic activity. It also implies the necessity of other signal propagating systems for optimal prolifer- ative responses.

Similarly, down-regulation of PKC by PDBu also reduced, but did not abolish, the stimulatory effects of bFGF on [3H]-thymidine incorporation (Table l) under a pre-treatment regime which is reported to inactivate com- pletely all of the enzymatic activity [23]. This result may again relate to the relative contribu- tion of other signal pathways to the maximal mitogenic response to bFGF.

A surprising result was obtained when we examined the effect of the corticosteroid dexamethasone upon bFGF-induced cell division. The mitogenic activity of bFGF can be potentiated by a number of substances including dexamethasone (Table 3). Dexamethasone increased the bFGF-induced response in 3T3 cells by almost 50%. However, when total

TABLE 3. EFFECTS OF DEXAMETHASONE ON FGF-sTIMULATED [3H]-THYMIDINE INCORPORATION AND [3H]-INOSITOL PHOSPHATE ACCUMULATION

Treatment

[3H]-thymidine incorporation

(dpm/well + S.E.M.) [aH]-inositol accumulation

(dpm/well + S.E.M.)

Control 379 + 65 549___ 12 50 ng/ml FGF 2394 + 191 942_+ 30 I ng/ml dexamethasone 516_ 20 627_+ 29 50 ng/ml FGF and I ng/ml

dexamethasone 3377 + 63 595 _ 19

3T3 cells were treated with 50ng/ml FGF in the presence and absence of I ng/ml dexamethasone. Dexamethasone was added directly to the culture medium 36 h and 30 rain respectively prior to stimulation and measurement of [3H]-thymidine incorporation and [3H]-inositol measurement. [3H]-thymidine incorporation and [3H]-inositol phosphates accumulation was measured as described in the Materials and Methods section. Results are the means_+S.E.M, of 6 and 3 replicate determinations respectively. Repeat experiments gave similar results.

CELLB 313-D

220 A. LOGAN and S. D. I.,OOA~

inositol phosphates formation was measured in bFGF-stimulated cells treated with dexamethasone there was an inhibition of inositol phosphates formation with complete inhibition occurring when 3T3 cells were pre-treated for more than 30 min with the corticosteroid. Thus, the mitogenic response to bFGF can be disso- ciated from the inositol phosphates signal and in fact can even be potentiated in the complete absence of any measurable formation of inositol phosphates. It seems then, that the inositol phosphates pathway may not be essen- tial for mitogenesis under some circumstances. There is evidence that other growth factors, such as PDGF, which also stimulate phosphoinositide hydrolysis, do not use the inositol phosphates pathway to signal growth [35]. How dexamethasone inhibits inositol phosphate formation, remains to be established, although a similar inhibitory effect has been noted by others in human HSWR fibroblasts chronically treated with dexamethasone and stimulated with growth factors [36]. They suggest that this may be due to dexamethasone inducing the putative phospholipase inhibitor lipomodulin. Dexamethasone potentiation of the mitogenic effect may simply reflect dexamethasone's influences on transcription, although there is no evidence to date to support this contention for FGF.

Which, if any, of the multiple membrane signal-transducing mechanisms implicated in this paper play a direct role in dictating whether a cell divides in response to bFGF, remains to be established. Although these results have not proved conclusive they do indicate the multipli- city of the signal pathways that can be linked to a receptor type. It seems that a complex network of positive and negative interactions exists between the multiple membrane-signalling pathways for bFGF and these include tyrosine kinase, phosphoinositidase C and adenylate cyclase. A number of other growth- promoting agents are also thought to modulate simultaneously phosphoinositidase C and adenylate cyclase, such as 5HT, thrombin, bombesin, vasopressin, bradykinin and phos- phatidic acid (as detailed in [29]). However, the

30O0

2 o

2000

.~ tO00

~, _. |

I ' " olo, o!t ~!o ~o!o Per tuu la toxin ng/ml

• Pertussls toxin and 50ng/ml FGF

• Pertusslll toxin alone

fcs

Fie. 2. Effects of FGF and pertussis toxin on [3H]-thymidine incorporation. 3T3 cells were treated for 60 h with pertussis toxin at increasing doses between 0 and 10 ng/ml in the presence and absence of 50 ng/ml FGF for the final 36 h. DNA synthesis in cultures was assessed by measuring [3H]-thymidine incorporation as described in the Materials and Methods section. Results are the means+S.E.M, of 6 replicate determinations from a single experiment. Repeat experiments gave similar results.

question remains whether the modulation of multiple signal pathways by bFGF is due to direct or indirect coupling to individual bFGF receptors.

If direct, whether these signal pathways are coupled to a single species of bFGF receptor or whether each receptor species implicated for bFGF (see Introduction) has a single pathway associated with it remains to be established; as is the mechanism which couples the receptor(s) to the effector systems which mediate the propagation of the mitogenic signal into the cell. It seems that G-proteins may be involved in this coupling but as yet the nature and roles of the G-proteins relevant to bFGF signalling have yet to be identified. Evidence that G-proteins do play some role in the mitogenic pathways for bFGF comes from the data presented in Fig. 2. This clearly demonstrates that pertussis toxin is a potent inhibitor of bFGF-induced DNA synthesis. Pertussis toxin reduces bFGF- stimulated [3H]-thymidine incorporation into


3T3 cells in a dose-related manner. The inhibitory G-protein, Gi, which modulates adenylate cyclase activity, is a known substrate for pertussis toxin. It seems that in bFGF-stimulated 3T3 cells pertussis toxin exhibits a similar action upon Gi since the fall in intracellular cAMP levels noted in FGF-stimulated cells is abolished by pertussis toxin (Table 2). Thus, Gi is implicated in coupling adenylate cyclase to the bFGF receptor(s).

A novel G-protein, Gp, has been proposed which links receptors to the inositol lipid signalling system (see Ref. 37). Gp is clearly distinct from Gi but may or may not be sensitive to pertussis toxin depending on the cell type from which it is isolated. This may reflect a hetero- geneity in this group of G-proteins. Although others [10] have reported that pertussis toxin can modulate phosphoinositidase C activity in cells such as neutrophils, it seems unlikely that in 3T3 cells this occurs since we could not measure any effects of pertussis toxin on the bFGF-stimulated formation of inositol phosphates. The unresponsiveness of inositol phosphates formation was evident even at pertussis toxin concentrations which maximally inhibit the stimulation of DNA synthesis (Table 4). This result suggests that a pertussis toxin-sensitive Gp-protein is not involved in coupling the bFGF receptor(s) to phosphoinositidase C.

Interestingly, when PKC is activated directly by TPA at a point in the pathway subsequent to the phosphoinositidase C catalysed diacylglycerol production, then pertussis toxin does inhibit the TPA-induced DNA synthesis at all of the TPA concentrations tested between 10-SM and 10-~2M (Fig. 1). Of interest with regard to this observation is the earlier report of Murayama and Ui, 1985 [38] that, in mouse 3T3 cells, receptor coupling to phosphoinositidase C was unaffected by pertussis toxin but several later events such as arachidonic acid release and uptake of Ca 2+ were inhibited. It is possible that pertussis toxin may be affecting PKC activity indirectly via its action on the adenylate cyclase Gi protein and its blockage of the cAMP signal, with consequent modulations of a post-diacylglycerol component of the pathway by cAMP. Alternatively, these results may represent modulation of PKC or its substrates directly by a pertussis toxin-sensitive G-protein, which may be Gi or a novel G-protein. Further experiments are underway to elucidate the mechanism of this interaction.

These results concur with the findings of Taylor et al. [14] concerning the effects of bFGF and pertussis toxin on DNA synthesis and inositol phosphates production, although these workers suggest that the effects of pertussis toxin are not linked directly to receptor

TABLE 4. EFFECTS OF PERTUSSIS TOXIN ON FGF-sTIMULATED [3H]-THYMIDINE INCORPORATION AND [3H]-INOSITOL PHOSPHATES ACCUMULATION

Treatment

[3H]-thymidine incorporation

(dpm/well ± S.E.M.)

[3H]-inositol phosphates accumulation

(dpm/well ± S.E.M.)

Control 379 ± 65 573 __ 29 50 ng/ml FGF 2394± 191 873 ± 18 10 ng/ml pertussis toxin 465 ± 53 550 ± 53 50 ng/ml FGF and 10 ng/ml

pertussis toxin 877 ± 113 847 _ 15

3T3 cells were stimulated for 60rain or 36 h with 50 ng/ml FGF in the presence or absence of 10 ng/ml pertussis toxin. Pertussis toxin was added to cultures 24 h previously and was continually present thereafter. Control cultures had additions of equivalent volumes of the vehicle, double distilled water. [3H]-inositol phosphate accumulation and [3H]-thymidine incorporation in cultures was measured as described in the Materials and Methods section. Results are the means + S.E.M. of 3 and 6 replicate determinations respectively from a single experiment. Repeat experiments gave similar results.

222 A. Loo^N and S. D. LOGAN

coupling. The results presented here suggest that in addition to an indirect action of pertussis toxin at post-phosphoinositidase C points in the phosphoinositide pathway, pertussis toxin could also modulate bFGF-induced DNA synthesis at the receptor level via its coupling to adenylate cyclase. These results do indicate that at least two G-proteins may be involved in the intracellular signalling pathways of bFGF. Gp, which in 3T3 cells may be a pertussis toxin- insensitive G-protein, linked to a phosphoinositidase, and pertussis toxin-sensitive Gi coupled negatively to adenylate cyclase.

The evidence for interactions of the multiple pathways emphasizes the complexity of the mitogenic signalling mechanisms of bFGF. Future experiments will address the question of direct vs indirect coupling of the b F G F receptor to these putative signal pathways. They will also further characterize the signal pathways for bFGF, their interactions and the regulatory G-proteins which may be involved in their regulation.

Acknowledgements--This work was supported by the MRC and the International Spinal Research Trust. Thanks are due to Dr PmL GALLIMORE and PETER GRAaHAM, Department of Cancer Studies, University of Birmingham, for assistance with the cAMP assays.

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