- Email: [email protected]
PDGF-AA effectively stimulates early events but has no mitogenic activity in AKR-2B mouse fibroblasts
EXPERIMENTAL
CELL
RESEARCH
201,
192-199 (19%)
PDGF-AA Effectively Stimulates Early Events but Has No Mitogenic Activity in AKR-2B Mouse Fibroblasts ANDREAS SIMM, VIVIANE HOPPE, DIETER TATJE, ANJA SCHENZINGER, AND J~JRGEN HOPPE’ Theodor-Boveri-Znstitut
jiir Biowissenschaften
(Biozentrum)
der Uniuersitiit,
The response of AKR-2B mouse fibroblasts, which express approximately equal numbers of platelet-derived growth factor (PDGF)-a! and $3 receptors on their surface (V. Hoppe et al. Eur. J. Biochem. 187, 207214, 1990) to all three isoforms of PDGF, was studied. All isoforms stimulated early events, i.e., receptor autophosphorylation on tyrosine, total cellular phosphorylation, increase in 32P-labeled phospholipid content, but there was no correlation between the extents measured for the different effects. Although rPDGF-AA effectively stimulated these early events, it was unable to induce [3H]thymidine incorporation and cell growth whereas rPDGF-BB and -AB stimulated the division of more than 90% of the cells. This activity was restored by addition of insulin-like growth factor I (IGF-I), which itself exhibited only a low mitogenic activity. rPDGF-AB or -BB did not require the presence of IGF-I to fully stimulate cells for [3H]thymidine incorporation and cell division. Apparently, rPDGF-AA induced only a “competence” state of the cells whereas rPDGF-AB or -BB was also able to initiate “progression.” It is speculated that some early events occuring during the competence phase might be part of a “maintenance” program elicited by growth factors. o 1st~. Academic PWS, IN.
INTRODUCTION Platelet-derived growth factor (PDGF)’ is a major mitogen in serum which promotes the proliferation of fibroblasts, glia cells, and smooth muscle cells in vitro [l-3]. The two homologous chains, termed A and B, which have been identified and sequenced, can combine to three different dimeric isoforms: AA, AB, and BB [4-81. The dimeric structure is stabilized by disulfide bridges, and after destruction of these bridges biological activity is lost [l-3, 91. This fact, that only dimers are biologically active, along with the discovery of two dif1 To whom correspondence and reprint requests should be addressed. Fax: (931) 888-4113. ’ Abbreviations used: FCS, fetal calf serum; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor; IGF-I, insulinlike growth factor I; SDS, sodium dodecyl sulfate. 0014-4827/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved
Physiologische
Chemie ZZ, Am Hublnnd,
8700 Wiirzburg,
Germany
ferent PDGF receptors (01and fi), led to the hypothesis that one dimeric PDGF may bind two receptor molecules (receptor subunit model) [lo-161. The model predicts that PDGF-AA binds only a-type PDGF receptors (receptors specific for PDGF), PDGF-AB binds one o(type and one P-type receptor, and PDGF-BB binds mainly P-type PDGF receptors. The two receptors share a common structure including an extracellular domain that is composed of five immunoglobulin-like repeats and a cytoplasmic portion that includes split tyrosine kinase sequences [IO-121. Despite extensive structural homology, the two receptors display considerable heterogeneity in their extracellular and cytoplasmic noncatalytic domains such as the kinase insertion stretches and the carboxy termini. These cytoplasmic regions participate in the interaction between the activated receptor and cytoplasmic proteins (for review see [17]) and thus may confer distinct substrate specifications to the homologous tyrosine kinases. A current open question related to the mechanism of signal transduction is the extent of functional overlap of mitogenic signals arising from the cell surface via homologous receptors. We have recently examined the effects of the three PDGF isoforms on the [Cazcli mobilization, inositol trisphosphate release, diacylglycerol production, and pH changes in cultured rat aortic smooth muscle cells [ 18-201. The marked differences found between the isoforms lead, together with previous results [13, 21-301, to the assumption that the a-type and the P-type receptors transmit different signals into the cell. On the other hand, other reports claim that both receptors may express the same spectrum of biological functions and differences must be related to unequal receptor populations [31-331. AKR-2B mouse fibroblasts that express similar numbers of CYor p receptors on their surface were chosen as a model system. Here we show that rPDGF-AA alone is unable to act as a mitogen although it stimulates effectively several early events, e.g., tyrosine autophosphorylation, total protein phosphorylation, and [32P]phospholipid synthesis. These results suggest that some early events may play a role in functions like “maintenance” [ 34-361. 192
EFFECTS
MATERIALS
AND
OF PDGF
METHODS
Materials PDGF isoforms were prepared as described [37]. IGF-I was from Bachem. Rabbit anti-mouse IgG, lipid standards, protein molecular weight standards, and p-coumaric acid were purchased from Sigma. [32P]Orthophosphate, 370 MBq/ml (carrier free), [sH]thymidine, 1.55 TBq/mmol, and Hyperfilm ECL were obtained from Amersham. Monoclonal antibody lG2 against phosphotyrosine was from Boehringer. Benzonase and thin-layer plates were purchased from Merck. Cell culture reagents were from GIBCO. Luminol was obtained from Fluka. Reinforced nitrocellulose was from Schleicher & Schiill and goat anti-rabbit peroxidase antibody from Dianova. Immobiline dry plates (pH 4 to pH 7) were from Pharmacia. Methods Growth-promoting actiuity. This was determined as described [38], using AKR-2B cells without insulin. (Cells were cultivated for less than 3 months to minimized fluctuations. During that time the responses to PDGF isoforms did not change.) Cell cycle analysis. After reaching confluence, cells were starved in MCDB 402 medium. After starvation for 48 h, fresh MCDB 402 medium, 60 PM bromodesoxyuridine, 60 PM desoxycytidine, and growth factors were added. Cells were harvested 44-46 h later by trypsin treatment and stored at -20°C in PBS containing 10% dimethyl sulfoxide and 10% FCS. Staining with ethidium bromide and Hoechst 33258 dye and analysis were performed as described [39] using an ICP22 flow cytometer (Phywe). The computer programs Multi-2D or M-Cycle (Phoenics) were used for quantification. Analytical methods. SDS-polyacrylamide gel electrophoresis was performed essentially as described [37]. Protein content was determined by the method of Redinbaugh and Turley [40] after calibration of the three PDGF isoforms by amino acid analysis. Autoradiograms were quantified by laser densitometry (Ultroscan, LKB). Data were analyzed by the computer program GraFit (Erithacus Software, London). Analysis of [32P]phospholipids [41]. AKR-PB cells were grown on g-cm’ petri discs for 5 days in McCoy medium containing 10% Hyclone. After reaching confluence they were synchronized by incubation in 2.5 ml MCDB 402 medium containing 50 PM orthophosphate for 2 days. Fifteen microcuries (32P]phosphate was added. Growth factors were added either immediately after the addition of the radioactivity or 60 min later. The reaction was terminated 15 or 30 min, respectively, after the addition of growth factors. Cells were washed three times with ice-cold PBS and then treated with 500 ~1 cold 30% trichloroacetic acid. Cells were scraped with a rubber policeman and transfered into a microtube. After washing twice with cold 30% trichloroacetic acid, 100 pl of 2.4 N hydrochloric acid and 300 pl of chloroform/methanol, l/2 (v/v), were added and the mixture was vigorously shaken. For phase separation 125 ~1 of chloroform and 125 ~1 of 0.1 N hydrochloric acid were added. After brief centrifugation, the upper phase was removed and discarded. The lower phase was washed twice with methanol/water/chloroform, 48/47/3 (v/v/v). Finally 100 ~1 of the lower phase was withdrawn, supplemented with 20 pg phospholipid standards, and dried under a nitrogen stream. Thin-layer chromatography of the extracted phospholipids. The dried samples were dissolved in 10 pl chloroform/methanol, 2/l (v/v). Five microliters was used for thin-layer chromatography. Analytical plates with a concentration zone were used. Plates were developed with chloroform/methanol/2 M aqueous ammonia, 4314818 (v/v/v), for 20-25 min. Standards were visualized by I, vapor. Radioactivity was detected by autoradiography using a Kodak XAR film. Stained areas were quantified by laser densitometry. Analysis of phosphoproteins. Cells were grown in 24-well plates (Greiner; 1.75~cm* surface/well) and arrested as described above for
193
ISOFORMS
the analysis of phospholipids. After a 2-h incubation with 50 &i [32P]phosphate in 300 ~1 MCDB medium containing 50 pM orthophosphate, growth factors were added for 10 min. The medium was removed and cells were lysed in 40 ~1 buffer containing 7 M urea, 2% Nonidet P-40, 2% 2-mercaptoethanol, 1 mM MgCl,, and 10 pi/ml benzonase solution for 5 min under vigorous shaking. Immobiline dry plates (pH 4-7) were cut into 0.5-cm strips and rehydrated in 8 M urea, 0.5% Nonidet P-40, 2 mM Tris base. Thirty microliters of the cell lysate was applied close to the cathode. Focusing was done at 2000 V for 24 h. The stripes were then soaked for 15 min in an equilibrium buffer (50 mMTris-Cl, pH 6.8,2% SDS, 6 Murea, 30% glycerin, some crystals of bromphenol blue) and mounted on top of the gel (16 X 12 cm, 0.7 mm thickness). The acrylamid content was 13.5% acrylamide, 0.36% N,N’methylene-bis-acrylamide. Gels were stained with Coomassie brilliant blue R to verify that equal amounts of proteins were loaded and that separation of proteins was identical. Autoradiography was done for 24 h using Kodak XAR film and intensifier screens. Analysis of tyrosine-phosphorylated proteins. Cells were grown and arrested as described above. One millimolar sodium orthovanadate was added for 15 min before the addition of growth factors. Stimulation was performed for 10 min at various concentrations. The medium was removed and for lysis 50 ~1 buffer containing 50 mM TrisCl, pH 6.7, 2% SDS, 2% mercaptoethanol, 1 mM sodium orthovanadate was added. After 5 min of vigorous shaking, 10 ~1 benzonase (6%) was added and shaking was continued for another 5 min. The entire solution was transferred into a microtest tube and 6 ~1 bromphenol blue in 50% glycerol was added. Thirty microliters was taken for SDS-PAGE (7.5% acrylamide-0.075% N,N’methylene-bisacrylamide) using a minigel system (Bio-Rad). Protein was transferred onto reinforced nitrocellulose by semidry blotting using 50 mM Caps, pH 10.0, 1 mM 3-mercaptopropionic acid, 0.1% SDS, and 10% methanol as blotting buffer for 2 h at 6 V. The nitrocellulose sheet was washed three times for 5 min with 50 mM Tris-Cl, pH 7.5, 150 mM NaCl (TS buffer). Saturation was performed overnight with 2% bovine serum albumin with 0.03% NaN, in TS. The sheets were incubated for 2 h with anti-phosphotyrosine antibody lG2 at 5 pg/ml (Boehringer, Mannheim, Germany) in TS containing 2% BSA, 0.2% Nonidet P-40, and 0.03% NaN, (TSBN). After washing three times with TS containing 0.5% BSA, 0.2% Nonidet P-40, 0.03% NaN, (washing buffer), the sheets were incubated for 1 h with rabbit antimouse IgG (l/500 dilution, Sigma) in TSBN. After washing three times with washing buffer, goat anti-rabbit IgG horse radish peroxydase-labeled antibody (Dianova) was added at a dilution of l/5000 for 1 h. The antibody was removed by washing three times and the sheets were soaked in 3 ml luminescence buffer containing 100 mM Tris-Cl, pH 8.5, 2.5 mM luminol (Fluka), 400 PM p-coumaric acid (Sigma), to which 3 ml hydrogen peroxide buffer was added (5.4 mM H,O,, 100 mM Tris-HCl, pH 8.5). After 1 min the sheets were quickly dried with filter paper and wrapped in a transparent foil. Light emission was detected with an ECL film (Amersham). Stock solutions of luminol (250 mM) and p-coumaric acid (90 mM) in DMSO were used.
RESULTS
Mitogenic
Activity
of rPDGF Isoforms
In the previously used protocols for [3H]thymidine incorporation into the DNA of AKR-2B mouse fibroblasts 2 @g/ml insulin was included into the basal protein-free MCDB 402 medium. Under these conditions rPDGF-BB and -AB stimulated [3H]thymidine incorporation to a similar extent; rPDGF-AA ‘was less active, reaching about 60% of the maximum level. E,, values for rPDGF-AB and -BB were in the range of 2 rig/ml, and that for rPDGF-AA was 10 rig/ml [37, 421. When
194
SIMM
ET AL.
insulin was omitted, rPDGF-AB and -BB stimulated [3H]thymidine incorporation still to the same extent but the E,, value was increased two- to three-fold (Fig. 1A). Remarkably rPDGF-AA alone exhibited only a residual activity (Fig. 1A). It was almost inactive at 10 rig/ml. Since insulin added at high concentrations might act via insulin-like growth factor I receptors it was replaced by insulin-like growth factor I (IGF-I). The low E,, value of 7 rig/ml indicated that IGF-I indeed acts via its receptors (insert, Fig. 1A). When IGF-I was added at 30 ng/ ml to various concentrations of rPDGF isoforms the formerly observed pattern in the presence of insulin was reconstituted; i.e., E,, values for rPDGF-AB and -BB were around 2-4 rig/ml and the maximum stimulation by rPDGF-AA reached 50% of that value observed with the two other isoforms. PDGF isoforms stimulated maximum DNA synthesis 21 h after the addition, whereas the maximum for IGF-I was at 17 h (Fig. 1B). Thus, the value obtained under standard assay conditions (analysis after 23 h [37, 381) underestimated the potency of IGF-I. Interestingly, together with PDGF-AA, IGF-I determined the rate of DNA synthesis as the maximum was found at 17 h. Both factors act synergistically under these conditions.
6000 4000 2000 0 0.1 m
6000 6000 4ooo 2ooo
Cell Cycle Analysis
0 1
0.1
10
100
growth factor [ ng / ml ]
20000
0
53-r
5
10
15
time of stimulation
20
3
25
30
[h ]
FIG. 1. (A) Stimulation of [3H]thymidine incorporation into DNA by various growth factors. Quiescent AKR-2B fibroblasts were stimulated by the indicated amounts of growth factors for 23 h; 0.1 pCi [3H]thymidine/well was added and after 45 min cells were harvested. W, rPDGF-BB; 0, rPDGF-BB + 30 rig/ml IGF-I; 0, rPDGFAB; 0, rPDGF-AB + 30 rig/ml IGF-I; A, rPDGF-AA; A, rPDGF-AA + 30 rig/ml IGF-I; (Insert) V, IGF-I + 25 rig/ml rPDGF-AA. Measurements were performed in triplicate. (B) Time-dependent [3H]thymidine incorporation into AKR-2B cells. At zero time growth fac-
[3H]Thymidine incorporation neither is a measurement for the percentage of cells that synthesize DNA nor does it indicate whether stimulated cells will divide. We have therefore performed a detailed cell cycle analysis using the double-staining technique [39]. Approximately 2 days after stimulation with either PDGF-BB or -AB (Fig. 2A) only 10% of the cells remained in the starting cycle (cycle 0). From those cells roughly 50% already had entered the first S-phase. The majority of the cells were recovered in cycle 1; i.e., they have completed one division. Some cells had already entered the second cycle. After treatment with PDGF-AA most of the cells remained in cycle 0. Remarkably, none of these cells had entered the S-phase. A few cells were found in the first cycle but none in the second cycle. These results thus show that PDGF-BB or -AB stimulated nearly all of the cells (>95%) to divide, whereas PDGFAA was almost inactive. Figure 2B demonstrates that PDGF-AA and IGF-I act synergistically also on cell growth. PDGF-AA stimulated maximally 18% of cell division and IGF-I alone 22%. Simultaneous addition resulted in the division of 64% of the cells.
tors were added to starved cultures of AKR-2B cells. (PDGF isoform, 33 rig/ml; IGF-I, 30 rig/ml.) [sH]Thymidine was added for 60 min at the indicated time. Measurements were performed in duplicate and repeated once with identical results. W, rPDGF-BB; 0, rPDGF-AB; A, rPDGF-AA + IGF-I; A, rPDGF-AA, V, IGF-I.
EFFECTS
.A.
cycle
t
cycle 1
0
cycle 2
1w
90
.’ 90
60
.’ 60
70
8
60
B
50
8
195
ISOFORMS
I loo
_
OF PDGF
50
30.00
40
40
20.00
30
30
10.00
20
20
0.00
10
10
0
0 AA
AB
BB
AA
+ IGF-I
AB
BB
+ IGF-I
.70
AA + IGF-I
PDGF-AA
-_U.“”
Wmll
FIG. 2. (A) Distribution of cells in various cell cycles. To starved cells bromodesoxyuridine and the three growth factors were added (PDGF isoforms, 100 rig/ml; IGF-I, 30 rig/ml). Cells were harvested after 41-46 h, stained, and analyzed by flow cytometry. Measurements were done in triplicate. (B) Synergistic effects of PDGF-AA and IGF-I. Cells were stimulated with the indicated amounts of PDGF-AA and IGF. Analysis was done as above. Standard deviation was less than 10%. The Z-axis indicates cell division as a percentage.
Stimulation
of Tyrosine Phosphorylation
All three isoforms stimulated tyrosine phosphorylation of a protein with an M, of 180,000 Da, which is generally believed to be the respective PDGF receptor (a- or P-type (Fig. 3A). The Ebo values (AB, 6 rig/ml; BB,
A
M.W.
c kd 1
15 rig/ml; AA, 25 rig/ml) correlate reasonably well with those obtained in the mitogenic assay without IGF-I (Fig. 3B). Again rPDGF-AB and -BB reached the same plateau. rPDGF-AA stimulated tyrosine autophosphorylation only to less than 25% compared with the other two isoforms. In all instances tyrosine phosphorylation
C AA BB AB B
180116-
840
200
400
600
growth factor [ rig/ml ] FIG. 3. Stimulation of tyrosine phosphorylation by various growth factors. Quiescent cells (-1.75 cm*) were stimulated for 10 min with the indicated amounts of growth factor. After lysis, 30 ~1 was subjected to SDS-polyacrylamide gel electrophoresis. Proteins were transferred onto nitrocellulose and phosphotyrosine was detected by immunodectection with monoclonal lG2 anti-phosphotyrosine antibody. Detection was done by chemiluminescence using Hyperfilm ECL (Amersham). Areas were quantified by laser densitometry. Curve fitting was performed assuming single-site occupation by the ligands. (A) Stimulation of tyrosine phosphorylation by PDGF isoforms (100 rig/ml); C, control. (B) Concentration dependence of a receptor autophosphorylation. n , rPDGF-BB; 0, rPDGF-AB; A, rPDGF-AA.
196
SIMM
c
AA A0 00
C AA A0 00
PI
C AA A0 00
PA
PIP
FIG. 4. Stimulation of lipid synthesis by various PDGF isoforms. To confluency-starved cells (7 cm2), [32P]phosphate (15 &i/petri disc) and growth factors were added simultaneously. Cells were harvested 30 min later and phospholipids were extracted and analyzed as detailed under Methods. For stimulation, 15 rig/ml PDGF-BB or PDGF-AB and 20 ng PDGF-AA were used. Similar results were obtained when the different conditions described under Methods were applied. Measurements were done in triplicate. C, control.
was maximum after 10 min and was below detection after 1 h (data not shown). IGF-I did not affect the autophosphorylation stimulated by the three isoforms of PDGF (data not shown). Stimulation of [32P]Phosphate Phospholipids
Incorporation
into
Measurements were done using two different approaches. First, [32P] radioactivity was added simultaneously with growth factor to the cells; second, cells were prelabeled for 1 h with [32P]phosphate, to exclude disturbance of the measurement caused by a stimulation of [32P]phosphate uptake. (Separate measurements showed that [32P]phosphate uptake is only slightly stimulated (1.2-fold) by the three PDGF isoforms.) Cells were harvested 15 or 30 min after the addition of growth factors. In all four instances the same pattern was observed, which was most pronounced under the conditions described in the legend to Fig. 4 (simultaneous addition and harvest after 30 min). rPDGF-BB was the most potent agent and substantially stimulated the formation of [32P]phosphatidylinositol and [32P]phosphatidic acid and slightly stimulated that of [32P]phosphatidylinositol monophosphate. Remarkably, rPDGF-AB was far less effective and the effects resembled those of rPDGF-AA. Stimulation
of Total Protein
Phosphorylation
Figure 5 shows the effect of the addition of rPDGF isoforms on the protein phosphorylation. Remarkably, all three isoforms substantially stimulated protein phosphorylation after a lo-min incubation. This was
ET AL.
quite unexpected since rPDGF-AA was much less effective in all preceding tests. Although the overall extent of total phosphorylation seems to be quite similar, there are distinct differences between the three isoforms. Proteins 1,2,3,9,10,11,13,16, and 20 were consistantly phosphorylated after the addition of all three isoforms. The following proteins were phosphorylated mainly in response to PDGF-BB: 6 and 14. Phosphorylation stimulated by PDGF-AA was very low or below detection in protein 4, 5, 14, and 15. Remarkably, protein 17 was phosphorylated in response to PDGF-AA and -AB and protein 12 selectively after treatment with PDGF-AA. Interestingly enough, rPDGF-AB effects seem to be more closely related to those of rPDGF-AA than to those of rPDGF-BB. DISCUSSION
There is a considerable debate whether the two types of receptors (a or p) which probably form three different complexes with the three isoforms of PDGF transduce different signals into the cell upon ligand binding. First reports [13,18-291 showed clear differences among the isoforms with regard to mitogenic potency, chemotaxis, and membrane ruffling in human fibroblasts. These results were challenged by Kazlauskas et al. [31], suggesting that the number of surface receptors plays a decisive role. This model was supported by Aarronson’s group [33], demonstrating that after transfection of the two individual receptors into naive cells qualitatively similar responses on [Ca2’] mobilization, PI turnover, and PI-3 kinase association were detected. We have previously reported different effects of the three isoforms on rat vascular smooth muscle cells. Although the lack of some events (i.e., [Ca2’] mobilization or IP, formation) may be attributed to the rather low number of titrable LY receptors there was a clear difference in the time-dependent formation of diacylglycerol. Remarkably rPDGF-AA stimulated the second phase as strongly as rPDGF-BB did [19]. To tackle the question whether the different effects observed with the three isoforms are due to unequal receptor populations we used AKR-2B mouse fibroblasts, which express 30,000 binding sites for rPDGFAA, 45,000 sites for rPDGF-BB, and 60,000 sites for rPDGF-AB on the cell surface [37]. This system thus appears to be suited to detecting subtle quantitative differences. Previous reports [37,56] showed that the number of (Y receptors did not change significantly during cultivation. rPDGF-AB and -BB caused a more than 20-fold stimulation of [3H]thymidine incorporation, indicative of a strong mitogenic potency (Fig. 1). E5,, were well in the expected range (110 rig/ml) and correlate with the reported KD values for binding [37]. Surprisingly, rPDGFAA had almost no effect, although, according to its KD
EFFECTS
OF PDGF
45 36 29 24 -
45 36 29 24
ISOFORMS
197
-
-
-
PH 4
PH 4
45 36 29 24
45 36 29 24
-
-
20 -
14 -
PH 4
PH 4
PH 6
FIG. 5. Stimulation of total cellular phosphorylation by the three isoforms of PDGF. Cells grown on a surface of 1.75 cm2 were made quiescent by incubation for 2 days in MCDB medium containing reduced phosphate content; 50 &i [32P]phosphate was added for 3 h. Cells were then stimulated for 10 min with 25 rig/ml PDGF-AA, 15 rig/ml PDGF-AB, and 15 rig/ml PDGF-BB. After lysis in 40 ~1 buffer an aliquot of 30 ~1 was taken for two-dimensional separation of proteins. pH range and M, range are indicated. Autoradiography was done for 16 h. Measurements were done in triplicate and repeated twice with similar results. (A) Control, (B) +PDGF-BB, (C) +PDGF-AB, (D) +PDGF-AA.
value of 0.2 nM (=6 rig/ml), a sufficient number of receptor sites must have been occupied under the applied experimental conditions. Insulin-like growth factor I effectively acted apparently synergistically with rPDGFAA, eliciting about 50-70% of the effect that rPDGF-AB evoked. IGF-I together with rPDGF-AB or -BB did not stimulate the [3H]thymidine incorporation to a higher extent than rPDGF-AB or -BB alone, indicating that rPDGF-AB or -BB caused maximum stimulation alone. Rather, the E,, values were approximately 2- to 3-fold reduced. The cell cycle analysis yielded the important results that rPDGF-BB or -AB stimulated nearly all of the cells to divide. This explains the observation that IGF-I did not enhance the maximum [3H]thymidine incorporation elicited by both PDGF isoforms. Possibly IGF-I acts on similar pathways, thus reducing the required amount of PDGF for full activity. The effects of
IGF-I and rPDGF-AA also were more than additive. The situation observed with rPDGF-AA is reminiscent of the model of “competence and progression” [43-461. Indeed IGF-I may be added up to 6 h after rPDGF-AA (data not shown). Thus, rPDGF-AA behaves like a competence factor and IGF-I is needed for progression. Clearly no such progression factor is required when cells are stimulated with rPDGF-AB or -BB. The almost complete lack of mitogenic activity of rPDGF-AA raised the interesting question whether the receptor was still able to stimulate some early events. We have studied, therefore, the effects of rPDGF-AA on several selected targets. Autophosphorylation of receptors on tyrosine residues is one of the earliest responses and is considered to play an important role in signal transduction [17]. All three rPDGF isoforms rapidly (tllZ - 2-5 min; data not
198
SIMM
shown) stimulate tyrosine phosphorylation of an - 180kDa protein that has been identified as a respective (Yor P-type receptor (Fig. 2). Remarkably, the &, values are close to those observed for [3H]thymidine incorporation and binding [37]. However, unlike in the mitogenic assay rPDGF-AA caused a reduced but distinct effect. The extent of tyrosine phosphorylation at saturation evoked by rPDGF-AA was 25% of that evoked by rPDGF-AB or -BB. Unexpectedly, tyrosine phosphorylation stimulated by rPDGF-AB was almost as high as that stimulated by rPDGF-BB. Assuming that rPDGFAB binds one (Y and one p receptor (c@ receptor), a reduced phosphorylation would have been anticipated. The tyrosine phosphorylation of the PP-receptor complex is well documented [17, 541, and there are some data for the aa-receptor complex [47,48] but so far the phosphorylation in an c&type receptor is unknown and the interpretation of the above results must await the identification of phosphorylation sites in this receptor complex. Following tyrosine autophosphorylation there is an increase in protein kinase activity, resulting in the phosphorylation of various cellular proteins on serine, threonine, or tyrosine residues. When the effects of the three isoforms are compared (Fig. 5) it is evident that the overall phosphorylation was quite similar and many common proteins were phosphorylated, but there were also distinct differences. Surprisingly, the patterns for rPDGF-AA and rPDGF-AB are closer related than that for rPDGF-BB, though in many other instances rPDGF-AB and -BB behave quite similarly. This unexpected result was also found when the effects of the three isoforms on the formation of 32P-labeled phospholipids were studied (Fig. 4). As expected [49], rPDGFBB effectively raised the levels of [32P]phosphatidylinositol and [32P]phosphatidic acid and to some extent [32P]phosphatidylinositol phosphate. rPDGF-AA and -AB caused only a moderate increase in the level of these lipids. In summary, the obtained results show that (Y receptors are less phosphorylated on tyrosine residues than fl receptors, but the extent of tyrosine autophosphorylation did not correlate with many other effects, i.e., the lack of mitogenic activity and the greater extent of total cellular phosphorylation. There seem to be distinct pathways which might be activated to various extents by the different types of receptors interacting at different steps in the cascade of signal transduction [50-531. The recent findings that tyrosine phosphorylations create recognition sites for SH, or SH, domains explain part of these observations. These domains apparently are specific for phosphotyrosine and its adjacent amino acid residues [17, 551. Two phosphotyrosines (PY 751 and PY 857) in the p receptor have been analyzed in detail. Interestingly, PY 751 lies in the interkinase domain of the p receptor and is responsible for the binding
ET AL.
to PI-3 kinase [54]. A corresponding homologous tyrosine at position 742 in the (Yreceptor is also responsible for the binding of PI-3 kinase [47]. The adjacent amino acids differ considerably, raising the possibility that they interact with different affinities with phosphotyrosine binding proteins (SH, or SH, domains). It is furthermore known that SH, domain proteins, e.g., PI, kinase, are expressed in various amounts in different cell types [54]. One might envisage a situation in which the binding is reduced and the proteins are less abundant. These quantitative changes will then result in qualitative effects, i.e., in the lack of mitogenic activity. It is tempting to speculate that some of the early events during the competence phase might be part of a survival program. It is interesting, in this regard, that a maintenance function has been postulated for PDGFAA based on the observation that PDGF-AA is highly expressed in aortic tissue without inducing proliferation. Our results corroborate well with these reports. Thus, AKR-2B cells might serve as a model to further discriminate between events leadingto competence, progression, or maintenance. We thank Dr. M. Poot for help during the cell cycle analysis and Mrs. R. Mayer for preparing the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft SFB 176 (TP AlO).
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Heldin, C.-H., and Westermark, B. (1984) Cell 37, 9-20. Deuel, T. F., Tong, B. D., and Huang, J. S. (1985) Curr. Top. Cell. Regul. 26, 51-64. Ross, R., Raines, E. W., and Bowen-Pope, D. F. (1986) Cell 46, 155-169. Doolittle, R. F., Hunkapiller, M. W., Hood, L. E., Devare, S. G., Robbins, K. C., Aaronson, S. A., and Antoniades, H. N. (1983) Science 22 1, 275-277. Waterfield, M. D., Scrace, G. T., Whittle, N., Stroobant, P., Johnsson, A., Wasteson, A., Westermark, B., Heldin, C.-H., Huang, J. S., and Deuel, T. F. (1983) Nature 304, 35-39. Weich, H. A., Sebald, W., Schairer, H.-U., and Hoppe, J. (1986) FEBS Lett. 198, 344-348. Betsholtz, C., Johnsson, A., Heldin, C.-H., Westermark, B., Lind, P., Urdea, M. S., Eddy, R., Shows, T. B., Philpott, K., Mellor, A. L., Knott, T. J., and Scott, J. (1986) Nature 320, 695-699. Hoppe, J., Schumacher, L., Eichner, W., and Weich, H. A. (1987) FEBS Lett. 223, 243-246. Jaumann, M., Hoppe, V., Tatje, D., Eichner, W., and Hoppe, J. (1991) Biochemistry 30,3303-3309. Yarden, Y., Escobedo, J. A., Kuang, W. J., Yang-Feng, T. L., Daniel,T. O.,Tremble, P. M., Chen,E.Y.,Ando, M.E., Harkins, R. N., Francke, U., Fried, V. A., Ullrich, A., and Williams, L. T. (1986) Nature 323, 226-232. Matsui, T., Heidaran, M., Miki, T., Popescu, N., La Rochelle, W., Kraus, M., Pierce, J., and Aaronson, S. (1989) Science 243, 800-804. Claesson-Welsh, L., Eriksson, A., Westermark, B., and Heldin, C. H. (1989) Proc. Natl. Acad. Sci. USA 86,4917-4921.
EFFECTS
OF PDGF
13.
Heldin, C.-H., Backstrom, G., &tman, A., Hammacher, Ronnstrand, L., Rubin, K., Nister, M., and Westermark, (1988) EMBO J. 7,1387-1393.
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M. G., and Turley,
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W. J. (1983) Mol. Cell. Endocrinol.
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H., and Hoppe, J.
CELL
RESEARCH
201,
192-199 (19%)
PDGF-AA Effectively Stimulates Early Events but Has No Mitogenic Activity in AKR-2B Mouse Fibroblasts ANDREAS SIMM, VIVIANE HOPPE, DIETER TATJE, ANJA SCHENZINGER, AND J~JRGEN HOPPE’ Theodor-Boveri-Znstitut
jiir Biowissenschaften
(Biozentrum)
der Uniuersitiit,
The response of AKR-2B mouse fibroblasts, which express approximately equal numbers of platelet-derived growth factor (PDGF)-a! and $3 receptors on their surface (V. Hoppe et al. Eur. J. Biochem. 187, 207214, 1990) to all three isoforms of PDGF, was studied. All isoforms stimulated early events, i.e., receptor autophosphorylation on tyrosine, total cellular phosphorylation, increase in 32P-labeled phospholipid content, but there was no correlation between the extents measured for the different effects. Although rPDGF-AA effectively stimulated these early events, it was unable to induce [3H]thymidine incorporation and cell growth whereas rPDGF-BB and -AB stimulated the division of more than 90% of the cells. This activity was restored by addition of insulin-like growth factor I (IGF-I), which itself exhibited only a low mitogenic activity. rPDGF-AB or -BB did not require the presence of IGF-I to fully stimulate cells for [3H]thymidine incorporation and cell division. Apparently, rPDGF-AA induced only a “competence” state of the cells whereas rPDGF-AB or -BB was also able to initiate “progression.” It is speculated that some early events occuring during the competence phase might be part of a “maintenance” program elicited by growth factors. o 1st~. Academic PWS, IN.
INTRODUCTION Platelet-derived growth factor (PDGF)’ is a major mitogen in serum which promotes the proliferation of fibroblasts, glia cells, and smooth muscle cells in vitro [l-3]. The two homologous chains, termed A and B, which have been identified and sequenced, can combine to three different dimeric isoforms: AA, AB, and BB [4-81. The dimeric structure is stabilized by disulfide bridges, and after destruction of these bridges biological activity is lost [l-3, 91. This fact, that only dimers are biologically active, along with the discovery of two dif1 To whom correspondence and reprint requests should be addressed. Fax: (931) 888-4113. ’ Abbreviations used: FCS, fetal calf serum; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor; IGF-I, insulinlike growth factor I; SDS, sodium dodecyl sulfate. 0014-4827/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved
Physiologische
Chemie ZZ, Am Hublnnd,
8700 Wiirzburg,
Germany
ferent PDGF receptors (01and fi), led to the hypothesis that one dimeric PDGF may bind two receptor molecules (receptor subunit model) [lo-161. The model predicts that PDGF-AA binds only a-type PDGF receptors (receptors specific for PDGF), PDGF-AB binds one o(type and one P-type receptor, and PDGF-BB binds mainly P-type PDGF receptors. The two receptors share a common structure including an extracellular domain that is composed of five immunoglobulin-like repeats and a cytoplasmic portion that includes split tyrosine kinase sequences [IO-121. Despite extensive structural homology, the two receptors display considerable heterogeneity in their extracellular and cytoplasmic noncatalytic domains such as the kinase insertion stretches and the carboxy termini. These cytoplasmic regions participate in the interaction between the activated receptor and cytoplasmic proteins (for review see [17]) and thus may confer distinct substrate specifications to the homologous tyrosine kinases. A current open question related to the mechanism of signal transduction is the extent of functional overlap of mitogenic signals arising from the cell surface via homologous receptors. We have recently examined the effects of the three PDGF isoforms on the [Cazcli mobilization, inositol trisphosphate release, diacylglycerol production, and pH changes in cultured rat aortic smooth muscle cells [ 18-201. The marked differences found between the isoforms lead, together with previous results [13, 21-301, to the assumption that the a-type and the P-type receptors transmit different signals into the cell. On the other hand, other reports claim that both receptors may express the same spectrum of biological functions and differences must be related to unequal receptor populations [31-331. AKR-2B mouse fibroblasts that express similar numbers of CYor p receptors on their surface were chosen as a model system. Here we show that rPDGF-AA alone is unable to act as a mitogen although it stimulates effectively several early events, e.g., tyrosine autophosphorylation, total protein phosphorylation, and [32P]phospholipid synthesis. These results suggest that some early events may play a role in functions like “maintenance” [ 34-361. 192
EFFECTS
MATERIALS
AND
OF PDGF
METHODS
Materials PDGF isoforms were prepared as described [37]. IGF-I was from Bachem. Rabbit anti-mouse IgG, lipid standards, protein molecular weight standards, and p-coumaric acid were purchased from Sigma. [32P]Orthophosphate, 370 MBq/ml (carrier free), [sH]thymidine, 1.55 TBq/mmol, and Hyperfilm ECL were obtained from Amersham. Monoclonal antibody lG2 against phosphotyrosine was from Boehringer. Benzonase and thin-layer plates were purchased from Merck. Cell culture reagents were from GIBCO. Luminol was obtained from Fluka. Reinforced nitrocellulose was from Schleicher & Schiill and goat anti-rabbit peroxidase antibody from Dianova. Immobiline dry plates (pH 4 to pH 7) were from Pharmacia. Methods Growth-promoting actiuity. This was determined as described [38], using AKR-2B cells without insulin. (Cells were cultivated for less than 3 months to minimized fluctuations. During that time the responses to PDGF isoforms did not change.) Cell cycle analysis. After reaching confluence, cells were starved in MCDB 402 medium. After starvation for 48 h, fresh MCDB 402 medium, 60 PM bromodesoxyuridine, 60 PM desoxycytidine, and growth factors were added. Cells were harvested 44-46 h later by trypsin treatment and stored at -20°C in PBS containing 10% dimethyl sulfoxide and 10% FCS. Staining with ethidium bromide and Hoechst 33258 dye and analysis were performed as described [39] using an ICP22 flow cytometer (Phywe). The computer programs Multi-2D or M-Cycle (Phoenics) were used for quantification. Analytical methods. SDS-polyacrylamide gel electrophoresis was performed essentially as described [37]. Protein content was determined by the method of Redinbaugh and Turley [40] after calibration of the three PDGF isoforms by amino acid analysis. Autoradiograms were quantified by laser densitometry (Ultroscan, LKB). Data were analyzed by the computer program GraFit (Erithacus Software, London). Analysis of [32P]phospholipids [41]. AKR-PB cells were grown on g-cm’ petri discs for 5 days in McCoy medium containing 10% Hyclone. After reaching confluence they were synchronized by incubation in 2.5 ml MCDB 402 medium containing 50 PM orthophosphate for 2 days. Fifteen microcuries (32P]phosphate was added. Growth factors were added either immediately after the addition of the radioactivity or 60 min later. The reaction was terminated 15 or 30 min, respectively, after the addition of growth factors. Cells were washed three times with ice-cold PBS and then treated with 500 ~1 cold 30% trichloroacetic acid. Cells were scraped with a rubber policeman and transfered into a microtube. After washing twice with cold 30% trichloroacetic acid, 100 pl of 2.4 N hydrochloric acid and 300 pl of chloroform/methanol, l/2 (v/v), were added and the mixture was vigorously shaken. For phase separation 125 ~1 of chloroform and 125 ~1 of 0.1 N hydrochloric acid were added. After brief centrifugation, the upper phase was removed and discarded. The lower phase was washed twice with methanol/water/chloroform, 48/47/3 (v/v/v). Finally 100 ~1 of the lower phase was withdrawn, supplemented with 20 pg phospholipid standards, and dried under a nitrogen stream. Thin-layer chromatography of the extracted phospholipids. The dried samples were dissolved in 10 pl chloroform/methanol, 2/l (v/v). Five microliters was used for thin-layer chromatography. Analytical plates with a concentration zone were used. Plates were developed with chloroform/methanol/2 M aqueous ammonia, 4314818 (v/v/v), for 20-25 min. Standards were visualized by I, vapor. Radioactivity was detected by autoradiography using a Kodak XAR film. Stained areas were quantified by laser densitometry. Analysis of phosphoproteins. Cells were grown in 24-well plates (Greiner; 1.75~cm* surface/well) and arrested as described above for
193
ISOFORMS
the analysis of phospholipids. After a 2-h incubation with 50 &i [32P]phosphate in 300 ~1 MCDB medium containing 50 pM orthophosphate, growth factors were added for 10 min. The medium was removed and cells were lysed in 40 ~1 buffer containing 7 M urea, 2% Nonidet P-40, 2% 2-mercaptoethanol, 1 mM MgCl,, and 10 pi/ml benzonase solution for 5 min under vigorous shaking. Immobiline dry plates (pH 4-7) were cut into 0.5-cm strips and rehydrated in 8 M urea, 0.5% Nonidet P-40, 2 mM Tris base. Thirty microliters of the cell lysate was applied close to the cathode. Focusing was done at 2000 V for 24 h. The stripes were then soaked for 15 min in an equilibrium buffer (50 mMTris-Cl, pH 6.8,2% SDS, 6 Murea, 30% glycerin, some crystals of bromphenol blue) and mounted on top of the gel (16 X 12 cm, 0.7 mm thickness). The acrylamid content was 13.5% acrylamide, 0.36% N,N’methylene-bis-acrylamide. Gels were stained with Coomassie brilliant blue R to verify that equal amounts of proteins were loaded and that separation of proteins was identical. Autoradiography was done for 24 h using Kodak XAR film and intensifier screens. Analysis of tyrosine-phosphorylated proteins. Cells were grown and arrested as described above. One millimolar sodium orthovanadate was added for 15 min before the addition of growth factors. Stimulation was performed for 10 min at various concentrations. The medium was removed and for lysis 50 ~1 buffer containing 50 mM TrisCl, pH 6.7, 2% SDS, 2% mercaptoethanol, 1 mM sodium orthovanadate was added. After 5 min of vigorous shaking, 10 ~1 benzonase (6%) was added and shaking was continued for another 5 min. The entire solution was transferred into a microtest tube and 6 ~1 bromphenol blue in 50% glycerol was added. Thirty microliters was taken for SDS-PAGE (7.5% acrylamide-0.075% N,N’methylene-bisacrylamide) using a minigel system (Bio-Rad). Protein was transferred onto reinforced nitrocellulose by semidry blotting using 50 mM Caps, pH 10.0, 1 mM 3-mercaptopropionic acid, 0.1% SDS, and 10% methanol as blotting buffer for 2 h at 6 V. The nitrocellulose sheet was washed three times for 5 min with 50 mM Tris-Cl, pH 7.5, 150 mM NaCl (TS buffer). Saturation was performed overnight with 2% bovine serum albumin with 0.03% NaN, in TS. The sheets were incubated for 2 h with anti-phosphotyrosine antibody lG2 at 5 pg/ml (Boehringer, Mannheim, Germany) in TS containing 2% BSA, 0.2% Nonidet P-40, and 0.03% NaN, (TSBN). After washing three times with TS containing 0.5% BSA, 0.2% Nonidet P-40, 0.03% NaN, (washing buffer), the sheets were incubated for 1 h with rabbit antimouse IgG (l/500 dilution, Sigma) in TSBN. After washing three times with washing buffer, goat anti-rabbit IgG horse radish peroxydase-labeled antibody (Dianova) was added at a dilution of l/5000 for 1 h. The antibody was removed by washing three times and the sheets were soaked in 3 ml luminescence buffer containing 100 mM Tris-Cl, pH 8.5, 2.5 mM luminol (Fluka), 400 PM p-coumaric acid (Sigma), to which 3 ml hydrogen peroxide buffer was added (5.4 mM H,O,, 100 mM Tris-HCl, pH 8.5). After 1 min the sheets were quickly dried with filter paper and wrapped in a transparent foil. Light emission was detected with an ECL film (Amersham). Stock solutions of luminol (250 mM) and p-coumaric acid (90 mM) in DMSO were used.
RESULTS
Mitogenic
Activity
of rPDGF Isoforms
In the previously used protocols for [3H]thymidine incorporation into the DNA of AKR-2B mouse fibroblasts 2 @g/ml insulin was included into the basal protein-free MCDB 402 medium. Under these conditions rPDGF-BB and -AB stimulated [3H]thymidine incorporation to a similar extent; rPDGF-AA ‘was less active, reaching about 60% of the maximum level. E,, values for rPDGF-AB and -BB were in the range of 2 rig/ml, and that for rPDGF-AA was 10 rig/ml [37, 421. When
194
SIMM
ET AL.
insulin was omitted, rPDGF-AB and -BB stimulated [3H]thymidine incorporation still to the same extent but the E,, value was increased two- to three-fold (Fig. 1A). Remarkably rPDGF-AA alone exhibited only a residual activity (Fig. 1A). It was almost inactive at 10 rig/ml. Since insulin added at high concentrations might act via insulin-like growth factor I receptors it was replaced by insulin-like growth factor I (IGF-I). The low E,, value of 7 rig/ml indicated that IGF-I indeed acts via its receptors (insert, Fig. 1A). When IGF-I was added at 30 ng/ ml to various concentrations of rPDGF isoforms the formerly observed pattern in the presence of insulin was reconstituted; i.e., E,, values for rPDGF-AB and -BB were around 2-4 rig/ml and the maximum stimulation by rPDGF-AA reached 50% of that value observed with the two other isoforms. PDGF isoforms stimulated maximum DNA synthesis 21 h after the addition, whereas the maximum for IGF-I was at 17 h (Fig. 1B). Thus, the value obtained under standard assay conditions (analysis after 23 h [37, 381) underestimated the potency of IGF-I. Interestingly, together with PDGF-AA, IGF-I determined the rate of DNA synthesis as the maximum was found at 17 h. Both factors act synergistically under these conditions.
6000 4000 2000 0 0.1 m
6000 6000 4ooo 2ooo
Cell Cycle Analysis
0 1
0.1
10
100
growth factor [ ng / ml ]
20000
0
53-r
5
10
15
time of stimulation
20
3
25
30
[h ]
FIG. 1. (A) Stimulation of [3H]thymidine incorporation into DNA by various growth factors. Quiescent AKR-2B fibroblasts were stimulated by the indicated amounts of growth factors for 23 h; 0.1 pCi [3H]thymidine/well was added and after 45 min cells were harvested. W, rPDGF-BB; 0, rPDGF-BB + 30 rig/ml IGF-I; 0, rPDGFAB; 0, rPDGF-AB + 30 rig/ml IGF-I; A, rPDGF-AA; A, rPDGF-AA + 30 rig/ml IGF-I; (Insert) V, IGF-I + 25 rig/ml rPDGF-AA. Measurements were performed in triplicate. (B) Time-dependent [3H]thymidine incorporation into AKR-2B cells. At zero time growth fac-
[3H]Thymidine incorporation neither is a measurement for the percentage of cells that synthesize DNA nor does it indicate whether stimulated cells will divide. We have therefore performed a detailed cell cycle analysis using the double-staining technique [39]. Approximately 2 days after stimulation with either PDGF-BB or -AB (Fig. 2A) only 10% of the cells remained in the starting cycle (cycle 0). From those cells roughly 50% already had entered the first S-phase. The majority of the cells were recovered in cycle 1; i.e., they have completed one division. Some cells had already entered the second cycle. After treatment with PDGF-AA most of the cells remained in cycle 0. Remarkably, none of these cells had entered the S-phase. A few cells were found in the first cycle but none in the second cycle. These results thus show that PDGF-BB or -AB stimulated nearly all of the cells (>95%) to divide, whereas PDGFAA was almost inactive. Figure 2B demonstrates that PDGF-AA and IGF-I act synergistically also on cell growth. PDGF-AA stimulated maximally 18% of cell division and IGF-I alone 22%. Simultaneous addition resulted in the division of 64% of the cells.
tors were added to starved cultures of AKR-2B cells. (PDGF isoform, 33 rig/ml; IGF-I, 30 rig/ml.) [sH]Thymidine was added for 60 min at the indicated time. Measurements were performed in duplicate and repeated once with identical results. W, rPDGF-BB; 0, rPDGF-AB; A, rPDGF-AA + IGF-I; A, rPDGF-AA, V, IGF-I.
EFFECTS
.A.
cycle
t
cycle 1
0
cycle 2
1w
90
.’ 90
60
.’ 60
70
8
60
B
50
8
195
ISOFORMS
I loo
_
OF PDGF
50
30.00
40
40
20.00
30
30
10.00
20
20
0.00
10
10
0
0 AA
AB
BB
AA
+ IGF-I
AB
BB
+ IGF-I
.70
AA + IGF-I
PDGF-AA
-_U.“”
Wmll
FIG. 2. (A) Distribution of cells in various cell cycles. To starved cells bromodesoxyuridine and the three growth factors were added (PDGF isoforms, 100 rig/ml; IGF-I, 30 rig/ml). Cells were harvested after 41-46 h, stained, and analyzed by flow cytometry. Measurements were done in triplicate. (B) Synergistic effects of PDGF-AA and IGF-I. Cells were stimulated with the indicated amounts of PDGF-AA and IGF. Analysis was done as above. Standard deviation was less than 10%. The Z-axis indicates cell division as a percentage.
Stimulation
of Tyrosine Phosphorylation
All three isoforms stimulated tyrosine phosphorylation of a protein with an M, of 180,000 Da, which is generally believed to be the respective PDGF receptor (a- or P-type (Fig. 3A). The Ebo values (AB, 6 rig/ml; BB,
A
M.W.
c kd 1
15 rig/ml; AA, 25 rig/ml) correlate reasonably well with those obtained in the mitogenic assay without IGF-I (Fig. 3B). Again rPDGF-AB and -BB reached the same plateau. rPDGF-AA stimulated tyrosine autophosphorylation only to less than 25% compared with the other two isoforms. In all instances tyrosine phosphorylation
C AA BB AB B
180116-
840
200
400
600
growth factor [ rig/ml ] FIG. 3. Stimulation of tyrosine phosphorylation by various growth factors. Quiescent cells (-1.75 cm*) were stimulated for 10 min with the indicated amounts of growth factor. After lysis, 30 ~1 was subjected to SDS-polyacrylamide gel electrophoresis. Proteins were transferred onto nitrocellulose and phosphotyrosine was detected by immunodectection with monoclonal lG2 anti-phosphotyrosine antibody. Detection was done by chemiluminescence using Hyperfilm ECL (Amersham). Areas were quantified by laser densitometry. Curve fitting was performed assuming single-site occupation by the ligands. (A) Stimulation of tyrosine phosphorylation by PDGF isoforms (100 rig/ml); C, control. (B) Concentration dependence of a receptor autophosphorylation. n , rPDGF-BB; 0, rPDGF-AB; A, rPDGF-AA.
196
SIMM
c
AA A0 00
C AA A0 00
PI
C AA A0 00
PA
PIP
FIG. 4. Stimulation of lipid synthesis by various PDGF isoforms. To confluency-starved cells (7 cm2), [32P]phosphate (15 &i/petri disc) and growth factors were added simultaneously. Cells were harvested 30 min later and phospholipids were extracted and analyzed as detailed under Methods. For stimulation, 15 rig/ml PDGF-BB or PDGF-AB and 20 ng PDGF-AA were used. Similar results were obtained when the different conditions described under Methods were applied. Measurements were done in triplicate. C, control.
was maximum after 10 min and was below detection after 1 h (data not shown). IGF-I did not affect the autophosphorylation stimulated by the three isoforms of PDGF (data not shown). Stimulation of [32P]Phosphate Phospholipids
Incorporation
into
Measurements were done using two different approaches. First, [32P] radioactivity was added simultaneously with growth factor to the cells; second, cells were prelabeled for 1 h with [32P]phosphate, to exclude disturbance of the measurement caused by a stimulation of [32P]phosphate uptake. (Separate measurements showed that [32P]phosphate uptake is only slightly stimulated (1.2-fold) by the three PDGF isoforms.) Cells were harvested 15 or 30 min after the addition of growth factors. In all four instances the same pattern was observed, which was most pronounced under the conditions described in the legend to Fig. 4 (simultaneous addition and harvest after 30 min). rPDGF-BB was the most potent agent and substantially stimulated the formation of [32P]phosphatidylinositol and [32P]phosphatidic acid and slightly stimulated that of [32P]phosphatidylinositol monophosphate. Remarkably, rPDGF-AB was far less effective and the effects resembled those of rPDGF-AA. Stimulation
of Total Protein
Phosphorylation
Figure 5 shows the effect of the addition of rPDGF isoforms on the protein phosphorylation. Remarkably, all three isoforms substantially stimulated protein phosphorylation after a lo-min incubation. This was
ET AL.
quite unexpected since rPDGF-AA was much less effective in all preceding tests. Although the overall extent of total phosphorylation seems to be quite similar, there are distinct differences between the three isoforms. Proteins 1,2,3,9,10,11,13,16, and 20 were consistantly phosphorylated after the addition of all three isoforms. The following proteins were phosphorylated mainly in response to PDGF-BB: 6 and 14. Phosphorylation stimulated by PDGF-AA was very low or below detection in protein 4, 5, 14, and 15. Remarkably, protein 17 was phosphorylated in response to PDGF-AA and -AB and protein 12 selectively after treatment with PDGF-AA. Interestingly enough, rPDGF-AB effects seem to be more closely related to those of rPDGF-AA than to those of rPDGF-BB. DISCUSSION
There is a considerable debate whether the two types of receptors (a or p) which probably form three different complexes with the three isoforms of PDGF transduce different signals into the cell upon ligand binding. First reports [13,18-291 showed clear differences among the isoforms with regard to mitogenic potency, chemotaxis, and membrane ruffling in human fibroblasts. These results were challenged by Kazlauskas et al. [31], suggesting that the number of surface receptors plays a decisive role. This model was supported by Aarronson’s group [33], demonstrating that after transfection of the two individual receptors into naive cells qualitatively similar responses on [Ca2’] mobilization, PI turnover, and PI-3 kinase association were detected. We have previously reported different effects of the three isoforms on rat vascular smooth muscle cells. Although the lack of some events (i.e., [Ca2’] mobilization or IP, formation) may be attributed to the rather low number of titrable LY receptors there was a clear difference in the time-dependent formation of diacylglycerol. Remarkably rPDGF-AA stimulated the second phase as strongly as rPDGF-BB did [19]. To tackle the question whether the different effects observed with the three isoforms are due to unequal receptor populations we used AKR-2B mouse fibroblasts, which express 30,000 binding sites for rPDGFAA, 45,000 sites for rPDGF-BB, and 60,000 sites for rPDGF-AB on the cell surface [37]. This system thus appears to be suited to detecting subtle quantitative differences. Previous reports [37,56] showed that the number of (Y receptors did not change significantly during cultivation. rPDGF-AB and -BB caused a more than 20-fold stimulation of [3H]thymidine incorporation, indicative of a strong mitogenic potency (Fig. 1). E5,, were well in the expected range (110 rig/ml) and correlate with the reported KD values for binding [37]. Surprisingly, rPDGFAA had almost no effect, although, according to its KD
EFFECTS
OF PDGF
45 36 29 24 -
45 36 29 24
ISOFORMS
197
-
-
-
PH 4
PH 4
45 36 29 24
45 36 29 24
-
-
20 -
14 -
PH 4
PH 4
PH 6
FIG. 5. Stimulation of total cellular phosphorylation by the three isoforms of PDGF. Cells grown on a surface of 1.75 cm2 were made quiescent by incubation for 2 days in MCDB medium containing reduced phosphate content; 50 &i [32P]phosphate was added for 3 h. Cells were then stimulated for 10 min with 25 rig/ml PDGF-AA, 15 rig/ml PDGF-AB, and 15 rig/ml PDGF-BB. After lysis in 40 ~1 buffer an aliquot of 30 ~1 was taken for two-dimensional separation of proteins. pH range and M, range are indicated. Autoradiography was done for 16 h. Measurements were done in triplicate and repeated twice with similar results. (A) Control, (B) +PDGF-BB, (C) +PDGF-AB, (D) +PDGF-AA.
value of 0.2 nM (=6 rig/ml), a sufficient number of receptor sites must have been occupied under the applied experimental conditions. Insulin-like growth factor I effectively acted apparently synergistically with rPDGFAA, eliciting about 50-70% of the effect that rPDGF-AB evoked. IGF-I together with rPDGF-AB or -BB did not stimulate the [3H]thymidine incorporation to a higher extent than rPDGF-AB or -BB alone, indicating that rPDGF-AB or -BB caused maximum stimulation alone. Rather, the E,, values were approximately 2- to 3-fold reduced. The cell cycle analysis yielded the important results that rPDGF-BB or -AB stimulated nearly all of the cells to divide. This explains the observation that IGF-I did not enhance the maximum [3H]thymidine incorporation elicited by both PDGF isoforms. Possibly IGF-I acts on similar pathways, thus reducing the required amount of PDGF for full activity. The effects of
IGF-I and rPDGF-AA also were more than additive. The situation observed with rPDGF-AA is reminiscent of the model of “competence and progression” [43-461. Indeed IGF-I may be added up to 6 h after rPDGF-AA (data not shown). Thus, rPDGF-AA behaves like a competence factor and IGF-I is needed for progression. Clearly no such progression factor is required when cells are stimulated with rPDGF-AB or -BB. The almost complete lack of mitogenic activity of rPDGF-AA raised the interesting question whether the receptor was still able to stimulate some early events. We have studied, therefore, the effects of rPDGF-AA on several selected targets. Autophosphorylation of receptors on tyrosine residues is one of the earliest responses and is considered to play an important role in signal transduction [17]. All three rPDGF isoforms rapidly (tllZ - 2-5 min; data not
198
SIMM
shown) stimulate tyrosine phosphorylation of an - 180kDa protein that has been identified as a respective (Yor P-type receptor (Fig. 2). Remarkably, the &, values are close to those observed for [3H]thymidine incorporation and binding [37]. However, unlike in the mitogenic assay rPDGF-AA caused a reduced but distinct effect. The extent of tyrosine phosphorylation at saturation evoked by rPDGF-AA was 25% of that evoked by rPDGF-AB or -BB. Unexpectedly, tyrosine phosphorylation stimulated by rPDGF-AB was almost as high as that stimulated by rPDGF-BB. Assuming that rPDGFAB binds one (Y and one p receptor (c@ receptor), a reduced phosphorylation would have been anticipated. The tyrosine phosphorylation of the PP-receptor complex is well documented [17, 541, and there are some data for the aa-receptor complex [47,48] but so far the phosphorylation in an c&type receptor is unknown and the interpretation of the above results must await the identification of phosphorylation sites in this receptor complex. Following tyrosine autophosphorylation there is an increase in protein kinase activity, resulting in the phosphorylation of various cellular proteins on serine, threonine, or tyrosine residues. When the effects of the three isoforms are compared (Fig. 5) it is evident that the overall phosphorylation was quite similar and many common proteins were phosphorylated, but there were also distinct differences. Surprisingly, the patterns for rPDGF-AA and rPDGF-AB are closer related than that for rPDGF-BB, though in many other instances rPDGF-AB and -BB behave quite similarly. This unexpected result was also found when the effects of the three isoforms on the formation of 32P-labeled phospholipids were studied (Fig. 4). As expected [49], rPDGFBB effectively raised the levels of [32P]phosphatidylinositol and [32P]phosphatidic acid and to some extent [32P]phosphatidylinositol phosphate. rPDGF-AA and -AB caused only a moderate increase in the level of these lipids. In summary, the obtained results show that (Y receptors are less phosphorylated on tyrosine residues than fl receptors, but the extent of tyrosine autophosphorylation did not correlate with many other effects, i.e., the lack of mitogenic activity and the greater extent of total cellular phosphorylation. There seem to be distinct pathways which might be activated to various extents by the different types of receptors interacting at different steps in the cascade of signal transduction [50-531. The recent findings that tyrosine phosphorylations create recognition sites for SH, or SH, domains explain part of these observations. These domains apparently are specific for phosphotyrosine and its adjacent amino acid residues [17, 551. Two phosphotyrosines (PY 751 and PY 857) in the p receptor have been analyzed in detail. Interestingly, PY 751 lies in the interkinase domain of the p receptor and is responsible for the binding
ET AL.
to PI-3 kinase [54]. A corresponding homologous tyrosine at position 742 in the (Yreceptor is also responsible for the binding of PI-3 kinase [47]. The adjacent amino acids differ considerably, raising the possibility that they interact with different affinities with phosphotyrosine binding proteins (SH, or SH, domains). It is furthermore known that SH, domain proteins, e.g., PI, kinase, are expressed in various amounts in different cell types [54]. One might envisage a situation in which the binding is reduced and the proteins are less abundant. These quantitative changes will then result in qualitative effects, i.e., in the lack of mitogenic activity. It is tempting to speculate that some of the early events during the competence phase might be part of a survival program. It is interesting, in this regard, that a maintenance function has been postulated for PDGFAA based on the observation that PDGF-AA is highly expressed in aortic tissue without inducing proliferation. Our results corroborate well with these reports. Thus, AKR-2B cells might serve as a model to further discriminate between events leadingto competence, progression, or maintenance. We thank Dr. M. Poot for help during the cell cycle analysis and Mrs. R. Mayer for preparing the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft SFB 176 (TP AlO).
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