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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:2273-2283

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:2273-2283.)
© 1995 American Heart Association, Inc.

Articles

Sodium Butyrate Inhibits Platelet-Derived Growth Factor–Induced Proliferation of Vascular Smooth Muscle Cells

Kasturi Ranganna; Trupti Joshi; Frank M. Yatsu

From the Department of Neurology, University of Texas Health Science Center at Houston (Tex).

Correspondence to Kasturi Ranganna, Department of Neurology, University of Texas Health Science Center at Houston, Houston, TX 77030.


*    Abstract
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 *Abstract
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Abstract Sodium butyrate (SB), a naturally occurring short-chain fatty acid, was investigated for its therapeutic value as an antiproliferative agent for vascular smooth muscle cells (SMCs). At 5-mmol/L concentration, SB had no significant effect on rat SMC proliferation. However, at the same concentration, SB inhibited platelet-derived growth factor (PDGF)-AA–, -AB–, and -BB–induced proliferation of SMCs. Exposure of SMCs to PDGF-BB resulted in activation of receptor intrinsic tyrosine kinase activity and autophosphorylation of ß-PDGF–receptor (ß-PDGFR). The activated ß-PDGFR physically associated and phosphorylated signaling molecules such as ras-GTPase activating protein (GAP) and phospholipase C{gamma} (PLC{gamma}). SB, in the absence of PDGF-BB, caused neither ß-PDGFR tyrosine phosphorylation nor phosphorylation and association of GAP and PLC{gamma} with ß-PDGFR. PDGF-BB–enhanced activation of receptor intrinsic tyrosine kinase activity and autophosphorylation of tyrosine residues of ß-PDGFR were unaffected by SB irrespective of whether SMCs were preincubated with SB before exposure to PDGF-BB plus SB or incubated concomitantly with PDGF-BB plus SB. Likewise, phosphorylation and association of GAP and PLC{gamma} with PDGF-BB–activated ß-PDGFR were unaffected. In addition, SB did not block PDGF-BB–stimulated, PLC{gamma}-mediated production of inositol triphosphate. Similarly, PDGF-BB–induced ß-PDGFR degradation was unaffected when SMCs were exposed to PDGF-BB plus SB, and SB by itself had no influence on ß-PDGFR degradation. Unlike ß-PDGFR kinase activity, mitogen-activated protein kinase (MAP-kinase) activity was stimulated by SB by about 2.7-fold. Exposure of SMCs to PDGF-BB caused an {approx}11.4-fold increase in MAP-kinase activity and this increase in activity was not significantly affected when cells were coincubated with PDGF-BB and SB (10.3-fold). However, pretreatment of SMCs with SB for 30 minutes and subsequent incubation in PDGF-BB plus SB abolished most of the PDGF-BB–induced MAP-kinase activity (4.6-fold). Transcription of growth response genes such as c-fos, c-jun, and c-myc were induced by PDGF-BB, and their induction was suppressed, particularly c-myc, by incubating SMCs with PDGF-BB plus SB. Similarly, preincubation of cells with SB for 30 minutes and subsequent incubation in PDGF-BB plus SB diminished PDGF-BB–induced transcription of c-fos, c-jun, and c-myc. However, SB by itself had no significant effect on c-fos, c-jun, and c-myc transcription. Our data suggest that the inhibition of PDGF-BB–induced proliferation of SMCs by SB involves MAP-kinase–regulated events as well as transcription of growth-response genes.


Key Words: sodium butyrate • proliferation • platelet-derived growth factor • smooth muscle cells


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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Atherosclerotic plaque development involves several synchronized events such as adhesion, migration, proliferation, and transformation of cells, and these events are mediated by growth factors and cytokines. A greater attention has been focused on PDGF in the origin of atherosclerosis because of its mitogenic,1 2 chemotactic,3 and vasoconstrictor4 effects on VSMCs. The implication that VSMC proliferation contributes to hypertension,5 6 atherosclerosis,7 8 and restenosis after angioplasty8 9 10 11 has led to a growing interest in the use of drugs to intercept this process.

SB, a natural bacterial fermentation product produced in mammalian colon,12 has been shown to be a differentiation promoter with a potent antiproliferative effect on several cancer cells.13 14 15 16 17 18 19 In addition to its antiproliferative activity, SB induces changes in cellular morphology,20 21 alters the expression of cellular genes,22 23 24 25 26 27 28 29 30 31 and modulates hormone action and hormone receptors23 25 26 27 28 29 30 31 as well as growth factor receptors.31 32 SB has also been shown to inhibit the high fat diet–induced mammary tumorigenesis.33 Additionally, SB has been used in preliminary clinical trials to treat certain acute leukemias34 35 and stable butyrate derivatives have been developed with a hope to use these compounds to treat mammary carcinoma.36 Since atherosclerotic plaque development involves proliferation of VSMCs, and SB is a naturally occurring short-chain fatty acid, it prompted us to investigate the potential therapeutic value of SB as an antiproliferative agent of VSMCs. In the present study we report that SB inhibited PDGF-induced proliferation of rat SMCs. We examined the effect of SB on various aspects of SMC proliferation and signal transduction pathways involved in the proliferative response to PDGF to understand the mechanism of inhibition of PDGF-induced proliferation of SMCs by SB. This information will provide clues to the step(s) that can be targeted for intervention of atherosclerotic plaque development.


*    Methods
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 up arrowIntroduction
 *Methods
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Materials
Recombinant PDGF-AA, -AB, and -BB were obtained from GIBCO BRL Life Technologies. SB came from Fluka. A7r5 rat aortic SMCs were obtained from American Type Culture Collection. Anti-phosphotyrosine, anti-ß–PDGFR, anti-GAP, and anti-PLC{gamma} were purchased from Upstate Biotechnology Inc. Anti-MAP–kinase and protein A/G PLUS-agarose conjugate were from Santa Cruz Biotechnology. [Methyl-3H]thymidine, Myo-[2-3H]inositol, and [{gamma}-32P]ATP and reagents for ECL were purchased from Amersham Corp.

Cell Cultures
Rat SMCs were grown in DMEM fortified with 10% FCS, 50 U/mL penicillin, and 50 µg/mL streptomycin. For proliferation assay, cells were plated in six-well trays. Cells were seeded into 25-cm2 flasks for myo-[3H]inositol incorporation studies. For other studies, cells were cultured in 150-mm culture dishes. Cells were grown to 90% confluence at 37°C in a humidified atmosphere of 5% CO2. Culture media were changed every other day. Experiments were performed using 90% confluent cells. Quiescent cells were obtained by incubating 90% confluent cells either in 0.1% serum containing medium for 48 hours or in serum-free medium for 24 hours. Different concentrations of PDGF-AA, -AB, or -BB were used for DNA synthesis measurements. Since 30 ng/mL PDGF-BB results in maximal induction of IP3 production in rat VSMCs,37 the same concentration was used in the present study for phosphoinositide assay. For protein phosphorylation, protein association, MAP-kinase, and gene transcription assays, 50 ng/mL PDGF-BB was used. These are short-term-exposure studies resulting in immediate early responses to PDGF-BB. To ensure efficient response, maximum concentration of PDGF-BB needed for full induction of SMC proliferation was used, {approx}20 to 50 ng/mL. In addition, almost all published signal transduction studies involving PDGF-BB used similar concentrations for early response studies (40 to 60 ng/mL). Concentration of SB used in the experiments was 5 mmol/L unless otherwise noted.

Mitogenic Assay
Quiescent cells were stimulated with PDGF in a serum-free medium containing 1 µCi/mL [3H]thymidine in the absence or presence of SB. After 24 hours of incubation, the cells were washed three times with PBS and fixed in 5% ice-cold TCA for 1 hour at 4°C. Fixed cells were washed three times with 5% ice-cold TCA and dissolved in 0.2N NaOH/0.1% SDS. Amount of radioactivity incorporated was measured.

Proliferation Assay
To measure proliferation, cells were grown to 90% confluence and then made quiescent by incubation in 0.1% FCS containing DMEM medium for 48 hours. Cells were then stimulated with serum-free DMEM containing 10 ng/mL PDGF-BB in the absence or presence of SB. After 24 hours of incubation, the cells were washed with PBS and dissociated with trypsin/EDTA. Dissociated cells were counted in triplicate with a hemocytometer. Cell viability was determined by trypan blue exclusion. Cell viability was more than 90% up to 5 mmol/L, both in the presence and absence of PDGF-BB. Between 5 and 10 mmol/L SB concentration, cell viability was reduced to 60% to 70%.

Phosphoinositide Assay
Quiescent cells prelabeled with myo-[3H]inositol (4 µCi/mL) for 48 hours were incubated in PBS containing 20 mmol/L lithium chloride for 10 minutes. After 10 minutes, phosphoinositide assay was initiated by the addition of test compounds and terminated after 1 minute by addition of 2 mL of 5% TCA. TCA supernatants collected by centrifugation were extracted three times with ether and neutralized before separation of inositol phosphates on anion exchange resin (AG 1X48; 200 to 400 mesh; Formate form). IPs, IP2s, and IP3s were separated according to the protocol described by Berridge et al.38 Radioactivity in each of these inositol phosphates was determined by liquid scintillation counting.

Immunoblots
Cells grown in 150-mm culture plates were washed with ice-cold PBS three times and lysed in 1 mL of RIPA buffer (20 mmol/L Tris, pH 7.4, 137 mmol/L NaCl, 10% glycerol, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 2 mmol/L EDTA, 1 mmol/L PMSF, 20 µmol/L leupeptin, and 1 mmol/L sodium orthovanadate) by gentle rocking for 20 minutes at 4°C.39 40 Lysates were then cleared by centrifugation at 10 000g for 15 minutes at 4°C. The clear lysates were normalized for total cellular protein, and an equal amount of protein from each lysate was fractionated on SDS-polyacrylamide gel. Polypeptides, separated by SDS-PAGE, were transferred to immobilon PVDF membrane and blocked for 1 hour in blocking buffer containing 5% nonfat dry milk and 0.1% Tween 20 in TBS. Blots were washed three times with TBS containing 0.1% Tween 20 and probed with appropriate primary antibody for 1 hour in TBS. Blots were washed three times with TBS containing 0.1% Tween 20 and then incubated with appropriate second antibody conjugated to horseradish peroxidase. Immunoreactive proteins were identified using an ECL detection kit from Amersham Corp.

Immunoprecipitation
Cell lysates were prepared as described above using RIPA buffer.39 40 Equal amounts of proteins were incubated with ß-PDGFR, GAP, PLC{gamma}, or P-Tyr antibody and 20 µL of protein A/G PLUS agarose-conjugate for about 12 hours at 4°C. Immunoprecipitated complexes were collected by centrifugation and washed once with RIPA buffer, twice with 0.5 mol/L LiCl in 0.1 mol/L Tris, pH 7.4, and once with 10 mmol/L Tris, pH 7.4. Immunoprecipitated proteins were eluted with SDS-PAGE buffer and separated on 7.5% gels. Separated proteins were transferred to immobilon PVDF membrane and used for immunoblotting as described above with suitable antibodies.

MAP-Kinase Assay
Cell lysates were prepared by using nonionic detergent buffer containing 1% Triton X-100, 20 mmol/L Tris, pH 8.0, 137 mmol/L NaCl, 10% glycerol, 2 mmol/L EDTA, 1 mmol/L PMSF, 20 µmol/L leupeptin, 50 mmol/L sodium fluoride, and 1 mmol/L sodium orthovanadate as described above.41 42 Similar amounts of proteins were incubated with polyclonal MAP-kinase antibody (C-16, ERK1, Santa Cruz Biotechnology) for 2 hours at 4°C, and 20 µL of protein A/G PLUS agarose-conjugate was added for the last 30 minutes. Immunocomplexes were collected and washed twice with nonionic detergent buffer followed by one wash in TBS supplemented with 1 mmol/L sodium orthovanadate and 5 mmol/L benzamidine. MAP-kinase activity was measured by addition of 40 µL of kinase reaction mix (30 mmol/L Tris, pH 8.0, 10 mmol/L MgCl2, 20 µmol/L ATP, 1 mmol/L DTT, 5 mmol/L benzamidine, 10 µCi of [{gamma}-32P]ATP and 6 µg of MBP) and incubation for 30 minutes at 37°C.41 42 Reaction was terminated by addition of 20 µL of 4x SDS-PAGE buffer. Reaction products were analyzed by SDS-PAGE on 12% gel followed by autoradiography. Phosphorylated MBP was excised from the dried gel, and radioactivity was measured by Cerenkov counting.

RNA Isolation and Northern Analysis
Quiescent SMCs grown in 150-mm culture dishes were exposed to different treatments for times indicated in the relevant "Results" section. Total RNA was isolated using TRI reagent method (Molecular Research Center). For Northern analysis, 15 µg of total RNA was denatured in sample loading buffer containing 1 µg/µL ethidium bromide and electrophoresed through 1% agarose formaldehyde gel.43 RNA was transferred to S&S MaxS nytran membrane by downward alkaline transfer method44 by using S&S Turbo Blotter transfer system. Transferred RNA was fixed to nytran membrane by ultraviolet irradiation. c-fos, c-jun, and c-myc cDNAs were labeled by random priming with the New England Biolabs NEBlot kit and {alpha}[32P]dCTP as recommended by the manufacturer. G3PDH oligonucleotide probe was labeled at the 5' end by the T4 polynucleotide kinase–catalyzed transfer of {gamma}-phosphate from {gamma}[32P]ATP to the 5' end of oligonucleotide. RNA blots were probed with random primer labeled c-fos, c-jun, c-myc, or 5' end-labeled 60-base–long G3PDH oligonucleotide probe.45 Blots were exposed to hyperfilm-MP (Amersham Corp) with intensifying screen at -70°C for 1 to 3 days. The autoradiographs were scanned with a BioRad GS-670 densitometer to quantify the intensity of hybridization signals.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Measurement of DNA Synthesis
Growth-arrested SMCs were exposed to PDGF-AA, -AB, or -BB with or without 5 mmol/L SB for 24 hours in the presence of [3H]thymidine. After 24 hours, the amount of [3H]thymidine incorporated was measured, and the results are shown in Fig 1Down. Although PDGF is a potent mitogen for SMCs, not all three isoforms are equally potent. PDGF-AA is the least potent and PDGF-BB is the most potent of the three isoforms of PDGF. Maximal mitogenic responses to PDGF-AB and -BB were approximately 5.3- and 6.2-fold above unstimulated values. The corresponding ED50 values for the mitogenic response to PDGF-AB and -BB were 7 and 5 ng/mL, respectively. However, stimulation of [3H]thymidine incorporation by PDGF-AA was only about 2.5-fold above unstimulated values at 20 ng/mL and remained the same above 20 ng/mL. SB at 5 mmol/L inhibited PDGF-AB– and -BB–induced DNA synthesis by more than 90%. On the other hand, PDGF-AA–induced [3H]thymidine incorporation was inhibited by about 65%.



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  		Figure 1. Graph shows mitogenic effect of PDGF isoforms on cultured rat SMCs. Quiescent SMCs were stimulated in serum-free medium containing [3H]thymidine with various concentrations of PDGF-AA, PDGF-AB, and PDGF-BB in the absence ({circ}, {triangleup}, {square}) or presence ({bullet}, {blacktriangleup}, {blacksquare}) of 5 mmol/L SB for 24 hours as described in "Methods." Data presented are mean of triplicates and representative of two independent experiments.

Since PDGF-BB is the most effective isoform of PDGF for SMCs, we extended the study to investigate the effective concentration of SB on PDGF-BB–induced proliferation (Tables 1Down and 2Down). Effect of SB on PDGF-BB–induced DNA synthesis (Table 1Down) and on increase in cell number (Table 2Down) was measured between the concentration of 1 mmol/L and 8 mmol/L. At 1 mmol/L SB there was about 25% inhibition of PDGF-BB–induced DNA synthesis. Concentrations of SB higher than 1 mmol/L exhibited significant inhibition of PDGF-BB–induced DNA synthesis. At 5-mmol/L concentration, SB completely inhibited PDGF-BB–induced DNA synthesis. To investigate whether similar inhibitory effects of SB are observed on PDGF-BB–induced increased cell number, we determined the number of cells after 24 hours' exposure to different concentrations of SB in the absence or presence of PDGF-BB (Table 2Down). As shown in Table 2Down, PDGF-BB induced an increase in cell number, and this increase in cell number was reduced significantly in the presence of SB irrespective of concentrations used. However, at up to 5-mmol/L SB concentration, more than 90% of the cells were viable in both the absence and presence of PDGF-BB. At an 8-mmol/L concentration of SB, cell viability was reduced to about 60% and 70% in the absence and presence of PDGF-BB, respectively.


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  		Table 1. Concentration-Dependent Effect of SB on PDGF-BB–Induced DNA Synthesis in Rat SMCs


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  		Table 2. Concentration-Dependent Effect of SB on PDGF-BB–Induced Proliferation of Rat SMCs

Although at a 5-mmol/L concentration PDGF-BB–induced DNA synthesis as well as cell number was completely inhibited by SB, PDGF-BB–stimulated protein and RNA contents were reduced by only about 40% (Table 3Down). In the absence of PDGF-BB, 5 mmol/L SB had no effect on protein and RNA content as in unstimulated cells. Since a 5-mmol/L concentration of SB almost completely inhibited PDGF-BB–induced DNA synthesis and cell proliferation without any adverse effect on SMCs, we selected this concentration of SB to extend our studies to understand the mechanism of inhibition of PDGF-BB–induced proliferation.


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  		Table 3. Effect of SB on Cellular Protein and RNA Content of Rat SMCs

PDGF-BB-Induced Protein Tyrosine Phosphorylation
Protein tyrosine phosphorylation has been implicated in mitogenic signal pathway. Therefore, we probed into PDGF-BB–stimulated protein tyrosine phosphorylation and its response to SB treatment (Fig 2Down). Although anti-P–tyr antibody recognized some bands in all the treatments, it identified distinct proteins that are highly phosphorylated on tyrosine residues on exposure to PDGF-BB compared with untreated cells. Coincubation of cells with SB and PDGF-BB caused no alteration in PDGF-BB–enhanced protein tyrosine phosphorylation profile. Similarly, pretreatment of cells for 30 minutes with SB and subsequent exposure to PDGF-BB plus SB had no influence on PDGF-BB–induced protein tyrosine phosphorylation profile. On the other hand, SB by itself caused no increased protein tyrosine phosphorylation similar to untreated cells.



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  		Figure 2. Blot shows influence of SB on PDGF-BB–induced protein tyrosine phosphorylation. Quiescent SMCs in serum-free DMEM were exposed for 5 minutes with no addition (Lane 1), 5 mmol/L SB (Lane 2), preincubation in 5 mmol/L SB for 30 minutes and then with 5 mmol/L SB plus 50 ng/mL PDGF-BB (Lane 3), 50 ng/mL PDGF-BB (Lane 4), or 5 mmol/L SB plus 50 ng/mL PDGF-BB (Lane 5) at 37°C. After incubation, cells were lysed as described in "Methods." Equal amounts of whole-cell proteins were separated by electrophoresis on 7.5% SDS-polyacrylamide gels, transferred to PVDF membrane, and immunoblotted with anti-P-Tyr antibody. Immunoreactivity was detected by ECL. PDGF-BB–induced tyrosine phosphorylation of cellular proteins is indicated by the arrows.

To assess whether PDGF-BB–induced ß-PDGFR autophosphorylation is modulated by SB, we analyzed anti-P–Tyr immunoprecipitates by immunoblotting with anti-ß-PDGFR antibody (Fig 3BDown). As in cellular proteins, PDGF-BB–induced ß-PDGFR tyrosine phosphorylation was unaffected by SB irrespective of whether the cells were exposed to SB before PDGF-BB plus SB or together with PDGF-BB. Cells exposed to no additions or SB alone exhibited no ß-PDGFR tyrosine phosphorylation. In addition, SB had no effect on ß-PDGFR level (Fig 3ADown), and it was similar irrespective of the treatment.



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  		Figure 3. Blots show effect of SB on PDGF-BB–activated ß-PDGFR autophosphorylation. Density-arrested cells were treated with similar additives as described in legend to Fig 2Up for 5 minutes at 37°C. Cell lysates were prepared and processed for immunoprecipitation and immunoblotting as described in "Methods." Similar amounts of cell lysates were used for immunoprecipitation with anti-ß–PDGFR or anti-P–Tyr. A, Anti-ß–PDGFR immunoblot of anti-ß–PDGFR immunoprecipitates. B, Anti-ß–PDGFR immunoblot of anti-P–Tyr immunoprecipitates.

Association of Signaling Molecules with ß-PDGFR
PDGF treatment of cells has been shown to result in PDGFR association and phosphorylation of signaling molecules such as PLC{gamma}, GAP, raf-I, and PI3-kinase. To ascertain whether inhibition of PDGF-BB–induced proliferation of SMCs by SB involved disruption of this signal transducing event, phosphorylation of GAP and PLC{gamma} and association of phosphorylated GAP and PLC{gamma} with activated ß-PDGFR were examined.

Although equal amounts of cell lysates were used for GAP immunoprecipitation, immunoblotting with anti-GAP antibody revealed that the amount of GAP immunoprecipitated was less in unstimulated and SB-treated cells compared with PDGF-BB–treated cells (Fig 4ADown). PDGF-BB–stimulated increase in GAP immunoprecipitation was not altered by exposing SMCs to SB before the addition of PDGF-BB plus SB or coincubation with PDGF-BB and SB. This differential effect of anti-GAP antibody on GAP immunoprecipitation suggests that the GAP antibody probably recognized PDGF-BB–activated GAP more efficiently than unstimulated GAP, as in no-addition control and in cells that were treated with SB alone (Fig 4ADown). To determine whether SB interferes with PDGF-BB–stimulated tyrosine phosphorylation of GAP, similar amounts of cell lysates were immunoprecipitated with anti-P-Tyr and immunoblotted with anti-GAP (Fig 4BDown). It was evident that SB did not alter PDGF-BB–enhanced tyrosine phosphorylation of GAP irrespective of whether the cells were exposed to SB before the addition of PDGF-BB plus SB or coincubated with PDGF-BB and SB. SB by itself caused no protein tyrosine phosphorylation as in no-addition treatment. Similar to tyrosine phosphorylation of GAP, association of activated GAP with ß-PDGFR was not modified by SB (Fig 4CDown). Immunoblotting of GAP immunoprecipitates with anti-ß–PDGFR revealed that ß-PDGFR was associated with GAP only in immunoprecipitates prepared from PDGF-BB–treated cells irrespective of the presence or absence of SB.



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  		Figure 4. Blots show effect of SB on PDGF-BB–stimulated tyrosine phosphorylation of GAP and its association with ß-PDGFR. Quiescent SMCs were incubated in serum-free DMEM with similar additions as described in legend to Fig 2Up. Equal amounts of cell lysates were immunoprecipitated with anti-GAP or anti-P-Tyr and processed for immunoblotting as detailed in "Methods." A, Anti-GAP immunoblot of anti-GAP immunoprecipitates. B, Anti-GAP immunoblot of anti-P-Tyr immunoprecipitates. C, Anti-ß-PDGFR immunoblot of GAP immunoprecipitates.

Effect of SB on PDGF-BB–induced protein tyrosine phosphorylation of PLC{gamma} and its association with activated ß-PDGFR was investigated by using P-Tyr and PLC{gamma} immunoprecipitates, respectively (Fig 5Down). Immunoblotting of PLC{gamma} immunoprecipitates with anti-PLC{gamma} revealed no significant effect on the levels of PLC{gamma} (Fig 5ADown). As in GAP, PDGF-BB stimulated tyrosine phosphorylation of PLC{gamma} that was not altered by SB irrespective of whether cells were pre-exposed to SB before the addition of PDGF-BB plus SB or coincubation with PDGF-BB and SB (Fig 5BDown). In addition, association of activated PLC{gamma} with ß-PDGFR was not modulated by exposing the cells to SB before the addition of PDGF-BB plus SB or concurrent incubation with PDGF-BB and SB (Fig 5CDown). These observations suggest that the mechanism of inhibition of PDGF-BB–induced SMC proliferation by SB does not involve activation of ß-PDGFR and its interaction with GAP and PLC{gamma}.



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  		Figure 5. Influence of SB on PDGF-BB–provoked tyrosine phosphorylation of PLC{gamma} and coprecipitation of ß-PDGFR with PLC{gamma}. Quiescent SMCs were treated as described in legend to Fig 2Up for 5 minutes at 37°C. Equal amounts of cell lysates were immunoprecipitated with anti-PLC{gamma} or anti-P-Tyr and processed for immunoblotting as described under "Methods." A, Anti-PLC{gamma} immunoblot of anti-PLC{gamma} immunoprecipitates. B, Anti-PLC{gamma} immunoblot of anti-P-Tyr immunoprecipitates. C, Anti-ß–PDGFR immunoblot of PLC{gamma} immunoprecipitates.

Influence of SB on PDGF-BB–Stimulated IP3 Production
Exposure of SMCs to SB caused no increase in IP3 formation compared with no-addition control (Table 4Down). Treatment of SMCs with PDGF-BB resulted in an increase of about 30% in IP3 production, which was increased to 60% by coincubating the cells with PDGF-BB and SB (Table 4Down). This observation suggests that activity of PLC{gamma} was unaffected by SB in the absence of PDGF-BB. But in the presence of PDGF-BB, SB stimulated PDGF-BB–induced IP3 formation.


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  		Table 4. Influence of SB on PDGF-BB–Induced Inositol Phosphate Production

PDGF-BB–Induced Downregulation of ß-PDGFR
To understand the mechanism of inhibition of PDGF-BB–induced proliferation by SB, we also investigated the influence of SB on PDGF-BB–stimulated ß-PDGFR downregulation. Cell lysates obtained from SMCs that were exposed to no-addition control, PDGF-BB, SB, or PDGF-BB plus SB for different lengths of time were analyzed by SDS-PAGE followed by immunoblotting with anti-ß-PDGFR to study the receptor downregulation (Fig 6Down). As shown in Fig 6Down, PDGF-BB–induced ß-PDGFR downregulation occurred after 30 minutes and was completely degraded after 2 hours of incubation of SMCs with PDGF-BB. This time-dependent, PDGF-BB–activated downregulation of ß-PDGFR was unaffected by SB when added together with PDGF-BB. Cells exhibited a pattern of ß-PDGFR downregulation similar to that in PDGF-BB–treated cells. Likewise, pretreatment of cells for 30 minutes with SB and subsequent exposure to PDGF-BB plus SB did not block PDGF-BB–stimulated downregulation of ß-PDGFR (data not shown). On the other hand, ß-PDGFR revealed no degradation when SMCs were exposed to SB alone, indicating that SB did not interfere with ligand-stimulated receptor downregulation.



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  		Figure 6. Blots show PDGF-BB–induced ß-PDGFR degradation. Serum-starved SMCs were incubated in serum-free DMEM containing no additions, 50 ng/mL PDGF-BB, 5 mmol/L SB, or 50 ng/mL PDGF-BB plus 5 mmol/L SB for indicated periods. Cell lysates were prepared and equal amounts of cell lysates were subjected to PAGE as detailed in "Methods." After electrophoresis, ß-PDGFR degradation was detected by probing with anti-ß–PDGFR antibody.

PDGF-BB–Induced MAP-Kinase Activity
MAP-kinases are serine and threonine kinases that are strongly activated by PDGF. Effect of SB on PDGF-BB–induced MAP kinase activity was measured by immunocomplex kinase assay. MAP-kinase was immunoprecipitated from the SMC cell lysates by using anti-MAP–kinase antibody, and the immunocomplex was subjected to in vitro kinase reaction by using MBP as a substrate (Fig 7Down). Surprisingly, exposure of SMCs to SB stimulated MAP-kinase activity by about 2.7-fold over no-addition control. Addition of PDGF-BB to SMCs stimulated MAP-kinase activity by about 11.4-fold. Coincubation of SMCs with PDGF-BB plus SB caused no significant effect on PDGF-BB–stimulated MAP-kinase activity (10.3-fold). But preincubation of SMCs with SB for 30 minutes and subsequent addition of PDGF-BB plus SB resulted in a significant decrease in PDGF-BB–induced MAP-kinase activity. There was only a 4.6-fold increase in activity over no-addition control, suggesting that MAP-kinase activities are differentially modified by SB.



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  		Figure 7. SB effect on PDGF-BB–induced and uninduced MAP-kinase activity. Density-arrested SMCs were exposed to treatments as described in legend to Fig 2Up for 5 minutes at 37°C. Cell lysates were prepared and equal amounts of cell lysates were used for immunoprecipitation of MAP-kinase. MAP kinase activities in the immunocomplexes were measured by incubation with MBP in the presence of [{gamma}-32P]ATP as described in "Methods." Proteins were separated by SDS-PAGE on 12% polyacrylamide gels and autoradiographed. The phosphorylated MBP bands are excised and incorporated radioactivities are measured. A, Autoradiogram of phosphorylated MBP. B, Graph shows quantification of MBP phosphorylation by Cerenkov counting.

Serum and PDGF-BB–Induced Transcription of c-fos, c-jun, and c-myc
Exposure of cells to serum or growth factors caused increased rapid transcription of nuclear proto-oncogenes. We investigated the effect of SB on serum and PDGF-BB–stimulated c-fos, c-jun, and c-myc transcription. The results of these experiments are depicted in Fig 8Down. Treatment of serum-starved SMCs with serum caused increased transcription of all three nuclear proto-oncogenes compared with no-addition control. Serum-induced effect was more dramatic on c-fos and c-jun than on c-myc. Addition of SB along with serum diminished the serum-induced transcription of c-fos (52%), c-jun (45%), and c-myc (30%), although SB by itself had no significant effect on the transcription of c-fos, c-jun, and c-myc. Similar to serum, PDGF-BB also induced the transcription of all three proto-oncogenes. SB diminished PDGF-BB–stimulated transcription of c-myc more strongly than c-fos and c-jun. PDGF-BB–induced transcription of c-myc was inhibited by about 70% to 80%, whereas c-fos and c-jun transcription was inhibited by about 45% irrespective of whether the cells were preexposed to SB and then coincubated with SB plus PDGF-BB or treated simultaneously with PDGF-BB plus SB.



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  		Figure 8. Blots show influence of SB on serum and PDGF-BB–induced transcription of c-fos, c-jun, and c-myc. Effect of SB on serum and PDGF-BB–induced transcription of c-fos (A), c-jun (B), and c-myc (C) is assessed at the time of their maximal transcription (c-fos, 30 minutes; c-jun and c-myc, 60 minutes). Serum-deprived SMCs in serum-free DMEM were incubated with no addition (Lane 3), 10% FCS (Lane 1), 10% FCS plus 5 mmol/L SB (Lane 2), 5 mmol/L SB (Lane 4), preincubation in 5 mmol/L SB for 30 minutes and subsequent incubation in 5 mmol/L SB plus 50 ng/mL PDGF-BB (Lane 5), 50 ng/mL PDGF-BB (Lane 6), or 50 ng/mL PDGF-BB plus 5 mmol/L SB (Lane 7) for 30 minutes or 60 minutes. After required incubation time, total RNA was isolated and the levels of c-fos, c-jun, and c-myc transcripts were measured as described in "Methods." The RNA blots were probed with G3PDH to normalize the quantity of RNA loaded onto each lane.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
SB treatment of SMCs caused inhibition of PDGF-AA–, -AB– and -BB–induced proliferation. Although PDGF is a major mitogen for many cells, response to three PDGF isoforms seemed to differ between cell types. VSMCs responded to three PDGF isoforms differently. In our study, PDGF-AA was least mitogenic of the three isoforms. It stimulated about a 2.5-fold increase in DNA synthesis whereas PDGF-AB and -BB stimulated increases in DNA synthesis of about 5.3-fold and 6.2-fold, respectively. These observations are in accordance with previous reports.37 46 47 It has been proposed that differences in the mitogenic sensitivity of the cells to different PDGF isoforms are attributable to both the absolute and relative number of {alpha}-PDGFR and ß-PDGFR, which differ depending on cell types.37 48 PDGF-AB and -BB interacted with both {alpha}- and ß-PDGFR. On the other hand, PDGF-AA interacted exclusively with {alpha}-PDGFR. On the basis of our DNA synthesis study, it appears that SMCs expressed both {alpha}- and ß-PDGFR, but expression of ß-PDGFR was higher than {alpha}-PDGFR.

SB at a 5-mmol/L concentration inhibited SMC proliferation induced by all three isoforms of PDGF, particularly PDGF-AB–induced and -BB–induced proliferation. The antiproliferative effect of SB was not mitogen-specific, because even serum-induced proliferation of several mammalian cells was inhibited by SB.16 17 Other studies have shown that insulin- and epidermal growth factor–induced DNA synthesis is inhibited by SB.49 These reports indicate that SB is specific for the process of proliferation rather than to the specific agent inducing proliferation. The reason for studying the effect of SB on PDGF-induced SMC proliferation is that PDGF is one of the key growth factors that induce SMC proliferation. Proliferation of SMCs is the hallmark of atherosclerosis.

Although the effective concentration of SB needed to inhibit PDGF-BB–induced SMC proliferation is in millimoles per liter, up to 5 mmol/L SB concentration, no toxic effects were observed. Cell viability was more than 90%. Moreover, the concentrations used in the present study are probably close to the physiological range.12 In addition, in the absence of PDGF-BB, SB had no significant effect on baseline proliferation (Tables 1Up and 2Up) as well as on cellular protein and RNA levels (Table 3Up). However, SB inhibited PDGF-BB–induced increase in cellular protein and RNA level by about 40% in contrast to DNA synthesis, which was completely inhibited. Since SB is an inducer of differentiation and inhibitor of cell cycle progression,15 50 51 it may be differently affecting the expression of cell differentiation and proliferation-specific genes in SMCs. Several reports indicate that SB can have different consequences on the induction of specific RNAs in the same cell.29 31 52 Previous studies have shown that the effects of SB are caused by structural alteration in chromatin15 53 54 as well as by regulation of transcription regulatory proteins.55 56 SB has been shown to cause hyperacetylation of histones15 53 and methylation of DNA,54 both of which may mediate SB effect on gene expression. Hyperacetylation of histones may lead to increased transcriptional activity, while DNA methylation may cause inhibition of gene expression resulting in increase or decrease in expression of specific genes depending on the status of the cells. In our study it appears that some of the genes that were induced by PDGF-BB were suppressed by SB, which may be cell-proliferation–related. Our unpublished data on G3PDH indicate that it is one of the PDGF-BB–induced genes inhibited by SB. Several other studies have shown increase in expression of differentiation-specific genes in response to SB treatment.22 23 24 25 26 27 28 29 30 31

How external growth signals carried by the growth factors are transmitted from the cell surface receptors to the nucleus is still vaguely understood. Substantial progress has been made in understanding the initial steps, with the delineation of the structure of growth factors and their receptor protein tyrosine kinases. PDGF, upon binding to the receptor, causes conformational changes of the receptor leading to receptor dimerization followed by activation of intrinsic tyrosine kinase activity.57 58 59 As a result, PDGFR undergoes autophosphorylation on tyrosines of the receptor cytoplasmic domain, causing the activated receptor to associate with signaling proteins that contain Src homology 2 domains60 61 such as PLC{gamma},62 63 P1-3 kinase,64 65 raf-166 and GAP.67 68 After activation and association with regulatory signaling molecules, PDGFR was rapidly internalized and degraded.65 69 70 71 We followed the effect of SB on these initial steps of PDGF-induced signal transduction to delineate the mode of action of SB. Our studies indicate that when SMCs are coincubated with SB and PDGF-BB, SB not only has no effect on PDGF-BB–stimulated tyrosine phosphorylation of ß-PDGFR (Fig 3BUp) but also appears to have no significant effects on PDGF-BB–induced tyrosine phosphorylation of cellular proteins resolved on the Western blot (Fig 2Up). Similarly, preincubation with SB and subsequent exposure to PDGF-BB plus SB had no effect on protein tyrosine phosphorylation. This observation suggests that SB effect is independent of PDGF-BB–induced protein tyrosine phosphorylation of ß-PDGFR. This observation is further substantiated by phosphorylation and association of signaling molecules such as GAP and PLC{gamma} with ß-PDGFR (Figs 4Up and 5Up). Exposure of SMCs to no addition or to SB caused no tyrosine phosphorylation or association of proteins. In the presence of PDGF-BB whether it was added with SB or subsequent to SB addition, ß-PDGFR autophosphorylation and its association with GAP and PLC{gamma} as well as their phosphorylation were unaffected. This suggests that mechanism of inhibition of PDGF-BB–induced SMC proliferation by SB is not through its effect on PDGF-BB binding–induced autophosphorylation on tyrosines of the receptor cytoplasmic domains and its association with signaling proteins GAP and PLC{gamma}.

Although there was no difference in the levels of ß-PDGFR (Fig 3AUp) and PLC{gamma} (Fig 5AUp) in different treatments, GAP immunoprecipitates exhibited differences in the levels of GAP (Fig 4AUp). Same amounts of proteins were used for immunoprecipitation of GAP from the cells exposed to different treatments. Yet we noticed an increase in GAP protein in GAP immunoprecipitates prepared from cells exposed to PDGF-BB, irrespective of whether SB was present or not. Since GAP undergoes tyrosine phosphorylation on exposure to PDGF-BB, it may cause conformational change in GAP, which may result in increased recognition by GAP antibody. Therefore, we observed an increase in GAP protein in GAP immunoprecipitates when cells are activated with PDGF-BB. Similar differences have been noted in other reports.68

Effect of SB on PDGF-BB binding to ß-PDGFR was determined by following ß-PDGFR downregulation. These studies revealed that SB has no effect on PDGF-BB–stimulated ß-PDGFR degradation. SMCs exhibited similar time-dependent PDGF-BB–induced ß-PDGFR downregulation both in the absence and presence of SB. On the other hand, SB by itself did not react with ß-PDGFR and exhibited no ß-PDGFR degradation (Fig 6Up). These observations suggest that the site of SB action may be downstream to the signaling pathway.

Analysis of IP3 reveals that SB by itself had no effect on PLC{gamma}-catalyzed IP3 formation. However, PDGF-BB stimulates IP3 formation. PDGF-BB–induced IP3 formation was not inhibited by exposing the SMCs to PDGF-BB plus SB. In fact, there was increased stimulation of IP3 formation when the cells were coincubated with PDGF-BB and SB than when they were exposed only to PDGF-BB. Although we do not know the mechanism by which PDGF-BB plus SB increased IP3 formation, there is an additional pathway that causes IP3 formation by PLC. This pathway was mediated by guanosine nucleotide protein, referred to as Gp.72 It is suggested that there was some form of interaction between PDGFR and Gp protein that caused an increase in IP3 formation from phosphatidylinositol 4,5-biphosphate by PLC. Increase in PDGF-BB–induced IP3 in PDGF-BB plus SB–treated cells was probably due to PLC activated by G-protein–mediated pathway. However, at the moment the significance of induction of IP3 in PDGF-BB plus SB–treated cells is not clear. Hydrolysis of phosphoinositide by PLC{gamma} released two second messengers: diacylglycerol, an activator of protein kinase C, and IP3, a generator of calcium signal, by releasing calcium from intracellular stores.73 74 Intracellular calcium is known to regulate biochemical processes either directly or indirectly through specific calcium binding proteins.75 However, we do not know whether calcium binding proteins have any role in SB-inhibited, PDGF-BB–induced SMC proliferation.

A key factor in the signaling pathway involved in transducing the signal from an activated protein tyrosine kinase to an intracellular response has been identified as the family of MAP-kinases. MAP-kinases, which are also called extracellular signal-regulated kinases (ERKs), are serine/threonine kinases. Although MAP-kinase 1 and 2 are the most extensively studied MAP-kinases, several other isoforms of MAP-kinase have recently been identified.76 MAP-kinases phosphorylate and regulate the activity of enzymes and transcription factors.77 Our studies revealed that SB stimulates MAP-kinase activity by about 2.7-fold compared with uninduced control cells. However, it is less than the MAP-kinase activity induced by PDGF-BB (11.4-fold). This PDGF-BB–stimulated activity is not significantly affected when cells are simultaneously exposed to PDGF-BB plus SB (10-fold). But the PDGF-BB–stimulated MAP-kinase activity is significantly inhibited if cells are preexposed to SB for 30 minutes before treatment with PDGF-BB plus SB, suggesting that preincubation with SB is blocking the activity that is activated by PDGF-BB. MAP-kinases are activated by concurrent phosphorylation of tyrosine and threonine residues. This double phosphorylation is mediated by the dual specificity MAP-kinase kinase (MKK or MEK).78 MEK is in turn regulated through phosphorylation by MAP-kinase kinase kinases (MEKK), which include the proto-oncogene product raf.79 In addition, MAP-kinase can undergo autophosphorylation on tyrosine residues.80 It is reasonable to think that the small amount of activation of MAP-kinase activity induced by SB may be the result of autophosphorylation of tyrosine residues. Similar induction of MAP-kinase activity was observed by Cook and McCormick.42 Protein phosphorylation and dephosphorylation are essential mechanisms in the pathways by which cell proliferation is regulated. For full activation of MAP-kinases, one or more distinct protein kinases are needed, and increased induction of MAP-kinase activity in PDGF-BB–treated cells could be due to activation of one or more of these distinct protein kinases. At the moment we do not know the mechanism of inhibition of MAP-kinase activity in cells that are pretreated with SB before treatment with PDGF-BB plus SB. It can be due to inactivation of a kinase that is supposed to activate MAP-kinase (MEK or MEKK?) or it can be due to the stimulation of a phosphatase that blocks the activation of MAP-kinase. Although MAP-kinase can be completely inactivated in vitro by treatment with either CD-45, a phosphotyrosine-specific phosphotyrosine phosphatase, or the catalytic subunit of phosphatase 2A, a predominantly serine/threonine-specific protein phosphatase, the mechanism of MAP-kinase inhibition in intact cells is not understood.81 But recently it has been reported that a growth-induced gene MKP1 encodes a dual specificity phosphatase that dephosphorylates and inactivates MAP-kinase more specifically than other phosphatases.82 Understanding of these phosphatases may provide clues to the mechanism of inhibition of MAP-kinase by SB.

One of the earliest genetic changes elicited by many mitogens is the induction of expression of immediate early growth-response genes.83 84 These genes couple early biochemical second-messenger signals to long-term changes in transcription, resulting in mitogenesis. Proto-oncogenes such as c-fos, c-jun, and c-myc are some of the immediate early growth-response genes that are induced in response to PDGF treatment.81 85 86 87 In our study SB inhibited PDGF-BB–stimulated c-fos, c-jun, and c-myc transcription irrespective of whether SMCs were preexposed to SB, before treatment with PDGF-BB plus SB, or treated concurrently with PDGF-BB plus SB. But SB inhibited PDGF-BB upregulated c-myc expression more severely than c-fos and c-jun expression. The reason for differences in extent of inhibition of PDGF-BB–stimulated expression of c-fos, c-jun, and c-myc is not clear. However, it is interesting that extent of inhibition of c-fos and c-jun expression by SB was similar. The importance of proto-oncogenes lies in the fact that they activate or suppress second-phase genes essential for cellular proliferation by interacting with their respective promoter regions. Since SB blocked the PDGF-BB–stimulated expression of proto-oncogenes, particularly c-myc, they may no longer function as effective nuclear signal transducers to bring about a cascade of gene-protein interactions that are manifested in cellular proliferation. Downregulation of c-myc expression has been observed in rectal carcinoma cells that were treated with SB.88 89 In these cells the inhibitory effect of SB on c-myc expression has been shown to be blocked by inhibitors of protein synthesis, suggesting that SB causes the synthesis of a protein(s) that downregulates c-myc expression.88 Although the mechanism of inhibition of c-myc by SB was not determined in our study, it probably involves a trans mechanism as in rectal carcinoma cells. However, at present we are investigating whether the effect of SB on PDGF-BB–induced c-myc upregulation is at transcriptional or posttranscriptional level. A considerable amount of constitutive expression of c-myc was observed in cells maintained in serum-free medium. Similar observations have been reported by others, and it is suggested that constitutive c-myc may represent a pool of functionally inactive, more stable, nonpolyadenylated molecules.90 91

Taken together, our findings reveal that SB has a potential antiatherosclerotic activity by suppressing PDGF-induced SMC proliferation. Elucidation of the antiproliferative effect of SB would greatly contribute to our understanding of the relationship between cell proliferation and atherosclerosis.


*    Selected Abbreviations and Acronyms
 
DMEM = Dulbecco's modified Eagle's medium
ECL = enhanced chemiluminescence
ERKs = extracellular signal-regulated kinases
FCS = fetal calf serum
G3PDH = glyceraldehyde-3-phosphate dehydrogenase
GAP = ras-GTPase activating protein
Gp = guanosine nucleotide protein
IP = inositol monophosphate
IP2 = inositol biphosphate
IP3 = inositol triphosphate
MAP = mitogen-activated protein
MBP = myelin basic protein
MEK = MAP-kinase kinase
MEKK = MAP-kinase kinase kinases
MKK = MAP-kinase kinase
PBS = phosphate-buffered saline
PDGF = platelet-derived growth factor
PDGFR = platelet-derived growth factor receptor
PI3-kinase = phosphatidylinositol 3-kinase
PLC = phospholipase C
PMSF = phenylmethylsulfonyl fluoride
P-Tyr = phosphotyrosine
RIPA = radioimmunoprecipitation assay buffer
SB = sodium butyrate
SDS = sodium dodecyl sulfate
SDS-PAGE = SDS-polyacrylamide gel electrophoresis
SMC(s) = smooth muscle cell(s)
TBS = Tris-buffered saline
TCA = trichloroacetic acid


*    Acknowledgments
 
This work was supported by a grant from The Clayton Foundation for Research, Houston, Tex. We thank Dr D.S. Loose-Mitchell for c-fos, c-jun, and c-myc probes, Dr P. Dash for G3PDH probe, and Elaine Marsh for the preparation of this manuscript.

Received January 11, 1995; accepted October 2, 1995.


*    References
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