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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3420-3427

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3420-3427.)
© 1997 American Heart Association, Inc.

Articles

Inhibition of Platelet-Derived Growth Factor BB–Induced Expression of Glyceraldehyde- 3-Phosphate Dehydrogenase by Sodium Butyrate in Rat Vascular Smooth Muscle Cells

Kasturi Ranganna; ; Frank M. Yatsu

From the Department of Neurology, University of Texas—Houston School of Medicine, Houston, Tex.

Correspondence to Kasturi Ranganna, Department of Neurology, University of Texas—Houston School of Medicine, 6431 Fannin, Suite 7.044, Houston TX 77030.


*    Abstract
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 *Abstract
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Abstract Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key regulatory enzyme of glycolysis, which exists in nuclei and functions as a DNA-binding protein as well as a nuclear protein, appears to be modulated by cellular activities. Exposure of quiescent rat smooth muscle cells (SMCs) to platelet-derived growth factor BB (PDGF-BB), which stimulates SMCs proliferation, caused a time-dependent increase in mRNA for GAPDH and its catalytic activity. Treatment of quiescent SMCs with sodium butyrate (SB), which is shown to inhibit PDGF-BB-induced SMC proliferation, caused a time- and concentration-dependent decrease in PDGF-BB-induced GAPDH mRNA expression and its catalytic activity. Nuclear run-on studies revealed that the PDGF-BB-induced rate of GAPDH gene transcription was reduced by about 50% in the presence of 5 mmol/L SB. The protein synthesis inhibitor, cycloheximide, failed to abolish the SB-inhibited PDGF-BB-induced rate of transcription of GAPDH, suggesting that SB is not dependent on ongoing protein synthesis to exert its effects on PDGF-BB-induced GAPDH transcription. Furthermore, measurement of GAPDH mRNA stability at various times after the inhibition of transcription with actinomycin D indicated that 5 mmol/L SB has no significant effect on the half-life of PDGF-BB-induced mRNA. The reduction in PDGF-BB-induced GAPDH expression by SB is probably caused by a cycloheximide-insensitive transcriptional mechanism. Thus, the inhibition of PDGF-BB-induced expression of GAPDH by SB suggests a link between SMC proliferation, energy consumption, and GAPDH gene upregulation.


Key Words: glyceraldehyde-3-phosphate dehydrogenase • sodium butyrate • platelet-derived growth factor • smooth muscle cells • mRNA


*    Introduction
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 *Introduction
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Proliferation of vascular SMCs is the key event in vascular proliferation disorders such as hypertension,1 2 3 atherosclerosis,2 3 4 and restenosis after balloon angioplasty.4 5 6 7 8 Many growth factors and cytokines modulate SMC proliferation. PDGF is considered as one of the predominant growth factors in the etiology of atherosclerosis, exhibiting mitogenic9 10 chemotactic,11 and vasoconstrictor12 effects on SMCs. Our previous study revealed that SB, a natural bacterial fermentation product of unabsorbed carbohydrates in mammalian colon,13 14 15 inhibits PDGF-induced rat SMC proliferation.16 Although SB has no effect on PDGF-BB-induced autophosphorylation of ß-PDGF-receptor and its association with phosphorylase C-{gamma} and ras GTPase, it inhibits PDGF-BB-induced c-myc expression significantly.

SB, a four-carbon short-chain fatty acid, has been shown to exhibit reversible pleiotropic effects on cells. Previous studies indicate that SB is a powerful promoter of cell differentiation17 18 19 20 21 with potent antiproliferative effects22 23 24 25 26 on a variety of normal and tumor cells. It is suggested that SB could be used for cell synchronization because it reversibly blocks the cell cycle at the early G1 phase.27 SB is also used in preliminary clinical trials to treat certain acute leukemias,28 29 and several stable butyrate derivatives have been developed to improve the therapeutic activity of SB.30 Furthermore, SB has been shown to inhibit high margarine diet-induced mammary tumorigenesis in rats.31 Additionally, SB has been shown to affect gene expression at a number of different levels including chromatin structure,23 32 33 34 transcription,35 36 37 38 mRNA stability,39 40 and transcriptional regulatory elements.41 42 43 44

GADPH, a glycolytic enzyme involved in cellular energy production, is generally thought to be an essential enzyme expressed ubiquitously at constant amounts under various physiological conditions. Lately, several studies revealed that expression of GAPDH is substantially and independently altered by cellular activities.45 46 47 48 49 50 During the course of our studies on inhibition of PDGF-BB-induced SMC proliferation by SB,16 we observed altered expression of GAPDH. In this study we describe the effect of SB on PDGF-BB-induced GAPDH expression and activity and discuss the possible significance of GAPDH expression in SMC proliferation.


*    Methods
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 *Methods
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Materials
Recombinant PDGF-BB was from Quality Controlled Biochemicals, Inc. Human GAPDH, 1.1-kb cDNA fragment, was from Clontech. Sodium butyrate was from Fluka. A7r5 rat aortic SMCs were from American Type Culture Collection. {alpha}-[32P]dCTP, {alpha}-[32P]UTP, and hyperfilm-MP were from Amersham Corporation. TRI reagent was from Molecular Research Center. Glyderaldehyde 3-phosphate, NAD, actinomycin D, and cycloheximide were from Sigma. CytoTox 96 was obtained from Promega.

Cell Culture
Rat SMCs were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 50 U/mL penicillin, and 50 µg/mL streptomycin, unless otherwise mentioned. The cells were grown to 80% to 90% confluence in a humidified atmosphere of 5% CO2 at 37°C, and culture media were changed every other day. For experiments, SMCs of third to eighth passages were used by plating onto 150-mm culture dishes. On reaching 80% to 90% confluence, cells were made quiescent by incubating in Dulbecco's modified Eagle's medium containing 0.2% fetal calf serum for 48 hours. Concentrations of PDGF-BB and sodium butyrate used in the studies were 20 ng/mL and 5 mmol/L, respectively, unless otherwise mentioned. All experiments were repeated a minimum of two times.

Cell Viability Assay
The CytoTox 96 colorimetric, nonradioactive cytotoxicity assay kit from Promega, was used for measuring cytotoxicity. The CytoTox 96 assay quantitatively measures LDH that is released due to cell lysis. To determine the cytotoxic effect of experimental additives, the culture media were removed at the end of the experiment, and cell monolayers were lysed using lysis buffer as described by the manufacturer. LDH activities in the culture media and in the corresponding cell lysates were measured using CytoTox 96 to estimate the percentage of total LDH released due to cell lysis and total LDH in the culture.

Preparation of Cell Extracts
Cells were washed twice with ice-cold phosphate-buffered saline and then scraped into ice-cold phosphate-buffered saline. Cell pellets were collected by centrifugation and maintained at 4°C during the rest of the procedure. Cell pellets were suspended in lysis buffer containing 0.01 mol/L Tris-HCl, pH 7.4, 0.25 mol/L sucrose, 0.001 mol/L EDTA, and 0.001 mol/L dithiothreitol48 and incubated on ice for 10 minutes. After incubation, the cell suspension was homogenized by repeated passage through a 27.5-gauge needle. The cell lysates were then centrifuged at 100 000g for 60 minutes at 4°C. The resulting cytosolic fractions were used for GAPDH assay and protein determination.

GAPDH Assay
GAPDH activity was determined by the modification of procedures described by Krebs51 by Pierce and Philipson.52 GAPDH activity was measured by following reduction of NAD to NADH spectrophotometrically. Required amounts of soluble cells extracts were preincubated in a medium containing 0.1 mol/L Tris, pH 8.5, 0.83 mmol/L NAD, 0.02 mol/L sodium fluoride, and 0.0033 mol/L L-cysteine for 5 minutes at room temperature. After preincubation, reactions were initiated by the addition of 0.0017 mol/L sodium arsenate and 0.23 mmol/L glyceraldehyde 3-phosphate. The reduction of NAD to NADH was recorded as a change in absorbance at 340 nm every 10 seconds over a 2-minute period. Control reactions were carried out similarly, except with no addition of 100 000g soluble protein extract. One unit of GAPDH activity was the amount that converts 1 nmol of substrate per minute at room temperature. Three different concentrations of each cytosolic fraction, in duplicate, were used for the measurement of GAPDH activity and the activity in the linear range was used for the analysis. The variation between the duplicate measurements was not >15%. The protein amounts were measured by using a Bio-Rad DC protein assay kit.

RNA Preparation and Northern Analysis
Total RNA was extracted using the TRI reagent method. Fifteen micrograms of total RNA was used for Northern analysis. Total RNA was denatured in sample-loading buffer containing ethidium bromide and electrophoresed through 1% agarose formaldehyde gel.53 RNAs were transferred to an Scheicher & Schuell MaxS nytran membrane by the downward alkaline transfer method54 using the Schleicher & Schuell turbo blotter transfer system. After transfer, RNA was fixed to the nytran membrane by ultraviolet cross-linking. The GAPDH cDNA probe was labeled by random priming using a New England Biolabs NEBlot kit and {alpha}-[32P]dCTP as recommended by the manufacturer. RNA blots were probed with random primer-labeled GAPDH cDNA.55 Autoradiography was performed using hyperfilm-MP with an intensifying screen at -70°C for 1 to 3 days. For quantitative comparison of mRNA levels, the hybridization signals were normalized to the quantities of 28 S rRNA in the samples. This was determined by scanning the intensity of ethidium bromide-stained 28 S rRNA using a Bio-Rad GS-670 densitometer. Quantitation of hybridization signals was accomplished by scanning the autoradiographs with the Bio-Rad GS-670 densitometer.

Nuclear Run-on Assay
Nuclei were isolated from cells in two 150-mm culture plates for each treatment using Nonidet P-40 lysis buffer.56 57 58 The nuclear pellets were suspended in 100 µL of 50 mmol/L Tris-HCl, pH 8.3, 40% (vol/vol) glycerol, 5 mmol/L MgCl2, and 0.1 mmol/L EDTA. Run-on transcription assays were initiated by adding 100 µL of reaction buffer containing 10 mmol/L Tris-HCl, pH 8.0, 5 mmol/L MgCl2, 0.3 mol/L KCl, 0.5 mmol/L each of ATP, CTP, and GTP, and 100 µCi of {alpha}-[32P]UTP (760 Ci/mmol). Run-on transcription assays were carried out at 37°C for 30 minutes. Reactions were terminated by adding 40 µL of 1 mg/mL RNase-free DNase I and incubation at 30°C for 5 minutes. Labeled RNA was purified as described in Current Protocols in Molecular Biology.58 Linearized GAPDH plasmid DNA from Ambion, Inc was denatured in 0.2 N NaOH for 30 minutes at room temperature, neutralized with 10 volumes of 6x SSC, and applied on to nitrocellulose membrane (2 µg/dot) using a Bio-Rad dot-blot apparatus. The cDNAs were fixed to the membrane by baking at 80°C under vacuum for 20 to 30 minutes. The blots were prehybridized for 3 hours at 65°C in 100 mmol/L TES-HCl buffer, pH 7.4, 0.2% sodium dodecyl sulfate, 10 mmol/L EDTA, 0.3 mmol/L NaCl, 1x Denhardt's solution and 200 µg/mL yeast tRNA. Hybridization was performed in the same buffer containing 0.5x106 cpm/mL of 32P-labeled nuclear RNA transcripts for 36 hours at 65°C. The blots were washed twice with 2x SSC at 65°C for 1 hour each. The blots were then incubated in 2x SSC containing 10 mg/mL of RNase A for 30 minutes at 37°C. After incubation, blots were washed once with 2x SSC for 1 hour at 37°C, air dried, and exposed to x-ray film at -70°C. Hybridization signals were quantitated using the Bio-Rad GS-670 densitometer.


*    Results
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 up arrowMethods
 *Results
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GAPDH mRNA Expression
Northern analyses were carried out to determine the concentration-dependent effect of SB on the expression of GAPDH mRNA in the absence and presence of PDGF-BB (Fig 1Down). Exposure of growth-arrested SMCs to PDGF-BB for 24 hours, induced the expression of GAPDH mRNA by about 2.4-fold over the no-addition control. SB inhibited this PDGF-BB induced GAPDH mRNA expression in a concentration-dependent manner, and at a 5 mmol/L concentration there was about 50% reduction in PDGF-BB-induced GAPDH mRNA upregulation. However, in the absence of PDGF-BB, SB had no significant effect on GAPDH mRNA level at any of the concentrations used.



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  		Figure 1. Concentration-dependent effect of SB on the GAPDH mRNA level. Serum-starved SMCs were treated with 0, 1, 3, and 5 mmol/L of SB, respectively, in the absence (lanes 1 to 4) or presence of PDGF-BB (lanes 5 to 8) for 24 hours. After incubation, total RNA was isolated and processed for Northern analysis as described in "Methods". A, Autoradiogram of Northern blot probed with GAPDH cDNA. B, Ethidium bromide-stained blot revealing 28 S rRNA. C, Densitometric values for GAPDH mRNAs were normalized to 28 S rRNA and expressed as fold difference compared with no-addition control.

To confirm that the downregulated PDGF-BB-induced GAPDH mRNA expression by butyrate is not due to a cytotoxic effect of SB, we measured the release of LDH as an indicator of cytotoxicity (Table 1Down). The percentage of total cellular LDH released due to cellular membrane damage appeared to be almost similar to that in SMCs treated with PDGF-BB in the absence or presence of different concentrations of SB. In the absence of PDGF-BB, there was an insignificant increase in LDH release, and this increase did not change in the presence of different concentrations of SB. These LDH release measurements and our previous cell viability analysis by trypan blue exclusion16 indicated that the concentrations of butyrate used in the study were not cytotoxic. Since 5 mmol/L SB was not cytotoxic and also inhibited PDGF-BB-induced SMC proliferation completely,16 we used this concentration for all further studies unless otherwise mentioned.


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  		Table 1. LDH Release From SMCs Treated With SB, PDGF-BB, or PDGF-BB Plus SB

Time-dependent studies were carried out to determine the time course of PDGF-BB-induced GAPDH mRNA expression and its response to SB (Fig 2ADown and Table 2Down). Growth-arrested SMCs exposed to PDGF-BB exhibited time-dependent effects on GAPDH mRNA expression. Although there was a decrease in the GAPDH mRNA level during the 0.5- and 1-hour incubations, its level increased with an increase in time of incubation. At the end of 20 hours of incubation in PDGF-BB, there was about a 3.9-fold increase in GAPDH mRNA level compared with the no-addition control. However, the PDGF-BB-induced increase in GAPDH mRNA level was significantly inhibited when cells were coincubated with PDGF-BB plus SB. At the end of 20 hours of incubation, there was about 60% inhibition of PDGF-BB-induced GAPDH mRNA expression. Similarly, preincubation of serum-starved SMCs with SB for 30 minutes followed by coincubation in SB plus PDGF-BB caused a similar extent of inhibition of the PDGF-BB-induced GAPDH mRNA level. On the other hand, incubation of SMCs with SB alone caused about a 20% decrease in the level of GAPDH mRNA at the end of a 20-hour incubation compared with the no-addition control.



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  		Figure 2. Time course of PDGF-BB-induced GAPDH gene expression and its inhibition by SB. Growth-arrested SMCs were treated for the indicated times with no addition (lane 1), SB (lane 2), preincubation in SB for 30 minutes and then with SB plus PDGF-BB (lane 3), PDGF-BB (lane 4), or PDGF-BB plus SB (lane 5) at 37°C. After the indicated time of incubation, total RNA was isolated and used for Northern analysis. A, Autoradiogram of GAPDH mRNA. B, Ethidium bromide-stained blot, revealing 28 S rRNA.


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  		Table 2. Summary of Time Course of GAPDH mRNA Expression Shown in Fig 1Up

GAPDH Activity
To determine whether the level of GAPDH mRNA was reflected in the level of GAPDH catalytic activity, cytosolic extracts were prepared for the measurement of GAPDH catalytic activity. We examined the effect of different concentrations of SB in the absence and presence of PDGF-BB on GAPDH activity of SMCs (Table 3Down). In the absence of PDGF-BB, serum-starved SMCs exhibited a slight increase in GAPDH activity in the presence of 1 to 3 mmol/L SB and no effect at 5 mmol/L SB. Exposure of SMCs to PDGF-BB resulted in about a 2.5-fold increase in GAPDH catalytic activity, and this activity was inhibited by butyrate in a concentration-dependent manner. At 5 mmol/L concentration, SB caused about 45% inhibition of PDGF-BB-enhanced activity.


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  		Table 3. Concentration-Dependent Effect of SB on PDGF-BB-Induced GAPDH Activity in Rat SMCs

Time course studies were carried out by exposing serum-starved SMCs to 5 mmol/L SB, in the absence and presence of PDGF-BB and measuring GAPDH activity at different time intervals of exposure up to 24 hours (Table 4Down). Cells exposed to PDGF-BB exhibited an increase in GAPDH activity after 6 hours of exposure and at the end of 24 hours of incubation there was about a 3-fold increase in GAPDH activity compared with no-addition control SMCs. Coincubation of SMC with PDGF-BB plus SB resulted in the inhibition of PDGF-BB-induced GAPDH activity throughout the exposure time, and at the end of 24 hours of incubation there was about 50% inhibition of PDGF-BB-induced GAPDH activity. However, compared with no-addition control, there was about a 1.5-fold increase in GAPDH activity. Similarly, preincubation of SMCs with SB for 30 minutes and subsequent incubation in PDGF-BB plus SB resulted in the inhibition of PDGF-BB-induced GAPDH activity throughout the incubation period. At the end of 24 hours of incubation, there was about a 1.3-fold increase in GAPDH activity over the no-addition control. In the absence of PDGF-BB, SB had no significant effect on GAPDH catalytic activity.


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  		Table 4. Time Course of GAPDH Activity in Rat SMCs

Transcription of GAPDH Gene
To determine whether the inhibition of PDGF-BB-induced GAPDH expression by SB is due to an altered rate of GAPDH gene transcription, nuclear run-on experiments were performed (Fig 3Down). Serum-starved SMCs exhibited an increase in the rate of transcription of GAPDH gene in response to PDGF-BB, and it was about 3-fold over the no-addition control. The PDGF-BB-stimulated increased rate of transcription of GAPDH gene was reduced by treating the cells with SB. The extent of reduction in the PDGF-BB-induced increased rate of transcription of the GAPDH gene was similar irrespective of whether SMCs were preexposed to SB or not before coincubation in PDGF-BB plus SB, and it was about 50%. However, SB by itself had no significant effect on GAPDH gene transcription.



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  		Figure 3. Effect of SB on GAPDH gene transcription. Quiescent SMCs were treated with no addition (lane 1), SB (lane 2), preincubation in SB for 30 minutes and then with PDGF-BB plus SB (lane 3), PDGF-BB (lane 4), or PDGF-BB plus SB (lane 5) for 24 hours. After incubation, nuclei were isolated and used for nuclear run-on assays as detailed in "Methods". In vitro labeled run-off RNA were used for probing the membranes containing immobilized GAPDH cDNA. Autoradiographs were quantified by densitometry and the relative rates of GAPDH transcription are presented.

To confirm that the inhibition of PDGF-BB-induced expression of GAPDH mRNA by SB was due to a transcriptional mechanism, investigations were carried out in the presence of inhibitors of transcription and protein synthesis (Fig 4Down). Exposure of SMCs to PDGF-BB for 24 hours caused an increase in GAPDH mRNA compared with the no-addition control. This increase in GAPDH mRNA was reduced to about 50% by treating SMCs with PDGF-BB plus SB. Treatment of SMCs with the transcription inhibitor, actinomycin D, alone caused about 20% reduction in GAPDH mRNA compared with the no-addition control. An almost similar extent of reduction in GAPDH mRNA level was observed in SB-, PDGF-BB-, and PDGF-BB plus SB-treated cells when they were coincubated with actinomycin D. In the presence of cycloheximide, a protein synthesis inhibitor, PDGF-BB-enhanced GAPDH mRNA was significantly reduced (Fig 4Down). However in PDGF-BB plus SB-treated cells, cycloheximide treatment did not relieve the inhibition of PDGF-BB-induced GAPDH mRNA level by SB. The extent of GAPDH mRNA expression in PDGF-BB plus SB-treated SMCs was almost similar in the absence and presence of cycloheximide. This observation suggests that inhibition of PDGF-BB-induced GAPDH mRNA level by SB could be due to an alteration in transcriptional mechanism(s), which may not depend on on-going protein synthesis.



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  		Figure 4. Effect of inhibition of transcription and protein synthesis on GAPDH mRNA levels. Growth-arrested SMCs were treated with no addition (lanes 1 to 3), SB (lanes 4 to 6), PDGF-BB (lanes 7 to 9), or PDGF-BB plus SB (lanes 10 to 12) in the absence (lanes 1, 4, 7, and 10) or presence of 1 µg/mL actinomycin D (lanes 2, 5, 8, and 11) or 1 µg/mL cyclohexamide (3, 6, 9, and 12) for 20 hours. Total RNA was isolated and subjected to Northern analysis using a GAPDH cDNA probe as described in "Methods." A, Extent of hybridization to the GAPDH cDNA probe was determined by densitometry. B, Results were normalized to the amount of 28 S rRNA. C, Data are presented as the fold difference in the extent of hybridization compared with no-addition control.

SB may also potentially destabilize PDGF-BB-induced GAPDH mRNA. To account for this possibility, SMCs were exposed to PDGF-BB and PDGF-BB plus SB in the presence of actinomycin D, and the rate of decline in GAPDH mRNA level was measured over a period of 24 hours (Fig 5Down). Both PDGF-BB- and PDGF-BB plus SB-treated cells exhibited a decline in the rate of GAPDH mRNA level in the presence of actinomycin D, and the rate of decline was not significantly different in PDGF-BB- and PDGF-BB plus SB-treated SMCs. The half-lives of GAPDH mRNA were about 22 and 17.5 hours in PDGF-BB-and PDGF-BB plus SB-treated SMCs, respectively. These observations indicate that inhibition of PDGF-BB-induced GAPDH mRNA expression by SB could not be due to destabilization of PDGF-BB-induced GAPDH mRNA.



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  		Figure 5. Stability of GAPDH mRNA in PDGF-BB- and PDGF-BB plus SB-treated rat smooth muscle cells. Serum-starved cells were treated with PDGF-BB or PDGF-BB plus SB in the presence of 1 µg/mL actinomycin D for the indicated time periods. Levels of GAPDH mRNA were determined by Northern analysis. Amounts of GAPDH mRNA were quantitated by densitometric scanning of the autoradiogram. The amounts of GAPDH mRNA at the indicated times were normalized to the respective 28 S rRNA. Autoradiographic optical densities were plotted as a function of time.

To be sure that the concentrations of actinomycin D and cycloheximide used in the investigations were not cytotoxic and the data obtained were not a reflection of their cytotoxicity, measurement of LDH release due to cell lysis were performed (Table 5Down). The percentage of total cellular LDH release was <27% for all the treatments except for the no-addition control cells treated with actinomycin D. The percentage of total LDH released in the presence of cycloheximide was comparatively less in all the treatments, which could be due to inhibition of LDH protein synthesis.


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  		Table 5. Measurement of Actinomycin D and Cycloheximide-Stimulated Cytotoxicity


*    Discussion
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 *Discussion
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In this study we have demonstrated that exposure of quiescent SMCs to PDGF-BB caused a time-dependent increase in GAPDH expression. These SMCs exhibited 2.5- to 3.9-fold increases in GAPDH mRNA levels and comparable 2.5- to 3.0- fold increases in the catalytic activity of GAPDH at the end of 20 to 24 hours of treatment with PDGF-BB. The increase in the GAPDH mRNA level appears to be due to an increased rate of GAPDH gene transcription. Nuclear run-on measurements revealed about a 3-fold increase in the rate of GAPDH gene transcription in PDGF-BB-treated SMCs compared with no-addition control SMCs. Treatment with SB significantly inhibited PDGF-BB-induced upregulation of GAPDH in a time- and concentration-dependent manner. At 5 mmol/L SB, the extent of inhibition of PDGF-BB-induced GAPDH mRNA was about 50% to 60%, and this decreased level of mRNA was reflected in an almost similar extent of inhibition of GAPDH catalytic activity at the end of 20 to 24 hours of incubation. This fall in the level of GAPDH expression appeared to be due to a reduction in the rate of GAPDH gene transcription. Nuclear run-on assays demonstrated that the rate of transcription of PDGF-BB-stimulated GAPDH gene was reduced to about 50% by SB, and the reduction in GAPDH expression was not due to cytotoxicity.

GAPDH is a key regulatory glycolytic enzyme that is involved in cellular metabolism. Until recently this enzyme was thought to be expressed constitutively at constant amounts in cells. In fact, expression of GAPDH mRNA was frequently used as an internal standard in gene transcription studies. However, several studies reported that expression of GAPDH mRNA was significantly altered due to increased cell growth both in normal45 and cancerous cells,46 47 on exposure to hormone,48 growth factors, serum,45 and drugs,49 and in response to hypoxia.50 Our studies indicate that GAPDH expression was altered depending on the proliferative state of SMCs. Exposure of quiescent SMCs to PDGF-BB, which caused 4- to 5-fold increases in SMC proliferation,16 also stimulated about a 3-fold increase in the rate of GAPDH gene transcription, 2.5- to 3.9-fold increases in GAPDH mRNA level, and 2.5- to 3-fold increases in GAPDH catalytic activity at the end of 24 hours of exposure. These observations suggest a relationship between SMC proliferation, cellular energy demand, and GAPDH expression. This relationship is substantiated by the effect of SB on PDGF-BB-induced SMC proliferation and GAPDH expression. SB inhibited both PDGF-BB-induced SMC proliferation16 and GAPDH expression in a concentration-dependent manner, suggesting that the GAPDH expression is associated with cell proliferation. Since cell proliferation is an energy-demanding process caused by increased cellular metabolism, it requires an energy-generating reaction. This need appears to be provided by the upregulation of GAPDH. Thus, the relevance of altered GAPDH expression in PDGF-BB- and PDGF-BB plus SB-treated cells appears to be evident. A similar relationship between cell proliferation, energy demand, and GAPDH expression was observed in normal, cancerous, and transformed cells.45 46 47 59 60 61 62 63 In fact, GAPDH was identified as one of the cancer-associated proteins.46 These observations indicate a link between increased expression of GAPDH, energy consumption, and rapid cell growth rate. This possible link could offer a means to suppress growth in cancer and atherosclerosis by diminishing the key reaction in glycolysis. In this regard, the use of SB appears to be promising. Although no other previous studies have shown an effect of SB on GAPDH expression, our present study revealed that SB significantly inhibited PDGF-BB-induced GAPDH mRNA expression as well as its activity, in addition to inhibition of PDGF-BB-induced SMC proliferation. This observation suggests that one of the possible mechanisms of inhibition of SMC proliferation could be by limiting the cellular energy generation through the regulation of GAPDH expression.

Although SB at a 5 mmol/L concentration inhibited PDGF-BB-induced SMC proliferation completely,16 it did not completely inhibit PDGF-BB-stimulated GAPDH expression, irrespective of whether SMCs were preexposed to SB or not. Because SB is a potent antiproliferative agent22 23 24 25 26 and a strong promoter of cell differentiation,17 18 19 20 21 it may promote the conversion of SMCs to the contractile phenotype by inhibiting the proliferative phenotype. Probably this process requires a certain amount of metabolic energy although not as much as may be required for cell proliferation. This possibility may explain the incomplete inhibition of PDGF-BB-induced GAPDH expression by SB. However, there may be other explanations for the incomplete inhibition of PDGF-BB-induced GAPDH expression by SB. Evidence indicates the presence of GAPDH in the nucleus, exhibiting functions that are unrelated to glycolysis. GAPDH is identified as a DNA-binding nuclear protein influencing transcription and replication of DNA.64 65 66 67 68 69 It is recognized as a DNA repair enzyme exhibiting uracil DNA glycosylase activity,67 which is cell cycle-regulated in human fibroblasts.68 In addition, GAPDH is shown to affect human parainfluenza virus type 3 gene expression through interaction with viral cis-acting regulatory RNAs.69 Because glycolysis does not occur within the nucleus, these observations suggest that GAPDH may have a dual role in cellular function: one related to glycolysis in the cytoplasm and the other associated with its presence in the nucleus. The observation that GAPDH is present in both cytoplasm and nucleus raises the possibility that it may shuttle between the two compartments. Although as yet there is no direct evidence that nuclear residence of GAPDH requires any protein modification, it is shown that only tyrosine-phosphorylated LDH, another glycolytic enzyme is restricted to the cell nuclei.70 At the moment, we do not know whether SMC nuclei contain GAPDH, but its presence as a nonhistone nuclear protein is shown in other cells.64 65 66 67 Because it is well known that SB causes acetylation and phosphorylation of chromatin proteins,17 18 19 20 21 22 23 24 71 72 it is possible that in PDGF-BB plus SB-treated SMCs, a fraction of GAPDH may be translocated to the nuclei as a result of protein modification,, thereby regulating DNA replication. This possibility could also explain why there was no complete inhibition of PDGF-BB-induced GAPDH expression by SB. Further investigations are required to identify potential nuclear GAPDH and its possible role in the inhibition of PDGF-BB-induced SMC proliferation by SB. In this context, it is interesting to note that replication of M-13 DNA is inhibited by GAPDH.66

Many reports indicate that SB modulates gene expression in a variety of cells, and within a cell, it causes different effects on specific mRNAs.36 43 73 Although in our study PDGF-BB-induced upregulation of GAPDH involves both transcriptional and translational mechanisms, inhibition of PDGF-BB-induced upregulation of GAPDH by SB appears to involve a transcriptional mechanism. Nuclear run-on analysis demonstrated that reduction in the PDGF-BB-induced GAPDH mRNA level in PDGF-BB plus SB-treated cells was caused by a reduction in the rate of transcription of GAPDH mRNA. Furthermore, transcription and translation inhibitor studies revealed that SB-inhibited, PDGF-BB-induced GAPDH expression was not due to altered message stability or activation of a labile "repressor" protein. In the absence of new transcription, the half-lives of GAPDH mRNAs of PDGF-BB- and PDGF-BB plus SB-treated SMCs were not significantly altered, and in the absence of new protein synthesis, the SB-inhibited GAPDH expression was not relieved. However, we do not know the exact transcriptional mechanism by which SB alters GAPDH expression. A large amount of the literature suggests that SB alters chromatin structure by modifying chromatin proteins such as histones and nonhistones by acetylation and phosphorylation.17 23 33 34 74 75 These and other changes in chromatin proteins are believed to disrupt nucleosome interaction with the key regulatory regions of DNA involved in transcriptional control, thus enabling transcription factors to gain access to their recognition sequences and activate transcription. Evidence also indicates that SB may alter gene activation by inducing both hypomethylation and hypermethylation of DNA.74 75 Other studies report that SB regulates transcription through modification of activities of selected transcription regulatory proteins, which affects their interaction with proteins bound to promoters. These observations suggest that specific sequences may be acted on in trans by SB-dependent factor modification.42 43 44 76 Because early growth response genes such as c-myc, c-fos, and c-jun affect cell proliferation by modulating expression of target genes by regulating their transcription rate, it is possible that our observation of inhibition of PDGF-BB-induced c-fos, c-jun, and particularly c-myc by SB16 may have significance in the inhibition of PDGF-BB-induced GAPDH expression as well as SMC proliferation. Although our present study did reveal cell proliferation-related regulation of GAPDH expression, we do not have any direct evidence that c-myc, c-fos, or c-jun may be involved in the regulation of GAPDH gene expression. In this context it is interesting to note that Mansur et al68 reported a cell cycle-regulated GAPDH, which exhibited a characteristic temporal sequence of expression in relation to DNA synthesis. However, further studies are required to determine which, if any, of the mechanisms mentioned mediated the inhibition of PDGF-BB-induced GAPDH expression by SB.


*    Selected Abbreviations and Acronyms
 
LDH = lactate dehydrogenase
PDGF = platelet-derived growth factor
SB = sodium butyrate
SMCs = smooth muscle cells


*    Acknowledgments
 
This work was supported by a grant from the Clayton Foundation for Research, Houston, Tex. We also thank Elaine Marsh and Tanaisha Johnson for the preparation of this manuscript and Trupti Joshi for technical assistance.

Received February 6, 1997; accepted August 12, 1997.


*    References
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