Neurochemical Research, Vol. 22, No. 4, 1997, pp. 351-362

Regulation of Antioxidant Enzyme Expression by NGF* Deepa Sampath1 and Regino Perez-Polo1,2 (Accepted September 9, 1996)

The rapid decreases in viability seen in H2O2-treated PC 12 cells reflect enhanced susceptibility of neural cell types to oxidant injury. The dose-response relationship between NGF concentration and survival after H2O2 treatment resembles that for NGF effects on PC 12 survival in serumless medium. Previously we have shown that NGF treatment enhances the activity of GSH-Px and catalase which catalyze the degradation of H2O2. Here in order to ascertain whether NGF stimulates transcription, affects mRNA stability, or acts post-transcriptionally, we measured catalase and GSHPx mKNA half-lives. While both catalase and GSH-Px transcripts are stable with a relatively long half life and a gradual decay in mRNA levels, NGF had different effects on their stability. NGF had marked effects on catalase mRNA stability. The catalase gene has a 3' flanking region with T-rich clusters and CA repeats known to be susceptible to regulation by destabilization or ubiquination. NGF maintained catalase mRNA levels of actinomycin D (ACT-D) treated PC 12 cells at twice that of cells exposed to ACT-D alone, delaying the rate of decay for catalase mRNA for 24 h. The NGF induction of GSH-Px and catalase mRNA was inhibited by cycloheximide (CHX) treatment with a slight decrease in their mRNA levels due to prolonged exposure to CHX. When the CHX treatment was delayed relative to the NGF treatment there was no effect on NGF effects on catalase and GSH-Px. The GSH-Px gene has conserved sequences in the open reading frame and 3' untranslated region which forms a stem-loop structure necessary for the incorporation of Se into this selenoprotein. While Se is important in stabilizing GSH-Px transcripts, it did not affect transcription rates or mRNA stability. These results are consistent with the hypothesis that NGF regulates catalase and GSH-Px expression via a primary effect on transcription factor pathways.

KEY WORDS: NGF; catalase; GSH; antioxidant; cell death.

emic tissue with free radical scavengers, superoxide dismutase (SOD), or catalase attenuates neuronal damage in experimental models of ischemia-reperfusion (79), despite the complications presented by the short half-life of these enzymes in circulation (9). Therefore, the therapeutic value of agents like nerve growth factor (NGF) which regulate cellular redox by stimulating GSH-Px and catalase (10-12) could be significant in the setting of ischemic injury. The PC 12 rat pheochromocytoma is a useful model for the study of free radical induced neuronal damage and death (10-12). The dose-response relationship between NGF concentration and survival after H2O2 treatment resembles that for NGF effects on PC 12 survival

INTRODUCTION While glutathione peroxidase (GSH-Px) and catalase are widely expressed in all tissues across species (1-3), the CNS has very low endogenous levels of these protective enzymes and is thought to have an increased susceptibility to oxidative damage due to its high levels of oxygen consumption and the essentially non-regenerative nature of CNS neurons (4-6). Infusion of isch1

University of Texas Medical Branch, Galveston, Texas 77555-0652. Address reprint requests to: J. Regino Perez-Polo, Ph.D., University of Texas Medical Branch, Galveston, Texas 77555-0652. Tel.: 409-7723668; fax: 409-772-8028; e-mail: [email protected]. * Special issue dedicated to Dr. Eduardo Soto.

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352 in serumless medium. NGF treatment for 5 days enhances the activity of GSH-Px and catalase which catalyze the degradation of H2O2, while LDH activity is decreased, consistent with an NGF-induced shift in PC 12 glucose metabolism from anaerobic glycolysis to the Krebs cycle (10-12). NGF-induced increases in catalase activity correlate with increased levels of catalase protein and mRNA by immunoblotting and RT-PCR analysis (10-12). Addition of NGF to PC12 cells increases GSH-Px and catalase transcripts by 2-3 fold over 72 hours (11,12). The mechanism by which NGF regulates GSH-Px and catalase expression is not known. A change in the abundance of a particular RNA may reflect a change in the transcriptional activity of the gene, a change in the rate of turnover of the specific RNA or both. The rate of degradation of a specific RNA can be measured when the synthesis of all RNA molecules is blocked. The metabolic stability of mRNA can be defined by its half life in the cell. Half lives are frequently measured by blocking mRNA synthesis with agents like Actinomycin D, a DNA intercalating agent which preferentially intercalates into transcriptionally active nucleosomes, isolating cytoplasmic RNA after different time intervals and monitoring the rate of loss of a particular transcript with a message specific probe. The decay rate constant is then obtained from the slope of a semilogarithmic plot of mRNA concentration as a function of time, the plot being defined as:

where [mRNA]t = mRNA concentration at time t, [mRNA]0 = mRNA concentration at time 0, and kobs = ln 2/t1/2 where t1/2 is the effective half life.

Further Stability of mRNA molecules along with transcription, mRNA processing, translational efficiency, and posttranslational modifications is an important determinant of protein levels in the cell. Unlike transcriptional control, the molecular mechanisms that govern post-transcriptional RNA processing are poorly understood. Much of the information regarding factors that regulate mRNA processing comes from prokaryotes. For several RNAs with a rapid turnover rate, sequences in the 3' untranslated regions that confer an inherent instability to the RNA have been detected. Other factors influencing

Sampath and Perez-Polo the rate of degradation of the mRNA molecule are, the length of the poly A tail (13,14), stem loop structures (15), metal responsive elements (16), sequences within the coding regions (17) and proteins with an ability to bind the RNA (18,19). While the regulatory elements that control the transcription of GSH-Px and catalase are well characterized, not much is known about the factors that regulate these mRNA species post-transcriptionally. NGF is known to generate cellular responses via a variety of signal transduction pathways and exerts posttranscription control on many mRNA species including those for growth associated protein (20), VGF (21), neuropeptide Y and neurofilament (22). However, the exact nature of the NGF-induced mechanism of post-transcription control, whether by direct interaction with conserved sequences on the mRNA or through the induction of trans-binding proteins, is unclear (20,23). Our primary aim was to determine the rate of degradation of GSH-Px and catalase mRNA and to determine the effects of NGF on the mRNA stability of GSH Px and catalase, if any. We found that the half life of GSH Px mRNA was ~45 hours while that of catalase was ~42 hours. It was also determined that NGF had no effects on the stability of GSH Px mRNA but stabilized that of catalase.

EXPERIMENTAL PROCEDURE RPMI 1640 medium was obtained from GIBCO (Grand island, NY). Fetal calf and horse sera were obtained from Irvine Scientific (Santa Ana, CA). Mouse 2.5S NGF was prepared as previously described (24). Moloney murine leukemia virus reverse transcriptase and DNA ladders were from GIBCO BRL (Grand Island, NY). Deoxynucleoside triphosphates were from Pharmacia (Piscataway, NJ). a32-P dCTP was obtained from ICN Biomedicals (Costa Mesa, CA). Primers for PCR reactions were obtained from the Oligonucleotide Synthesis Laboratory at UTMB. Actinomycin D and Oligo dT15 was purchased from Boehringer Mannheim (Indianapolis, IN). Taq DNA polymerase was from Perkin Elmer Cetus (Norwalk, CT). Ultra pure gelatin, sodium dodecyl sulphate, acrylamide, N.N-methylene-bis-acrylamide, ammonium persulphate, and N,N,N',N'-tetramethylenediamine (TEMED) were obtained from Bio-Rad (Richmond, CA). All other reagents were obtained from Sigma Chemical Co. Cell Culture. The PC 12 pheochromocytoma line was the generous gift of Dr. Lloyd Greene. Stock cultures of PC 12 cells were maintained in a medium consisting of RPMI 1640 supplemented with (V/V) 5% fetal calf serum and 5% horse serum which is called complete medium. At alternate feedings, medium containing PSN antibiotic mixture (5 mg penicillin + 5 mg streptomycin + 10 mg neomycin/ml) was used at a concentration of 1% (V/V). Cells were grown in a humidified atmosphere containing 5% CO2. Cultures were split once weekly in a 1:2 ratio by vigorous shaking and trituration. Time Course of Actinomycin D Treatment on PC12 Cells. Cells were plated at a density of 6 x 106 cells/T250 flask (Falcon). Acti-

NGF and Antioxidant Enzymes nomycin D (Act D) was dissolved in absolute ethanol and added at a concentration of 2 (ig/ml to PC 12 cells at various times such that a time course of Actinomycin D treatment for 48 hours, 40 hours, 32 hours, 24 hours, 16 hours, 8 hours and 0 hour was obtained. Cells were harvested at the same time, and the flasks frozen at -80°C for subsequent RNA insolation. Effect of Co-Treatment with NGF and Actinomycin D Treatment on PC12 Cells. Cells were plated at a density of 6 X 106 cells/1250 flask (Falcon). For the experiment one flask of PC12 cells served as the control, one flask of PC 12 cells received Actinomycin D in 100% ethanol (2 ng/ml) for 24 hours while another flask received the same for 48 hours. A third flask of PC 12 cells was co-treated with both Actinomycin D in 100% ethanol (2 ug/ml) and NGF (100 ng/ml) for 24 hours and a fourth flask of PC 12 cells was cotreated with Actinomycin D in 100% ethanol (2 Hg/ml) and NGF (100 ng/m) for 48 hours. Cells were harvested at the same time, and the flasks frozen at -80°C for subsequent RNA insolation. RNA Isolation. RNA isolation was performed using the method of Ceca and Chomczynski 1986. Briefly, the cells were resuspended in 650ul of guanidine thiocyanate containing B mercaptoethanol (solution D). Then equal volumes of phenol (H2O saturated phenol), 0.1 volume of 3M NaOAc pH 5.1, and 0.2 volume of chloroform: isoamylalcohol (49:1) was added. The mixture was vortexed vigorously and kept on ice for 15 min. after which it was centrifuged at 14,000 g for 20 min in a microfuge to separate the aqueous from the phenol phase. The aqueous phase was then transferred to a fresh tube and an equal volume of 100% ice-cold isopropanol was added to precipitate the RNA. The RNA pellet was redissolved in solution D and precipitated with an equal volume of ice-cold isopropanol. The pellet was washed with 70% ethanol and dissolved in Tris-Edta pH 8.0. The amount of RNA contained in each sample was determined by absorbance at 260 run using a spectrophotometer, while the quality of the RNA sample was analyzed on 1.2% formaldehyde agarose gel. Reverse Transcription and PCR Amplification. In general all RTPCR determinations were carried out using linear ranges of amplifications and standards as outlined in previous studies (10-12). 2.4 ug total RNA was reverse transcribed using the 3' reverse primer for the catalase mRNA, and using oligo dT15 for the GSH Px and cyclophilin mRNA species. The reaction mixture contained 50 mM KC1, 10 mM Tris (pH 8.3), 2.0 mM MgC12, 0.01% gelatin, 15 pmol 3' catalase reverse primer or 0.16 ug oligo dT15, 10 units Rnasin, 200 units MMLV-RT and dNTP's at a concentration of 0.5 mM each. The reaction was incubated at 37°C for 1.5 hours and then terminated by heating to 90°C for 5 minutes. PCR amplification of the cDNA was then performed by adding 15 pmol each of the forward and reverse primers for catalase, GSH Px, and cyclophilin, 1 X PCR buffer, 200 mM of each dNTP, and 2.5 units of Taq DNA polymerase per assay. To monitor the reaction 5 (iCi of A32-P dCTP was added to each reaction tube. After PCR amplification, the mineral oil was removed by chloroform extraction and the samples analyzed by electrophoresis. The gels were stained with ethidium bromide, photographed, and exposed to Kodak XAR film. The cDNA length was estimated by comparison of Rf values with those observed for 123 bp ladder fragments. The catalase forward and reverse primers amplified a 493 bp segment from nucleotide 1079 to 1572 of the rat catalase cDNA sequence (25), while the GSH Px PCR product was 362 bp ranging from nucleotide 529 to 891 of the rat GSH Px cDNA sequence (26). The cyclophilin PCR product was 224 bp ranging from nucleotide 204 to 429 of the rat cyclophilin cDNA. For quantification, PCR bands on the autoradiogram were scanned using a densitometer in transparent mode and the GSH-Px and catalase signal normalized to the corresponding cyclophilin signal from the same RNA, which was then expressed as a

353 percentage of the value obtained for control PC12 cells (taken as 100%). Effect of Cycloheximide Pretreatment on the Induction of GSHPx and Catalase mRNA by NGF in PC12 Cells. PC12 cells were plated at 6 X 106 cells/T250 flask into five flasks coated with Poly D lysine. Cells were fed every other day. Addition of cycloheximide, NGF, or both was done at every media change, depending on the experimental requirements. In each set of experiments, a three day protocol with the following conditions were set up. One flask was treated with cycloheximide (5 ug/ml) on day 1, followed by addition of NGF (100 ng/ml) for day 2 and 3; a second flask received NGF (100ng/ml) on day 1, with addition of cycloheximide (5 ug/ml) on day 2, and NGF (100ng/ml) again on day 3 a third flask received NGF (100 ng/ml) on days 1 and 2, with addition of cycloheximide (5 ug/ml) on day 3. A fourth flask was used as a reference point and did not receive any NGF or cycloheximide, while a fifth flask was treated with NGF (100 ng/ml) on days 1, 2, and 3. After 3 days the flasks were set aside for RNA isolation. Effect of a Co-Treatment of NGF and Cycloheximide on the NGF Induction of GSH-Px and Catalase mRNA. PC 12 cells were plated at 6 X 106 cells/T250 flask into a total of eleven flasks and the following conditions were set up. One flask was treated with NGF 100 ng/ml 24 hours, another flask was cotreated with NGF (100 ng/ml) and cycloheximide (5 ug/ml) for 24 hours, a third flask was used as an reference point and did not receive NGF or cycloheximide, and lastly a time course of cycloheximide treatment (5 (ig/ml) for 8, 16, or 24 hours was established. After 24 hours, cells were harvested for RNA insolation. Preparation of Nitrocellulose Filters. 5 ug of pCat10, a plasmid containing the rat catalase cDNA, 5 ug of p123, a plasmid containing ribosomal DNA sequences and 5 ug of the PCR amplimer for GSH Px (obtained by RT-PCR of 2.5ug of total RNA from PC 12 cells) were denatured at 99°C for 5 min, and applied to the nitrocellulose in a slot-blot manifold and allowed to filter onto the membrane for 30 min. The membranes were then washed with one time with 6 X SSC and UV irradiated at 1200 joules to cross link the DNA to the filter.

RESULTS Rates of Degradation for GSH-Px mRNA. The rate of degradation of GSH-Px mRNA was measured in PC 12 cells after addition of 2 ug/ml Actinomycin D at various times (Fig. 1). There was a steady decline in GSH Px mRNA levels over 48 hours. The data shown are a sample of three independent experimental measurements and are relative to the signal obtained for the corresponding cyclophilin mRNA (ie. GSHPx/Cyclophilin) and expressed as a percentage of the control value. In this experiment the half-life for GSHPx was estimated to be ~ 45 hours. After 48 hours of treatment with Actinomycin D, the levels of GSH-Px mRNA declined to 48% of the control. Rates of Degradation for Catalase mRNA. The rate of decay for catalase mRNA also showed a steady decline over time up to 48 hours and was estimated to be ~ 42 hours. Data are a sample representative of three

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Fig. 1. a). Represents the PCR products for GSH-Px (367bp) and cyclophilin (246bp) mRNA in PC 12 cells treated with 0 ug/ml Actinomycin D (lane 1) or 2 ug/ml Actinomycin D for 8 hours (lane 2), 16 hours (lane 3), 24 hours (lane 4), 32 hours (lane 5), 40 hours (lane 6), and 48 hours (lane 7). The data shown are a sample of three independent experimental measurements, b). Represents the rate of decay for GSH-Px mRNA over 48 hours in PC 12 cells treated with 2 ug/ml Actinomycin D. Data shown are the mean of three independent experimental measurements. For quantification, PCR bands on the autoradiogram were scanned using a densitometer in transparent mode and the GSH-Px signal normalized to the corresponding cyclophilin signal from the same RNA, which was then expressed as a percentage of the value obtained for control PC 12 cells (taken as 100%).

independent experimental measurements and are expressed relative to the signal obtained for the cyclophilin from the same RNA (ie. catalase/ Cyclophilin) and expressed as a percentage of the control. After 48 hours of treatment with Actinomycin D, the levels of GSH-Px mRNA declined to 41% of the control (Fig. 2). Effect of NGF on the Rates of Degradation of GSHPx mRNA. The rate of degradation for GSH-Px mRNA in PC 12 cells treated with 2 ug/ml Actinomycin D for 24 or 48 hours were compared to the decay rates of GSH-Px mRNA in PC 12 cells co-treated with 2 jig/ml Actinomycin D and 100 ng/ml NGF for 24 or 48 hours; as well as to the levels of GSH-Px mRNA in PC 12 cells that served as a control.

The level of GSH-Px mRNA in Actinomycin treated cells at 24 hours was 35% of control as compared to 40% in cells co-treated with Actinomycin D and NGF. At 48 hours, Actinomycin D treated cells had 25% of the control value of GSH-Px mRNA as compared to 32% in cells co-treated with Actinomycin D and NGF. We conclude that addition of NGF had no effect on the rate of degradation of GSH-Px mRNA in the Actinomycin D treated PC 12 cells at either 24 or 48 hours. The data shown are a sample of two independent experimental measurements and are relative to the signal obtained for the corresponding cyclophilin mRNA (ie. HPx/Cyclophilin) and expressed as a percentage of the control value (Figure 3).

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Fig. 2. a). Represents the PCR products for catalase mRNA (492bp) in PC 12 cells treated with 2.0 (ig/ml Actinomycin D (lane 1) or 2 ug/ml Actinomycin D for 8 hours (lane 2), 16 hours (lane 3), 24 hours (lane 4), 32 hours (lane 5), 40 hours (lane 6), and 48 hours (lane 7). The data shown are a sample of three independent experimental measurements, b). Represents the rate of decay for catalase mRNA over 48 hours in PC 12 cells treated with 2 ug/ml Actinomycin D. Data shown are the mean of three independent experimental measurements. For quantification, PCR bands on the autoradiogram were scanned using a densitometer in transparent mode and the catalase signal normalized to the corresponding cyclophilin signal from the same RNA, which was then expressed as a percentage of the value obtained for control PC12 cells (taken as 100%).

Effect of NGF on the Rates of Degradation of Catalase mRNA. In PC 12 cells treated with 2 ug/ml Actinomycin D for 24 or 48 hours, the levels of catalase mRNA declined steadily while cells co-treated with 100 ng/ml NGF and 2 ug/ml Actinomycin D showed a retardation in the rate of decay over 24 hours. At 24 hours, the levels of catalase mRNA in PC 12 cells co-treated with NGF and Actinomycin D was twice as much (72% of control) as compared to catalase mRNA levels in PC 12 cells treated with Actinomycin D alone (37% of control). However, the levels of catalase mRNA did not differ significantly from each other after 48 hours of either co-treatment with Actinomycin D and NGF or treat-

ment with Actinomycin D alone. Actinomycin D is toxic to PC 12 cells over time. Cells cotreated with NGF and Actinomycin D for 48 hours are protected from cell death due to the protective effects of NGF, but may no longer be able to respond to the stimulatory effects of NGF on gene expression. Data are a sample representative of two independent experimental measurements and are expressed relative to the signal obtained for the cyclophilin from the same RNA (ie. catalase/Cyclophilin) and expressed as a percentage of the control (Fig. 4). Effect of Cydoheximide Pretreatment on the Induction of GSH-Px and Catalase mRNA by NGF. The re-

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Fig. 3. a). Represents the PCR products for GSH-Px (367bp) and cyclophilin (246bp) mRNA in PC12 cells used as a control (lane 1), PC12 cells treated with 2 ug/ml Actinomycin D for 24 (lane 2) or 48 hours (lane 4), and PC 12 cells co-treated with 2 ug/ml Actinomycin D and 100 ng/ml NGF for 24 (lane 3) or 48 hours (lane 5). The data shown are a sample of two independent experimental measurements, b). Represents the effect of NGF on the rate of decay of GSH-Px mRNA. Data shown are the mean of two independent experimental measurements. For quantification, PCR bands on the autoradiogram were scanned using a densitometer in transparent mode shown are the mean of two independent experimental measurements. For quantification, PCR bands on the autoradiogram were scanned using a densitometer in transparent mode and the GSH-Px signal normalized to the corresponding cyclophilin signal from the same RNA, which was then expressed as a percentage of the value obtained for control PC12 cells (taken as 100%).

suits of two different treatment protocols are presented in Fig. 5. The first paradigm involved i) addition of cycloheximide to PC 12 cells on day 1, which was then washed off by changing the media prior to the addition of NGF on day 2 and 3. This resulted in a complete inhibition of NGF induction of GSH-Px and catalase mRNA (lanes 2 and 6) as compared to the value in control PC 12 cells (lanes 1 and 5). In fact, the amounts of GSH-Px and catalase mRNA in these cells were lower than the control value (lanes 2 and 6). ii) A second set of PC 12 cells received NGF on day 1, which was

washed off by media change prior to the addition of cycloheximide on day 2, which was washed off again prior to the readdition of NGF on day 3 and had GSHPx mRNA that was 50% higher than the control value (lane 3) and catalase mRNA that was 30% higher than the control value (lane 7). iii) The third set of PC 12 cells received NGF on day 1 and 2, followed by the addition of cycloheximide on day three and had GSH-Px mRNA that was 120% higher than the control value (lane 4) and catalase mRNA that was 100% higher than the control value (lane 8). This increase in GSH-Px and catalase

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Fig. 4. a). Represents the PCR products for catalase (492bp) mRNA in PC 12 cells used as a control (lane 1), PC 12 cells treated with 2 )J.g/ml Actinotnycin D for 24 (lane 2) or 48 hours (lane 4), and PC 12 cells co-treated with 2 ug/ml Actinomycin D and 100 ng/ml NGF for 24 (lane 3) or 48 hours (lane 5). The data shown are a sample of two independent experimental measurements, b). Represents the effect of NGF on the rate of decay of catalase mRNA. Data shown are the mean of two independent experimental measurements. For quantification, PCR bands on the autoradiogram were scanned using a densitometer in transparent mode and the catalase signal normalized to the corresponding cyclophilin signal from the same RNA, which was then expressed as a percentage of the value obtained for control PC12 cells (taken as 100%).

mRNA reflects the length of exposure to NGF prior to addition of cycloheximide. In the second experimental paradigm additions of cycloheximide and NGF were made exactly as described in the first, that the additional media changes to wash off one agent prior to the addition of the next agent were deleted. In this paradigm, the media was replenished strictly as per the cell maintenance schedule. PC 12 cells treated with NGF (100 ng/ml) for three days were used as a reference (lanes 10 and 15), as were PC 12 cells maintained under basal conditions without addition of NGF or cycloheximide (lanes 9 and 14). Pretreatment with cycloheximide on day 1, prior to the addition of NGF on day 2, blocked GSH-Px

and catalase mRNA induction by NGF (lanes 11 and 16). Treatment with NGF on day 1, prior to the addition of cycloheximide on day 2, with readdition of NGF on day 3 without removal of cycloheximide; or treatment with NGF on day 1 and 2, with addition of cycloheximide on day 3 resulted in partial increases in GSH-Px (lanes 12 and 13) and catalase (lanes 17 and 18) mRNA commensurate with the length of exposure to NGF prior to addition of cycloheximide. Thus, regardless of the paradigm employed, the results obtained were identical. Effect of Co-Treatment of PC12 Cells with Cycloheximide (5 ug/ml) and NGF (100 ng/ml) on the NGF Induction of GSH-Px mRNA. Co-treatment of PC 12 cells

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Fig. 5. a). Represents the PCR products for GSH-Px (367bp), cyclophilin (246bp), and addition of cycloheximide (5 ng/ml) on day 2 which was again washed off, prior to addition of NGF (100ng/ml) on day 3 are shown in (lanes 3 and 7); PC12 cells treated with NGF (100ng/ml) on day land 2 which was washed off, before adding cycloheximide (5 ug/ml) on day 3 are shown in (lanes 4 and 8). PC 12 cells used as a reference are shown in lanes (1 and 5). Lanes 9-18 represent the effects of the same treatment protocol as above except that PC12 cells were treated with cycloheximide (5 ug/ml) on day 1, followed by the addition of NGF (100ng/ml) on day 2 and 3 in presence of cycloheximide (lanes 11 and 16), PC12 cells treated with NGF (100ng/ml) on day1, with addition of cycloheximide (5 ug/ml) in presence of NGF (100ng/ml) on day2, followed by readdition of NGF (100ng/ml) on day 3 (lanes 12 and 17); PC12 cells with NGF (100ng/ml) on days 1 and 2, followed by addition of cycloheximide in presence of NGF (100 ng/ml) on day 3 (lanes 13 and 18). PC 12 cells used as a reference are shown in lanes 9 and 14, and those treated with NGF (100ng/ml) for 3 days are shown in lanes 10 and 15. b). Represents the effect of cycloheximide pretreatment on the NGF induction of GSHPx and catalase mRNA. For quantification, PCR bands on the autoradiogram were scanned using a densitometer in transparent mode and the GSHPx and catalase signal normalized to the corresponding cyclophilin signal from the same RNA, which was then expressed as a percentage of the value obtained for control PC 12 cells (taken as 100%).

with cycloheximide (5 ug/ml) and NGF (100 ng/ml) for 24 hours blocked the NGF induction of GSH Px mRNA (Fig. 6 lane 6). A time course of cycloheximide (5 ug/ml) treatment for 8, 16, or 24 hours (lanes 5, 4, 3) did not alter GSH-Px mRNA significantly from the control value (lane 1). PC 12 cells treated with NGF for 24 NGF demonstrated a small but significant increase in GSH-Px mRNA (lane 7) as compared to the control value. Effect of Co-Treatment of PC 12 Cells with Cycloheximide (5 ug/ml) and NGF (100 ug/ml) on the NGF Induction of Catalase mRNA. Co-treatment of PC 12 cells with cycloheximide (5 ug/ml) and NGF (100 ng/ml) for 3 days and 1 day blocked the NGF induction of catalase mRNA (Fig. 7, lanes 1 and 2). PC 12 cells treated with NGF (100 ng/ml) for three days had significantly higher amounts of catalase mRNA (lane 3) as compared to the control value (lane 4). A time course

of cycloheximide treatment for 3 and 2 days decreased catalase mRNA to some extent (lanes 5 and 6), while treatment for 1 day with cycloheximide (5 (ig/ml) did not significantly alter catalase mRNA (lane 7) as compared to the control value (lane 8).

DISCUSSION Depending on the method used for mRNA detection, measurements of half life can represent a range of events, temporally, from the rate of initial cleavage of the RNA chain to the rate at which it is converted to small fragments. Degradative events within a specific region of the mRNA, spanned by oligonucleotide primers used for the PCR amplification, can be detected by RTPCR measurements, which are then used as a measure of the rate of decay of the mRNA species. The rates of

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Fig. 6. a). Represents the PCR products for GSH-Px (367) and cyclophilin (246bp) in PC12 cells treated with cycloheximide (5 ug/ml) for 8, 16, or 24 hours (lanes 3, 4, and 5) or cycloheximide (5 ug/ml) + NGF (100ng/ml) for 24 hours (lane6), or NGF alone for 24 hours (lane 7). Control PC 12 cells used as a reference are shown in lanes 1 and 2. The data shown are a sample of three independent experimental measurements, b). Represents the effect of a cotreatment of cycloheximide and NGF on the NGF induction of GSH-Px mRNA. For quantification, PCR bands on the autoradiogram were scanned using a densitometer in transparent mode and the catalase signal normalized to the corresponding cyclophilin signal from the same RNA, which was then expressed as a percentage of the value obtained for control PC 12 cells (taken as 100%).

degradation of GSH-Px and catalase transcripts were estimated to be ~45 hours and ~42 hours respectively indicating that both mRNA species had unusually long half lives.

Other mRNA species with long half lives have been reported such as the vasopressin days (28), pm383 encoded L1 mRNA with a half life of 26 hours (29), the neurofilament mRNA from dorsal root ganglia with a

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Fig. 7. a). Represents the PCR products for catalase (492bp) in PC12 figure 6cells treated with cycloheximide (5 ug/ml) + NGF (100ng/ml) for 3 and 1 day (lanes 1 and 2), or NGF alone for 3 days (lane 3). Control PC 12 cells used as a reference are shown in lane 4. PC 12 cells treated with cycloheximide (5 ug/ml) for 3, 2, or 1 day are shown in (lanes 5, 6, and 7). PC12 cells used as a reference are shown in lane 8. The data shown are a sample of three independent experimental measurements, b). Represents the effect of a cotreatment of cycloheximide and NGF on the NGF induction of catalase mRNA. For quantification, PCR bands on the autoradiogram were scanned using a densitometer in transparent mode and the catalase signal normalized to the corresponding cyclophilin signal from the same RNA, which was then expressed as a percentage of the value obtained for control PC 12 cells (taken as 100%).

half life of 4 days (30), the adipocyte fatty acid binding protein mRNA with a half life of 4.6 days (31) and the 16S rRNA of chloroplasts with a half life of 40 hours (32). In selenoproteins like GSH-Px, the nutrient, Selenium was found to regulate GSH-Px mRNA turnover by stabilizing the mRNA species (33). Analysis of the structure of the GSH-Px gene revealed the presence of a sin-

gle stem loop structure in the 3' untranslated region (34,26) which was found to be important in the incorporation of Selenium. The effect of this structure on the stability of this RNA is not known. However, many studies have indicated that stem loop structures have a stabilizing effect on upstream mRNA's (35,36) The structural characteristics of GSH-Px mRNA, along with the effect of Selenium on its stability, makes the GSH-

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NGF and Antioxidant Enzymes Px mRNA, a stable long lived species, which decays gradually when KNA synthesis is blocked. Analysis of the catalase gene sequences did not reveal any sequence or structural motifs that could potentially regulate mRNA stability. However, it seems logical that the mRNA species for catalase is a stable molecule based on its role as a constitutively expressed antioxidant enzyme. The decay kinetics for both GSHPx and catalase transcripts (Fig. 1 and 2) follow a pattern that might be expected for stable slow decaying mRNA species that code for constitutively expressed housekeeping enzymes. When the rates of decay of GSH-Px transcripts were compared in PC 12 cells treated with either Actinomycin D alone or with NGF and Actinomycin D, for 24 hours, it was found that NGF did not have any effect on the rate of degradation of GSH Px mRNA (Fig. 3). For catalase, however, in a similar comparison, PC 12 cells cotreated with NGF and Actinomycin D had twice the levels of catalase mRNA as compared to the levels of catalase mRNA in cells treated with Actinomycin D alone (Fig. 4). This indicated that NGF-mediated stabilization of catalase mRNA contributed to the overall increases in the levels of this transcript following NGF treatment. By 48 hours, there were no significant differences in catalase or GSH Px mRNA in cells treated either with Actinomycin D or co-treated with Actinomycin D and NGF. It is evident that de novo protein synthesis is a necessary intermediate in the induction of GSH-Px and catalase mRNA by NGF in PC12 cells. Pretreatment of PC 12 cells with cycloheximide on day 1, could deplete labile early gene products within 24 hours (37). The complete absence of new protein synthesis, would result in a lack of secondary proteins needed to induce GSHPx and catalase mRNA. This in turn, would result not only in the inhibition of NGF induction of GSH-Px and catalase expression, but in a decrease in the amounts of these transcripts as compared to the control (due to mRNA decay). The progressively increasing levels of GSH-Px and catalase mRNA seen in PC 12 cells treated with NGF for 24 and 48 hours prior to the addition of cycloheximide reflects the length of time available to NGF in which to induce these transcripts, before a blockade is introduced by cycloheximide treatment. When PC 12 cells are co-treated with cycloheximide and NGF for 1 day the NGF induction of GSH-Px is blocked, while PC 12 cells treated with NGF for 1 day had a small, but significant increase in GSH-Px mRNA as compared to the control value. Since PC12 cells treated with NGF for 3 days but not 1 day had significant increases in catalase mRNA we had to cotreat PC 12 cells

with cycloheximide and NGF for 3 days in order to assess the importance of intermediate protein synthesis on the NGF induction catalase. The results obtained identify catalase as displaying a classic late gene effect to NGF stimulation.

ACKNOWLEDGMENTS Thanks to K. Werrbach-Perez for excellent technical assistance and G. Jackson for helpful suggestions. This work was supported in part by NINDS grant NS-18708.

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