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Glucocorticoids elevate GTP cyclohydrolase I mRNA levels in vivo and in PC12 cells
Molecular Brain Research 48 Ž1997. 251–258
Research report
Glucocorticoids elevate GTP cyclohydrolase I mRNA levels in vivo and in PC12 cells Lidia Serova a , Bistra Nankova a , Mark Rivkin b, Richard Kvetnansky c , Esther L. Sabban a
a,)
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA b North Dakota State UniÕersity, Fargo, ND 58102, USA c Institute of Experimental Endocrinology, SloÕak Academy of Sciences, BratislaÕa, SloÕakia Accepted 11 February 1997
Abstract GTP cyclohydrolase I ŽGTPCH. is the rate-limiting enzyme in the formation of tetrahydrobiopterin, the cofactor for catecholamine, indolamine and nitric oxide biosynthesis. The effect of glucocorticoids on GTPCH gene expression was examined by direct infusion of cortisol to rats and by incubation of PC12 cells with glucocorticoids. Northern blot analysis revealed that infusion of cortisol for 1 or 7 days elevated levels of the 3.6 kb GTPCH mRNA species in rat adrenal medulla, while the 1.2 kb mRNA species were only increased by 1 day cortisol. Cortisol administration to hypophysectomized animals elicited a 4–5-fold elevation in both forms of GTPCH mRNA. These results indicate that glucocorticoids may be directly involved in the regulation of adrenomedullary GTPCH mRNA levels by physiological stress. Incubation of PC12 cells with plasma from immobilized, but not control, animals increased the level of the 3.6 kb mRNA. Treatment of PC12 cells with dexamethasone for 12–48 h elicited a 4–6-fold elevation in both GTPCH mRNAs. Using the nuclear run-on assay, increased transcription of the GTPCH gene was observed in the rat adrenal medulla with immobilization stress, or in PC12 cells treated with dexamethasone. This is the first report that glucocorticoids can alter GTPCH expression. Keywords: GTP cyclohydrolase I mRNA; Transcription; Glucocorticoids; Stress; PC12 cells; Adrenal medulla; Hypophysectomy
1. Introduction GTP cyclohydrolase I ŽGTPCH. is the rate-limiting enzyme in the de novo biosynthesis of tetrahydrobiopterin ŽBH4., which is a cofactor for hydroxylation of tyrosine, phenylalanine, tryptophan w2,20x and also in nitric oxide biosynthesis w34x. Regulation of BH4 levels appears to play a crucial role in catecholamine and other neurotransmitter biosynthesis. Changes in BH4 concentration within the central nervous system have been demonstrated in several diseases, such as inborn errors of BH4 metabolism w17x, Parkinson’s disease, Shy-Drager syndrome, Alzheimer’s disease w51x, hyperphenylalaninemia and dystonia w5,35,36x. Some of these disorders are related to defects in BH4 synthesis and in GTPCH. Thus, deficiencies in GTPCH activity are crucial in hyperphenylalaninemia and defects
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Corresponding author. Fax: q1 Ž914. 993-4058; E-mail: [email protected]
in the GTPCH gene have been found to cause dystonia in some patients w18x. Regulation of GTPCH enzyme activity is tissue-specific and controlled by several mechanisms, including Ž1. BH4dependent feedback inhibition w11x, Ž2. regulation through changes in intracellular GTP levels w13x and Ž3. cytokine induced activation w50x. In rat adrenals, reserpine or insulin induced hypoglycemia increase GTPCH enzyme activity w49x. In PC12 cells, GTPCH enzyme activity was found to be stimulated by increased cAMP levels or by membrane depolarization w1,45x and subject to end-product inhibition w45x. Mechanisms responsible for the regulation of GTPCH gene expression are just beginning to be identified. GTPCH mRNA levels are raised by nerve growth factor treatment w15x, while they are decreased by leukemia inhibitory factor or ciliary neurotrophic factor in cultures of neonatal rat superior cervical ganglia w46x. cAMP analogs elevate GTPCH mRNA levels in primary cultures containing embryonic rat brain mesencephalic or hypothalamic
0169-328Xr97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 0 9 8 - 3
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dopaminergic neurons w55x. Treatment of rats with reserpine revealed elevations in GTPCH mRNA in noradrenergic and dopaminergic neurons, suggesting that the level of expression of GTPCH mRNA may be coupled to changes in nerve impulses and their corresponding second messenger-signaling pathways w14x. We have recently shown that a single or repeated immobilization stress elicited elevation of both forms of GTPCH mRNA in the rat adrenal medulla w44x. Stress is known to stimulate the hypothalamic-pituitary-adrenal ŽHPA. axis and the sympathoadrenal system causing elevations of ACTH and glucocorticoids in plasma and activation of synthesis and release of catecholamines in the central and peripheral nerve systems w9,12,21– 23,24a,25b,26x. Mechanisms involved in stress triggered regulation of genes encoding catecholamine biosynthetic enzymes, as well as co-expressed neuropeptides, are intensively studied w16,28,32,33,39,40,42,54x. Our previous study is the first to begin to identify mechanisms of stress elicited induction of GTPCH mRNAs w44x. Splanchnic nerve section did not affect induction of the 3.6 kb GTPCH mRNA by immobilization. While hypophysectomy was found to have no effect on basal levels, it prevented the stress elicited rise in both 3.6 and 1.2 kb GTPCH mRNAs. These finding indicate that hormonal stimuli may be important for the regulation of GTPCH gene expression by stress, but did not enable us to determine which hormones play a more crucial role in this regulation and whether the effects are direct. Here we examine the effect of glucocorticoids directly by administration of cortisol to rats, and by addition of glucocorticoids to cultured cells. This is the first study to show that glucocorticoids can regulate induction of GTPCH mRNA in vivo and elevate GTPCH mRNA levels in cultured cells, most probably by affecting the transcription. The results suggest that glucocorticoids may be involved in mediating stress-induced changes in GTPCH gene expression and in altering the neurotransmitter biosynthetic pathways that utilize tetrahydrobiopterin.
2. Materials and methods 2.1. Reagents
a w 32 PxUTP Ž800 Cirmmol. and a w 32 PxdCTP Ž) 6000 Cirmmol. and Gene-Screen Plus nylon membranes were purchased from Du Pont-New England Nuclear. The enzymes EcoRI and BamHI were from Promega, the RNA transcription kit was from Ambion, Plasmid Mega kit was from Quagen, RNAzol was from Tel-Test, Nuc Trap columns were from Stratagene and the Megaprime random primer labeling kit was from Amersham. RPMI 1640 and dialyzed fetal bovine and horse sera was from Gibco-BRL. Dexamethasone and cortisol were from Sigma ŽSt. Louis, MO.. All other chemicals were of molecular biology grade.
2.2. In ÕiÕo experiments All animal experiments were approved by the Animal Care and Use Committee. Adult, pathogen-free, male Sprague–Dawley rats Ž250–320 g. were purchased from Taconic Farms ŽGermantown, NY. and housed fourrcage. They were maintained under controlled conditions of 12 h light–dark cycle at 23 " 28C. Animals were given food and water ad libitum. Cortisol Žhydrocortisone-21-hemisuccinate, sodium salt crystalline; Sigma. dissolved in saline at 464 mgrml was administered via osmotic minipumps Žmodel 2001, Alzet, Palo, CA.. The minipump, inserted s.c. in the interscapular area, delivered 25 mg cortisolrkgrday at a rate of f 1.0 m lrh for 1 or 7 days. Control animals were administered the same volume of saline under identical conditions. Hypophysectomy was performed by the vendor. Experiments were carried out 14 days after surgery and complete removal of hypophysis was confirmed at the end of the experiment. Hypophysectomized animals were provided with saline instead of water. Immobilization stress was applied for 2 h as described in detail previously w21,31x. All experiments were performed between 08:00 and 13:00 h with 6–8 animalsrgroup. Animals were euthanized by decapitation, blood was collected in heparinized tubes, centrifuged and plasma was transferred in new tubes. Adrenal medulla were immediately frozen in liquid nitrogen. 2.3. In Õitro experiments PC12 cells were grown as previously described w38x in RPMI 1640 medium, 10% heat-inactivated donor horse serum, 5% heat-inactivated fetal calf serum, and 50 m grml of streptomycin-penicillin Žcomplete medium. in 7% CO 2 . In some experiments, the serum in the media was replaced with plasma from control rats or rats exposed to 2 h immobilization stress and cells grown for an additional 24 h. All experiments with dexamethasone were performed in medium in which the sera were replaced by dialyzed sera. The cells were pre-incubated for 2 days in this medium, before exposure to 1 m M dexamethasone. Parallel cultures Ž4–5. were used for each experimental group and all experiments were repeated at least once. 2.4. Isolation of RNA and Northern blots The relative levels of GTPCH, TH and cyclophilin mRNAs were determined by Northern analysis as described previously w31,44x. Total RNA was isolated according to the procedure of Chomczynski and Sacchi w3x using RNAzol. 15 m g total RNA from PC12 cells or punched out adrenal medulla were loaded on 1.2% agarose gel. The RNA was transferred to Gene-Screen Plus membranes. Hybridizations were performed with the rat GTPCH cRNA
L. SeroÕa et al.r Molecular Brain Research 48 (1997) 251–258
probe, TH and cyclophilin cDNA probes or a DNA probe for 18S rRNA as described earlier w33,44x. The singlestrand antisense cRNA probe for GTPCH was transcribed with T7 RNA polymerase with w 32 Px a-UTP Ž800 Cirmmol. using RNA transcription kit ŽAmbion. and purified by phenol-chloroform extraction. The TH and cyclophilin cDNA probes or the probe for 18S rRNA were labeled with w 32 Px a-dCTP Ž6000 Cirmmol. by the random primer method ŽMegaprime, Amersham. and purified on Nuc Trap columns ŽStratagene.. Hybridization was performed at 428C, in a solution containing 5 = SSPE Ž0.15 M NaCl, 10 mM NaH 2 PO4 , 1 mM EDTA., 50% formamide, 5 = Denhardt’s, and 1% sodium dodecyl sulfate ŽSDS., and 10 6 dpm of 32 P-labeled probes. After hybridization with the GTPCH cRNA probe, the filters were washed twice in 2 = SSPE at room temperature, then once in 0.1 = SSPE, 1% SDS at 558 for 60 min. Blots were stripped in boiling solution of 10 mM Tris–HCl pH 8, 1 mM EDTA, 1% SDS, then hybridized consecutively with DNA probes. The hybridization with the DNA probes and washing of the filters were as previously described w33x. Following exposure to X-ray film ŽKodak. within the linear range of the signal, autoradiograms were scanned and analyzed by Image-Pro-Analysis software. 2.5. Nuclear run-on assays The rate of GTPCH gene transcription in adrenal medulla and in PC12 cells was measured as described by Fossom et al. w7x. Nuclei were isolated from: Ž1. freshly dissected adrenal medulla of controls and rats exposed for different time to immobilization stress; and Ž2. PC12 cells incubated for 6–24 h with 1 m M dexamethasone. The tissues or cells were homogenized in buffer containing 140 mM NaCl, 1.5 mM MgCl 2 , 10 mM Tris pH 7.5, 0.2% Triton X-100, 1 mM dithiothreitol, then centrifuged over sucrose cushion Ž1 M sucrose, 50 mM NH 4 Cl, 0.1% Triton X-100, 5 mM MgCl 2 , 10 mM Tris pH 7.5.. Pellets were re-suspended gently in 50% glycerol, 0.1 mM EDTA, 5 mM MgCl 2 , 50 mM HEPES pH 7.5. Equal amounts of nuclei was used in each reaction, and the in vitro labeled RNAs were hybridized to plasmids encoding rat GTPCH or cyclophilin cDNAs or vector DNA immobilized on nitrocellulose membranes at two 5-fold different concentrations. The results from two independent experiments, with 6–8 animals or 5 Petri dishesrgroup, were summarized and normalized to the levels, obtained with nuclei from the control group.
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2.7. Statistics Results are presented as means " S.E.M. One-way ANOVA test with Fisher’s test or Student’s t-test were used for the statistical evaluation of the data.
3. Results 3.1. Effect of cortisol on GTPCH mRNA leÕels in rat adrenal medulla We examined the effect of exogenously administered glucocortocoids on the steady-state levels of GTPCH mRNAs in the adrenal medulla. Infusion of cortisol for 1 day was found to double the levels of both the 3.6 and 1.2 kb GTPCH mRNA species. Prolonged infusion of cortisol for 7 days resulted in increases only in the 3.6 kb GTPCH mRNA ŽFig. 1. while the 1.2 kb form was not significantly altered. The effect of cortisol on the 3.6 kb mRNA was similar with 1 or 7 days. However, the rise with cortisol was smaller than the 3–5-fold rise attained with immobilization stress w44x. Since exogenously administered cortisol might interfere with effects of endogenous glucocorticoids, ACTH or other pituitary hormones, we carried out experiments in hypophysectomized animals. Hypophysectomy, as we reported previously w44x, did not significantly influence the steady-state levels of both forms of GTPCH mRNA ŽFig. 2.. Cortisol administration to hypophysectomized animals induced a 4–5-fold elevation of 3.6 and 1.2 kb GTPCH mRNAs. These results indicate that glucocorticoids may be directly involved in the regulation of GTPCH mRNA levels.
2.6. Identification of promoter regions homologous to glucocorticoid response element (GRE) The sequences of the GTPCH genes in Gene Bank w52,19x Žaccession number L29478 for human and D38601 for mouse. were compared to the consensus GRE Ž5X-TGGTACAAATGTTCT-3X . using PC GENE, 15 bases at a time, with a threshold of 85% identity.
Fig. 1. Effect of cortisol infusions on GTPCH mRNA levels in the adrenal medulla. Animals were infused with cortisol or saline Žcontrol. for 1 or 7 days by minipump. Adrenal medullary GTPCH mRNA levels were assessed by Northern blots. The data are normalized to 18S rRNA and presented as fold induction over controls. ) P - 0.05 vs. controls.
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Fig. 2. Effect of cortisol infusions to hypophysectomized rats. Summary of Northern blot data from individual animals Ž6–7rgroup.. ) P - 0.05 comparing Hypox control to Hypox cortisol.
3.2. Effect of plasma from immobilized rats and dexamethasone on expression of GTPCH mRNAs in PC12 cells To begin to identify the humoral factors, such as glucocorticoids, that might be directly involved in induction of GTPCH mRNA by stress, we incubated PC12 cells with plasma from immobilized rats, which have greatly elevated corticosterone w4,27x. Levels of the 3.6 kb form of GTPCH mRNA were significantly higher in PC12 cells exposed to plasma from immobilized animals ŽFig. 3.. In contrast, incubation with plasma from control animals did not alter the steady-state levels of both GTPCH mRNA forms. Since plasma contains many factors that could elevate GTPCH expression, we examined the effect of the synthetic glucocorticoid, dexamethasone. PC12 cells were treated with 1 m M dexamethasone for 6–48 h, and the levels of GTPCH mRNA analyzed by Northern blot. For comparison, the same filters were reprobed to determine TH mRNA and cyclophilin levels ŽFig. 4.. The induction of both GTPCH mRNAs was dependent on the time of
Fig. 4. Time course of GTPCH mRNAs induction in PC12 cells treated with dexamethasone ŽA. Northern blot, ŽB. descriptive statistics. Cells were incubated for 2 days in medium with dialyzed sera and then for 6–48 h with a fresh aliquot of the same medium containing 1 m M dexamethasone. Total RNA was isolated at indicated time points and levels of GTPCH, TH and cyclophilin mRNAs analyzed by Northern blot as described in Materials and methods. ) P - 0.05 compared to its respective untreated controls.
incubation. With 6 h of incubation, only the levels of 3.6 kb GTPCH mRNA were significantly changed. Both GTPCH mRNAs were significantly increased after 12 h with dexamethasone. The maximum elevation was observed by 48 h. With longer times of exposure the levels of both GTPCH mRNA species were increased 4–6-fold by dexamethasone ŽFig. 4.. The TH mRNA levels were also elevated after 12 h of incubation with dexamethasone and were further increased by 24–48 h. The induction of GTPCH mRNA paralleled the increase in TH mRNA levels. In contrast, dexamethasone did not alter cyclophilin mRNA levels, which served as a control. 3.3. Transcriptional effect of immobilization stress and dexamethasone on GTPCH gene
Fig. 3. Plasma from immobilized rats induced GTPCH mRNA levels. PC12 cells were incubated with plasma from rats immobilized for 2 h and from controls. Total RNA was isolated after 24 h and the GTPCH mRNA levels determined by Northern blot. ) P - 0.05 compared to plasma from controls.
The observed elevation of GTPCH mRNA by glucocorticoids may directly reflect increased levels of transcription or enhanced stability of the transcripts or both. To study if increases in GTPCH mRNA by glucocorticoids are due at least in part to activated transcription of the gene, we performed nuclear run-on assays. In the adrenal medulla,
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Fig. 5. Run-on assay of GTPCH transcription rate. The time courses of relative GTPCH transcription is shown on left for rat adrenal medulla ŽA. and PC12 cells ŽB.. Representative hybridizations are shown on right for nuclei from: ŽA. adrenal medullae from control rats ŽC. or after 30 min immobilization ŽIMO. hybridized to 5-fold different concentrations of GTPCH plasmid Žhigh, low. or to vector Žlow, high. as control; ŽB. PC12 cells untreated controls ŽC. or after 24 h dexamethasone ŽDex. with two concentrations Žlow, high. of GTPCH or cyclophilin cDNA plasmids.
the incorporation of radiolabeled UTP into GTPCH-RNA was almost double the control levels by 30 min immobilization stress and it was further increased with 60 and 120 min of immobilization ŽFig. 5A.. In PC12 cells treated with dexamethasone, the rate of transcription also was raised but only after 12 h of incubation and remained f 2-fold higher than the control levels at 24 h ŽFig. 5B.. We did not observe any changes in transcription rate for cyclophilin. The observed increased transcription in response to stress or dexamethasone treatment indicates that the GTPCH gene may contain GREŽs.. Computer analysis was carried out to examine if the sequences of the GTPCH genes in the Gene Bank database have regions homologous to a consensus GRE. As shown in Fig. 6, sequences homologous to the complement of the consensus GRE
Fig. 6. Putative GRE sites detected by comparison of consensus GRE to human and mouse promoter sequences.
were found at positions y1548 to y1533 and y919 to y905 in the human promoter w52x and at position y523 to y513 in the mouse promoter w19x.
4. Discussion The results of this study indicate that GTPCH gene expression is regulated in vivo and in vitro at least partially by glucocorticoids. Infusion of cortisol to rats during 1 or 7 days caused a small, but significant, elevation in the adrenal medullary 3.6 kb GTPCH mRNA, which is considered to be encoding the active enzyme w10x. It was shown previously that administration of cortisol to rats for 1 day had no significant effect on basal TH and PNMT mRNA levels, however, 7 days cortisol infusion significantly raised adrenomedullary PNMT levels w27x. Although hypophysectomy was shown to prevent the stress-elicited induction of GTPCH and PNMT mRNA levels, the effect of exogenously administered cortisol was less pronounced for both genes w27,44x. Administration of the exogenous cortisol to hypophysectomized animals produced approximately the same rise in the levels of both GTPCH mRNA species as attained by immobilization stress. Cortisol infusion was effective in induction of GTPCH mRNA in hypophysectomized rats, indicating that glucocorticoids appear to be able to directly influence GTPCH gene expression. Previous results revealed that hypophysectomy blocked induction of adrenal GTPCH, PNMT and
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preproenkephalin mRNAs by immobilization stress w40,44,48x. The expression of PNMT gene has been shown to be regulated by glucocorticoids w53,54x. A GRE is present in the PNMT promoter w37x, and mutation of this site prevents hormonal induction w53x. However, hypophysectomy did not effect the induction of adrenal TH and DBH mRNAs by immobilization stress w33,40x although glucocorticoids can also greatly elevate their levels in adrenal chromaffin cells and PC12 cells w29,32,47x. Increases in TH expression by glucocorticoids are transcriptionally mediated w7,30x, however, the GREŽs. in the TH gene has not yet been identified. The involvement of glucocorticoids in the regulation of GTPCH gene expression was confirmed by in vitro experiments. In the present study, we have shown that in PC12 cells the rise of GTPCH mRNA can be induced by incubation with plasma from immobilized rats, which contains the endogenous glucocorticoid, corticosterone w4,27x, or with the synthetic exogenous glucocorticoid, dexamethasone. Both treatments resulted in increased levels of the 3.6 kb GTPCH mRNA. It was elevated after 12 h incubation with dexamethasone. GTPCH mRNA levels were increased and remained high for as long as 48 h. The time course of TH induction was similar as measured here and reported in previous studies and also is at least in part, transcriptionally mediated w29,32,47x. Parallel regulation of TH and GTPCH gene expression was also found in response to leukemia inhibitory factor and ciliary neurotrophic factor treatments in cultured superior cervical ganglia cells w46x. Although the increase in expression on GTPCH mRNA paralleled the rise of TH mRNA levels in PC12 cells they may differ in vivo since hypophysectomy abolished stress elicited rise of GTPCH but not of TH mRNA w33,44x. Glucocorticoids were found to directly affect rate of transcription of GTPCH gene in response to stress in adrenal medulla and in PC12 cells with dexamethasone treatment. The increased transcription was already 2-fold higher after 30 min of stress while the rise in PC12 cells was much slower. The time course for elevation of the rate of transcription in PC12 cells treated by dexamethasone was similar to increases in GTPCH mRNA levels. The reason for the faster response in vivo is unclear, however, glucocorticoids may indirectly modulate Ženhance or inhibit, depending on the biological circumstances. expression through their control of other transcription factors, such as AP1 and Egr1 w6,54x, or by changing the transcript stability or protein turnover. Alterations in glucocorticoid receptor levels w43x also contribute to the multiple pathways of exerting glucocorticoid effects in the cell. In addition to glucocorticoids, cAMP analogs and membrane depolarization also trigger parallel up-regulation of both GTPCH and TH gene expression w41x. Interestingly, while cAMP analogs and dexamethasone were not additive in their effects on TH transcription and mRNA levels in PC12 cells, combined treatment yielded higher TH activity
than either treatment alone w7,47x. It is attractive to speculate that there might be cross talk between cAMP and glucocorticoid mediated pathways with respect to induction of GTPCH, and that the increase, observed in TH activity may result from their additive effects on inductions in GTPCH gene expression and BH4 levels. In contrast to the longer GTPCH mRNA, the 1.2 kb GTPCH mRNA was induced by 1, but not 7, days cortisol infusion. These results indicate that under some conditions the two GTPCH mRNAs may be regulated differently. There is not yet enough information available regarding the differences between the two GTPCH mRNA species in the rat. It is not known how many rat GTPCH genes exist. It remains to be determined if the two mRNAs arise from differential splicing or from transcription at different promoters. If these mRNAs differ, as in human, solely by splicing at their 3X-end, glucocorticoids, as well as splanchnic denervation, may be affecting this splicing. The differential regulation of the 1.2 kb mRNAs could also be indirect. After long-term elevation of glucocorticoids in plasma, several mechanisms can be involved in the regulation of gene expression. Glucocorticoids besides their own effect on gene expression can activate negative feedback loops acting at the level of the pituitary, hypothalamus and higher nerve centers through their own receptors w12x or affect on neurotransmitters synthesis, turnover and release w8x. These mechanisms may prevent a rise in 1.2 kb GTPCH mRNA after chronic hormone treatment. Glucocorticoids may directly stimulate GTPCH gene expression by classical mechanisms via binding to a glucocorticoid receptor and interacting with positive GREs within the promoter. The sequences of human and part of the mouse, but not the rat, GTPCH promoters have been determined w19,52x. Comparison of these to the consensus GRE revealed two sequences in human GTPCH promoter and one in the mouse promoter with ) 85% homology to the complement of the consensus GRE. Whether the rat gene contains GRE-like sequences and whether these putative GREs are involved in the induction of GTPCH gene expression by glucocorticoids remain to be determined. This is the first study to our knowledge about the regulation of GTPCH mRNA by glucocorticoids. Given that GTPCH is the rate-limiting enzyme in BH4 biosynthesis, whose deficiency is associated with several neurological disorders w5,17,35,36,52x, our results indicate that glucocorticoids may modulate BH4 levels with corresponding changes in BH4-dependent reactions.
Acknowledgements This work was supported by Grant NS 32166 from the National Institute of Health. We thank Drs. Bargava Hiremagalur and Irwin Kopin for their help and useful suggestions.
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Research report
Glucocorticoids elevate GTP cyclohydrolase I mRNA levels in vivo and in PC12 cells Lidia Serova a , Bistra Nankova a , Mark Rivkin b, Richard Kvetnansky c , Esther L. Sabban a
a,)
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA b North Dakota State UniÕersity, Fargo, ND 58102, USA c Institute of Experimental Endocrinology, SloÕak Academy of Sciences, BratislaÕa, SloÕakia Accepted 11 February 1997
Abstract GTP cyclohydrolase I ŽGTPCH. is the rate-limiting enzyme in the formation of tetrahydrobiopterin, the cofactor for catecholamine, indolamine and nitric oxide biosynthesis. The effect of glucocorticoids on GTPCH gene expression was examined by direct infusion of cortisol to rats and by incubation of PC12 cells with glucocorticoids. Northern blot analysis revealed that infusion of cortisol for 1 or 7 days elevated levels of the 3.6 kb GTPCH mRNA species in rat adrenal medulla, while the 1.2 kb mRNA species were only increased by 1 day cortisol. Cortisol administration to hypophysectomized animals elicited a 4–5-fold elevation in both forms of GTPCH mRNA. These results indicate that glucocorticoids may be directly involved in the regulation of adrenomedullary GTPCH mRNA levels by physiological stress. Incubation of PC12 cells with plasma from immobilized, but not control, animals increased the level of the 3.6 kb mRNA. Treatment of PC12 cells with dexamethasone for 12–48 h elicited a 4–6-fold elevation in both GTPCH mRNAs. Using the nuclear run-on assay, increased transcription of the GTPCH gene was observed in the rat adrenal medulla with immobilization stress, or in PC12 cells treated with dexamethasone. This is the first report that glucocorticoids can alter GTPCH expression. Keywords: GTP cyclohydrolase I mRNA; Transcription; Glucocorticoids; Stress; PC12 cells; Adrenal medulla; Hypophysectomy
1. Introduction GTP cyclohydrolase I ŽGTPCH. is the rate-limiting enzyme in the de novo biosynthesis of tetrahydrobiopterin ŽBH4., which is a cofactor for hydroxylation of tyrosine, phenylalanine, tryptophan w2,20x and also in nitric oxide biosynthesis w34x. Regulation of BH4 levels appears to play a crucial role in catecholamine and other neurotransmitter biosynthesis. Changes in BH4 concentration within the central nervous system have been demonstrated in several diseases, such as inborn errors of BH4 metabolism w17x, Parkinson’s disease, Shy-Drager syndrome, Alzheimer’s disease w51x, hyperphenylalaninemia and dystonia w5,35,36x. Some of these disorders are related to defects in BH4 synthesis and in GTPCH. Thus, deficiencies in GTPCH activity are crucial in hyperphenylalaninemia and defects
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Corresponding author. Fax: q1 Ž914. 993-4058; E-mail: [email protected]
in the GTPCH gene have been found to cause dystonia in some patients w18x. Regulation of GTPCH enzyme activity is tissue-specific and controlled by several mechanisms, including Ž1. BH4dependent feedback inhibition w11x, Ž2. regulation through changes in intracellular GTP levels w13x and Ž3. cytokine induced activation w50x. In rat adrenals, reserpine or insulin induced hypoglycemia increase GTPCH enzyme activity w49x. In PC12 cells, GTPCH enzyme activity was found to be stimulated by increased cAMP levels or by membrane depolarization w1,45x and subject to end-product inhibition w45x. Mechanisms responsible for the regulation of GTPCH gene expression are just beginning to be identified. GTPCH mRNA levels are raised by nerve growth factor treatment w15x, while they are decreased by leukemia inhibitory factor or ciliary neurotrophic factor in cultures of neonatal rat superior cervical ganglia w46x. cAMP analogs elevate GTPCH mRNA levels in primary cultures containing embryonic rat brain mesencephalic or hypothalamic
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dopaminergic neurons w55x. Treatment of rats with reserpine revealed elevations in GTPCH mRNA in noradrenergic and dopaminergic neurons, suggesting that the level of expression of GTPCH mRNA may be coupled to changes in nerve impulses and their corresponding second messenger-signaling pathways w14x. We have recently shown that a single or repeated immobilization stress elicited elevation of both forms of GTPCH mRNA in the rat adrenal medulla w44x. Stress is known to stimulate the hypothalamic-pituitary-adrenal ŽHPA. axis and the sympathoadrenal system causing elevations of ACTH and glucocorticoids in plasma and activation of synthesis and release of catecholamines in the central and peripheral nerve systems w9,12,21– 23,24a,25b,26x. Mechanisms involved in stress triggered regulation of genes encoding catecholamine biosynthetic enzymes, as well as co-expressed neuropeptides, are intensively studied w16,28,32,33,39,40,42,54x. Our previous study is the first to begin to identify mechanisms of stress elicited induction of GTPCH mRNAs w44x. Splanchnic nerve section did not affect induction of the 3.6 kb GTPCH mRNA by immobilization. While hypophysectomy was found to have no effect on basal levels, it prevented the stress elicited rise in both 3.6 and 1.2 kb GTPCH mRNAs. These finding indicate that hormonal stimuli may be important for the regulation of GTPCH gene expression by stress, but did not enable us to determine which hormones play a more crucial role in this regulation and whether the effects are direct. Here we examine the effect of glucocorticoids directly by administration of cortisol to rats, and by addition of glucocorticoids to cultured cells. This is the first study to show that glucocorticoids can regulate induction of GTPCH mRNA in vivo and elevate GTPCH mRNA levels in cultured cells, most probably by affecting the transcription. The results suggest that glucocorticoids may be involved in mediating stress-induced changes in GTPCH gene expression and in altering the neurotransmitter biosynthetic pathways that utilize tetrahydrobiopterin.
2. Materials and methods 2.1. Reagents
a w 32 PxUTP Ž800 Cirmmol. and a w 32 PxdCTP Ž) 6000 Cirmmol. and Gene-Screen Plus nylon membranes were purchased from Du Pont-New England Nuclear. The enzymes EcoRI and BamHI were from Promega, the RNA transcription kit was from Ambion, Plasmid Mega kit was from Quagen, RNAzol was from Tel-Test, Nuc Trap columns were from Stratagene and the Megaprime random primer labeling kit was from Amersham. RPMI 1640 and dialyzed fetal bovine and horse sera was from Gibco-BRL. Dexamethasone and cortisol were from Sigma ŽSt. Louis, MO.. All other chemicals were of molecular biology grade.
2.2. In ÕiÕo experiments All animal experiments were approved by the Animal Care and Use Committee. Adult, pathogen-free, male Sprague–Dawley rats Ž250–320 g. were purchased from Taconic Farms ŽGermantown, NY. and housed fourrcage. They were maintained under controlled conditions of 12 h light–dark cycle at 23 " 28C. Animals were given food and water ad libitum. Cortisol Žhydrocortisone-21-hemisuccinate, sodium salt crystalline; Sigma. dissolved in saline at 464 mgrml was administered via osmotic minipumps Žmodel 2001, Alzet, Palo, CA.. The minipump, inserted s.c. in the interscapular area, delivered 25 mg cortisolrkgrday at a rate of f 1.0 m lrh for 1 or 7 days. Control animals were administered the same volume of saline under identical conditions. Hypophysectomy was performed by the vendor. Experiments were carried out 14 days after surgery and complete removal of hypophysis was confirmed at the end of the experiment. Hypophysectomized animals were provided with saline instead of water. Immobilization stress was applied for 2 h as described in detail previously w21,31x. All experiments were performed between 08:00 and 13:00 h with 6–8 animalsrgroup. Animals were euthanized by decapitation, blood was collected in heparinized tubes, centrifuged and plasma was transferred in new tubes. Adrenal medulla were immediately frozen in liquid nitrogen. 2.3. In Õitro experiments PC12 cells were grown as previously described w38x in RPMI 1640 medium, 10% heat-inactivated donor horse serum, 5% heat-inactivated fetal calf serum, and 50 m grml of streptomycin-penicillin Žcomplete medium. in 7% CO 2 . In some experiments, the serum in the media was replaced with plasma from control rats or rats exposed to 2 h immobilization stress and cells grown for an additional 24 h. All experiments with dexamethasone were performed in medium in which the sera were replaced by dialyzed sera. The cells were pre-incubated for 2 days in this medium, before exposure to 1 m M dexamethasone. Parallel cultures Ž4–5. were used for each experimental group and all experiments were repeated at least once. 2.4. Isolation of RNA and Northern blots The relative levels of GTPCH, TH and cyclophilin mRNAs were determined by Northern analysis as described previously w31,44x. Total RNA was isolated according to the procedure of Chomczynski and Sacchi w3x using RNAzol. 15 m g total RNA from PC12 cells or punched out adrenal medulla were loaded on 1.2% agarose gel. The RNA was transferred to Gene-Screen Plus membranes. Hybridizations were performed with the rat GTPCH cRNA
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probe, TH and cyclophilin cDNA probes or a DNA probe for 18S rRNA as described earlier w33,44x. The singlestrand antisense cRNA probe for GTPCH was transcribed with T7 RNA polymerase with w 32 Px a-UTP Ž800 Cirmmol. using RNA transcription kit ŽAmbion. and purified by phenol-chloroform extraction. The TH and cyclophilin cDNA probes or the probe for 18S rRNA were labeled with w 32 Px a-dCTP Ž6000 Cirmmol. by the random primer method ŽMegaprime, Amersham. and purified on Nuc Trap columns ŽStratagene.. Hybridization was performed at 428C, in a solution containing 5 = SSPE Ž0.15 M NaCl, 10 mM NaH 2 PO4 , 1 mM EDTA., 50% formamide, 5 = Denhardt’s, and 1% sodium dodecyl sulfate ŽSDS., and 10 6 dpm of 32 P-labeled probes. After hybridization with the GTPCH cRNA probe, the filters were washed twice in 2 = SSPE at room temperature, then once in 0.1 = SSPE, 1% SDS at 558 for 60 min. Blots were stripped in boiling solution of 10 mM Tris–HCl pH 8, 1 mM EDTA, 1% SDS, then hybridized consecutively with DNA probes. The hybridization with the DNA probes and washing of the filters were as previously described w33x. Following exposure to X-ray film ŽKodak. within the linear range of the signal, autoradiograms were scanned and analyzed by Image-Pro-Analysis software. 2.5. Nuclear run-on assays The rate of GTPCH gene transcription in adrenal medulla and in PC12 cells was measured as described by Fossom et al. w7x. Nuclei were isolated from: Ž1. freshly dissected adrenal medulla of controls and rats exposed for different time to immobilization stress; and Ž2. PC12 cells incubated for 6–24 h with 1 m M dexamethasone. The tissues or cells were homogenized in buffer containing 140 mM NaCl, 1.5 mM MgCl 2 , 10 mM Tris pH 7.5, 0.2% Triton X-100, 1 mM dithiothreitol, then centrifuged over sucrose cushion Ž1 M sucrose, 50 mM NH 4 Cl, 0.1% Triton X-100, 5 mM MgCl 2 , 10 mM Tris pH 7.5.. Pellets were re-suspended gently in 50% glycerol, 0.1 mM EDTA, 5 mM MgCl 2 , 50 mM HEPES pH 7.5. Equal amounts of nuclei was used in each reaction, and the in vitro labeled RNAs were hybridized to plasmids encoding rat GTPCH or cyclophilin cDNAs or vector DNA immobilized on nitrocellulose membranes at two 5-fold different concentrations. The results from two independent experiments, with 6–8 animals or 5 Petri dishesrgroup, were summarized and normalized to the levels, obtained with nuclei from the control group.
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2.7. Statistics Results are presented as means " S.E.M. One-way ANOVA test with Fisher’s test or Student’s t-test were used for the statistical evaluation of the data.
3. Results 3.1. Effect of cortisol on GTPCH mRNA leÕels in rat adrenal medulla We examined the effect of exogenously administered glucocortocoids on the steady-state levels of GTPCH mRNAs in the adrenal medulla. Infusion of cortisol for 1 day was found to double the levels of both the 3.6 and 1.2 kb GTPCH mRNA species. Prolonged infusion of cortisol for 7 days resulted in increases only in the 3.6 kb GTPCH mRNA ŽFig. 1. while the 1.2 kb form was not significantly altered. The effect of cortisol on the 3.6 kb mRNA was similar with 1 or 7 days. However, the rise with cortisol was smaller than the 3–5-fold rise attained with immobilization stress w44x. Since exogenously administered cortisol might interfere with effects of endogenous glucocorticoids, ACTH or other pituitary hormones, we carried out experiments in hypophysectomized animals. Hypophysectomy, as we reported previously w44x, did not significantly influence the steady-state levels of both forms of GTPCH mRNA ŽFig. 2.. Cortisol administration to hypophysectomized animals induced a 4–5-fold elevation of 3.6 and 1.2 kb GTPCH mRNAs. These results indicate that glucocorticoids may be directly involved in the regulation of GTPCH mRNA levels.
2.6. Identification of promoter regions homologous to glucocorticoid response element (GRE) The sequences of the GTPCH genes in Gene Bank w52,19x Žaccession number L29478 for human and D38601 for mouse. were compared to the consensus GRE Ž5X-TGGTACAAATGTTCT-3X . using PC GENE, 15 bases at a time, with a threshold of 85% identity.
Fig. 1. Effect of cortisol infusions on GTPCH mRNA levels in the adrenal medulla. Animals were infused with cortisol or saline Žcontrol. for 1 or 7 days by minipump. Adrenal medullary GTPCH mRNA levels were assessed by Northern blots. The data are normalized to 18S rRNA and presented as fold induction over controls. ) P - 0.05 vs. controls.
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Fig. 2. Effect of cortisol infusions to hypophysectomized rats. Summary of Northern blot data from individual animals Ž6–7rgroup.. ) P - 0.05 comparing Hypox control to Hypox cortisol.
3.2. Effect of plasma from immobilized rats and dexamethasone on expression of GTPCH mRNAs in PC12 cells To begin to identify the humoral factors, such as glucocorticoids, that might be directly involved in induction of GTPCH mRNA by stress, we incubated PC12 cells with plasma from immobilized rats, which have greatly elevated corticosterone w4,27x. Levels of the 3.6 kb form of GTPCH mRNA were significantly higher in PC12 cells exposed to plasma from immobilized animals ŽFig. 3.. In contrast, incubation with plasma from control animals did not alter the steady-state levels of both GTPCH mRNA forms. Since plasma contains many factors that could elevate GTPCH expression, we examined the effect of the synthetic glucocorticoid, dexamethasone. PC12 cells were treated with 1 m M dexamethasone for 6–48 h, and the levels of GTPCH mRNA analyzed by Northern blot. For comparison, the same filters were reprobed to determine TH mRNA and cyclophilin levels ŽFig. 4.. The induction of both GTPCH mRNAs was dependent on the time of
Fig. 4. Time course of GTPCH mRNAs induction in PC12 cells treated with dexamethasone ŽA. Northern blot, ŽB. descriptive statistics. Cells were incubated for 2 days in medium with dialyzed sera and then for 6–48 h with a fresh aliquot of the same medium containing 1 m M dexamethasone. Total RNA was isolated at indicated time points and levels of GTPCH, TH and cyclophilin mRNAs analyzed by Northern blot as described in Materials and methods. ) P - 0.05 compared to its respective untreated controls.
incubation. With 6 h of incubation, only the levels of 3.6 kb GTPCH mRNA were significantly changed. Both GTPCH mRNAs were significantly increased after 12 h with dexamethasone. The maximum elevation was observed by 48 h. With longer times of exposure the levels of both GTPCH mRNA species were increased 4–6-fold by dexamethasone ŽFig. 4.. The TH mRNA levels were also elevated after 12 h of incubation with dexamethasone and were further increased by 24–48 h. The induction of GTPCH mRNA paralleled the increase in TH mRNA levels. In contrast, dexamethasone did not alter cyclophilin mRNA levels, which served as a control. 3.3. Transcriptional effect of immobilization stress and dexamethasone on GTPCH gene
Fig. 3. Plasma from immobilized rats induced GTPCH mRNA levels. PC12 cells were incubated with plasma from rats immobilized for 2 h and from controls. Total RNA was isolated after 24 h and the GTPCH mRNA levels determined by Northern blot. ) P - 0.05 compared to plasma from controls.
The observed elevation of GTPCH mRNA by glucocorticoids may directly reflect increased levels of transcription or enhanced stability of the transcripts or both. To study if increases in GTPCH mRNA by glucocorticoids are due at least in part to activated transcription of the gene, we performed nuclear run-on assays. In the adrenal medulla,
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Fig. 5. Run-on assay of GTPCH transcription rate. The time courses of relative GTPCH transcription is shown on left for rat adrenal medulla ŽA. and PC12 cells ŽB.. Representative hybridizations are shown on right for nuclei from: ŽA. adrenal medullae from control rats ŽC. or after 30 min immobilization ŽIMO. hybridized to 5-fold different concentrations of GTPCH plasmid Žhigh, low. or to vector Žlow, high. as control; ŽB. PC12 cells untreated controls ŽC. or after 24 h dexamethasone ŽDex. with two concentrations Žlow, high. of GTPCH or cyclophilin cDNA plasmids.
the incorporation of radiolabeled UTP into GTPCH-RNA was almost double the control levels by 30 min immobilization stress and it was further increased with 60 and 120 min of immobilization ŽFig. 5A.. In PC12 cells treated with dexamethasone, the rate of transcription also was raised but only after 12 h of incubation and remained f 2-fold higher than the control levels at 24 h ŽFig. 5B.. We did not observe any changes in transcription rate for cyclophilin. The observed increased transcription in response to stress or dexamethasone treatment indicates that the GTPCH gene may contain GREŽs.. Computer analysis was carried out to examine if the sequences of the GTPCH genes in the Gene Bank database have regions homologous to a consensus GRE. As shown in Fig. 6, sequences homologous to the complement of the consensus GRE
Fig. 6. Putative GRE sites detected by comparison of consensus GRE to human and mouse promoter sequences.
were found at positions y1548 to y1533 and y919 to y905 in the human promoter w52x and at position y523 to y513 in the mouse promoter w19x.
4. Discussion The results of this study indicate that GTPCH gene expression is regulated in vivo and in vitro at least partially by glucocorticoids. Infusion of cortisol to rats during 1 or 7 days caused a small, but significant, elevation in the adrenal medullary 3.6 kb GTPCH mRNA, which is considered to be encoding the active enzyme w10x. It was shown previously that administration of cortisol to rats for 1 day had no significant effect on basal TH and PNMT mRNA levels, however, 7 days cortisol infusion significantly raised adrenomedullary PNMT levels w27x. Although hypophysectomy was shown to prevent the stress-elicited induction of GTPCH and PNMT mRNA levels, the effect of exogenously administered cortisol was less pronounced for both genes w27,44x. Administration of the exogenous cortisol to hypophysectomized animals produced approximately the same rise in the levels of both GTPCH mRNA species as attained by immobilization stress. Cortisol infusion was effective in induction of GTPCH mRNA in hypophysectomized rats, indicating that glucocorticoids appear to be able to directly influence GTPCH gene expression. Previous results revealed that hypophysectomy blocked induction of adrenal GTPCH, PNMT and
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preproenkephalin mRNAs by immobilization stress w40,44,48x. The expression of PNMT gene has been shown to be regulated by glucocorticoids w53,54x. A GRE is present in the PNMT promoter w37x, and mutation of this site prevents hormonal induction w53x. However, hypophysectomy did not effect the induction of adrenal TH and DBH mRNAs by immobilization stress w33,40x although glucocorticoids can also greatly elevate their levels in adrenal chromaffin cells and PC12 cells w29,32,47x. Increases in TH expression by glucocorticoids are transcriptionally mediated w7,30x, however, the GREŽs. in the TH gene has not yet been identified. The involvement of glucocorticoids in the regulation of GTPCH gene expression was confirmed by in vitro experiments. In the present study, we have shown that in PC12 cells the rise of GTPCH mRNA can be induced by incubation with plasma from immobilized rats, which contains the endogenous glucocorticoid, corticosterone w4,27x, or with the synthetic exogenous glucocorticoid, dexamethasone. Both treatments resulted in increased levels of the 3.6 kb GTPCH mRNA. It was elevated after 12 h incubation with dexamethasone. GTPCH mRNA levels were increased and remained high for as long as 48 h. The time course of TH induction was similar as measured here and reported in previous studies and also is at least in part, transcriptionally mediated w29,32,47x. Parallel regulation of TH and GTPCH gene expression was also found in response to leukemia inhibitory factor and ciliary neurotrophic factor treatments in cultured superior cervical ganglia cells w46x. Although the increase in expression on GTPCH mRNA paralleled the rise of TH mRNA levels in PC12 cells they may differ in vivo since hypophysectomy abolished stress elicited rise of GTPCH but not of TH mRNA w33,44x. Glucocorticoids were found to directly affect rate of transcription of GTPCH gene in response to stress in adrenal medulla and in PC12 cells with dexamethasone treatment. The increased transcription was already 2-fold higher after 30 min of stress while the rise in PC12 cells was much slower. The time course for elevation of the rate of transcription in PC12 cells treated by dexamethasone was similar to increases in GTPCH mRNA levels. The reason for the faster response in vivo is unclear, however, glucocorticoids may indirectly modulate Ženhance or inhibit, depending on the biological circumstances. expression through their control of other transcription factors, such as AP1 and Egr1 w6,54x, or by changing the transcript stability or protein turnover. Alterations in glucocorticoid receptor levels w43x also contribute to the multiple pathways of exerting glucocorticoid effects in the cell. In addition to glucocorticoids, cAMP analogs and membrane depolarization also trigger parallel up-regulation of both GTPCH and TH gene expression w41x. Interestingly, while cAMP analogs and dexamethasone were not additive in their effects on TH transcription and mRNA levels in PC12 cells, combined treatment yielded higher TH activity
than either treatment alone w7,47x. It is attractive to speculate that there might be cross talk between cAMP and glucocorticoid mediated pathways with respect to induction of GTPCH, and that the increase, observed in TH activity may result from their additive effects on inductions in GTPCH gene expression and BH4 levels. In contrast to the longer GTPCH mRNA, the 1.2 kb GTPCH mRNA was induced by 1, but not 7, days cortisol infusion. These results indicate that under some conditions the two GTPCH mRNAs may be regulated differently. There is not yet enough information available regarding the differences between the two GTPCH mRNA species in the rat. It is not known how many rat GTPCH genes exist. It remains to be determined if the two mRNAs arise from differential splicing or from transcription at different promoters. If these mRNAs differ, as in human, solely by splicing at their 3X-end, glucocorticoids, as well as splanchnic denervation, may be affecting this splicing. The differential regulation of the 1.2 kb mRNAs could also be indirect. After long-term elevation of glucocorticoids in plasma, several mechanisms can be involved in the regulation of gene expression. Glucocorticoids besides their own effect on gene expression can activate negative feedback loops acting at the level of the pituitary, hypothalamus and higher nerve centers through their own receptors w12x or affect on neurotransmitters synthesis, turnover and release w8x. These mechanisms may prevent a rise in 1.2 kb GTPCH mRNA after chronic hormone treatment. Glucocorticoids may directly stimulate GTPCH gene expression by classical mechanisms via binding to a glucocorticoid receptor and interacting with positive GREs within the promoter. The sequences of human and part of the mouse, but not the rat, GTPCH promoters have been determined w19,52x. Comparison of these to the consensus GRE revealed two sequences in human GTPCH promoter and one in the mouse promoter with ) 85% homology to the complement of the consensus GRE. Whether the rat gene contains GRE-like sequences and whether these putative GREs are involved in the induction of GTPCH gene expression by glucocorticoids remain to be determined. This is the first study to our knowledge about the regulation of GTPCH mRNA by glucocorticoids. Given that GTPCH is the rate-limiting enzyme in BH4 biosynthesis, whose deficiency is associated with several neurological disorders w5,17,35,36,52x, our results indicate that glucocorticoids may modulate BH4 levels with corresponding changes in BH4-dependent reactions.
Acknowledgements This work was supported by Grant NS 32166 from the National Institute of Health. We thank Drs. Bargava Hiremagalur and Irwin Kopin for their help and useful suggestions.
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