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Apomorphine induction of AP-1 DNA binding in the rat striatum after dopamine depletion
151
Molecular Brain Research, 15 (1992) 151-155 Elsevier Science Publishers B.V.
BRESM 80139
Apomorphine induction of AP-1 DNA binding in the rat striatum after dopamine depletion K.R. Pennypacker, W.Q. Zhang, H. Ye and J.S. Hong Neuropharmacology Section, Laboratory of Molecular and Integrative Neuroscience, National Institute of Em'ironmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 (USA) (Accepted 19 May 1992)
Key words: Transcription factor; Striatum; 6-Hydroxydopamine; Jun protein, Fos-related antigen; c-Fos protein; AP-1 DNA binding
The expression of AP-1 transcription factors was assessed in the dopamine-depleted rat striatum over a 1 week period of repeated apomorphine injections. A single injection of apomorphine increased the expression of a 35 kDa Fos-related antigen and Jun proteins and their expression continued to increase until day 3 of repeated apomorphine treatment in dopamine-depleted striata. Apomorphine induces AP-1 transcription factors which may be involved in modulating gene expression in the striatum.
The transcription factors, c-Fos and Fos-related antigens (Fra), are induced in the rat striatum by administration of dopamine receptor agonists9'2°'24. After unilateral substantia nigral lesions, dopaminergic agonists selective for D1, but not D 2, receptors induce the expression of c-Fos and Fra immunoreactivities in the striatum e°. These transcription factors complex with one of the jun transcription factors, forming protein dimers that bind to the AP-1 element in the promotor region of target genes 15. Binding of these protein complexes to AP-1 DNA elements can either enhance or inhibit gene transcription. The genes of protachykinin1, prodynorphin3 and proenkephalin e contain potential AP-1 elements in their promotor regions. The levels of these neuropeptides are differentially modulated by dopamine in the rat striatum 7. Denervation of the nigrostriatal pathway increases [MetS]-enkephalin peptide and mRNA levels, but decreases or fails to alter peptide and mRNA of substance P and dynorphin7'11,1s. Repeated injections of the dopamine agonist, apomorphine (APO), at concentrations that activate D 1 receptors, increases both substance P and dynorphin synthesis with little effect on [MetS]-enkephalin synthesis7,12,13,14. 6-Hydroxydopamine (6-OHDA) lesions, which induce dopamine
receptor supersensitivity, followed by APO administration leads to a dramatic increase in prodynorphin message 7'14. The available evidence suggests that stimulation of the D 1 receptor up-regulates protachykinin and prodynorphin expression 8'1°, while proenkephalin expression may be regulated by the D z receptor 8. Thus, dopamine may regulate neuropeptide expression in the striatum by inducing c-Fos and Fra expression. Previously, we have found increased levels of a 35 kDa Fra in the 6-OHDA-lesioned striatum after injections (twice a day, 5 mg/kg s.c.) of APO for 7 days25. The 35 kDa Fra and dynorphin immunoreactivities were co-localized suggesting that the Fra transcription factors may be involved in peptide gene expression 25. In this report, we examined both Fra and Jun immunoreactivity in dopamine-depleted striata during the time course of APO treatment and utilized gel shift assays to assess AP-1 DNA binding activity. The striatal dopamine pathway of male Fischer rats (Charles River, Raleigh, NC) was lesioned by unilateral stereotaxic injection of 6-OHDA in artificial cerebral spinal fluid delivered into the substantia nigra, using coordinates based on Bregma: A-0.58; L + 0.16; V0.76 cm t9. The contralateral substantia nigra was injected with artificial cerebral spinal fluid. After 2 weeks,
Correspondence: K.R. Pennypacker, NIEHS MD 14-06, P.O. Box 12233, Research Triangle Park, NC 27709, USA. Fax: (1) 919-541-0841.
152 the rats were given bidaily injections of APO (5 m g / k g s.c., Sigma St. Louis, MO), a concentration that stimulates D~ receptors as well as D 2, for indicated times. Rats were decapitated 90 rain after the final injection, striata removed and the tissue was frozen on dry ice. Striata from treated rats were homogenized and nuclear protein extracts were prepared according to Sonnenburg et al. 2~. Fifty /xg of protein extract was separated on 12% SDS-polyacrylamide gels, transferred onto nitrocellulose membranes and the resulting blot blocked in 3% skim milk containing phosphate buffered saline (PBS) for 30 min. Blots were incubated with antibodies that recognize c-Fos and Fra (gift from Dr. Michael Iadarola, NIH) 24 at a 1:500 dilution or antibodies that cross-react with the Jun proteins (Oncogene Science) at a 1:50 dilution overnight at 4°C. The antibody against the Jun proteins was produced against a peptide containing the DNA binding domain of the c-Jun protein. After washing in PBS, membranes were incubated in anti-rabbit IgG alkaline phosphatase for 2 h at room temperature. After washing, the blots were developed in 5-bromo-4-chloro-3-indoyl phosphate p-toluidine s a l t / N i t r o blue tetrazolium (Sigma) or Lumi-Phos (Bohringer-Mannheim). With Lumi-Phos, blots were exposed to autoradiography film for 1 h at room temperature, while blots developed at least 30 min and up to 24 h in the colorimetric reagent. Oligomers used in this study were purchased from Research Genetics (Huntsville, Alabama). An 8-mer oligo was annealed to oligomers for the incorporation [c~-32p]dATP. Annealing was achieved by incubating 3-4 x the amount of 8-met with each single-stranded oligomer in 10 mM Tris-HCl, pH 8.0, 100 mM NaCI for l(J min at 65°C. After cooling to room temperature, 25 ng was labeled by using 10 units T7 polymerase, 100 mM dTTP, 100 mM dCTP, 100 mM d G T P and 30/zCi [a--~2p]dATP. The AP-1 element (22-mer) contains the consensus sequence (5'-TGAGTCA-3') in 5'-CTAGTGATGAGTCAGCCGGATC-3' while the 8-mer sequence is 5'-GATCCGGC-3'. Binding reactions (30/zl) were performed at room temperature and reaction mixtures contained 50 ~g protein, 20 mM Tris-HCl, pH 7.8, 100 mM NaC1, 5 mM MgCI> 1 mM EDTA, 5 mM dithiothreitol, 50 /~g/ml bovine serum albumin (BSA), 11)0 /xg/ml poly(dIdC), 10% glycerol, and approximately 0.1 ng (5 × 10 4 cpm) of specified probe. P r o t e i n - D N A complexes were separated on a 5% nondenaturing polyacryamide gel. Gels were run at 150 V in 50 mM Tris, 50 mM boric acid, 1 mM EDTA, dried and autoradiographed. Two weeks after lesioning of the substantia nigra, basal levels of 35 kDa Fra and 39 kDa Jun immunoreactivity were increased in the dopamine-depleted ipsi-
A.
No Trt. i
APO I
|r
Cont. Ips. Cont. Ips. r~11
u
II
I
kDa
kDa
69-47--
- - 55 (c-Fos) - - 35 (Fra)
30--
gq
No Trt. I
APO II
I
Cont. Ips. Cont. Ips. I
II
II
II
J
kDa
69-47--
- - 3 9 kDa (Jun)
30--
Fig. I. Induction of AP-1 proteins in the rat striatum after 6-OHDA lesion alone and/or following a single APO injection. Nuclear protein extracts (50/~g) from dopamine-depleted striata or 90 min after a single APO injection (5 mg/kg) were analyzed on Western blots for c-Fos and Fra immunoreactivity (A) or Jun immunoreactivity (13). Extracts from control striata (contralateral to the lesion; No Trt., Cont.) did not contain detectable Jun or Fra immunoreactivity while extracts from dopamine-depleted striata contained immunoreactivity to both proteins lips.). A single APO injection induced Jun immunoreactivity without increasing Fra expression in control striata (APO; Cont.). After APO treatment, extracts from dopamine-depleted striata contained increased levels of both Fra and Jun immunoreactivity (APO; Ips.). Each lane contained striatal extracts from 2 rats and these data are representative of 3 experiments.
lateral striatum (Fig. IA, B; Ips.). Control striata contralateral to the lesion contained undetectable levels of these proteins (Fig. 1A, B; Cont.). One injection of APO caused an increase in the expression of the 35 kDa Fra (Fig. 1A) and 39 kDa Jun protein (Fig. 1B) as well as inducing the c-Fos protein at 55 kDa (Fig. 1A) in lesioned striatal tissue after 90 min. Less intense Jun immunoreactive bands appear at 50 and 34 kDa (Fig. 1B) which may represent posttranslational modifications of the protein or other species from the jun protein family. In contralateral control striatal tissue, APO treatment caused a smaller increase in the level of Jun immunoreactivity (Fig. 1B, Cont.) with no detectable effect on c-Fos or Fra immunoreactivity (Fig.
153 control striata during the above time course of repeated A P O treatment. Fra immunoreactivity at 35 kDa was barely detectable (Fig. 3) and blots had to be developed overnight to visualize protein bands. The level of the 35 kDa Fra remained constant after the first injection of A P O (Day 0) until day 7 when the expression of this protein appeared to increase slightly. The expression of the Jun protein gradually increased during the treatment time course (Fig. 3B). Longer development times were necessary to detect AP-1 D N A binding (Fig. 3C). Very little binding was observed after a single injection (Day 0) or one day of treatment. Binding was detectable after 3 days of treatment and remained at similar levels at day 5 and day 7. Other investigators have described a profile of Fosimmunoreactive bands at 55 kDa (c-Fos), 46 kDa and 35 kDa in brains of rodents treated with seizure-inducing agents 21'22 and in striatum acutely treated with cocaine 24. The identities of the 46 and 35 kDa Fra's are unknown. The APO-induced profile in the striatum
1A). The 35 k D a Fra was detectable only with overexposure of the blot (data not shown). The time course of Jun and Fra immunoreactivities and AP-1 D N A binding in Striatal tissue after 6 - O H D A lesioning was followed during the 7 day (twice a day, 5 m g / k g s.c.) treatment regimen with APO. The level of the 35 k D a Fra gradually increased and peaked after 3 days of treatment (Fig. 2A). The c-Fos protein was induced only after 1 injection of A P O (Fig. 2A, Day 0) and disappeared thereafter. Jun immunoreactivity (Fig. 2B) peaked after 3 days of treatment and decreased with subsequent treatments. Binding to the canonical AP-1 D N A sequence increased after one day of treatment (Fig. 2C) and remained relatively constant thereafter. Antibodies against Fra proteins (Fig. 2C; at Fra) and Jun proteins (at Fra) inhibited AP-1 D N A binding in striatal extracts indicating that these proteins are involved in binding to this D N A sequence. Levels of Fra and Jun immunoreactivity and AP-1 D N A binding were also examined in the contralateral
A.
B=
Day
0
1 I
3 II
5 II
7 II
Day
I
kDa
69-47--
-- 55 (c-Fos)
47--
1 I
kDa 69--
0
3 II
5 II
7 II
I
kDa
-- 39 kDa (Jun)
30--
- - 35 {Fra) 30--
Co 0
Day I
1 i
3 I
5 in
7 i
NRS ~tFra cxJun HI
II
|1
I
Fig. 2. Time course of the expression of AP-I proteins and DNA binding activity in dopamine-depleted striata (ipsilateral to the 6-OHDA lesion) during injections (twice a day, 5 mg/kg s.c.) of APO. Nuclear protein extracts (50/zg) after 1 APO injection (Day 0)), 1 day, 3 days, 5 days or 7 days after APO administration (5 mg/kg) were analyzed on Western blots (A,B) or gel electrophoresis DNA binding assays (C). Striatal extracts were prepared 90 min after the last injection. Western blots were probed with antibodies against c-Fos and Fra (A) or Jun proteins (B). C shows the time course of binding to the canonical AP-1 DNA element. The AP-1 binding was specific since normal rabbit serum did not affect binding (C; NRS), while antibodies against the Fra ( ct Fra) or Jun proteins ( ct Jun) inhibited binding. Each lane contained extracts of striata from 2 rats and these data are representive of 3 experiments.
154
A.
g.
Day
I
i[m
kDa
5
3
7 "
Ii
Day ~ kOa - - 3 5 (Fra)
69-47--
0
kDa 69 -47 --
30--
1 I
3 II
5 n
7 I t - - I
- - 3 9 k D a (Jun)
30--
Cm
Day
I
0
II
1
i
3
n
5
li
7
B
Ill'==1
Fig. 3. Time course of the expression of AP-1 proteins and DNA binding activity in control striatal tissue (contralateral to the lesion) during bidaily APO injections. Nuclear extracts (50/~g) from striata contralateral to the lesion were prepared after 1 injection (Day 0), 1 day, 3 days, 5 days and 7 days after APO treatments (twice a day, 5 mg/kg s.c.). Striatal tissue was harvested 90 rain after the last injection. Western blot analysis shows the distribution of Fra (A) and Jun (B) immunoreactivities. DNA binding activity was analyzed with the canonical AP-1 element (C). As a control for non-specific binding, the probe was run in the presence of BSA (B). Each lane contained striatal extracts from 2 rats and these data are representative of 3 experiments.
differs in that the 35 kDa Fra is expressed without 46 kDa Fra. However, the basis for these differences is unknown and more studies are needed to elucidate the pharmacology of the expression of this protein. The 55 kDa c-Fos protein was only detected after one injection of APO indicating that c-Fos expression could not be induced after the initial drug treatment. A refractory period for c-Fos expression after initial expression has been described previously t6'23. Gene expression of c-Fos has been found to be down-regulated by other members of the Fra family suggesting that the 35 kDa Fra could be inhibiting Fos expression 17. Lesioning of the substantia nigra and APO treatment both increased Fra and Jun immunoreactivity in the striatum. It was not surprizing that lesioning elevated Fra and Jun expression since the Fos-like immunoreactivity is increased with D 2 r e c e p t o r inhibiton 4'5 and after 6-OHDA lesioning of the substantia nigra 4. The combination of these two treat-
ments acted in concert to further enhance the expression of these transcription factors. The neurons of dopamine-depleted striata are supersensitive to dopamine agonists accounting for the dramatic increase in Fra and Jun immunoreactivity. In the intact contralateral striatum, APO treatment increased Jun immunoreactivity to a greater extent than the 35 kDa Fra. Since the Fra expression is restricted to the striatal patches after APO administration 25, Jun expression may occur either in more cells throughout the stiatum or be induced to a greater extent per cell. AP-1 D N A binding as well as Fra expression was greater in the dopamine-depleted striatum after repeated APO treatment. In the dopamine-depleted striatum after treatment with dopamine agonists, Fra immunoreactivity is distributed homogenously throughout this brain region 9'z5, whereas, in the control side, only neurons in striatal compartments termed patches or striosomes, contain Fra immunoreactivity. The
155 g r e a t e r d i s t r i b u t i o n of F r a i m m u n o r e a c t i v i t y o n the d o p a m i n e - d e p l e t e d side a c c o u n t s for the higher levels of AP-1 D N A b i n d i n g . AP-1 D N A b i n d i n g , as well as J u n a n d F r a imm u n o r e a c t i v i t y r e a c h e d maximal levels in s t r i a t u m ipsilateral to the lesion after 1 - 3 days of A P O t r e a t m e n t . P r e l i m i n a r y results show that p r o d y n o r p h i n m R N A levels do n o t significantly increase u n t i l day 3 of A P O t r e a t m e n t a n d c o n t i n u e to increase until day 7, suggesting that AP-1 p r o t e i n expression may be involved in the e n h a n c e m e n t of this n e u r o p e p t i d e gene. Previously, we have shown that the 35 k D a F r a is co-localized with b o t h d y n o r p h i n a n d s u b s t a n c e P p e p t i d e s 25. T h e p r o t a c h y k i n i n gene c o n t a i n s a canonical AP-1 site 1, while the p r o d y n o r p h i n g e n e c o n t a i n s a site that differs by 1 base 3. T h e activation of d o p a m i n e receptors i n d u c e s c-Fos a n d F r a expression 2°'24 as well as i n c r e a s i n g p e p t i d e levels 8'1~. T h e results p r e s e n t e d here indicate that d o p a m i n e agonists can also i n d u c e J u n expression. T h e r e f o r e , activation of d o p a m i n e receptors i n d u c e s AP-1 t r a n s c r i p t i o n factor expression which may b i n d to target sites that r e g u l a t e p e p t i d e gene expression. We would like to thank Drs. Jeffrey Kitzler and L.H. Lazarus for their critical reading of this manuscript. 1 Carter, M.S. and Krause, J.E., Structure, expression, and some regulatory mechanisms of the rat preprotachykinin gene encoding substance P, neurokinin A, neuropeptide k, and neuropeptide gamma, J. Neurosci., 10 (1990) 2203-2214. 2 Comb M., Birnberg, N.C., Seasholtz, A., Herbert, E. and Goodman H., A cyclic AMP-phorbol ester inducible DNA element, Nature, 323 (1986) 353-356. 3 Douglass, J., McMurray, C.T., Garrett, J.E., Adelman, J.P. and Calavetta, L., Characterization of the rat dynorphin gene, Mol. Endocrinol., 3 (1989) 2070-2078. 4 Dragunow, M., Robertson, G.S., Faull, R.L.M., Robertson, H.A. and Jansen, K., D 2 dopamine receptor antagonists induce fos and and related proteins in rat striatal neurons, Neuroscience, 32 (1990) 287-294. 5 Dragunow, M., Williams, M. and Faull, R.L.M., Haloperidol induces fos related molecules in intrastriatal grafts derived from fetal striatal priomordia, Brain Res., 530 (1990) 309-311. 6 Dragunow, M., Leah, J.D. and Faull, R.L.M., Prolonged and selective induction of Fos-related antigens in striatal neurons after 6-hydroxydopamine lesion of the rat substantia nigra pars compacta, Mol. Brain Res., 10 (1991) 355-358. 7 Gerfen, C.R., Engber, T.M., Mahan, L.C., Susal, Z., Chase, T.N., Monsma, F.J. Jr. and Sibley, D.R., D I and D 2 dopamine receptor-regulated gene expression of striatonigral and striatopallidal neurons, Science, 7 (1990) 1429-1432. 8 Gerfen, C.R., McGinty, J.F. and Young W.S. III., Dopamine differentially regulates dynorphin, substance P, and enkephal.in
expression in striatal neurons: in situ hybridization histochemical analysis, J. Neurosci.7 11 (1991) 1016-1031. 9 Graybeil, A.M., Moratella, R. and Robertson, H.A., Amphetamine and cocaine induce drug-specific activation of the c-los gene in striosome-matrix compartments and limbic subdivisions of the striatum, Proc. Natl. Acad. Sci. USA, 87 (1990) 6912-6916. 10 Jiang, H.-K., McGinty, J.F. and Hong, J.S., Differential modulation of striatonigral dynorphin and enkephalin by dopamine receptor subtypes, Brain Res., 507 (1990) 57-64. 11 Li, S.J., Sivam, S.P. and Hong, J.S., Regulation of the concentration of dynorphin(1-8) in the striatonigral pathway by the dopaminergic system, Brain Res., 398 (1986) 390-392. 12 Li, S.J., Sivam, S.P., McGinty, J.F., Huang, Y.S. and Hong, J.S., Dopaminergic regulation of tachykinin metabolism in the striatonigral pathway, J. PharmacoL Exp. Ther., 243 (1987) 792-798. 13 Li, S.J., Sivam, S.P., McGinty, J.F., Douglass, J., Calvetta, L. and Hong, J.S., Regulation of the metabolism of striatal dynorphin by the dopaminergic system, J. Pharmacol Exp. Ther., 246 (1988) 403-408. 14 Li, S.J., Jiang, H.K., Stachowiak, M.K., Hudson, P.M., Owyang, V., Nanry, K., Tilson, H.A. and Hong, J.S., Influence of nigrostriatal dopaminergic tone on the biosynthesis of dynorphin and enkephalin in rat striatum, Mol. Brain Res., 8 (1990) 219-225. 15 Morgan, J.I. and Curran, T., Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun, Annu. Rev. Neurosci., 14 (1991) 421-451. 16 Morgan, J.I., Cohen, D.R., Hempstead, J.L. and Curran, T., Mapping patterns of c-fos expression in the central nervous system after seizure, Science, 237 (1987) 192-197. 17 Nakabeppu, Y. and Nathans, D., A naturally occurring truncated form of FosB that inhibits Fos/Jun transcriptional activity, Cell, 64 (1991) 751-759. 18 Normand, E., Popovich, T., Onteniente, B., Fellmann, D., Piatier-Tonneau, D., Auffray, C. and Bloch, B., Dopaminergic neurons of the substantla nigra modulate proenkephalin A gene in rat striatal neurons, Brain Res., 439 (1988) 39-46. 19 Paxinos, B. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, London, 1986. 20 Robertson, G.S., Herrera, D.G., Dragunow, M. and Robertson, H.A., L-DOPA activates c-los in the striatum ipsilateral to a 6-hydroxydopamine lesion of the substantia nigra, Eur. J. Pharmacol., 159 (1989) 99-100. 21 Sonnenberg, J.L., Macgregor-Leon, P.F., Curran, T. and Morgan, J.I., Dynamic alterations occur in the levels and composition of transcription factor AP-1 complexes after seizure, Neuron, 3 (1989a) 359-365. 22 Sonnenberg, J.L., Mitchelmore, C., Macgregor-Leon, P.F., Hempstead, J., Morgan, J.l. and Curran, T., Glutamate receptor agonists increase the expression of Fos, Fra, and AP-1 DNA binding activity in the mammmalian brain, Z Neurosci Res. 24 (1989b) 72-80. 23 Winston, S.M., Hayward, M.D., Nestler, E.J. and Duman, R.S., Chronic electroconvulsive seizures down-regulate expression of the immediate-early genes c-fos and c-jun in rat cerebral cortex, J. Neurochem., 54 (1990) 1920-1925. 24 Young, S.T., Porrino, L.J. and Iadarola, M.J., Cocaine induces striatal c-fos-immunoreactive proteins via dopaminergic D 1 receptors, Proc. Natl. Acad. Sci. USA, 88 (1991) t291-1295. 25 Zhang, W.Q., Pennypacker, K.R., Ye, H., Merchenthaler, I.J., Grimes, L., Iadarola, M.J. and Hong, J.S., A 35 kDa fos-related antigen is co-localized with substance P and dynorphin in striatal neurons, Brain Res., 577 (1992) 312-317.
Molecular Brain Research, 15 (1992) 151-155 Elsevier Science Publishers B.V.
BRESM 80139
Apomorphine induction of AP-1 DNA binding in the rat striatum after dopamine depletion K.R. Pennypacker, W.Q. Zhang, H. Ye and J.S. Hong Neuropharmacology Section, Laboratory of Molecular and Integrative Neuroscience, National Institute of Em'ironmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 (USA) (Accepted 19 May 1992)
Key words: Transcription factor; Striatum; 6-Hydroxydopamine; Jun protein, Fos-related antigen; c-Fos protein; AP-1 DNA binding
The expression of AP-1 transcription factors was assessed in the dopamine-depleted rat striatum over a 1 week period of repeated apomorphine injections. A single injection of apomorphine increased the expression of a 35 kDa Fos-related antigen and Jun proteins and their expression continued to increase until day 3 of repeated apomorphine treatment in dopamine-depleted striata. Apomorphine induces AP-1 transcription factors which may be involved in modulating gene expression in the striatum.
The transcription factors, c-Fos and Fos-related antigens (Fra), are induced in the rat striatum by administration of dopamine receptor agonists9'2°'24. After unilateral substantia nigral lesions, dopaminergic agonists selective for D1, but not D 2, receptors induce the expression of c-Fos and Fra immunoreactivities in the striatum e°. These transcription factors complex with one of the jun transcription factors, forming protein dimers that bind to the AP-1 element in the promotor region of target genes 15. Binding of these protein complexes to AP-1 DNA elements can either enhance or inhibit gene transcription. The genes of protachykinin1, prodynorphin3 and proenkephalin e contain potential AP-1 elements in their promotor regions. The levels of these neuropeptides are differentially modulated by dopamine in the rat striatum 7. Denervation of the nigrostriatal pathway increases [MetS]-enkephalin peptide and mRNA levels, but decreases or fails to alter peptide and mRNA of substance P and dynorphin7'11,1s. Repeated injections of the dopamine agonist, apomorphine (APO), at concentrations that activate D 1 receptors, increases both substance P and dynorphin synthesis with little effect on [MetS]-enkephalin synthesis7,12,13,14. 6-Hydroxydopamine (6-OHDA) lesions, which induce dopamine
receptor supersensitivity, followed by APO administration leads to a dramatic increase in prodynorphin message 7'14. The available evidence suggests that stimulation of the D 1 receptor up-regulates protachykinin and prodynorphin expression 8'1°, while proenkephalin expression may be regulated by the D z receptor 8. Thus, dopamine may regulate neuropeptide expression in the striatum by inducing c-Fos and Fra expression. Previously, we have found increased levels of a 35 kDa Fra in the 6-OHDA-lesioned striatum after injections (twice a day, 5 mg/kg s.c.) of APO for 7 days25. The 35 kDa Fra and dynorphin immunoreactivities were co-localized suggesting that the Fra transcription factors may be involved in peptide gene expression 25. In this report, we examined both Fra and Jun immunoreactivity in dopamine-depleted striata during the time course of APO treatment and utilized gel shift assays to assess AP-1 DNA binding activity. The striatal dopamine pathway of male Fischer rats (Charles River, Raleigh, NC) was lesioned by unilateral stereotaxic injection of 6-OHDA in artificial cerebral spinal fluid delivered into the substantia nigra, using coordinates based on Bregma: A-0.58; L + 0.16; V0.76 cm t9. The contralateral substantia nigra was injected with artificial cerebral spinal fluid. After 2 weeks,
Correspondence: K.R. Pennypacker, NIEHS MD 14-06, P.O. Box 12233, Research Triangle Park, NC 27709, USA. Fax: (1) 919-541-0841.
152 the rats were given bidaily injections of APO (5 m g / k g s.c., Sigma St. Louis, MO), a concentration that stimulates D~ receptors as well as D 2, for indicated times. Rats were decapitated 90 rain after the final injection, striata removed and the tissue was frozen on dry ice. Striata from treated rats were homogenized and nuclear protein extracts were prepared according to Sonnenburg et al. 2~. Fifty /xg of protein extract was separated on 12% SDS-polyacrylamide gels, transferred onto nitrocellulose membranes and the resulting blot blocked in 3% skim milk containing phosphate buffered saline (PBS) for 30 min. Blots were incubated with antibodies that recognize c-Fos and Fra (gift from Dr. Michael Iadarola, NIH) 24 at a 1:500 dilution or antibodies that cross-react with the Jun proteins (Oncogene Science) at a 1:50 dilution overnight at 4°C. The antibody against the Jun proteins was produced against a peptide containing the DNA binding domain of the c-Jun protein. After washing in PBS, membranes were incubated in anti-rabbit IgG alkaline phosphatase for 2 h at room temperature. After washing, the blots were developed in 5-bromo-4-chloro-3-indoyl phosphate p-toluidine s a l t / N i t r o blue tetrazolium (Sigma) or Lumi-Phos (Bohringer-Mannheim). With Lumi-Phos, blots were exposed to autoradiography film for 1 h at room temperature, while blots developed at least 30 min and up to 24 h in the colorimetric reagent. Oligomers used in this study were purchased from Research Genetics (Huntsville, Alabama). An 8-mer oligo was annealed to oligomers for the incorporation [c~-32p]dATP. Annealing was achieved by incubating 3-4 x the amount of 8-met with each single-stranded oligomer in 10 mM Tris-HCl, pH 8.0, 100 mM NaCI for l(J min at 65°C. After cooling to room temperature, 25 ng was labeled by using 10 units T7 polymerase, 100 mM dTTP, 100 mM dCTP, 100 mM d G T P and 30/zCi [a--~2p]dATP. The AP-1 element (22-mer) contains the consensus sequence (5'-TGAGTCA-3') in 5'-CTAGTGATGAGTCAGCCGGATC-3' while the 8-mer sequence is 5'-GATCCGGC-3'. Binding reactions (30/zl) were performed at room temperature and reaction mixtures contained 50 ~g protein, 20 mM Tris-HCl, pH 7.8, 100 mM NaC1, 5 mM MgCI> 1 mM EDTA, 5 mM dithiothreitol, 50 /~g/ml bovine serum albumin (BSA), 11)0 /xg/ml poly(dIdC), 10% glycerol, and approximately 0.1 ng (5 × 10 4 cpm) of specified probe. P r o t e i n - D N A complexes were separated on a 5% nondenaturing polyacryamide gel. Gels were run at 150 V in 50 mM Tris, 50 mM boric acid, 1 mM EDTA, dried and autoradiographed. Two weeks after lesioning of the substantia nigra, basal levels of 35 kDa Fra and 39 kDa Jun immunoreactivity were increased in the dopamine-depleted ipsi-
A.
No Trt. i
APO I
|r
Cont. Ips. Cont. Ips. r~11
u
II
I
kDa
kDa
69-47--
- - 55 (c-Fos) - - 35 (Fra)
30--
gq
No Trt. I
APO II
I
Cont. Ips. Cont. Ips. I
II
II
II
J
kDa
69-47--
- - 3 9 kDa (Jun)
30--
Fig. I. Induction of AP-1 proteins in the rat striatum after 6-OHDA lesion alone and/or following a single APO injection. Nuclear protein extracts (50/~g) from dopamine-depleted striata or 90 min after a single APO injection (5 mg/kg) were analyzed on Western blots for c-Fos and Fra immunoreactivity (A) or Jun immunoreactivity (13). Extracts from control striata (contralateral to the lesion; No Trt., Cont.) did not contain detectable Jun or Fra immunoreactivity while extracts from dopamine-depleted striata contained immunoreactivity to both proteins lips.). A single APO injection induced Jun immunoreactivity without increasing Fra expression in control striata (APO; Cont.). After APO treatment, extracts from dopamine-depleted striata contained increased levels of both Fra and Jun immunoreactivity (APO; Ips.). Each lane contained striatal extracts from 2 rats and these data are representative of 3 experiments.
lateral striatum (Fig. IA, B; Ips.). Control striata contralateral to the lesion contained undetectable levels of these proteins (Fig. 1A, B; Cont.). One injection of APO caused an increase in the expression of the 35 kDa Fra (Fig. 1A) and 39 kDa Jun protein (Fig. 1B) as well as inducing the c-Fos protein at 55 kDa (Fig. 1A) in lesioned striatal tissue after 90 min. Less intense Jun immunoreactive bands appear at 50 and 34 kDa (Fig. 1B) which may represent posttranslational modifications of the protein or other species from the jun protein family. In contralateral control striatal tissue, APO treatment caused a smaller increase in the level of Jun immunoreactivity (Fig. 1B, Cont.) with no detectable effect on c-Fos or Fra immunoreactivity (Fig.
153 control striata during the above time course of repeated A P O treatment. Fra immunoreactivity at 35 kDa was barely detectable (Fig. 3) and blots had to be developed overnight to visualize protein bands. The level of the 35 kDa Fra remained constant after the first injection of A P O (Day 0) until day 7 when the expression of this protein appeared to increase slightly. The expression of the Jun protein gradually increased during the treatment time course (Fig. 3B). Longer development times were necessary to detect AP-1 D N A binding (Fig. 3C). Very little binding was observed after a single injection (Day 0) or one day of treatment. Binding was detectable after 3 days of treatment and remained at similar levels at day 5 and day 7. Other investigators have described a profile of Fosimmunoreactive bands at 55 kDa (c-Fos), 46 kDa and 35 kDa in brains of rodents treated with seizure-inducing agents 21'22 and in striatum acutely treated with cocaine 24. The identities of the 46 and 35 kDa Fra's are unknown. The APO-induced profile in the striatum
1A). The 35 k D a Fra was detectable only with overexposure of the blot (data not shown). The time course of Jun and Fra immunoreactivities and AP-1 D N A binding in Striatal tissue after 6 - O H D A lesioning was followed during the 7 day (twice a day, 5 m g / k g s.c.) treatment regimen with APO. The level of the 35 k D a Fra gradually increased and peaked after 3 days of treatment (Fig. 2A). The c-Fos protein was induced only after 1 injection of A P O (Fig. 2A, Day 0) and disappeared thereafter. Jun immunoreactivity (Fig. 2B) peaked after 3 days of treatment and decreased with subsequent treatments. Binding to the canonical AP-1 D N A sequence increased after one day of treatment (Fig. 2C) and remained relatively constant thereafter. Antibodies against Fra proteins (Fig. 2C; at Fra) and Jun proteins (at Fra) inhibited AP-1 D N A binding in striatal extracts indicating that these proteins are involved in binding to this D N A sequence. Levels of Fra and Jun immunoreactivity and AP-1 D N A binding were also examined in the contralateral
A.
B=
Day
0
1 I
3 II
5 II
7 II
Day
I
kDa
69-47--
-- 55 (c-Fos)
47--
1 I
kDa 69--
0
3 II
5 II
7 II
I
kDa
-- 39 kDa (Jun)
30--
- - 35 {Fra) 30--
Co 0
Day I
1 i
3 I
5 in
7 i
NRS ~tFra cxJun HI
II
|1
I
Fig. 2. Time course of the expression of AP-I proteins and DNA binding activity in dopamine-depleted striata (ipsilateral to the 6-OHDA lesion) during injections (twice a day, 5 mg/kg s.c.) of APO. Nuclear protein extracts (50/zg) after 1 APO injection (Day 0)), 1 day, 3 days, 5 days or 7 days after APO administration (5 mg/kg) were analyzed on Western blots (A,B) or gel electrophoresis DNA binding assays (C). Striatal extracts were prepared 90 min after the last injection. Western blots were probed with antibodies against c-Fos and Fra (A) or Jun proteins (B). C shows the time course of binding to the canonical AP-1 DNA element. The AP-1 binding was specific since normal rabbit serum did not affect binding (C; NRS), while antibodies against the Fra ( ct Fra) or Jun proteins ( ct Jun) inhibited binding. Each lane contained extracts of striata from 2 rats and these data are representive of 3 experiments.
154
A.
g.
Day
I
i[m
kDa
5
3
7 "
Ii
Day ~ kOa - - 3 5 (Fra)
69-47--
0
kDa 69 -47 --
30--
1 I
3 II
5 n
7 I t - - I
- - 3 9 k D a (Jun)
30--
Cm
Day
I
0
II
1
i
3
n
5
li
7
B
Ill'==1
Fig. 3. Time course of the expression of AP-1 proteins and DNA binding activity in control striatal tissue (contralateral to the lesion) during bidaily APO injections. Nuclear extracts (50/~g) from striata contralateral to the lesion were prepared after 1 injection (Day 0), 1 day, 3 days, 5 days and 7 days after APO treatments (twice a day, 5 mg/kg s.c.). Striatal tissue was harvested 90 rain after the last injection. Western blot analysis shows the distribution of Fra (A) and Jun (B) immunoreactivities. DNA binding activity was analyzed with the canonical AP-1 element (C). As a control for non-specific binding, the probe was run in the presence of BSA (B). Each lane contained striatal extracts from 2 rats and these data are representative of 3 experiments.
differs in that the 35 kDa Fra is expressed without 46 kDa Fra. However, the basis for these differences is unknown and more studies are needed to elucidate the pharmacology of the expression of this protein. The 55 kDa c-Fos protein was only detected after one injection of APO indicating that c-Fos expression could not be induced after the initial drug treatment. A refractory period for c-Fos expression after initial expression has been described previously t6'23. Gene expression of c-Fos has been found to be down-regulated by other members of the Fra family suggesting that the 35 kDa Fra could be inhibiting Fos expression 17. Lesioning of the substantia nigra and APO treatment both increased Fra and Jun immunoreactivity in the striatum. It was not surprizing that lesioning elevated Fra and Jun expression since the Fos-like immunoreactivity is increased with D 2 r e c e p t o r inhibiton 4'5 and after 6-OHDA lesioning of the substantia nigra 4. The combination of these two treat-
ments acted in concert to further enhance the expression of these transcription factors. The neurons of dopamine-depleted striata are supersensitive to dopamine agonists accounting for the dramatic increase in Fra and Jun immunoreactivity. In the intact contralateral striatum, APO treatment increased Jun immunoreactivity to a greater extent than the 35 kDa Fra. Since the Fra expression is restricted to the striatal patches after APO administration 25, Jun expression may occur either in more cells throughout the stiatum or be induced to a greater extent per cell. AP-1 D N A binding as well as Fra expression was greater in the dopamine-depleted striatum after repeated APO treatment. In the dopamine-depleted striatum after treatment with dopamine agonists, Fra immunoreactivity is distributed homogenously throughout this brain region 9'z5, whereas, in the control side, only neurons in striatal compartments termed patches or striosomes, contain Fra immunoreactivity. The
155 g r e a t e r d i s t r i b u t i o n of F r a i m m u n o r e a c t i v i t y o n the d o p a m i n e - d e p l e t e d side a c c o u n t s for the higher levels of AP-1 D N A b i n d i n g . AP-1 D N A b i n d i n g , as well as J u n a n d F r a imm u n o r e a c t i v i t y r e a c h e d maximal levels in s t r i a t u m ipsilateral to the lesion after 1 - 3 days of A P O t r e a t m e n t . P r e l i m i n a r y results show that p r o d y n o r p h i n m R N A levels do n o t significantly increase u n t i l day 3 of A P O t r e a t m e n t a n d c o n t i n u e to increase until day 7, suggesting that AP-1 p r o t e i n expression may be involved in the e n h a n c e m e n t of this n e u r o p e p t i d e gene. Previously, we have shown that the 35 k D a F r a is co-localized with b o t h d y n o r p h i n a n d s u b s t a n c e P p e p t i d e s 25. T h e p r o t a c h y k i n i n gene c o n t a i n s a canonical AP-1 site 1, while the p r o d y n o r p h i n g e n e c o n t a i n s a site that differs by 1 base 3. T h e activation of d o p a m i n e receptors i n d u c e s c-Fos a n d F r a expression 2°'24 as well as i n c r e a s i n g p e p t i d e levels 8'1~. T h e results p r e s e n t e d here indicate that d o p a m i n e agonists can also i n d u c e J u n expression. T h e r e f o r e , activation of d o p a m i n e receptors i n d u c e s AP-1 t r a n s c r i p t i o n factor expression which may b i n d to target sites that r e g u l a t e p e p t i d e gene expression. We would like to thank Drs. Jeffrey Kitzler and L.H. Lazarus for their critical reading of this manuscript. 1 Carter, M.S. and Krause, J.E., Structure, expression, and some regulatory mechanisms of the rat preprotachykinin gene encoding substance P, neurokinin A, neuropeptide k, and neuropeptide gamma, J. Neurosci., 10 (1990) 2203-2214. 2 Comb M., Birnberg, N.C., Seasholtz, A., Herbert, E. and Goodman H., A cyclic AMP-phorbol ester inducible DNA element, Nature, 323 (1986) 353-356. 3 Douglass, J., McMurray, C.T., Garrett, J.E., Adelman, J.P. and Calavetta, L., Characterization of the rat dynorphin gene, Mol. Endocrinol., 3 (1989) 2070-2078. 4 Dragunow, M., Robertson, G.S., Faull, R.L.M., Robertson, H.A. and Jansen, K., D 2 dopamine receptor antagonists induce fos and and related proteins in rat striatal neurons, Neuroscience, 32 (1990) 287-294. 5 Dragunow, M., Williams, M. and Faull, R.L.M., Haloperidol induces fos related molecules in intrastriatal grafts derived from fetal striatal priomordia, Brain Res., 530 (1990) 309-311. 6 Dragunow, M., Leah, J.D. and Faull, R.L.M., Prolonged and selective induction of Fos-related antigens in striatal neurons after 6-hydroxydopamine lesion of the rat substantia nigra pars compacta, Mol. Brain Res., 10 (1991) 355-358. 7 Gerfen, C.R., Engber, T.M., Mahan, L.C., Susal, Z., Chase, T.N., Monsma, F.J. Jr. and Sibley, D.R., D I and D 2 dopamine receptor-regulated gene expression of striatonigral and striatopallidal neurons, Science, 7 (1990) 1429-1432. 8 Gerfen, C.R., McGinty, J.F. and Young W.S. III., Dopamine differentially regulates dynorphin, substance P, and enkephal.in
expression in striatal neurons: in situ hybridization histochemical analysis, J. Neurosci.7 11 (1991) 1016-1031. 9 Graybeil, A.M., Moratella, R. and Robertson, H.A., Amphetamine and cocaine induce drug-specific activation of the c-los gene in striosome-matrix compartments and limbic subdivisions of the striatum, Proc. Natl. Acad. Sci. USA, 87 (1990) 6912-6916. 10 Jiang, H.-K., McGinty, J.F. and Hong, J.S., Differential modulation of striatonigral dynorphin and enkephalin by dopamine receptor subtypes, Brain Res., 507 (1990) 57-64. 11 Li, S.J., Sivam, S.P. and Hong, J.S., Regulation of the concentration of dynorphin(1-8) in the striatonigral pathway by the dopaminergic system, Brain Res., 398 (1986) 390-392. 12 Li, S.J., Sivam, S.P., McGinty, J.F., Huang, Y.S. and Hong, J.S., Dopaminergic regulation of tachykinin metabolism in the striatonigral pathway, J. PharmacoL Exp. Ther., 243 (1987) 792-798. 13 Li, S.J., Sivam, S.P., McGinty, J.F., Douglass, J., Calvetta, L. and Hong, J.S., Regulation of the metabolism of striatal dynorphin by the dopaminergic system, J. Pharmacol Exp. Ther., 246 (1988) 403-408. 14 Li, S.J., Jiang, H.K., Stachowiak, M.K., Hudson, P.M., Owyang, V., Nanry, K., Tilson, H.A. and Hong, J.S., Influence of nigrostriatal dopaminergic tone on the biosynthesis of dynorphin and enkephalin in rat striatum, Mol. Brain Res., 8 (1990) 219-225. 15 Morgan, J.I. and Curran, T., Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun, Annu. Rev. Neurosci., 14 (1991) 421-451. 16 Morgan, J.I., Cohen, D.R., Hempstead, J.L. and Curran, T., Mapping patterns of c-fos expression in the central nervous system after seizure, Science, 237 (1987) 192-197. 17 Nakabeppu, Y. and Nathans, D., A naturally occurring truncated form of FosB that inhibits Fos/Jun transcriptional activity, Cell, 64 (1991) 751-759. 18 Normand, E., Popovich, T., Onteniente, B., Fellmann, D., Piatier-Tonneau, D., Auffray, C. and Bloch, B., Dopaminergic neurons of the substantla nigra modulate proenkephalin A gene in rat striatal neurons, Brain Res., 439 (1988) 39-46. 19 Paxinos, B. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, London, 1986. 20 Robertson, G.S., Herrera, D.G., Dragunow, M. and Robertson, H.A., L-DOPA activates c-los in the striatum ipsilateral to a 6-hydroxydopamine lesion of the substantia nigra, Eur. J. Pharmacol., 159 (1989) 99-100. 21 Sonnenberg, J.L., Macgregor-Leon, P.F., Curran, T. and Morgan, J.I., Dynamic alterations occur in the levels and composition of transcription factor AP-1 complexes after seizure, Neuron, 3 (1989a) 359-365. 22 Sonnenberg, J.L., Mitchelmore, C., Macgregor-Leon, P.F., Hempstead, J., Morgan, J.l. and Curran, T., Glutamate receptor agonists increase the expression of Fos, Fra, and AP-1 DNA binding activity in the mammmalian brain, Z Neurosci Res. 24 (1989b) 72-80. 23 Winston, S.M., Hayward, M.D., Nestler, E.J. and Duman, R.S., Chronic electroconvulsive seizures down-regulate expression of the immediate-early genes c-fos and c-jun in rat cerebral cortex, J. Neurochem., 54 (1990) 1920-1925. 24 Young, S.T., Porrino, L.J. and Iadarola, M.J., Cocaine induces striatal c-fos-immunoreactive proteins via dopaminergic D 1 receptors, Proc. Natl. Acad. Sci. USA, 88 (1991) t291-1295. 25 Zhang, W.Q., Pennypacker, K.R., Ye, H., Merchenthaler, I.J., Grimes, L., Iadarola, M.J. and Hong, J.S., A 35 kDa fos-related antigen is co-localized with substance P and dynorphin in striatal neurons, Brain Res., 577 (1992) 312-317.