Angiotensin II induces a complex activation of transcription factors in the rat brain: expression of Fos, Jun and Krox proteins

NeuroscienceVol. 65, No. 1, pp. 9349, 1995 ~

Pergamon

0306-4522(94)00482-X

Elsevier ScienceLtd Copyright © 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00

ANGIOTENSIN II INDUCES A COMPLEX ACTIVATION OF TRANSCRIPTION FACTORS IN THE RAT BRAIN: EXPRESSION OF FOS, JUN A N D KROX PROTEINS C. J. L E B R U N , * t A. B L U M E , * T. H E R D E G E N , ~ K. S E I F E R T , * R. BRAVO§ and T. U N G E R H *Department of Pharmacology, Im Neuenheimer Feld 366 and :~II. Institute of Physiology, Im Neuenheimer Feld 326, University of Heidelberg, 69120 Heidelberg, Germany §Bristol-Myers Squibb Pharmaceutical Research Institute, Molecular Biology Department, Princeton, NJ, 08543, U.S.A. IlDepartment of Pharmacology, University of Kiel, HospitalstraBe 4, 24105 Kiel, Germany Abstract-- We investigated the effects of intracerebroventricular injection of angiotensin II on neuronal

immediate early gene-encoded protein synthesis in the brain of conscious rats. The expression of seven immediate early gene-encoded transcription factors (c-Fos, FosB, c-Jun, JunB, JunD, Krox-20 (Egr-2) and Krox-24 (NGFI-A, Egr-1, Zif/268) was assessed simultaneously. Angi0tensin II (1, 10, 100 ng) induced a dose-dependent expression of c-Fos and Krox-24 in the subfornical organ, the median preoptic area and in the paraventricular nucleus and supraoptic nucleus of the hypothalamus, regions known to be involved in the central osmoregulatory and neuroendocrine actions of angiotensin II. FosB expression was induced four hours after icv injection of the highest dose of angiotensin II in the median preoptic area and paraventricular nucleus; c-Jun expression was restricted to the median preoptic area, subfornical organ and paraventricular nucleus, and JunB was only induced in the median preoptic area and subfornical organ. In these above mentioned regions, JunD exhibited a high basal staining, which was not visibly altered by angiotensin II. Krox-20 was not induced by angiotensin II. Intracerebroventricular injections of isotonic saline did not induce immediate early gene expression in any of the above brain areas. The angiotensin II-AT1 receptor antagonist, losartan, applied intracerebroventricular five minutes prior to angiotensin II, prevented the angiotensin II-induced immediate early gene protein expression. Losartan alone had no effects on immediate early gene expression. Our data show for the first time that stimulation of central periventricular angiotensin II-AT 1 receptors induces a finely tuned temporospatial expression of various immediate early gene-encoded transcription factors in distinct regions of the forebrain involved in blood pressure regulation and body fluid homeostasis. Thus, angiotensin II, in addition to its short-term regulatory actions, can participate through these transcription factors in neuroplastic processes.

mediated by the angiotensin ATl-receptor. 28 In the CNS, angiotensin II exerts defined neuroendocrine and osmoregulatory actions such as vasopressin and oxytocin secretion, release of adrenocorticotropic hormone, stimulation of drinking behaviour and natriuresis, and it participates in central autonomic control. 38'48 In adult individuals, brain structures involved in these central effects of A N G II such as the organum vasculosum of the lamina terminalis, the subfornical organ (SFO), the median preoptic area (MnPO), the paraventricular and supraoptic nucleus of the hypothalamus (PVN, SON), the area postrema or the nucleus tractus solitarii contain predominantly angiotensin receptors of the AT1 subtype, while the AT2 subtype dominates in the cerebellum, the locus coeruleus, the septum and the nuclei of the inferior olive. 34'45'46 In contrast to peripheral tissues, little is known about the effects of the stimulation of central angiotensin II receptors on the expression of IEGs. 16 Although adult neurons do not retain mitogenic ability, l E G s are expressed in neurons following a variety of stimuli, tlA7'2°'3°'3~'42It has been proposed

Angiotensin II is known to be not only involved in blood pressure and body fluid homeostasis, but may also mediate trophic changes in vascular smooth muscle cells. 37 Treatment with angiotensin II can result in cellular hypertrophy, e.g. in vascular smooth muscle cells of compliance vessels from normotensive rats, or in cellular hyperplasia, e.g. in vascular smooth muscle cells of mesenteric arteries from spontaneously hypertensive rats. 12'36An important step in cellular growth processes is the induction of immediate early genes (lEG) like c-fos and c-jun and their respective protein products, c-Fos and c-Jun which act as transcription factors. 1,4,31 Expression of c-fos, c-jun and c-myc m R N A by angiotensin II was observed in vascular smooth muscle cells. 23,32,47 The angiotensin II-induced c-fos m R N A expression is

tTo whom correspondence should be addressed. Abbreviations: AP-1, activator protein-l; i.c.v., intracerebroventricular; lEG, immediate early gene; MnPO, median preoptic area; PVN, paraventricular nucleus; SFO, subfornical organ; SON, supraoptic nucleus. NSC 65/I--D

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by several a u t h o r s t h a t in adult b r a i n tissue the e x p r e s s i o n o f I E G s m a y result in e s t a b l i s h i n g n e u r o n a l plasticity by linking p r i m a r y stimuli to n e u r o a n a t o m i c a l c h a n g e s via a l t e r a t i o n s in gene expression. 3j T h e c - f o s - e n c o d e d p r o t e i n (c-Fos) b i n d s to the A P - I r e c o g n i t i o n site f o u n d in the p r o m o t e r regions o f m a n y genes, b u t d o e s so efficiently only after h e t e r o d i m e r i z a t i o n with c - J u n o r related p r o reins, s'13'41 F u r t h e r m o r e , the I E G e x p r e s s i o n features t e m p o r a l - s p a t i a l p a t t e r n s w h i c h define t r a n s c r i p tional o p e r a t i o n s J ~'2°'26'4~ T h e r e f o r e , the effect o f c - F o s a n d related t r a n s c r i p t i o n f a c t o r s in regulating gene e x p r e s s i o n d e p e n d s o n f u n c t i o n a l l y related p a r t ners for the f o r m a t i o n o f t r a n s c r i p t i o n c o m p l e x e s . In the p r e s e n t study, we e x a m i n e d the synthesis o f seven I E G - e n c o d e d p r o t e i n s in distinct b r a i n areas following c e n t r a l a n g i o t e n s i n II r e c e p t o r s t i m u l a t i o n .

Experimental protocol All experiments were performed between 9 a.m. and l p.m. Rats were randomly allocated to six groups. Group 1 (n = 5) did not receive any icv injection. Group 2 (n - 8 ) received 5/11 0.9% NaCI, i.c.v, and served as vehicle control. The animals of group 3 received different icv injections of angiotensin lI (l.0ng ( n - 4 ) ; 10ng ( n - 4 ) ; 100ng (n = 10)). The animals of group 4 (n = 5) were pretreated with the specific angiotensin II-ATI receptor antagonist, losartan, (5 ~g, icv) 5min prior to the icv injection of angiotensin II (100 ng). Group 5 (n = 4) received an i.c.v. injection of 0.9% NaC1 followed by an i.c.v, injection of angiotensin II (100 ng). Group 6 (n = 4) received an i.c.v. injection of losartan (5/~g) followed by an icv injection of 0.9% NaC1. Ninety minutes after the last injection, rats were anesthetized with diethylether and perfused intracardially with 200 ml 4% paraformaldehyde in phosphate buffer at 4'C. Brains were removed, postfixed for 24h in 4% paraformaldehyde and then incubated for 72 h in 30% sucrose for cryoprotection.

lmmunohistochemistrv EXPERIMENTAL PROCEDURES

Animals Experiments were performed in conscious male Wistar rats weighing 250-300 g (Dr K. Thomae GmbH, Biberach, Germany). Animals were kept under controlled conditions with respect to temperature, humidity and light periodicity. For intracerebroventricular (i.c.v.) injections, a chronic cannula (PP20, Portex, U.K.) was implanted into the right lateral ventricle under chloralhydrate (400mg/kg i.p.) anesthesia. Rats were then housed individually for a sevenday-postoperative recovery period. To reduce unspecific stress-induced expression of transcription factors, animals were handled daily until the experiments were performed.

Cryostat-cut coronal sections (50/~m) were processed free-floating for immunocytochemistry using the conventional avidin-biotin complex peroxidase reaction as described in detail previously. ~v The dilutions of antibodies were as follows: anti-c-Fos 1:25,000; anti-FosB 1:2000; anti-c-Jun 1 : 30,000; anti-Jun B 1 : 4000; anti-JunD 1 : 8000; anti-Krox-20 1:4000; anti-Krox-24 1:4000. Generation and proofs of specificity of the polyclonal antibodies have been described previously) 9'25

Quantitati~'e analysis For each animal, one or two sections of each specifically labelled area (MnPO, SFO, SON and PVN) that contained the structure of interest at about the same level were chosen.

B

"| 4~

Fig. 1. Dose-dependent expression of c-Fos in the MnPO following icv injection of." (A), 0.9% NaCI as control; (B), 1 ng A N G II; (C), l0 ng angiotensin II; (D), 100 ng angiotensin II.

Angiotensin II in the rat brain The regions of interest were photographed and the number of stained neurons was counted by a person who was unaware of the treatment. The numbers presented in Table 1 refer to the averages and standard deviations of each experimental group. Data were subjected to a one-way analysis of variance (ANOVA). Student's t-test was used as a follow-up test to analyse differences between groups. Results were considered to be significantly different when P < 0.05.

Materials Angiotensin II (Bachem, Bubendorf, Switzerland) was dissolved in 0.9% NaCI and stored in 0.1% albumin-coated vials at -20°C until use. Aliquots were diluted as required on the day of the experiment. Losartan, a generous gift from Dr R. D. Smith (Dupont Merck Pharmaceutical Company, Wilmington, Delaware, U.S.A.) was dissolved in 0.9% NaC1 and stored at -20~'C until use. RESULTS

Control experiments (Groups 1 and 2) All antisera produced substantial basal labeling in some brain areas, e.g. cortex and striatum, as has been described elsewhere98 In the investigated areas, we found an expression of J u n D in MnPO, SFO, PVN and S O N of untreated rats. This basal labeling reflects the rather constitutive expression of J u n D that is also considered to act as housekeeping protein in many cell types.

Effect of intracerebroventricular tensin H (Group 3)

injections of angio-

Angiotensin II was injected icv at doses of 1 ng, 10ng and 100ng, respectively, l E G protein expression was assessed 90 min later. Since FosB has a delayed maximum of expression, 17'2°'26 this protein was additionally assessed 4 h later (n = 3). All immunoreactivities were exclusively nuclear. c-Fos. Angiotensin II administered icv at doses of 10 ng and 100ng induced a specific and dosedependent bilateral expression of c-Fos in the subfornical organ (SFO), the median preoptic area (MnPO), and in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus, whereas labeling was not affected in other brain areas (Fig. 1, Table 1). Following 1 ng angiotensin II, a weak suprabasal c-Fos immunoreactivity was observed in the S F O and in the M n P O in one animal. FosB. N o expression of FosB in the described regions was observed 90 min after icv injection of angiotensin II. However, FosB became clearly visible in the M n P O and PVN 4 h after the injection of 100 ng angiotensin II (Fig. 2A, Table 1). c-Jun, c-Jun was expressed dose-dependently after icv injections of 10 and 100 ng angiotensin II in the MnPO, S F O and PVN but not in the SON (Fig. 2B, Table 1). JunB. The expression of JunB was dosedependently stimulated in the M n P O and S F O following icv injection of angiotensin II, while the PVN and the S O N remained unstained (Fig. 2C, Table 1). JunD. M a n y neurons showed a distinct J u n D

95

Table 1. Expression of Fos, Jun and Krox proteins following intracerebroventricular injections of angiotensin II NaC1 c-Fos SFO MnPO PVN SON

--

ANGII 1 ng

ANGII 10 ng

ANGII 100 ng

6 ± 3*

18 ± 6"? 9*t 33 ± 7*t 8 ± 4*t

135 ± 22"t~ 40 ± 1 2 * t 220± 81*t~ 15 ± 4*t

--

12 ± 4 *

--

--

--

--

31 ±

FosB SFO MnPO PVN SON c-Jun SFO MnPO PVN SON JunB SFO MnPO PVN SON Krox-24 SFO MnPO PVN SON

3 ± 1"¢~ 7 ± 3"?~

19 ± 4*t 56 ± 25"t 44 ± 7*t

47 ± 5*t~ 83 ± l l * t 194 ± 26"~

9 ± 5*t 10 ± 5*t

30 ± 5*t~ 39 ± 6*t~

43 ± 7*? 41 ± 18*t 67 ± 8*t 23 ± 3*t

229 ± 79 ± 284 ± 76±

30*t~ 14*t 107*t~ 35"t

Data show the numbers of neurons (mean ___S.D.) stained or c-Fos, FosB, c-Jun, JunB and Krox-24 in the subfornical organ (SFO), median preoptic area (MnPO), paraventricular nucleus (PVN) and supraoptic nucleus (SON) following icv injections of angiotensin (ANG II) (l, 10, 100ng). *P <0.05 when compared to NaC1, t P <0.05 when compared to 1 ng, ~P <0.05 when compared to 10ng (ANOVA followed by Student's t-test). immunoreactivity in the SFO, MnPO, PVN and SON. Following icv injection of angiotensin II (100ng), there was no visible difference in J u n D immunoreactivity in these regions when compared to control animals injected with 0.9% NaCI (Fig. 2D). Krox-20. Angiotensin II injected icv did not induce any Krox-20 immunoreactivity above basal expression which was clearly present in the cortex and striatum. Krox-24. A specific, dose-dependent expression was observed in the MnPO, SFO, PVN and S O N following icv angiotensin II (10, 100 ng) (Table 1). After 1 ng A N G II, no specific staining could be detected in these regions.

Effect of the angiotensin II-AT1 receptor antagonist losartan on the angiotensin H-induced immediate early gene protein expression (Groups 4, 5, 6) Icv pretreatment with the angiotensin II-AT1 receptor antagonist, losartan, completely prevented the angiotensin II-induced expression of the Fos, Jun and Krox-24 proteins in the SFO, MnPO, PVN and SON in each animal (Fig. 3A). Animals of group 5 received an icv injection of 0.9% NaCI followed by an icv injection of angiotensin II (100 ng). The pattern of

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expression was similar to the one observed in animals treated with 100 ng angiotensin II alone (Fig. 3C). Losartan applied i.c.v. 5 min prior to the injection of 0.9% NaC1 (group 6) did not induce any expression of the Fos, Jun and Krox 24 proteins (Fig. 3B).

DISCUSSION

This study describes the expression of the IEGinduced transcription factors c-Fos, FosB, c-Jun, JunB, JunD, Krox-20 and Krox-24 in the rat CNS following stimulation of periventricular angiotensin II receptors in the brain. Apart from Krox-20 and JunD, all proteins were dose-dependently and exclusively stimulated in specific forebrain areas containing angiotensin II receptor fields, namely SFO, MnPO, PVN and SON. The angiotensin II-induced expression of Jun, Fos and Krox-24 proteins could be prevented by icy pretreatment with the angiotensin II-ATI receptor antagonist, losartan, demonstrating the involvement of angiotensin II-AT1 receptors.

Distribution pattern of immediate early gene-induced proteins following angiotensin H The SFO and the MnPO of the lamina terminalis, which contain a high level of angiotensin II-AT1 receptors, are involved in osmo- and cardiovascular regulation27'38'48 and, together with the PVN and

A

SON, in the release of neurohypophyseal hormones. 5'39'49 Angiotensinergic pathways between the SFO and the MnPO have been described. 27 Furthermore, there is considerable evidence for the existence of neuronal angiotensin II-containing pathways from circumventricular organs such as the SFO to the PVN a n d S O N . 27 Finally, the SFO and MnPO are target structures through which brain angiotensin II can induce the release of vasopressin from neurosecretory neurons in the PVN and S O N . 39'49 Since the AT1 receptor-containing PVN is located in close vicinity to the wall of the third ventricle, angiotensin II injected i.c.v, could diffuse from the third ventricle to the PVN and act directly or via interneurons on IEG expression in this nucleus. However, the absence of IEG protein expression in the periventricular area along the third ventricle renders this possibility unlikely. One could imagine a cascade of IEG stimulation through a neuronal pathway originating in the lamina terminalis and continuing to the PVN and SON. On the other hand, it is also possible that separate populations of cells respond to different concentrations of angiotensin II in either area. A similar pattern of expression of c-Fos was observed in response to icv injections of angiotensin II in a previous study26 In contrast to our data, these authors reported that c -Fos was also expressed in the OVLT. This finding may be attributed to the fact that the stimulus used in their study was stronger since a

D

Fig. 2. Following i.c.v, injection of 100 ng angiotensin II, immunohistochemistry demonstrates a distinct accumulation of (A) FosB in the MnPO, (B) c-Jun in the MnPO, (C) JunB in the SFO; (D) shows high basal expression of JunD in the SON after 0.9% NaC1.

Angiotensin II in the rat brain

Fig. 3. Inhibition of Krox-24 expression in the PVN following icv injection of the angiotensin II-ATI receptor antagonist, losartan (5 #g, icy), 5 min prior to the i.c.v, injection of 100 ng ANG II. (A) Losartan (5/~g)/angiotensin II (100 ng), (B) Losartan (5 #g)/0.9% NaC1; (C) 0.9% NaC1/angiotensin II (I00 ng). relatively high dose of 250ng angiotensin II was injected i.c.v.

Differential expression of immediate early geneinduced proteins The Fos and Jun families comprise c-Fos, FosB, Fra-l, Fra-2 as well as c-Jun, JunB and JunD. Members of the two families can form various homoand heterodimeric complexes which bind to so-called activator protein-I (AP-1) and Ca2+/cAMP response element (CRE) consensus sequences of the DNA. 21'24'25'26'41The transcriptional regulation which results from this binding is modified by several factors: 31'4t the binding affinity of the dimers is depen-

97

dent on the partners forming them; the AP-1 flanking sequences play an important role in the binding to this site and the resulting regulatory activity; some transcription factors have an inhibitory potency, e.g. JunB inhibits the c-Jun promoter, and the c-Fos:JunB dimer can act as a repressor at AP-1 sites. 7:°'43 Another feature of IEG proteins is their varying stability which depends on the partner to form dimers and, in consequence, results in different time courses of transcriptional activity.26,4~Thus, the variability of both, target DNA-sites and DNA binding complexes, allows an extremely fine regulation of gene transcription. Differential temporo-spatial patterns of IEG expression have already been observed under various experimental conditions. 11,17,18,2°With angiotensin II, we observed distinct patterns of lEGs induced in different brain regions: in the MnPO, expression of c-Fos, c-Jun, JunB and Krox-24 was induced after 90 min, while JunD, being expressed constitutively, was not increased above basal levels. Thus, the first dimers to be formed can be composed of c-Fos, on one hand, and three different Jun proteins on the other hand. As mentioned above, one of these heterodimers (c-Fos:JunB) has a negative regulatory potency. After 4 h, FosB joined in. FosB complexes exhibit the highest DNA binding affinity and the strongest transcriptional activity in vitro compared to the other AP-1 proteins. 41'51 A truncated form of FosB has been shown to inhibit the regulatory effects of c-Fos:Jun heterodimers. 33 Furthermore, FosB also suppresses gene expression. 33 Therefore, the late appearance of FosB could contribute to the termination of gene expression. After the lowest dose of I ng angiotensin II, AP-1 dimers were composed only of stimulated c-Fos and basal JunD; in this complex the lifetime of c-Fos is markedly prolonged: 1 The SFO exhibited an expression of c-Fos, c-Jun, JunB, JunD and Krox-24 after 90min like the MnPO, but no expression of FosB was induced after 4h. The two hypothalamic regions, SON and PVN, also featured patterns of angiotensin II-induced expression which differed from the others: in the SON, neither c-Jun nor JunB were induced; FosB was not expressed after 4 h. In the PVN, no expression of JunB occurred, but Krox-24 was expressed in a high number of neurons, and FosB appeared after 4 h. Since none of the above mentioned regions exhibited the same pattern of IEG protein expression, our results suggest that each of these areas plays a specific role in the regulatory circuit which is stimulated by angiotensin II by activating different sets of target genes. Another interesting point is the finding that two kinds of structurally totally different transcription factors such as c-Fos/c-Jun, on one hand, and Krox-24 which contains a so-called zinc-finger motif and binds to another consensus sequence,6 on the other hand, showed a congruent pattern of expression.

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Krox-20 has initially been described as an I E G protein induced by growth factor stimulation a n d plays a n i m p o r t a n t role in development. 6a'5° In the adult rat, Krox-20 is only induced by intense stimuli e.g. epileptic seizureJ 9~29 In our study in adult rats, Krox-20 expression was not stimulated by angiotensin II in the brain. Physiological significance expression

o f immediate

earl)" gene

In peripheral tissues, angiotensin II can induce growth in s m o o t h muscle a n d o t h e r cells, a process in which I E G s play an i m p o r t a n t role. 12'36"37M a m m a l i a n b r a i n cells are classically assumed to be postmitotic. Thus, icy t r e a t m e n t with angiotensin II a n d subsequent induction o f I E G s is not likely to result in g r o w t h o f n e u r o n a l structures. The expression o f transcription factors p r o b a b l y plays a role in n e u r o n a l plasticity. 3° Previous studies have s h o w n t h a t intraperitoneally or intravenously injected hypertonic saline can induce the expression of c-Fos in regions like the M n P O , P V N a n d

S O N . 14'35'44 Peripheral injection of hypertonic saline changes the m o r p h o l o g y of the S O N such as the extent of gila cell contact with magnocellular n e u r o n a l m e m b r a n e s , the n u m b e r of contacts between magnocellular neurones, the size of cell bodies, nucleoli a n d the golgi apparatus. 2 A n g i o t e n s i n II can be released in distinct hypothalamic nuclei u p o n h y p e r o s m o l a r stimulation, 15'~° a n d there is evidence for a participation of central angiotensinergic p a t h w a y s in the various responses to osmotic stimuli. 3"22'48Thus, it is reasonable to hypothetize that angiotensin II, by inducing the expression of transcription factors, plays a role in functional and morphological responses to osmotic stress. Investigation o f target genes of l E G s , which form the link between transcription factors and n e u r o n a l plasticity, will be the next step to address this hypothesis.

Acknowledgements~This study was supported by a grant to Thomas Unger from the Deutsche Forschungsgemeinschaft (DFG) (Zi 110/22-2). Annegret Blume is a recipient of a training grant from the DFG (Graduiertenkolleg "Molecular and Cellular Neurobiology").

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