The Journal of Neuroscience, March 1995, 7~73): 1854-1868 Neuronal Regulation of C-fos, C-jun, and junB Immediate-Early Genes in Rat Adrenal Medulla Markku Pelto-Huikko,’ Ake Dagerlind,2 Juha Kononen,’ Jan M. Lundberg,3 Marcello Villar,2 Jari Koistinaho,’ Rodrigo Bravo,4 and Tomas Hlikfelt” ‘Department of Biomedical Sciences, University of Tampere, Tampere, Finland, Departments of 2Neuroscience and 3Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden, and 4Department of Molecular Biology, Bristol-Meyers Squibb Pharmaceutical Research Institute, Princeton, New Jersey We have applied in situ hybridization histochemistry, it is possible that also another neurotransmitter(s), in ad- Northern analysis, and immunocytochemistry to study the dition to ACh, released from splanchnic nerve terminals is regulation of the immediate-early gene (IEG) c-fos, c-jun, involved in the regulation of their expression, especially of and junB mRNAs and the respective proteins in the rat ad- c-jun and junB. renal medulla. Electrophoresis mobility shift assay was [Key words: chromaffin ceils, preganglionic sympathetic used to examine changes in AP-1 DNA binding activity. In neurons, innervation, ACh, transcription, gene regulation] nontreated rats the mRNA and protein levels for these three IEGs were low. Reflex stimulation of adrenal medulla Gene transcription is regulated by the combined action of several elicited by a single capsaicin injection induced a rapid and truns-acting factors on distinct regulatory elements in target marked elevation in the mFlNA levels for these IEGs. Stim- genes (Dynan 1989; Lin et al., 1990). The complexity of the ulation with nicotine also caused a drastic increase in the intracellular signaling pathways involved in the control of gene mRNA levels, whereas muscarine only induced moderate expression after extracellular stimulation reflects the heteroge- elevations. c-fos and c-jun were induced strongly in adren- neity of truns-acting factor/enhancer interactions. Several genes aline cells and only weakly in noradrenaline cells. junB was coding for transcriptionally active proteins have been character- upregulated mainly in adrenaline cells. The AP-1 DNA bind- ized during the last decade (Sheng and Greenberg, 1990). One ing activity was low in control adrenals, whereas a marked of these transcription factors is the activator protein-l (AP-I) increase was observed after nicotine treatment. (Angel et al., 1987; Lee et al., 1987), which is formed by dimeric Treatment of the animals with a nicotinic (chlorisonda- complexes of the proteins (reviewed in Curran and Franza, 1988; mine) or a muscarinic (atropine) receptor antagonist did Abate and Curran, 1990; Vogt and Bos, 1990; Angel and Karin, not change the expression of IEGs studied. The combina- 1991) encoded by thefts (c--OS, fosB, f&l, fru-2) and jun (c- tion of the two drugs, however elevated the mRNA levels jun, junB, junD) families of proto-oncogenes (Bohman et al., for all three IEGs, especially for junB. Pretreatment of the 1987; Cohen and Curran, 1988; Hirai et al., 1989; Ryder et al., rats with chlorisondamine alone or in combination with at- 1989; Zerial et al., 1989; Nishina et al., 1990). Since a great ropine diminished the capsaicin-induced increase in c-fos, variety of extracellular stimuli elicits their rapid and transient whereas atropine alone was less efficient. Increase in c-jun induction in a protein synthesis-independent manner, they have mRNA was not affected by these drugs. The capsaicin-in- also been referred to as cellular immediate-early genes (IEGs). duced elevation of junB mRNA levels was not influenced The induction of the various members of ,fos and jun families by chlorisondamine or atropine alone, whereas both com- exhibit differential time course after stimulations, and thus the bined potentiated the effect of capsaicin. compositions of AP-1 complexes exhibit dynamic temporal The present results demonstrate that neurotransmitters changes (Morgan and Curran, 199 1). These complexes vary with released from splanchnic nerve terminals induce expres- regard to their binding affinity and specificity for the variants of sion of c-fos, c-jun, and junB in adrenal chromaffin cells AP-1 sites (Rysek and Bravo, 1991). The AP-I factors have also which results in increased AP-1 DNA binding activity. Al- been shown to interact with other nuclear proteins which may though stimulation of both nicotinic and muscarinic cholin- modify their target specificity and transcriptional activity (Hai ergic receptors may mediate the induction of these IEGs, and Curran, 1991; Schtile and Evans, 1991). This constitutes an operative basis for a large diversity of the transcriptional control, which allows a flexible and precise regulation of the expression Received Mar. 9, 1994; revised Aug. 22, 1994; accepted Sept. 6, 1994. of a specific subset of genes in response to extracellular stimuli. The skillful technical assistance of Ms. Hannele Ylitie, Ms. Katarina Aman, Ms. Siv Nilsson, Ms. Katarina Friberg, and Ms. Marketta Vuorinen is greatly The adrenal medulla responds to stimulation exerted by pre- appreciated. Drs. Tiina Henttinen and Antero Salminen are thanked for their ganglionic sympathetic nerve terminals by secretion of catecho- help in setting up the EMSA. This study was supported by the Swedish Medical lamines and costored bioactive peptides to meet the adaptive Research Council (04X-2887), Marcus and Marianne Wallenbergs Stiftelse, Sigurd and Elsa Goljes Minne, Svenska SZllskapet fGr Medicinsk Forskning changes needed. ACh is the major transmitter in preganghonic och Svenska LBkarsPllskapet. nerve terminals, and it binds to nicotinic receptors on chromaffin Correspondence should be addressed to Markku Pelto-Huikko, Department cells to elicit secretion (Ungar and Philips, 1983). This activation of Biomedical Sciences, University of Tampere, Box 607, FIN-33 IO1 Tampere, Finland. is followed by selective long-term induction of gene expression. Copyright 0 I995 Society for Neuroscience 0270-6474/95/l 5 1854-15$05.00/O Transsynaptic regulation of catecholamine synthesizing enzymes The Journal of Neuroscience, March 1995, 15(3) 1855 (Axelrod, 197 1; Thoenen, 1975; Zigmond, 1985) and more re- Rats in group 2 (n = 10) were anesthetized with chloral hydrate, and cently coexisting peptides has been studied by several research the left adrenal gland was surgically denervated. After 4 weeks, five of them received an injection of capsaicin (25 mg/kg, s.c.) 30 min before groups (LaGamma et al., 1989; Stachowiak and Got, 1992; Wan decapitation. et al., 1992; Dagerlind et al., 1994). Some results in these studies Group 3 rats (n = 40) were injected intraperitoneally with nicotine have been controversial, especially those dealing with the reg- (2 mglkg, Sigma), and the rats were decapitated after 5, 10, 20, 30 min, ulation of preproenkephalin (ENK) mRNA levels. Thus, de- and 1, 2, and 6 hr. Group 4 (n = 35) received an injection of muscarine (0.1 mg/kg, creased impulse activity after denervation and explanting the i.u.: Sigma) IO. 20. 30 min. and I. 2. and 6 hr before decaoitation. adrenal medulla increase ENK peptide (Schultzberg et al., 1979; * Group S’animals (n = 40) were g&en an intraperitoneal injection of Bohn et al., 1983; LaGamma et al., 1984; Dagerlin et al., 1994) chlorisondamine (5 mglkg, Ciba-Geigy), atropine (1 mg/kg; Leiras, Hel- and mRNA levels (Kilpatrick et al., 1984; LaGamma et al., sinki, Finland), or a combination of chlorisondamine and atropine 1 hr 1985, 1989; Dagerlind et al., 1994). Other researchers have dem- before injection with capsaicin (25 mglkg, s.c.). They were sacrificed 30 min after the capsaicin injection. onstrated an increase in ENK mRNA and peptide levels after After excision the adrenals were placed in ice-cold saline. All adre- direct and reflex stimulation of the adrenal medulla (Kanamatsu nals from each group were mounted together with controls on the same et al., 1986; Stachowiak and Got, 1992). chuck and frozen on dry ice. Cryostat sections (14 pm thick) were cut AP-1 binding sites are present in several of the enzyme and in a Microm HM 500 cryostat (Heidelberg, Germany) and thawed onto peptide genes expressed in adrenal chromaffin cells, and it has Probe-On (Fischer Scientific, Pittsburgh, PA) or Super Frost (Menzel, Germany) glasses and stored at -20°C until used. been suggested that AP- 1 binding proteins play an important role For immunocytochemical demonstration of the respective proteins in the regulation of adrenal gene expression (Stachowiak et al., rats (n = 30) were injected with capsaicin (25 mg/kg, s.c.) I, 2, 4, and 1990a,b; Gog et al., 1992; Icard-Liepkalns et al., 1992; Sta- 6 hr before sacrifice. Nicotine (2 mg/kg, i.p.) and muscarine (0. I mg/kg, chowiak and Got, 1992). Levels of c-fos, c-jun, and junB m- i.p.) were given 1 and 2 hr before perfusion. The rats were anesthesized RNAs and proteins are normally low or undetectable in the ad- with an overdose of chloral hydrate and perfused transcardially first with 100 ml of physiological saline followed by 2% paraformaidehyde in renal medulla, whereas a high expression of c-jun mRNA is seen _ohosohate-buffered _ saline (PBS) for 5 min. The adrenals were excised in the adrenal cortex (Koistinaho, 1991; Koistinaho et al., 1993; and postfixed in the same fixative for 1 hr. The tissues were cryopro- Pelto-Huikko et al., 1991; Pennypacker et al., 1992; Wessel and tected with 10% sucrose in PBS, frozen with carbon dioxide gas, and Joh, 1992). Stimulation of adrenal chromaffin cells in vivo or in sectioned at 14 km. The sections were thawn onto chromgelatin-subbed glass slides and processed directly for immunocytochemistry. vitro with nicotine or angiotensin (Stachowiak et al., 1990a,b; For Northern blotting and electrophoresis mobility shift assay rats (n Koistinaho, 1991; Koistinaho et al., 1993) and reflex activation = 10) were injected intraperitoneally with 2 mg/kg nicotine and sacri- of splanchnic nerve with reserpine, insulin or capsaicin induce ficed 30 min and 2 hr after the injection, respectively.