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Modulation of angiotensin II type 2 receptor mRNA in rat hypothalamus and brainstem neuronal cultures by growth factors
Molecular Brain Research 47 Ž1997. 229–236
Research report
Modulation of angiotensin II type 2 receptor mRNA in rat hypothalamus and brainstem neuronal cultures by growth factors X.-C. Huang, U.V. Shenoy, E.M. Richards, C. Sumners
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Department of Physiology, College of Medicine, UniÕersity of Florida, GainesÕille, FL 32610, USA Accepted 23 December 1996
Abstract This study investigates the regulatory effects of growth factors upon angiotensin II type 2 ŽAT2 . mRNA levels in neurons co-cultured from newborn rat hypothalamus and brainstem. Incubation of cultured neurons with nerve growth factor ŽNGF; 5–50 ngrml. caused time-dependent changes in the steady-state levels of AT2 receptor mRNA. Short-term Ž0.5–1.0 h. incubations with NGF resulted in significant increases in AT2 receptor mRNA, whereas longer-term incubations Ž4–24 h. caused significant decreases. Activation of NGF receptors is known to stimulate phospholipase C-g and subsequently activate protein kinase C ŽPKC.. Incubation of cultures with the PKC activator, phorbol-12-myristate-13-acetate ŽPMA; 100 nM., caused temporal changes in AT2 receptor mRNA levels similar to those observed with NGF. By contrast, insulin Ž0.1–10 m grml. elicited only significant decreases in AT2 receptor mRNA levels. The observed abilities of NGF and insulin to regulate the expression of AT2 receptor mRNA are consistent with the fact that the AT2 receptor gene promoter region contains several cis DNA regulatory elements that respond to growth factor-stimulated transcription factors. These novel observations which show that NGF and insulin can regulate AT2 receptor mRNA in neurons derived from neonatal rat CNS lend support to the idea that AT2 receptors have a role in development and differentiation. q 1997 Elsevier Science B.V. Keywords: Nerve growth factor; Insulin; Protein kinase C; Angiotensin II type 2 receptor; Neuron
1. Introduction The brain contains both major subtypes of receptors for the octapeptide angiotensin II ŽAng II. w31,36x. Ang II acts at Ang II type 1 ŽAT1 . receptors in the hypothalamus and brainstem to elicit the well-documented physiological responses, such as increased blood pressure, fluid and salt intake, and vasopressin release w11,20,29,37x. By contrast, the physiological roles of Ang II type 2 ŽAT2 . receptors in the brain are not established. The occurrence of high levels of AT2 receptors in tissues from neonatal animals, including brain, led to the suggestion that these sites have a role in development andror differentiation w8,25,36x. More direct evidence for a role of AT2 receptors in these processes has come from studies in both non-neural and neural cells. For example, activation of AT2 receptors inhibits neointimal formation following arterial injury w17x and inhibits proliferation of coronary endothelial cells w32x. Further) Department of Physiology, Box 100274, University of Florida, Gainesville, FL 32610, USA. Fax: q1 Ž352. 846-0270; E-mail: [email protected]
0169-328Xr97r$17.00 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 4 7 - 8
more, transfection of the AT2 receptor gene into ballooninjured rat arteries attenuated the stimulatory effects of Ang II Žvia AT1 receptors. on cell growth and proliferation w26x. The implication from these studies is that AT1 and AT2 receptors have antagonistic actions on cell growth and proliferation. With respect to neural cells, a recent study has linked AT2 receptors to programmed cell death w39x. In that study, activation of AT2 receptors in PC-12W pheochromocytoma cells Žan in vitro model of rat sympathetic neurons. led to apoptosis w39x. Studies in mutant mice lacking the gene encoding the AT2 receptor suggest that these sites have a role in centrally mediated behavior, since these mice exhibited decreased spontaneous movements and exploratory behavior compared with wild type animals w10,16x. Thus, AT2 receptors may have a role in the central control of certain behaviors as well as having roles in development, differentiation and apoptosis. Further evidence for a role of AT2 receptors in growth processes has come from two other areas of investigation. First, studies on the intracellular signal transduction pathways that are modulated by AT2 receptors have revealed interactions with pathways that are normally modulated by
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growth factors. For example, it is well known that mitogen-activated protein ŽMAP. kinases are critical components of the cellular processes of growth, differentiation and apoptosis w2,22,38x and it is clear that MAP kinases, such as Erk 1 and Erk 2, are activated by growth factors w28x. We have recently determined, using neuronal cultures prepared from newborn rat hypothalamus and brainstem, that selective stimulation of neuronal AT2 receptors elicits an inhibition of Erk 1 and Erk 2 activities w12x. Similar inhibitory effects of AT2 receptors on MAP kinase have been demonstrated in PC-12W cells w39x. Thus, the modulation of MAP kinase activity by AT2 receptor stimulation suggests a mechanism by which these receptors are involved in growth and developmental processes. Studies on the regulation of AT2 receptors have also supported this idea, since it has been observed both in peripheral tissues and cultured peripheral cells that growth factors are powerful regulators of AT2 receptor expression. Nerve growth factor ŽNGF., epidermal growth factor ŽEGF., bombesin, insulin, interleukin-1 b and serum starvation have all been shown to modulate the expression of AT2 receptors or AT2 receptor mRNA on R3T3 fibroblasts and on PC-12W cells w6,9,13–15,18,21x. These observations are perhaps not surprising considering that cloning and sequencing of the promoter region of the AT2 receptor gene has revealed the presence of several potential cis DNA elements that respond to hormones or transcription factors w13,19,24x. These include an activator protein-1 ŽAP-1. site which responds to protein kinase C ŽPKC. activation and an insulin-response sequence ŽIRS. similar to that of the phosphoenol pyruvate carboxy kinase ŽPEPCK. gene. Based on the above findings, we have investigated the effects of growth factors on the regulation of AT2 receptor mRNA in neurons cultured from newborn rat hypothalamus and brainstem. In extensive prior studies, we have demonstrated that these cells contain AT2 receptors which are identical to those in other tissues and cells from biochemical, molecular and pharmacological standpoints w33x. The data presented here indicate that NGF, insulin and PKC activation by phorbol-12-myristate-13-acetate ŽPMA. regulate the expression of AT2 receptors in cultured neurons, albeit in a different manner than in peripheral neural cells w21x. This is the first demonstration that growth factors can modulate AT2 receptors in cells derived from the brain and provides a further indication that the AT2 receptor system has importance in growth and development processes in the neonatal nervous system. 2. Materials and methods 2.1. Materials Newborn Sprague–Dawley rats were obtained from our breeding colony which originated from Charles River Farms ŽWilmington, MA.. Dulbecco’s modified Eagle’s medium ŽDMEM. and TRIzol reagent were purchased
from Gibco-BRL Life Technologies. Plasma-derived horse serum ŽPDHS. was from Central Biomedia ŽIrwin, MO.. Trypsin Ž1 = crystallized. was from Worthington Biochemicals ŽFreehold, NJ.. RT-PCR kits were purchased from Perkin Elmer Biotechnologies ŽNorwalk, CT.. Nerve growth factor was purchased from Promega Biotechnology ŽMadison, WI.. Deoxyribonuclease 1 ŽDNase 1., cytosine arabinoside ŽARC ., phorbol-12-myristate-13-acetate ŽPMA., 4a-phorbol Ž4a-PE. and insulin were from Sigma ŽSt. Louis, MO.. All other chemicals were purchased from Fisher Scientific ŽPittsburgh, PA. and were of analytical grade or higher. Primers for the AT2 receptor and b-actin genes were synthesized in the DNA facility of the Interdisciplinary Center for Biotechnology Research, University of Florida. The sequences of these primers are as follows. AT2 receptor gene Sense primer: 5X-CAATCTGGCTGTGGCTGACT-3X Antisense primer: 5X-ATGACAATCTCCTGCCAACG-3X
b-Actin gene Sense primer: 5X-CTCATGAAGATCCTGACCGA-3X Antisense primer: 5X-TACGGATGTCAACGTCACAC-3X 2.2. Neuronal cultures Neuronal co-cultures were prepared from the brainstem and hypothalamic block of newborn Sprague–Dawley rats exactly as described previously w12,33x. Trypsin and DNase 1-dissociated cells were re-suspended in DMEM containing 10% PDHS and were plated in poly-L-lysine-coated 60-mm Nunc plastic tissue culture dishes. Cells were grown for 3 days at 378C in a humidified incubator with 95% airr5% CO 2 and were then treated with 1 m M ARC for 2 days. After this, ARC was removed and the cells incubated with fresh DMEMr5% PDHS for 9–12 days before use. At this time, cultures consisted of ) 90% neurons and - 10% astrogliarmicroglia. 2.3. Extraction of total RNA Growth media were removed from control or drugtreated neuronal cultures which were then washed once with ice-cold phosphate-buffered saline ŽpH 7.4.. Following this, cultured cells were lysed in TRIzol reagent Ž0.5 mlrdish. and transferred to a sterile tube. The lysates were incubated for 5 min at room temperature to permit the complete dissociation of nucleoprotein complexes. Following this, 0.2 ml chloroformr1 ml TRIzol reagent was added to each tube and samples were shaken vigorously by hand for 15 s. Samples were then incubated at room temperature for 3 min, followed by centrifugation at 12 000 = g for 15 min at 48C. The upper aqueous phase
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was transferred to a fresh tube and RNA was precipitated by mixing with an equal volume of isopropyl alcohol at room temperature for 10 min, followed by centrifugation. The RNA pellet was washed once with 75% ethanol and RNA samples were stored in the same solvent. The RNA concentration was determined by spectrophotometry. Before use, 20 m g RNA was incubated with 1 U DNAase 1 at 378C for 15 min and then the RNA was re-extracted using the method described above. 2.4. ReÕerse transcriptase polymerase chain reaction (RTPCR) RT-PCR was performed by using RNA PCR kits from Perkin Elmer Biotechnology. Briefly, reverse transcription
Fig. 2. Stimulatory effects of NGF on neuronal AT2 receptor mRNA levels. Neuronal cultures were treated with control solution or the indicated concentrations of NGF for 30 min at 378C, followed by analyses of AT2 receptor and b-actin mRNAs as detailed in Materials and methods. Top: representative ethidium bromide-stained gels showing the RT-PCR DNA products which correspond to the AT2 receptor and b-actin mRNAs. Bottom: quantification of the DNA bands that correspond to the AT2 receptor mRNA. Data are expressed as mean"S.E.M. from 3 independent experiments. ) P - 0.05.
Fig. 1. Effects of NGF on neuronal AT2 receptor mRNA levels as a function of incubation time. Neuronal cultures were treated with control solution or NGF Ž5 ngrml. at 378C for the indicated times, followed by analyses of AT2 receptor and b-actin mRNAs as detailed in Materials and methods. Top: representative ethidium bromide stained gels showing the RT-PCR DNA products which correspond to AT2 receptor Ž1.05 kb. and b-actin Ž0.3 kb. mRNAs. Bottom: quantification of the DNA bands corresponding to the AT2 receptor mRNA. Data are expressed as the ratio of AT2 receptor mRNAr b-actin mRNA in each treatment situation. Results are mean"S.E.M. from 3 independent experiments. ) P - 0.05.
was carried out in a 20 m l volume containing 5 mM MgCl 2 , 50 mM KCl, 10 mM Tris–HCl ŽpH 8.3., 1 mM dGTP, 1 mM dATP, 1 mM dTTP, 1 mM dCTP, 1 Urm l RNase inhibitor, 2.5 Urm l MuLV reverse transcriptase, 2.5 m M random hexamers and 1 m g of the total RNA sample. The mixture was incubated at 258C for 10 min, then 428C for 30 min, 998C for 5 min and finally 58C for 5 min. Following this, 10 m l Žfor AT2 receptor. or 5 m l Žfor b-actin. of reverse transcription reaction product were
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combined with 40 m l Žfor AT2 receptor. or 20 m l Žfor b-actin. of PCR master mix containing 50 mM KCl, 10 mM Tris–HCl ŽpH 8.3., AmpliTaq DNA polymerase 2.5 Ur100 m l and 0.2 pgrm l each of the upstream and downstream primers. PCR was performed at 948C for 4 min, followed by 30 cycles Žfor AT2 receptor. or 20 cycles Žfor b-actin. at 948C for 1 min, 558C for 1 min, 728C for 1 min. After final extension at 728C for 8 min, PCR products were electrophoresed on a 1% agarose gel containing 1 m grml of ethidium bromide. The densities of PCR products were analyzed by Imagequant and Microsoft Excel softwares. The relative AT2 receptor mRNA levels were compared by a ratio of band densities representing AT2 receptor mRNA over the band densities of the corresponding b-actin mRNA. 2.5. Experimental groups and data analysis All results are expressed as mean " S.E.M. and were obtained by combining data from individual experiments. Comparisons of multiple means were made using an analysis of variance, followed by a Newman–Keuls test to assess statistical significance between individual means. Statistical analyses were performed using Sigma Stat Software ŽJandel, San Rafael, CA..
3. Results In the experiments described here, we utilized RT-PCR to assess the levels of AT2 receptor mRNA in neuronal cultures. In preliminary experiments, we determined that production of the 1.05-kb AT2 receptor-specific DNA by PCR was linear between 27 and 33 cycles of amplification. Furthermore, production of the 1.05-kb AT2 receptor DNA increased linearly as a function of RT volume from 5–15 m l, at 30 cycles of PCR amplification Ždata not shown.. Based upon this, in subsequent studies we performed 30 cycles of PCR with 10 m l RT volume for the AT2 receptor mRNA amplification. We also determined that production of a 0.3-kb b-actin-specific DNA from the PCR was linear between 10 and 25 cycles of amplification and from 2.5 to 7.5 m l of RT volume, at 20 cycles of PCR amplification Ždata not shown.. Thus, in subsequent studies, we performed 20 cycles of PCR with 5 m l RT volume for the b-actin mRNA amplification. The relative levels of AT2 receptor mRNA levels in control and drug-treated neuronal cultures were compared following quantification of the 1.05-kb AT2 receptor and 0.3-kb b-actin bands. Incubation of neuronal cultures with NGF Ž5 ngrml. resulted in a time-dependent alteration in the intensity of the 1.05-kb AT2 receptor band Žcorresponding to AT2 receptor mRNA.. The data presented in Fig. 1 show that NGF induced a significant increase in AT2 receptor mRNA levels during the first 2 h after treatment, with a maximal increase at 30 min. By contrast, longer incubation times
Fig. 3. Inhibitory effects of NGF on neuronal AT2 receptor mRNA levels. Neuronal cultures were treated with control solution or the indicated concentrations of NGF for 24 h at 378C, followed by analyses of AT2 receptor and b-actin mRNAs as detailed in Materials and methods. Top: representative ethidium bromide-stained gels showing the RT-PCR DNA products which correspond to the AT2 receptor and b-actin mRNAs. Bottom: quantification of the DNA bands that correspond to the AT2 receptor mRNA. Data are expressed as mean"S.E.M. from 3 independent experiments. ) P - 0.05.
with NGF Ž4–24 h. elicited a significant decrease in AT2 receptor mRNA levels and a maximal reduction was obtained 12–24 h after treatment. The effects of NGF on AT2 receptor mRNA in neuronal cultures were also concentration-dependent. A maximal stimulatory effect was elicited by 5–50 ngrml NGF ŽFig. 2., whereas a maximal inhibitory effect was obtained with 50 ngrml NGF ŽFig. 3.. In all cases, treatment of neuronal cultures with NGF
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failed to elicit significant changes in b-actin mRNA ŽFigs. 1–3.. Activation of NGF receptors leads to a stimulation of phospholipase C-g ŽPLC-g . w35x and subsequent activation of PKC w3,4,27x. PKC can regulate gene transcription via an activator protein-1 ŽAP-1. site w1,7,34x and the presence of an AP-1 site within the AT2 receptor promotor has been documented w13,19,24x. Based on this, we analyzed the effects of activation of PKC on AT2 receptor mRNA levels in neuronal cultures. Incubation of neuronal cultures with the PKC activator PMA Ž100 nM. resulted in a time-dependent change in AT2 receptor mRNA similar to that observed with NGF. The data presented in Fig. 4
Fig. 5. Effects of insulin on neuronal AT2 receptor mRNA levels as a function of incubation time. Neuronal cultures were treated with control solution or insulin Ž10 m grml. at 378C for the indicated times, followed by analyses of the AT2 receptor and b-actin mRNAs as detailed in Materials and methods. Top: representative ethidium bromide-stained gels showing the RT-PCR DNA products which correspond to the AT2 receptor and b-actin mRNAs. Bottom: quantification of the DNA bands that correspond to the AT2 receptor mRNA. Data are expressed as mean"S.E.M. from 3 independent experiments. ) P - 0.05.
Fig. 4. Effects of PMA on neuronal AT2 receptor mRNA levels as a function of incubation time. Neuronal cultures were treated with control solution or PMA Ž100 nM. for the indicated times at 378C, followed by analysis of AT2 receptor and b-actin mRNAs as detailed in Materials and methods. Top: representative ethidium bromide-stained gels showing the RT-PCR DNA products which correspond to the AT2 receptor and b-actin mRNAs. Bottom: quantification of the DNA bands that correspond to the AT2 receptor mRNA. Data are expressed as mean"S.E.M. from 3 independent experiments. ) P - 0.05.
show that PMA induced a significant increase in the levels of AT2 receptor mRNA during the first 2 h after treatment, with a maximal increase obtained at 30 min. Longer incubation times with PMA elicited a significant decrease in AT2 receptor mRNA levels, with a maximal reduction obtained between 12 and 24 h ŽFig. 4.. PMA treatment did not alter b-actin mRNA levels ŽFig. 4. and AT2 receptor mRNA levels were not altered by treatment of cultures with 100 nM 4-a phorbol ester which does not activate PKC Ždata not shown.. The AT2 receptor gene also contains a nucleotide sequence similar to the IRS which occurs in the promotor
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region of the PEPCK gene w13x. Based upon this, in the next series of experiments we investigated the effects of insulin on AT2 receptor mRNA levels in neuronal cultures in order to test whether the IRS has a role in AT2 receptor gene transcription. Incubation of neuronal cultures with insulin Ž10 m grml. induced a significant time-dependent decrease in the levels of AT2 receptor mRNA ŽFig. 5.. This effect became apparent following 4 h of treatment
Fig. 6. Inhibitory effects of insulin on neuronal AT2 receptor mRNA levels. Neuronal cultures were treated with control solution or the indicated concentrations of insulin for 24 h at 378C, followed by analysis of AT2 receptor mRNA levels as detailed in Materials and methods. Top: representative ethidium bromide-stained gels showing the RT-PCR DNA products which correspond to the AT2 receptor and b-actin mRNAs. Bottom: quantification of the DNA bands that correspond to the AT2 receptor mRNA. Data are expressed as mean"S.E.M. from 3 independent experiments. ) P - 0.05.
with insulin and was maximal after 24 h ŽFig. 5.. The inhibitory effects of insulin were also concentration-dependent, with maximal effects obtained at 1–10 m grml insulin ŽFig. 6..
4. Discussion The data presented in these studies show that the steady-state level of mRNA for AT2 receptors on brainstem and hypothalamic neurons co-cultured from 1-day-old rats is regulated by growth factors. NGF and insulin alter steady-state levels of AT2 receptor mRNA. PKC activation with PMA, mimicking one of the signal transduction pathways activated by NGF w3,4,27x, also affected steady-state mRNA levels of the AT2 receptor. Insulin caused time- and dose-dependent decreases in steady-state levels of AT2 receptor mRNA. Since the promotor region of the mouse AT2 receptor gene contains a sequence somewhat homologous to the IRS of the PEPCK gene w13x, these findings are perhaps not surprising. It should be noted, however, that this sequence was not reported to be present in the promotor region of the AT2 receptor gene of the rat w19x. Insulin has previously been shown to regulate steady-state levels of mRNA for the AT2 receptor in R3T3 cells w15x, but in that study insulin increased both mRNA for the AT2 receptor and the number of AT2 receptor sites on the cells. Several possible reasons for the differences between the two studies exist. First, the growth conditions and treatment paradigms used. R3T3 cells were grown to confluency, a situation in which AT2 receptor mRNA expression is increased w6x. Following this, the serum-containing medium was removed and replaced with insulin-containing serum-free media for 2 days. Neuronal cultures, which do not grow to confluency as they are not continuously dividing cells, were grown for 9–12 days in media containing 5% serum and then insulin was added to the media without otherwise altering it. Second, the cell lineages are very different, i.e. primary rat neurons compared to a mouse fibroblast cell line. Previous studies have suggested that cell lineage could be important because some DNA-binding proteins interacting with the promotor region of the AT2 receptor gene are different in PC-12W and vascular smooth muscle cells w14x, cell lines of very different cell lineages. Further studies will be needed to resolve these differences. NGF and PMA caused similar changes in the expression of mRNA for the AT2 receptor. The effects were time- and dose-dependent but biphasic. NGF and PMA caused in the short-term increases in AT2 receptor mRNA and in the long-term Ž) 4 h incubation. decreases in AT2 receptor mRNA. There are several consensus DNA sequences in the promotor region of the rat AT2 receptor gene which might be influenced by NGF and PMA application to the cells. There are two AP-1 sequences in the rat
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AT2 receptor promotor region w19x as well as AP-1 sites in the mouse AT2 receptor gene w13,19,24x. AP-1 sequences bind heterodimers of c-fos and c-jun which are activated following stimulation of PKC activity w34x. There is also a Myc-binding site in the rat AT2 receptor gene which can bind c-myc. AT1 receptor stimulation by AngII leads to increases in c-fos, c-jun and c-myc levels w5,30x. Thus, AT1 receptor stimulation may decrease AT2 receptor transcription as has previously been suggested w19x. In view of previous findings showing opposite actions of AT1 and AT2 receptors on the activities of the important growth regulating molecules, the MAP kinases w12,26x, it may be important for activation of AT1 receptors to decrease synthesis of the opposing AT2 receptor so that the AT1mediated activation of MAP kinase by angiotensin II could predominate. For example, PMA stimulation of neuronal cells results in increased c-fos and c-jun. This increases AT1 receptor mRNA transcription w23x and decreases steady-state levels of AT2 receptor mRNA, suggesting that under these conditions the AT1 receptor mediated effects of angiotensin would override the AT2 receptor-induced effects. This could occur for example following long-term Ž) 4 h. exposure of cells to PMA or NGF. This would add another layer of complexity to the opposing interactions of the AT1 and AT2 receptor subtypes and also suggests that other trophic agents may similarly regulate the AT2 receptor. These and other possibilities await further study. In conclusion, AT2 receptor mRNA levels in cultured neurons are regulated by growth factors. This regulation by growth factors was occasionally different than had been reported in other cells suggesting that tissue-specific regulation of AT2 receptor mRNA levels may occur. Acknowledgements We thank Jennifer Moore for preparation of cultured neurons and Jennifer Brock for typing the manuscript. This work was supported by Grant NIH NS-19441.
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References w20x w1x Angel, P., Imagawa, M., Chiu, R., Stein, B., Imbra, R.J., Ranmsdorf, H.J., Jouat, C., Herruch, P. and Karin, M., Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor, Cell, 49 Ž1987. 729–739. w2x Ahn, N.G., Seger, R. and Krebs, E.G., The mitogen-activated protein kinase activator, Curr. Opin. Cell Biol., 4 Ž1992. 992–999. w3x Balbi, D. and Allen, J.M., Role of protein kinase C in mediating NGF effect on neuropeptide Y expression in PC12 cells, Mol. Brain Res., 23 Ž1994. 310–316. w4x Bargatti, P., Mazzoni, M., Carini, C., Neri, L.M., Marchisio, M., Bertolaso, L., Previati, M., Zauli, G. and Capitani, S., Changes of nuclear protein kinase C activity and isotype composition in PC12 cell proliferation and differentiation, Exp. Cell Res., 224 Ž1996. 72–78. w5x Bottari, S.P., De Gasparo, M., Steckelings, U. and Levens, N.R., Angiotensin II receptor subtypes: characterization, signalling mecha-
w21x
w22x
w23x
w24x
235
nisms and possible physiological implications, Front. Neuroendocrinol., 14 Ž1993. 123–171. Camp, H.S. and Dudley, D.T., Modulation of angiotensin II receptor ŽAT2 . mRNA levels in R3T3 cells, Receptor, 5 Ž1995. 123–132. Chiu, R., Boyer, W.J., Meek, J., Hunter, T. and Karin, M., The c-Fos protein interacts with c-JunrAP-1 to stimulate transcription of AP-1 responsive gene, Cell, 54 Ž1988. 541–552. Cook, V.I., Grove, K.L., McMenamin, K.M., Carter, M.R., Harding, J.W. and Speth, R.C., The AT2 angiotensin receptor subtype predominates in the 19 day gestation fetal rat brain, Brain Res., 560 Ž1991. 334–336. Dudley, D.T. and Summerfelt, R.M., Regulated expression of angiotensin II ŽAT2 . binding sites in R3T3 cells, Regul. Peptides, 44 Ž1993. 199–206. Hein, L., Barsh, G.S., Pratt, R.E., Dzau, V.J. and Kobilka, B.K., Behavioral and cardiovascular effects of disrupting the angiotensin II type-2 receptor in mice, Nature, 377 Ž1995. 744–747. Hogarty, D.C., Speakman, E.A., Puig, V. and Phillips, M.I. The role of angiotensin, AT1 and AT2 receptors in the pressor, drinking and vasopressin responses to central angiotensin, Brain Res., 586 Ž1993. 289–294. Huang, X.-C., Richards, E.M. and Sumners, C., Mitogen-activated protein kinase in rat brain neuronal cultures are activated by angiotensin II type 1 receptors and inhibited by angiotensin II type 2 receptors, J. Biol. Chem., 271 Ž1996. 15635–15641. Ichiki, T. and Inagami, T., Expression, genomic organization, and transcription of the mouse angiotensin II type 2 receptor gene, Circ. Res., 76 Ž1995. 693–700. Ichiki, T. and Inagami, T., Transcriptional regulation of the mouse angiotensin II type 2 receptor gene, Hypertension, 25 Ž1995. 720– 725. Ichiki, T., Kambayashi, Y. and Inagami, T., Multiple growth factors modulate mRNA expression of angiotensin II type-2 receptor in R3T3 cells, Circ. Res., 77 Ž1995. 1070–1076. Ichiki, T., Labosky, P.A., Suiota, C., Okuyama, S., Imagawa, Y., Fogo, A., Niimura, F., Ichikawa, I., Hogan, B.L. and Inagami, T., Effects on blood pressure and exploratory behavior in mice lacking angiotensin II type 2 receptor, Nature, 377 Ž1995. 748–750. Janiak, P., Pillon, A., Prost, J.F. and Vilaine, J.P., Role of angiotensin subtype 2 receptor in neointima formation after vascular injury, Hypertension, 20 Ž1992. 737–745. Kambayashi, Y., Bardhan, S. and Inagami, T., Peptide growth factors markedly decrease the ligand binding of angiotensin II type 2 receptor in rat cultured vascular smooth muscle, Biochem. Biophys. Res. Commun., 194 Ž1993. 478–482. Kobayashi, S.-I., Ohnishi, J., Nibu, Y., Nishimatsu, S.I., Umemura, S., Ishi, M., Murakami, K. and Miyazaki, H., Cloning of the rat angiotensin II type 2 receptor gene and identification of its functional promotor region, Biochim. Biophys. Acta, 1262 Ž1995. 155– 158. Koepke, J.P., Bovy, P.R., McMahon, E.G., Olins, G., Reitz, D.B., Salles, K., Schun, J.R., Trapani, A.J. and Blaine, E.D., Central and peripheral actions of a nonpeptide angiotensin II receptor antagonist, Hypertension, 15 Ž1990. 841–847. Kijima, K., Matsubara, H., Murasawa, S., Maruyama, K., Mori, Y. and Inada, M., Gene transcription of angiotensin II type 2 receptor is repressed by growth factors and glucocorticoids in PC12 cells, Biochem. Biophys. Res. Commun., 216 Ž1995. 359–366. Lenormand, P., Pages, G., Sardet, C., l’Allenrain, G., Meloche, S. and Pouyssegur, J., MAP kinases: activation, subcellular localization and role in the control of cell proliferation, AdÕ. Second Messenger Phosphoprotein Res., 28 Ž1993. 237–244. Lu, D., Sumners, C. and Raizada, M.K., Regulation of angiotensin II type 1 receptor messenger ribonucleic acid in neuronal cultures of normotensive and spontaneously hypertensive rat brains by phorbol esters and forskolin, J. Neurochem., 62 Ž1994. 2079–2084. Martin, M.M. and Elton, T.S., The sequence and genomic organiza-
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w25x
w26x
w27x
w28x
w29x
w30x
w31x
X.-C. Huang et al.r Molecular Brain Research 47 (1997) 229–236 tion of the human type 2 angiotensin II receptor, Biochem. Biophys. Res. Commun., 209 Ž1995. 554–562. Millan, M., Jacobowitz, D.M., Aguilera, G. and Catt, K.J., Differential distribution of AT1 and AT2 angiotensin II receptor subtypes in the rat brain during development, Proc. Natl. Acad. Sci. USA, 88 Ž1992. 11440–11444. Nakajima, M., Hutchinson, H.G., Fujinaga, M., Hayashida, W., Morishita, R., Zhang, L., Horiuchi, M., Pratt, R.E. and Dzau, V.J., The angiotensin II type 2 ŽAT2 . receptor antagonizes the growth effects of the AT1 receptor: gain-of-function study using gene transfer, Proc. Natl. Acad. Sci. USA, 92 Ž1995. 10663–10667. O’Driscoll, K.R., Teng, K.K., Fabbro, D., Greene, L.A. and Weinstein, I.B., Selective translocation of protein kinase C-delta in PC12 cells during nerve growth factor-induced neuritogenesis, Mol. Biol. Cell, 6 Ž1995. 449–458. Pages, G., Lenormand, P., L’Allenrain, G., Chambard, J.C., Meloche, S. and Pouyssegur, J., Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation, Proc. Natl. Acad. Sci. USA, 90 Ž1993. 8319–8323. Qadri, F., Culman, J., Veltmar, A., Maas, K., Rascher, W. and Unger, T., Angiotensin II-induced vasopressin release is mediated through alpha-1 adrenoceptors and angiotensin II AT1 receptors in the supraoptic nucleus, J. Pharmacol. Exp. Ther., 267 Ž1993. 567– 574. Sadoshima, J. and Izumo, S., Signal transduction pathways of angiotensin II-induced c-fos gene expression in cardiac myocytes in vitro. Roles of phospholipid-derived second messengers, Circ. Res., 73 Ž1993. 424–438. Song, K.A., Allen, A., Paxinos, G. and Mendelsohn, F.A.O., Mapping of angiotensin II receptor subtype heterogeneity in rat brain, J. Comp. Neurol., 316 Ž1992. 467–490.
w32x Stoll, M., Steckelings, U.M., Paul, M., Bottari, S.P., Metzger, R. and Unger, T., The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells, J. Clin. InÕest., 95 Ž1995. 651–657. w33x Sumners, C., Raizada, M.K., Kang, J., Lu, D. and Posner, P., Receptor-mediated effects of angiotensin II on neurons, Front. Neuroendocrinol., 15 Ž1994. 203–230. w34x Takeuchi, K., Nakamura, N., Cook, N.S., Pratt, R.E. and Dzau, V.J., Angiotensin II can regulate gene expression by the AP-1 binding sequence via a protein kinase C-dependent pathway, Biochem. Biophys. Res. Commun., 72 Ž1990. 1189–1192. w35x Torres, M. and Bogenmann, E., Nerve growth factor induces a multimeric TrkA receptor complex in neuronal cells that includes CrK, SHC and PLC-gamma but excludes P13ØCAS, Oncogene, 12 Ž1996. 77–86. w36x Tsutsumi, K. and Saavedra, J.M., Differential development of angiotensin II receptor subtypes in the rat brain, Endocrinology, 128 Ž1991. 630–632. w37x Wong, P.C., Hart, S.D., Zaspel, A.M., Chiu, A.T., Ardecky, R.J., Smith, R.D. and Timmermans, P.B.M.W.M., Functional studies of non-peptide angiotensin II receptor specific subtype specific ligands: Dup 753 ŽAII-1. and PD123177 ŽAII-2., J. Pharmacol. Exp. Ther., 255 Ž1990. 584–592. w38x Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J. and Greenberg, M.E., Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis, Science, 270 Ž1995. 1326–1331. w39x Yamada, T., Horiuchi, M. and Dzau, V.J., Angiotensin II type 2 receptor mediates programmed cell death, Proc. Natl. Acad. Sci. USA, 93 Ž1996. 156–160.
Research report
Modulation of angiotensin II type 2 receptor mRNA in rat hypothalamus and brainstem neuronal cultures by growth factors X.-C. Huang, U.V. Shenoy, E.M. Richards, C. Sumners
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Department of Physiology, College of Medicine, UniÕersity of Florida, GainesÕille, FL 32610, USA Accepted 23 December 1996
Abstract This study investigates the regulatory effects of growth factors upon angiotensin II type 2 ŽAT2 . mRNA levels in neurons co-cultured from newborn rat hypothalamus and brainstem. Incubation of cultured neurons with nerve growth factor ŽNGF; 5–50 ngrml. caused time-dependent changes in the steady-state levels of AT2 receptor mRNA. Short-term Ž0.5–1.0 h. incubations with NGF resulted in significant increases in AT2 receptor mRNA, whereas longer-term incubations Ž4–24 h. caused significant decreases. Activation of NGF receptors is known to stimulate phospholipase C-g and subsequently activate protein kinase C ŽPKC.. Incubation of cultures with the PKC activator, phorbol-12-myristate-13-acetate ŽPMA; 100 nM., caused temporal changes in AT2 receptor mRNA levels similar to those observed with NGF. By contrast, insulin Ž0.1–10 m grml. elicited only significant decreases in AT2 receptor mRNA levels. The observed abilities of NGF and insulin to regulate the expression of AT2 receptor mRNA are consistent with the fact that the AT2 receptor gene promoter region contains several cis DNA regulatory elements that respond to growth factor-stimulated transcription factors. These novel observations which show that NGF and insulin can regulate AT2 receptor mRNA in neurons derived from neonatal rat CNS lend support to the idea that AT2 receptors have a role in development and differentiation. q 1997 Elsevier Science B.V. Keywords: Nerve growth factor; Insulin; Protein kinase C; Angiotensin II type 2 receptor; Neuron
1. Introduction The brain contains both major subtypes of receptors for the octapeptide angiotensin II ŽAng II. w31,36x. Ang II acts at Ang II type 1 ŽAT1 . receptors in the hypothalamus and brainstem to elicit the well-documented physiological responses, such as increased blood pressure, fluid and salt intake, and vasopressin release w11,20,29,37x. By contrast, the physiological roles of Ang II type 2 ŽAT2 . receptors in the brain are not established. The occurrence of high levels of AT2 receptors in tissues from neonatal animals, including brain, led to the suggestion that these sites have a role in development andror differentiation w8,25,36x. More direct evidence for a role of AT2 receptors in these processes has come from studies in both non-neural and neural cells. For example, activation of AT2 receptors inhibits neointimal formation following arterial injury w17x and inhibits proliferation of coronary endothelial cells w32x. Further) Department of Physiology, Box 100274, University of Florida, Gainesville, FL 32610, USA. Fax: q1 Ž352. 846-0270; E-mail: [email protected]
0169-328Xr97r$17.00 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 4 7 - 8
more, transfection of the AT2 receptor gene into ballooninjured rat arteries attenuated the stimulatory effects of Ang II Žvia AT1 receptors. on cell growth and proliferation w26x. The implication from these studies is that AT1 and AT2 receptors have antagonistic actions on cell growth and proliferation. With respect to neural cells, a recent study has linked AT2 receptors to programmed cell death w39x. In that study, activation of AT2 receptors in PC-12W pheochromocytoma cells Žan in vitro model of rat sympathetic neurons. led to apoptosis w39x. Studies in mutant mice lacking the gene encoding the AT2 receptor suggest that these sites have a role in centrally mediated behavior, since these mice exhibited decreased spontaneous movements and exploratory behavior compared with wild type animals w10,16x. Thus, AT2 receptors may have a role in the central control of certain behaviors as well as having roles in development, differentiation and apoptosis. Further evidence for a role of AT2 receptors in growth processes has come from two other areas of investigation. First, studies on the intracellular signal transduction pathways that are modulated by AT2 receptors have revealed interactions with pathways that are normally modulated by
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growth factors. For example, it is well known that mitogen-activated protein ŽMAP. kinases are critical components of the cellular processes of growth, differentiation and apoptosis w2,22,38x and it is clear that MAP kinases, such as Erk 1 and Erk 2, are activated by growth factors w28x. We have recently determined, using neuronal cultures prepared from newborn rat hypothalamus and brainstem, that selective stimulation of neuronal AT2 receptors elicits an inhibition of Erk 1 and Erk 2 activities w12x. Similar inhibitory effects of AT2 receptors on MAP kinase have been demonstrated in PC-12W cells w39x. Thus, the modulation of MAP kinase activity by AT2 receptor stimulation suggests a mechanism by which these receptors are involved in growth and developmental processes. Studies on the regulation of AT2 receptors have also supported this idea, since it has been observed both in peripheral tissues and cultured peripheral cells that growth factors are powerful regulators of AT2 receptor expression. Nerve growth factor ŽNGF., epidermal growth factor ŽEGF., bombesin, insulin, interleukin-1 b and serum starvation have all been shown to modulate the expression of AT2 receptors or AT2 receptor mRNA on R3T3 fibroblasts and on PC-12W cells w6,9,13–15,18,21x. These observations are perhaps not surprising considering that cloning and sequencing of the promoter region of the AT2 receptor gene has revealed the presence of several potential cis DNA elements that respond to hormones or transcription factors w13,19,24x. These include an activator protein-1 ŽAP-1. site which responds to protein kinase C ŽPKC. activation and an insulin-response sequence ŽIRS. similar to that of the phosphoenol pyruvate carboxy kinase ŽPEPCK. gene. Based on the above findings, we have investigated the effects of growth factors on the regulation of AT2 receptor mRNA in neurons cultured from newborn rat hypothalamus and brainstem. In extensive prior studies, we have demonstrated that these cells contain AT2 receptors which are identical to those in other tissues and cells from biochemical, molecular and pharmacological standpoints w33x. The data presented here indicate that NGF, insulin and PKC activation by phorbol-12-myristate-13-acetate ŽPMA. regulate the expression of AT2 receptors in cultured neurons, albeit in a different manner than in peripheral neural cells w21x. This is the first demonstration that growth factors can modulate AT2 receptors in cells derived from the brain and provides a further indication that the AT2 receptor system has importance in growth and development processes in the neonatal nervous system. 2. Materials and methods 2.1. Materials Newborn Sprague–Dawley rats were obtained from our breeding colony which originated from Charles River Farms ŽWilmington, MA.. Dulbecco’s modified Eagle’s medium ŽDMEM. and TRIzol reagent were purchased
from Gibco-BRL Life Technologies. Plasma-derived horse serum ŽPDHS. was from Central Biomedia ŽIrwin, MO.. Trypsin Ž1 = crystallized. was from Worthington Biochemicals ŽFreehold, NJ.. RT-PCR kits were purchased from Perkin Elmer Biotechnologies ŽNorwalk, CT.. Nerve growth factor was purchased from Promega Biotechnology ŽMadison, WI.. Deoxyribonuclease 1 ŽDNase 1., cytosine arabinoside ŽARC ., phorbol-12-myristate-13-acetate ŽPMA., 4a-phorbol Ž4a-PE. and insulin were from Sigma ŽSt. Louis, MO.. All other chemicals were purchased from Fisher Scientific ŽPittsburgh, PA. and were of analytical grade or higher. Primers for the AT2 receptor and b-actin genes were synthesized in the DNA facility of the Interdisciplinary Center for Biotechnology Research, University of Florida. The sequences of these primers are as follows. AT2 receptor gene Sense primer: 5X-CAATCTGGCTGTGGCTGACT-3X Antisense primer: 5X-ATGACAATCTCCTGCCAACG-3X
b-Actin gene Sense primer: 5X-CTCATGAAGATCCTGACCGA-3X Antisense primer: 5X-TACGGATGTCAACGTCACAC-3X 2.2. Neuronal cultures Neuronal co-cultures were prepared from the brainstem and hypothalamic block of newborn Sprague–Dawley rats exactly as described previously w12,33x. Trypsin and DNase 1-dissociated cells were re-suspended in DMEM containing 10% PDHS and were plated in poly-L-lysine-coated 60-mm Nunc plastic tissue culture dishes. Cells were grown for 3 days at 378C in a humidified incubator with 95% airr5% CO 2 and were then treated with 1 m M ARC for 2 days. After this, ARC was removed and the cells incubated with fresh DMEMr5% PDHS for 9–12 days before use. At this time, cultures consisted of ) 90% neurons and - 10% astrogliarmicroglia. 2.3. Extraction of total RNA Growth media were removed from control or drugtreated neuronal cultures which were then washed once with ice-cold phosphate-buffered saline ŽpH 7.4.. Following this, cultured cells were lysed in TRIzol reagent Ž0.5 mlrdish. and transferred to a sterile tube. The lysates were incubated for 5 min at room temperature to permit the complete dissociation of nucleoprotein complexes. Following this, 0.2 ml chloroformr1 ml TRIzol reagent was added to each tube and samples were shaken vigorously by hand for 15 s. Samples were then incubated at room temperature for 3 min, followed by centrifugation at 12 000 = g for 15 min at 48C. The upper aqueous phase
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was transferred to a fresh tube and RNA was precipitated by mixing with an equal volume of isopropyl alcohol at room temperature for 10 min, followed by centrifugation. The RNA pellet was washed once with 75% ethanol and RNA samples were stored in the same solvent. The RNA concentration was determined by spectrophotometry. Before use, 20 m g RNA was incubated with 1 U DNAase 1 at 378C for 15 min and then the RNA was re-extracted using the method described above. 2.4. ReÕerse transcriptase polymerase chain reaction (RTPCR) RT-PCR was performed by using RNA PCR kits from Perkin Elmer Biotechnology. Briefly, reverse transcription
Fig. 2. Stimulatory effects of NGF on neuronal AT2 receptor mRNA levels. Neuronal cultures were treated with control solution or the indicated concentrations of NGF for 30 min at 378C, followed by analyses of AT2 receptor and b-actin mRNAs as detailed in Materials and methods. Top: representative ethidium bromide-stained gels showing the RT-PCR DNA products which correspond to the AT2 receptor and b-actin mRNAs. Bottom: quantification of the DNA bands that correspond to the AT2 receptor mRNA. Data are expressed as mean"S.E.M. from 3 independent experiments. ) P - 0.05.
Fig. 1. Effects of NGF on neuronal AT2 receptor mRNA levels as a function of incubation time. Neuronal cultures were treated with control solution or NGF Ž5 ngrml. at 378C for the indicated times, followed by analyses of AT2 receptor and b-actin mRNAs as detailed in Materials and methods. Top: representative ethidium bromide stained gels showing the RT-PCR DNA products which correspond to AT2 receptor Ž1.05 kb. and b-actin Ž0.3 kb. mRNAs. Bottom: quantification of the DNA bands corresponding to the AT2 receptor mRNA. Data are expressed as the ratio of AT2 receptor mRNAr b-actin mRNA in each treatment situation. Results are mean"S.E.M. from 3 independent experiments. ) P - 0.05.
was carried out in a 20 m l volume containing 5 mM MgCl 2 , 50 mM KCl, 10 mM Tris–HCl ŽpH 8.3., 1 mM dGTP, 1 mM dATP, 1 mM dTTP, 1 mM dCTP, 1 Urm l RNase inhibitor, 2.5 Urm l MuLV reverse transcriptase, 2.5 m M random hexamers and 1 m g of the total RNA sample. The mixture was incubated at 258C for 10 min, then 428C for 30 min, 998C for 5 min and finally 58C for 5 min. Following this, 10 m l Žfor AT2 receptor. or 5 m l Žfor b-actin. of reverse transcription reaction product were
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combined with 40 m l Žfor AT2 receptor. or 20 m l Žfor b-actin. of PCR master mix containing 50 mM KCl, 10 mM Tris–HCl ŽpH 8.3., AmpliTaq DNA polymerase 2.5 Ur100 m l and 0.2 pgrm l each of the upstream and downstream primers. PCR was performed at 948C for 4 min, followed by 30 cycles Žfor AT2 receptor. or 20 cycles Žfor b-actin. at 948C for 1 min, 558C for 1 min, 728C for 1 min. After final extension at 728C for 8 min, PCR products were electrophoresed on a 1% agarose gel containing 1 m grml of ethidium bromide. The densities of PCR products were analyzed by Imagequant and Microsoft Excel softwares. The relative AT2 receptor mRNA levels were compared by a ratio of band densities representing AT2 receptor mRNA over the band densities of the corresponding b-actin mRNA. 2.5. Experimental groups and data analysis All results are expressed as mean " S.E.M. and were obtained by combining data from individual experiments. Comparisons of multiple means were made using an analysis of variance, followed by a Newman–Keuls test to assess statistical significance between individual means. Statistical analyses were performed using Sigma Stat Software ŽJandel, San Rafael, CA..
3. Results In the experiments described here, we utilized RT-PCR to assess the levels of AT2 receptor mRNA in neuronal cultures. In preliminary experiments, we determined that production of the 1.05-kb AT2 receptor-specific DNA by PCR was linear between 27 and 33 cycles of amplification. Furthermore, production of the 1.05-kb AT2 receptor DNA increased linearly as a function of RT volume from 5–15 m l, at 30 cycles of PCR amplification Ždata not shown.. Based upon this, in subsequent studies we performed 30 cycles of PCR with 10 m l RT volume for the AT2 receptor mRNA amplification. We also determined that production of a 0.3-kb b-actin-specific DNA from the PCR was linear between 10 and 25 cycles of amplification and from 2.5 to 7.5 m l of RT volume, at 20 cycles of PCR amplification Ždata not shown.. Thus, in subsequent studies, we performed 20 cycles of PCR with 5 m l RT volume for the b-actin mRNA amplification. The relative levels of AT2 receptor mRNA levels in control and drug-treated neuronal cultures were compared following quantification of the 1.05-kb AT2 receptor and 0.3-kb b-actin bands. Incubation of neuronal cultures with NGF Ž5 ngrml. resulted in a time-dependent alteration in the intensity of the 1.05-kb AT2 receptor band Žcorresponding to AT2 receptor mRNA.. The data presented in Fig. 1 show that NGF induced a significant increase in AT2 receptor mRNA levels during the first 2 h after treatment, with a maximal increase at 30 min. By contrast, longer incubation times
Fig. 3. Inhibitory effects of NGF on neuronal AT2 receptor mRNA levels. Neuronal cultures were treated with control solution or the indicated concentrations of NGF for 24 h at 378C, followed by analyses of AT2 receptor and b-actin mRNAs as detailed in Materials and methods. Top: representative ethidium bromide-stained gels showing the RT-PCR DNA products which correspond to the AT2 receptor and b-actin mRNAs. Bottom: quantification of the DNA bands that correspond to the AT2 receptor mRNA. Data are expressed as mean"S.E.M. from 3 independent experiments. ) P - 0.05.
with NGF Ž4–24 h. elicited a significant decrease in AT2 receptor mRNA levels and a maximal reduction was obtained 12–24 h after treatment. The effects of NGF on AT2 receptor mRNA in neuronal cultures were also concentration-dependent. A maximal stimulatory effect was elicited by 5–50 ngrml NGF ŽFig. 2., whereas a maximal inhibitory effect was obtained with 50 ngrml NGF ŽFig. 3.. In all cases, treatment of neuronal cultures with NGF
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failed to elicit significant changes in b-actin mRNA ŽFigs. 1–3.. Activation of NGF receptors leads to a stimulation of phospholipase C-g ŽPLC-g . w35x and subsequent activation of PKC w3,4,27x. PKC can regulate gene transcription via an activator protein-1 ŽAP-1. site w1,7,34x and the presence of an AP-1 site within the AT2 receptor promotor has been documented w13,19,24x. Based on this, we analyzed the effects of activation of PKC on AT2 receptor mRNA levels in neuronal cultures. Incubation of neuronal cultures with the PKC activator PMA Ž100 nM. resulted in a time-dependent change in AT2 receptor mRNA similar to that observed with NGF. The data presented in Fig. 4
Fig. 5. Effects of insulin on neuronal AT2 receptor mRNA levels as a function of incubation time. Neuronal cultures were treated with control solution or insulin Ž10 m grml. at 378C for the indicated times, followed by analyses of the AT2 receptor and b-actin mRNAs as detailed in Materials and methods. Top: representative ethidium bromide-stained gels showing the RT-PCR DNA products which correspond to the AT2 receptor and b-actin mRNAs. Bottom: quantification of the DNA bands that correspond to the AT2 receptor mRNA. Data are expressed as mean"S.E.M. from 3 independent experiments. ) P - 0.05.
Fig. 4. Effects of PMA on neuronal AT2 receptor mRNA levels as a function of incubation time. Neuronal cultures were treated with control solution or PMA Ž100 nM. for the indicated times at 378C, followed by analysis of AT2 receptor and b-actin mRNAs as detailed in Materials and methods. Top: representative ethidium bromide-stained gels showing the RT-PCR DNA products which correspond to the AT2 receptor and b-actin mRNAs. Bottom: quantification of the DNA bands that correspond to the AT2 receptor mRNA. Data are expressed as mean"S.E.M. from 3 independent experiments. ) P - 0.05.
show that PMA induced a significant increase in the levels of AT2 receptor mRNA during the first 2 h after treatment, with a maximal increase obtained at 30 min. Longer incubation times with PMA elicited a significant decrease in AT2 receptor mRNA levels, with a maximal reduction obtained between 12 and 24 h ŽFig. 4.. PMA treatment did not alter b-actin mRNA levels ŽFig. 4. and AT2 receptor mRNA levels were not altered by treatment of cultures with 100 nM 4-a phorbol ester which does not activate PKC Ždata not shown.. The AT2 receptor gene also contains a nucleotide sequence similar to the IRS which occurs in the promotor
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region of the PEPCK gene w13x. Based upon this, in the next series of experiments we investigated the effects of insulin on AT2 receptor mRNA levels in neuronal cultures in order to test whether the IRS has a role in AT2 receptor gene transcription. Incubation of neuronal cultures with insulin Ž10 m grml. induced a significant time-dependent decrease in the levels of AT2 receptor mRNA ŽFig. 5.. This effect became apparent following 4 h of treatment
Fig. 6. Inhibitory effects of insulin on neuronal AT2 receptor mRNA levels. Neuronal cultures were treated with control solution or the indicated concentrations of insulin for 24 h at 378C, followed by analysis of AT2 receptor mRNA levels as detailed in Materials and methods. Top: representative ethidium bromide-stained gels showing the RT-PCR DNA products which correspond to the AT2 receptor and b-actin mRNAs. Bottom: quantification of the DNA bands that correspond to the AT2 receptor mRNA. Data are expressed as mean"S.E.M. from 3 independent experiments. ) P - 0.05.
with insulin and was maximal after 24 h ŽFig. 5.. The inhibitory effects of insulin were also concentration-dependent, with maximal effects obtained at 1–10 m grml insulin ŽFig. 6..
4. Discussion The data presented in these studies show that the steady-state level of mRNA for AT2 receptors on brainstem and hypothalamic neurons co-cultured from 1-day-old rats is regulated by growth factors. NGF and insulin alter steady-state levels of AT2 receptor mRNA. PKC activation with PMA, mimicking one of the signal transduction pathways activated by NGF w3,4,27x, also affected steady-state mRNA levels of the AT2 receptor. Insulin caused time- and dose-dependent decreases in steady-state levels of AT2 receptor mRNA. Since the promotor region of the mouse AT2 receptor gene contains a sequence somewhat homologous to the IRS of the PEPCK gene w13x, these findings are perhaps not surprising. It should be noted, however, that this sequence was not reported to be present in the promotor region of the AT2 receptor gene of the rat w19x. Insulin has previously been shown to regulate steady-state levels of mRNA for the AT2 receptor in R3T3 cells w15x, but in that study insulin increased both mRNA for the AT2 receptor and the number of AT2 receptor sites on the cells. Several possible reasons for the differences between the two studies exist. First, the growth conditions and treatment paradigms used. R3T3 cells were grown to confluency, a situation in which AT2 receptor mRNA expression is increased w6x. Following this, the serum-containing medium was removed and replaced with insulin-containing serum-free media for 2 days. Neuronal cultures, which do not grow to confluency as they are not continuously dividing cells, were grown for 9–12 days in media containing 5% serum and then insulin was added to the media without otherwise altering it. Second, the cell lineages are very different, i.e. primary rat neurons compared to a mouse fibroblast cell line. Previous studies have suggested that cell lineage could be important because some DNA-binding proteins interacting with the promotor region of the AT2 receptor gene are different in PC-12W and vascular smooth muscle cells w14x, cell lines of very different cell lineages. Further studies will be needed to resolve these differences. NGF and PMA caused similar changes in the expression of mRNA for the AT2 receptor. The effects were time- and dose-dependent but biphasic. NGF and PMA caused in the short-term increases in AT2 receptor mRNA and in the long-term Ž) 4 h incubation. decreases in AT2 receptor mRNA. There are several consensus DNA sequences in the promotor region of the rat AT2 receptor gene which might be influenced by NGF and PMA application to the cells. There are two AP-1 sequences in the rat
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AT2 receptor promotor region w19x as well as AP-1 sites in the mouse AT2 receptor gene w13,19,24x. AP-1 sequences bind heterodimers of c-fos and c-jun which are activated following stimulation of PKC activity w34x. There is also a Myc-binding site in the rat AT2 receptor gene which can bind c-myc. AT1 receptor stimulation by AngII leads to increases in c-fos, c-jun and c-myc levels w5,30x. Thus, AT1 receptor stimulation may decrease AT2 receptor transcription as has previously been suggested w19x. In view of previous findings showing opposite actions of AT1 and AT2 receptors on the activities of the important growth regulating molecules, the MAP kinases w12,26x, it may be important for activation of AT1 receptors to decrease synthesis of the opposing AT2 receptor so that the AT1mediated activation of MAP kinase by angiotensin II could predominate. For example, PMA stimulation of neuronal cells results in increased c-fos and c-jun. This increases AT1 receptor mRNA transcription w23x and decreases steady-state levels of AT2 receptor mRNA, suggesting that under these conditions the AT1 receptor mediated effects of angiotensin would override the AT2 receptor-induced effects. This could occur for example following long-term Ž) 4 h. exposure of cells to PMA or NGF. This would add another layer of complexity to the opposing interactions of the AT1 and AT2 receptor subtypes and also suggests that other trophic agents may similarly regulate the AT2 receptor. These and other possibilities await further study. In conclusion, AT2 receptor mRNA levels in cultured neurons are regulated by growth factors. This regulation by growth factors was occasionally different than had been reported in other cells suggesting that tissue-specific regulation of AT2 receptor mRNA levels may occur. Acknowledgements We thank Jennifer Moore for preparation of cultured neurons and Jennifer Brock for typing the manuscript. This work was supported by Grant NIH NS-19441.
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References w20x w1x Angel, P., Imagawa, M., Chiu, R., Stein, B., Imbra, R.J., Ranmsdorf, H.J., Jouat, C., Herruch, P. and Karin, M., Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor, Cell, 49 Ž1987. 729–739. w2x Ahn, N.G., Seger, R. and Krebs, E.G., The mitogen-activated protein kinase activator, Curr. Opin. Cell Biol., 4 Ž1992. 992–999. w3x Balbi, D. and Allen, J.M., Role of protein kinase C in mediating NGF effect on neuropeptide Y expression in PC12 cells, Mol. Brain Res., 23 Ž1994. 310–316. w4x Bargatti, P., Mazzoni, M., Carini, C., Neri, L.M., Marchisio, M., Bertolaso, L., Previati, M., Zauli, G. and Capitani, S., Changes of nuclear protein kinase C activity and isotype composition in PC12 cell proliferation and differentiation, Exp. Cell Res., 224 Ž1996. 72–78. w5x Bottari, S.P., De Gasparo, M., Steckelings, U. and Levens, N.R., Angiotensin II receptor subtypes: characterization, signalling mecha-
w21x
w22x
w23x
w24x
235
nisms and possible physiological implications, Front. Neuroendocrinol., 14 Ž1993. 123–171. Camp, H.S. and Dudley, D.T., Modulation of angiotensin II receptor ŽAT2 . mRNA levels in R3T3 cells, Receptor, 5 Ž1995. 123–132. Chiu, R., Boyer, W.J., Meek, J., Hunter, T. and Karin, M., The c-Fos protein interacts with c-JunrAP-1 to stimulate transcription of AP-1 responsive gene, Cell, 54 Ž1988. 541–552. Cook, V.I., Grove, K.L., McMenamin, K.M., Carter, M.R., Harding, J.W. and Speth, R.C., The AT2 angiotensin receptor subtype predominates in the 19 day gestation fetal rat brain, Brain Res., 560 Ž1991. 334–336. Dudley, D.T. and Summerfelt, R.M., Regulated expression of angiotensin II ŽAT2 . binding sites in R3T3 cells, Regul. Peptides, 44 Ž1993. 199–206. Hein, L., Barsh, G.S., Pratt, R.E., Dzau, V.J. and Kobilka, B.K., Behavioral and cardiovascular effects of disrupting the angiotensin II type-2 receptor in mice, Nature, 377 Ž1995. 744–747. Hogarty, D.C., Speakman, E.A., Puig, V. and Phillips, M.I. The role of angiotensin, AT1 and AT2 receptors in the pressor, drinking and vasopressin responses to central angiotensin, Brain Res., 586 Ž1993. 289–294. Huang, X.-C., Richards, E.M. and Sumners, C., Mitogen-activated protein kinase in rat brain neuronal cultures are activated by angiotensin II type 1 receptors and inhibited by angiotensin II type 2 receptors, J. Biol. Chem., 271 Ž1996. 15635–15641. Ichiki, T. and Inagami, T., Expression, genomic organization, and transcription of the mouse angiotensin II type 2 receptor gene, Circ. Res., 76 Ž1995. 693–700. Ichiki, T. and Inagami, T., Transcriptional regulation of the mouse angiotensin II type 2 receptor gene, Hypertension, 25 Ž1995. 720– 725. Ichiki, T., Kambayashi, Y. and Inagami, T., Multiple growth factors modulate mRNA expression of angiotensin II type-2 receptor in R3T3 cells, Circ. Res., 77 Ž1995. 1070–1076. Ichiki, T., Labosky, P.A., Suiota, C., Okuyama, S., Imagawa, Y., Fogo, A., Niimura, F., Ichikawa, I., Hogan, B.L. and Inagami, T., Effects on blood pressure and exploratory behavior in mice lacking angiotensin II type 2 receptor, Nature, 377 Ž1995. 748–750. Janiak, P., Pillon, A., Prost, J.F. and Vilaine, J.P., Role of angiotensin subtype 2 receptor in neointima formation after vascular injury, Hypertension, 20 Ž1992. 737–745. Kambayashi, Y., Bardhan, S. and Inagami, T., Peptide growth factors markedly decrease the ligand binding of angiotensin II type 2 receptor in rat cultured vascular smooth muscle, Biochem. Biophys. Res. Commun., 194 Ž1993. 478–482. Kobayashi, S.-I., Ohnishi, J., Nibu, Y., Nishimatsu, S.I., Umemura, S., Ishi, M., Murakami, K. and Miyazaki, H., Cloning of the rat angiotensin II type 2 receptor gene and identification of its functional promotor region, Biochim. Biophys. Acta, 1262 Ž1995. 155– 158. Koepke, J.P., Bovy, P.R., McMahon, E.G., Olins, G., Reitz, D.B., Salles, K., Schun, J.R., Trapani, A.J. and Blaine, E.D., Central and peripheral actions of a nonpeptide angiotensin II receptor antagonist, Hypertension, 15 Ž1990. 841–847. Kijima, K., Matsubara, H., Murasawa, S., Maruyama, K., Mori, Y. and Inada, M., Gene transcription of angiotensin II type 2 receptor is repressed by growth factors and glucocorticoids in PC12 cells, Biochem. Biophys. Res. Commun., 216 Ž1995. 359–366. Lenormand, P., Pages, G., Sardet, C., l’Allenrain, G., Meloche, S. and Pouyssegur, J., MAP kinases: activation, subcellular localization and role in the control of cell proliferation, AdÕ. Second Messenger Phosphoprotein Res., 28 Ž1993. 237–244. Lu, D., Sumners, C. and Raizada, M.K., Regulation of angiotensin II type 1 receptor messenger ribonucleic acid in neuronal cultures of normotensive and spontaneously hypertensive rat brains by phorbol esters and forskolin, J. Neurochem., 62 Ž1994. 2079–2084. Martin, M.M. and Elton, T.S., The sequence and genomic organiza-
236
w25x
w26x
w27x
w28x
w29x
w30x
w31x
X.-C. Huang et al.r Molecular Brain Research 47 (1997) 229–236 tion of the human type 2 angiotensin II receptor, Biochem. Biophys. Res. Commun., 209 Ž1995. 554–562. Millan, M., Jacobowitz, D.M., Aguilera, G. and Catt, K.J., Differential distribution of AT1 and AT2 angiotensin II receptor subtypes in the rat brain during development, Proc. Natl. Acad. Sci. USA, 88 Ž1992. 11440–11444. Nakajima, M., Hutchinson, H.G., Fujinaga, M., Hayashida, W., Morishita, R., Zhang, L., Horiuchi, M., Pratt, R.E. and Dzau, V.J., The angiotensin II type 2 ŽAT2 . receptor antagonizes the growth effects of the AT1 receptor: gain-of-function study using gene transfer, Proc. Natl. Acad. Sci. USA, 92 Ž1995. 10663–10667. O’Driscoll, K.R., Teng, K.K., Fabbro, D., Greene, L.A. and Weinstein, I.B., Selective translocation of protein kinase C-delta in PC12 cells during nerve growth factor-induced neuritogenesis, Mol. Biol. Cell, 6 Ž1995. 449–458. Pages, G., Lenormand, P., L’Allenrain, G., Chambard, J.C., Meloche, S. and Pouyssegur, J., Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation, Proc. Natl. Acad. Sci. USA, 90 Ž1993. 8319–8323. Qadri, F., Culman, J., Veltmar, A., Maas, K., Rascher, W. and Unger, T., Angiotensin II-induced vasopressin release is mediated through alpha-1 adrenoceptors and angiotensin II AT1 receptors in the supraoptic nucleus, J. Pharmacol. Exp. Ther., 267 Ž1993. 567– 574. Sadoshima, J. and Izumo, S., Signal transduction pathways of angiotensin II-induced c-fos gene expression in cardiac myocytes in vitro. Roles of phospholipid-derived second messengers, Circ. Res., 73 Ž1993. 424–438. Song, K.A., Allen, A., Paxinos, G. and Mendelsohn, F.A.O., Mapping of angiotensin II receptor subtype heterogeneity in rat brain, J. Comp. Neurol., 316 Ž1992. 467–490.
w32x Stoll, M., Steckelings, U.M., Paul, M., Bottari, S.P., Metzger, R. and Unger, T., The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells, J. Clin. InÕest., 95 Ž1995. 651–657. w33x Sumners, C., Raizada, M.K., Kang, J., Lu, D. and Posner, P., Receptor-mediated effects of angiotensin II on neurons, Front. Neuroendocrinol., 15 Ž1994. 203–230. w34x Takeuchi, K., Nakamura, N., Cook, N.S., Pratt, R.E. and Dzau, V.J., Angiotensin II can regulate gene expression by the AP-1 binding sequence via a protein kinase C-dependent pathway, Biochem. Biophys. Res. Commun., 72 Ž1990. 1189–1192. w35x Torres, M. and Bogenmann, E., Nerve growth factor induces a multimeric TrkA receptor complex in neuronal cells that includes CrK, SHC and PLC-gamma but excludes P13ØCAS, Oncogene, 12 Ž1996. 77–86. w36x Tsutsumi, K. and Saavedra, J.M., Differential development of angiotensin II receptor subtypes in the rat brain, Endocrinology, 128 Ž1991. 630–632. w37x Wong, P.C., Hart, S.D., Zaspel, A.M., Chiu, A.T., Ardecky, R.J., Smith, R.D. and Timmermans, P.B.M.W.M., Functional studies of non-peptide angiotensin II receptor specific subtype specific ligands: Dup 753 ŽAII-1. and PD123177 ŽAII-2., J. Pharmacol. Exp. Ther., 255 Ž1990. 584–592. w38x Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J. and Greenberg, M.E., Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis, Science, 270 Ž1995. 1326–1331. w39x Yamada, T., Horiuchi, M. and Dzau, V.J., Angiotensin II type 2 receptor mediates programmed cell death, Proc. Natl. Acad. Sci. USA, 93 Ž1996. 156–160.