Journal of Neuroscience Research 3152-57 (1992)

Dexamethasone Blocks Nerve Growth Factor Induction of Nerve Growth Factor Receptor mRNA in PC12 Cells P. J. Foreman, G. Taglialatela, G.R. Jackson, and J.R. Perez-Polo Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston (P.J.F., G.T., G.R.J., J.R.P.-P.), and Institute for Research on Senescence, Sigma Tau, Pomezia, Italy (G.T.) Glucocorticoids and nerve growth factor (NGF) have been shown to have antagonistic effects on chromaffin cells in vivo. Here we determined the effect of the synthetic glucocorticoid, dexamethasone, on levels of mRNA for the nerve growth factor receptor (NGFR) in rat PC12 pheochromocytoma cells. Following administration of dexamethasone (1 pM) there is a decline in NGFR mRNA expression. More importantly, administration of dexamethasone appears to block the NGF-mediated induction of NGFR when both agents are administered simultaneously. These data support the hypothesis that glucocorticoids and NGF act in opposition in determination of the phenotype of chromaffin cells. Key words: glucocorticoid, PC12, chromaffin INTRODUCTION Adrenal chromaffin cells and sympathetic ganglion cells derived from the sympathoadrenal region of the neural crest are thought to arise from a common neuroendocrine precursor (Landis and Patterson, 198 1). The local adrenal medullary environment provides the hormonal cues that determine the final phenotypic cellular commitment (Anderson and Axel, 1986; Anderson and Michelson, 1989). This choice is in part determined by the relative levels of glucocorticoid and nerve growth factor (NGF) during development (Landis and Patterson, 1981; Doupe et al., 1985). NGF induces and maintains the survival of these precursors as sympathetic neurons (Unsicker et al., 1978; Aloe and Levi-Montalcini, 1979), whereas glucocorticoids induce the formation of a chromaffin cell phenotype (Anderson and Axel, 1986). Similarly, NGF treatment of the rat PC12 pheochromocytoma cells (Greene and Tischler, 1976), derived from an adrenal medullary tumor, results in the acquisition of a sympathetic neuronal phenotype (Greene and Tischler, 1982) and glucocorticoids may direct these cells toward a more “chromaffin-like” state as measured by the induction of tyrosine hydroxylase transcription and activity 0 1992 Wiley-Liss, Inc.

(Lewis et al., 1983, 1987; Edgar and Thoenen, 1978). Thus, NGF and glucocorticoids direct development of the adrenal medulla in two different pathways. The synthetic glucocorticoid, dexamethasone, decreases expression of the NGF receptor (NGFR) p75 NGFR in PC12 (Tocco et al., 1988). However, only the low-affinity, high-capacity NGF binding activity decreases following dexamethasone treatment. Given that there are two mRNAs that code for the high- and lowaffinity NGFR and that most glucocorticoid effects are mediated through transcriptional regulation, it was of interest to examine the effects of dexamethasone on NGFR mRNA in PC12 cells. In a recent report it was shown that dexamethasone decreases NGFR mRNA in PC12 (Yakolev et al., 1990). Since NGF increases both NGFR protein (Bernd and Greene, 1984) and mRNA in PC12 cells (Doherty et al., 1988), while there is not increase in NGF binding activity after co-administration of NGF and dexamethasone (Tocco et al., 1988), it was of further interest to measure the effect of dexamethasone on the NGF induction of NGFR mRNA. In the first part of this study, we replicated the demonstration of a dexamethasone-induced NGFR decrease as determined by crosslinking ‘251-betaNGF to NGFR and then immunoprecipitating the NGF/NGFR complex for analysis on SDS-PAGE and autoradiography. In the second part we show that dexamethasone decreases NGFR mRNA expression in PC12 and blocks the ability of NGF to induce NGFR mRNA as determined by northern analysis.

MATERIALS AND METHODS Cell Culture PC12 cells were used in all studies (kindly donated by Dr. Lloyd Greene). Cultures were raised in RPMI Received April 15, 1991; revised July 17, 1991; accepted July 18, 1991. Address reprint requests to J.R. Perez-Polo, Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77550.

NGFR mRNA and Dexamethasone

1640 (GIBCO, Grand Island, NY), 5% fetal bovine serum, and 5% donor horse serum (Irvine Scientific Sales, Irvine, CA) at 37°C in a humidified incubator containing 5% CO,. Half the media was replaced 3 X weekly with or without a 1% penicillin-streptomycin-neomycin (PSN) antibiotic mixture (GIBCO, Grand Island, NY) supplement, alternatively. Cultures were split 1: 1 and grown to confluency before treatments began. Dexamethasone-treated cells were cultured in charcoalstripped serum (Armelin et al., 1974) from 24 hr prior to the start of treatments until assayed. Dexamethasone (FLUKA, Buchs, Switzerland) dissolved in 95% ethanol at a final concentration of 1 pM. The final concentration of ethanol (0.05%) had no detectable effect on any of the parameters tested. Controls received the same volume of 95% ethanol. NGF was diluted in RPMI and administered in 0.05% media volume at a final concentration of 3.7 nM. Treatments were for 3 days and were administered on day 1 and on the subsequent feeding 48 hr later. Each experiment was performed at least twice.

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processed for SDS-polyacrilamide gel electrophoresis by one of the three methods indicated above.

Northern Analysis Total RNA was isolated by the guanidine-acid phenol method (Chomczynski and Sacchi, 1987). Hybridization of a random primer labeled 32P-cNGFR (gift of Dr. Eric Shooter) to NGFR mRNA was performed by the method of Radeke et al. (1987). Following treatment, PC 12 cells were dislodged and washed once with PBS. Pellets were solubilized in 1.2 ml lysis buffer and processed in duplicates of 600 pl. OD260,280ratios between 1.9 and 2.1 were typically observed with 100-200 pg of total RNA recovered per flask of cells. Equivilant samples of total RNA were loaded on a 1% agarose-formaldehyde denaturing RNA, electrophoresed, and blotted to nitrocellulose membranes by standard methods (Asubel et al., 1987). Blots were hybridized for 72 hr with 1 X lo6 c p d m l of a random primer labeled 32P-cNGFR, which had been purified on a Stratagene GENECLEAN column (specific activity of probe: 5 x lo8 d p d p g NGF Isolation and Iodination DNA). Membranes were washed 4 X in 0.2% SSC/O.1% Mouse submaxillary beta-NGF was purified and SDS for 30 min at 65°C and autoradiographed for 3 to 6 tested for purity and bioactivity , as described elsewhere days. After autoradiography membranes were rehybrid(Mobley et al., 1981; Marchetti and Perez-Polo, 1987; ized with a random primer labeled chicken beta-actin Greene, 1977). NGF was iodinated by the lactoperoxi- 32P-cDNA. dase method (Olender and Stach, 1980). Specific activity was 2,000-3,500 cpm/fmol with 80-95% acid precipiRESULTS table counts. Immunoprecipitation of NGFR revealed that in all NGFR Immunoprecipitation cases there was specific labeling of a 100 kDa protein (83 Changes in NGFR protein following dexametha- kDa to 110 kDa) which was abolished with a 300-fold sone treatment were analyzed by modifications of a excess of unlabeled NGF (Fig. 1). This is consistent with crosslinking and immunoprecipitation procedure (Tani- findings for the major rat NGFR species (Buxer et al., uchi et al., 1986). Three methods to isolate the NGFR 1983; Hosang and Shooter, 1985; Grob et al., 1983; were used, all of which rely on the autoradiographic Green and Greene, 1986). We also occasionally obvisualization of '251-NGF crosslinked to NGFR, but dif- served a 150-160 kDa NGFR protein that corresponds to fer as to which protein component of the NGF/NGFR the high molecular weight NGFR species reported in the complex is precipitated (Fig. 1): crosslinking and immu- literature (Fig. 1). Three treatments of PC 12 cells were examined: noprecipitation with the rat NGFR specific monoclonal antibody, 192-IgG (Fig. la); crosslinking and immuno- Cells were treated with regular media containing horse precipitation with a polyclonal sheep anti-NGF antisera and fetal bovine sera (C; control), steroid-stripped media (Fig. 1b); and crosslinking with no immunoprecipitation (S; stripped), or steroid-stripped media that contained 1 (not shown). This multiple approach avoided artifacts pM dexamethasone (D; dexamethasone) . Treatment of that might be present in a single approach. After dexa- PC12 cells with 1 p M dexamethasone for 3 days resulted methasone treatment, cells were dislodged, washed 2 X in decreased NGFR protein as measured by crosslinking with PBS (pH 7.4), suspended in 2 ml of 10 nM 1251- and subsequent immunoprecipitation of the 1251-NGF/ NGF/PBS or with an additional 3 p M cold NGF at 37°C NGFR complexes with mAb 192 (Fig. 1A) or sheep antifor 60 min and crosslinked with 10 mM 1,3-ethyl-di- NGF antisera (Fig. 1B). Image analysis of the autoramethylisopropyl-carbodiimide(Sigma Chemical Co., St. diographs revealed a decline of 40% (anti NGF antisera) Louis, MO) for 20 min at 25°C followed by 50 mM to 60% (mAb 192) in the labeled NGFR present in dexaTris-HC1 (pH 7.0). Samples, in triplicate, were washed methasone-treated cells as compared to either of the once in PBS and solubilized in 0.5% Nonidet p40/1 mM controls used (Fig. 2). There was no difference between phenylmethylsulfonylfluoride/PBS for 2 hr at 4°C and the levels of NGFR detected in PC12 cells grown in

Foreman et al.

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C

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Fig. 1 . SDS-PAGE autoradiographs of '251-NGF-labeIedPC12 NGFR isolated by (A) immunoprecipitation with 192-IgG and (B) immunoprecipitation with anti-NGF antisera. Control

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Fig. 2. Image analysis of autoradiographs from Figure 1. regular media as compared to steroid-stripped media. However, there was no consistent dexamethasoneinduced decrease in NGFR as measured here for PC12 cells grown in media containing sera which had not been charcoal-stripped of steroids (data not shown). Hybridization of a rat NGFR 32P-cDNA probe to

media (C); stripped media (S); 1 p M dexamethasone for 3 days in stripped media (D). Lanes marked ( + ) have 300-fold excess unlabeled NGF.

40 p g of PC12 total RNA revealed the presence of a single RNA species of approximately 3.7 kb in all treatment groups (Figs. 3 and 4A). Six conditions were analyzed: control PC12 (C), stripped media control (S), control with 3.7 nM NGF (CN), stripped control with 3.7 nM NGF (SN), stripped media with 1 p M dexamethasone (D), and stripped media with both 3.7 nM NGF and 1 IJ.M dexamethasone (SDN). Figure 5 graphically displays the result of scanning densitometry of the individual lanes from the same blot. NGF treatment reproducibly increased expression of NGFR mRNA in PC12 grown in control or stripped media for 3 days (Fig. 4A). This increase was much more robust at 6 days (Fig. 3). Dexamethasone treatment of these cells decreased the expression of NGFR mRNA in PC12 grown in stripped media and also blocked the increase in NGFR mRNA observed in NGF-treated PC12 cells (Fig. 4A). There are also less NGFR mRNA in the PC 12 grown in stripped media as compared to controls. These differences were not due to variations in total RNA applied to the gel as the 18s and 28s ribosomal RNA bands are essentially identical in all conditions (Fig. 4B). Rehybridization of

NGFR mRNA and Dexamethasone

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U ' Control

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Fig. 3. Hibridization of the 32P-cNGFR probe to PC12 RNA alone (control) or treated with 3.7 nM NGF for 3 days (+NGF). 2.3+

A C

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Fig. 4. A: Hybridization of the 32P-NGFR probe to PC12 RNA. Lanes are as in Figure 1. B: Pattern of RNA on gels 25 in A showing amount of total RNA present.

the membranes with a random primer labeled chicken beta-actin 32P-cDNA showed similar changes among treatment groups with respect to NGF effects but not dexamethasone (Fig. 6).

DISCUSSION As expected, dexamethasone decreased NGFR protein expression in PC12 cells. Three methods were used to extract the receptor and all of them gave similar results. The decrease in NGFR reported here appears to be

Fig. 6. Hybridization of a 32P-betaactin probe to same blot as shown in Figure 3, right side. Blot was stripped of cNGFR probe in 0.1 x SSC/O.1% SDS for 10 min at 85°C and probed for beta-actin mRNA by conditions identical to those given for NGFR mRNA.

the result of inhibition of NGFR expression at the transcriptional level since 3 days exposure to dexamethasone also decreased NGFR mRNA. This is consistent with some effects of glucocorticoids on PC12 cells (Leonard et al., 1987; Yakolev et al., 1990). An explanation for these findings is that the initial decrease in transcription of NGFR mRNA reduces the amount of low-affinity NGFR protein available to bind NGF and that this reduction in bound NGF further reduces transcription of NGFR mRNA synthesis. This would imply that the signal for NGF induction of NGFR mRNA synthesis is mediated by the low-affinity NGF receptor. Since NGF and dexamethasone were administered simultaneously, it is not likely that this sequence of events takes place. Another explanation is that dexamethasone impairs further transcription of NGFR mRNA even in the presence of NGF stimulation, regardless of whether the signal is mediated by the high-affinity NGFR or low-affinity NGFR. Thus, additional NGFR mRNA synthesis does not occur in the presence of NGF. This hypothesis is compatible with the in vivo antagonistic effects of NGF and dexamethasone on neural crest derived adrenal medullary precursor cells where gluco-

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Foreman et al.

corticoids can direct the previously sympathetic neuronal phenotype towards a chromaffin phenotype (Anderson and Axel, 1986), and NGF can convert chromaffin cells to sympathetic cells (Unsicker et al., 1978) as well as the in vitro inhibitory effects on some PC12 mRNAs (Leonard et al., 1987). Hence, one effect of dexamethasone may be to specifically block NGFR transcription in PC 12 thereby reducing the ability of NGF to induce the sympathetic phenotype, while facilitating the acquisition of a chromaffin cell phenotype. By decreasing the available NGFR from the low affinity receptor reservoir along with the ability to synthesize new NGFR, the chromaffin phenotype would be more likely to be realized. Lastly, it is possible that steroids selectively affect the stability of NGFR mRNA. This is not likely given the lack of appropriate consensus sequences in the NGFR mRNA sequence.

ACKNOWLEDGMENTS The authors wish to thank Dr. Eric Shooter and Dr. Eugene Johnson for the very kind gifts of the NGFR cDNA probe and the monoclonal antibody 192, respectively. Thanks to Zhaohui Pan for preparation of the NGF, Karin Werrbach-Perez for excellent technical assistance, and Donna Masters for manuscript preparation. This work was supported in part by NINDS grant NS18708 and a grant from the Sigma Tau Company, Pomezia, Italy.

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NGFX mRNA and Dexamethasone Unsicker K, Krisch B, Otten J, Thoenen H (1978): Nerve growth factor-induced fiber outgrowth from isolated rat adrenal chromaffin cells: impairment by glucocorticoids. Proc Natl Acad Sci USA 75:3498-3502.

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Yakovelev AG, De Bernardi MA, Fabrazzo M, Brooker G, Costa E, Mochetti I(1990): Regulation of nerve growth factor receptor mRNA content by dexamethasone: In vitro and in vivo studies. Neurosi Lett 116:216-220.