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Stress-induced glucocorticoids suppress the antisense molecular regulation of FGF-2 expression
ARTICLE IN PRESS Psychoneuroendocrinology (2007) 32, 376–384
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/psyneuen
Stress-induced glucocorticoids suppress the antisense molecular regulation of FGF-2 expression Matthew G. Frank, Andre Der-Avakian, Sondra T. Bland, Linda R. Watkins, Steven F. Maier Department of Psychology and Center for Neuroscience, Campus Box 345, University of Colorado, Boulder, CO 80309-0345, USA Received 18 April 2006; received in revised form 19 January 2007; accepted 1 February 2007
KEYWORDS Fibroblast growth factor; Hippocampus; Corticosterone
Summary Psychological stress can upregulate basic fibroblast growth factor (FGF-2) expression. Because glucocorticoids can also upregulate FGF-2 expression, the present studies investigated whether stress-induced glucocorticoids mediate the effects of stress on FGF-2. FGF-2 is regulated by an FGF-2 antisense (AS) molecular mechanism and so the present experiments also, for the first time, assessed the effects of stress on FGF-2-AS mRNA, as well as the mediating role of glucocorticoids. The effects of either escapable shock (ES) or yoked-inescapable tail shock (IS) on FGF-2 and FGF-2-AS were determined. To test whether glucocorticoids mediate the effect of stress on FGF-2 and FGF-2-AS, animals were pretreated with temporary corticosterone (CORT) synthesis inhibitors and exposed to IS. To test whether glucocorticoids are sufficient to modulate FGF-2 and FGF-2-AS mRNA, animals were injected with CORT and mRNA measured. ES and IS similarly downregulated FGF-2-AS mRNA at 0 h post-stress and upregulated FGF-2 mRNA 2 h post-stress. Inhibition of CORT synthesis abrogated the effect of IS on both FGF-2-AS and FGF-2 mRNA. Exogenous CORT mimicked the effects of ES and IS on FGF-2, but not FGF-2-AS mRNA. The present study demonstrates that glucocorticoids mediate the effects of stress on FGF-2 and FGF-2-AS. & 2007 Elsevier Ltd. All rights reserved.
1. Introduction Basic fibroblast growth factor (FGF-2) is widely and abundantly expressed throughout the CNS and plays an Corresponding author. Tel.: +1 303 919 8116;
fax: +1 303 492 2967. E-mail address: [email protected] (M.G. Frank). 0306-4530/$ - see front matter & 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.psyneuen.2007.02.001
important role in adult neurogenesis and neuroprotection (Reuss and von Bohlen und Halbach, 2003). Glucocorticoids and glucocorticoid analogues upregulate FGF-2 expression at both the protein (Chadi et al., 1993; Mocchetti et al., 1996) and mRNA level (Mocchetti et al., 1996; Colangelo et al., 1998; Hansson et al., 2000) throughout the CNS. In addition, reduction of basal glucocorticoid levels by adrenalectomy attenuates expression of FGF-2 (Follesa and Mocchetti, 1993; Chao and McEwen, 1994; Riva et al.,
ARTICLE IN PRESS Stress-induced glucocorticoids and FGF-2 1995a) indicating that glucocorticoids exert tonic control over FGF-2 expression. Glucocorticoids and glucocorticoid analogues also induce FGF-2 expression in astrocytes (Riva et al., 1995b; Niu et al., 1997; Magnaghi et al., 2000), which are considered the predominant source of FGF-2 in the CNS (Woodward et al., 1992; Gonzalez et al., 1995; Weickert et al., 2005). Glucocorticoid regulation of FGF-2 may be particularly relevant in conditions characterized by glucocorticoidmediated neurotoxicity. Excess glucocorticoid levels such as those induced under acute and chronic stress have numerous effects on the CNS, particularly the hippocampus, which is abundant in both mineralocorticoid and glucocorticoid receptors (McEwen, 1999). The effects of glucocorticoids can be deleterious to the CNS (Sapolsky, 1999) insofar as excess glucocorticoids disrupt learning, memory, and plasticity (McEwen and Sapolsky, 1995), inhibit neurogenesis (Gould, 1994), induce atrophy of neuronal processes (Sapolsky, 1996), and can be neurotoxic with prolonged exposure (Sapolsky, 1996). Similar to the effects of glucocorticoids, acute and chronic stress upregulate FGF-2 expression throughout the CNS (Molteni et al., 2001; Fumagalli et al., 2005). Early life stress both upregulates and downregulates basal FGF-2 expression in adults depending on the brain region examined (Fumagalli et al., 2005). While it is reasonable to assume that the effects of stress on FGF-2 expression are mediated by glucocorticoids, this assumption has not been experimentally tested. Of greater relevance is whether glucocorticoids and/or stress modulate a unique antisense (AS) molecular mechanism, which directly regulates translation of FGF-2. Unlike the genomic organization of most mammalian genes, the FGF-2 gene locus contains an RNA transcribed from the AS strand (FGF-2 antisense; FGF-2-AS), which is complementary to 464 nucleotides present in the 30 untranslated region of the FGF-2 gene (Knee et al., 1997). FGF-2-AS and FGF-2 exhibit inverse transcriptional expression (Li et al., 1996; Knee et al., 1997) suggesting that FGF2-AS may be involved in the post-transcriptional regulation of FGF-2 expression. In addition, the coordinated expression of FGF-2-AS and FGF-2 suggests that both genes may be regulated by common signaling pathways and molecules. FGF-2-AS is expressed in the rat CNS and inhibits FGF-2 translation in vitro (Li and Murphy, 2000). Further, in some cell types cellular FGF-2 protein levels are negatively correlated with FGF-2-AS mRNA levels demonstrating FGF2-AS translation control over FGF-2. Unlike FGF-2, FGF-2-AS exhibits rapid and transient expression patterns, which precede alterations in FGF-2 (Baguma-Nibasheka et al., 2005). Given the AS molecular regulation of FGF-2 expression, the present investigation examined whether glucocorticoid and stress effects on FGF-2 expression may be mediated, in part, through modulation of FGF-2-AS. In light of the inverse transcriptional expression of FGF-2 and FGF-2-AS, we initially tested whether stress downregulates FGF-2-AS mRNA, while upregulating FGF-2 mRNA. As a strong test of this prediction, we utilized a stress paradigm in which animals are exposed to escapable shock (ES) or yoked-inescapable tail shock (IS). Animals exposed to ES and IS simultaneously receive the same number, duration,
377 and intensity of shock, except that animals exposed to ES control the offset of each shock. Termination of shock by the ES animal automatically results in the offset of shock for the IS animal. Both ES and IS induce comparable systemic elevations in corticosterone (CORT) and adrenocorticotropin releasing hormone (Maier et al., 1986) under the exact conditions used here, as well as equivalent expression of neuropeptides, including corticotropin releasing hormone that drive HPA axis activation (Helmreich et al., 1999). Therefore, if glucocorticoids mediate the stress effects on FGF-2 and/or FGF-2-AS, ES and IS should induce similar changes in the expression of these genes even though they produce quite different effects on other hormones and transmitters (e.g. 5-HT; Maier and Watkins, 2005). If glucocorticoids mediate the effects of stress on FGF-2 and FGF-2-AS mRNA, inhibition of glucocorticoid synthesis should abrogate the effects of stress on expression of these genes. To test this notion, animals were pretreated with temporary CORT synthesis inhibitors, exposed to IS and FGF-2 and FGF2-AS mRNA measured. A final experiment was conducted to test whether glucocorticoids are sufficient to alter the expression of FGF-2 and FGF-2-AS mRNA. In this experiment, the effects of exogenous CORT on FGF-2 and FGF-2-AS mRNA were measured. The present investigation focused on hippocampal FGF-2 and FGF-2-AS mRNA given the particular vulnerability of this structure to the neurotoxic effects of excess glucocorticoids (Sapolsky, 1999).
2. Materials and methods 2.1. Animals Male Sprague–Dawley rats (60–90 d old; Harlan SpragueDawley, Inc., Indianapolis, IN, USA) were pair-housed with food and water available ad libitum. The colony was maintained at 25 1C on a 12 h light/dark cycle (lights on at 0700 h). All experimental procedures were conducted in accord with the University of Colorado Institutional Animal Care and Use Committee. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques.
2.2. Experimental design 2.2.1. Experiment 1: effect of ES and IS on hippocampal FGF-2 and FGF-2-AS mRNA Details of the stressor controllability protocol have been published previously (Bland et al., 2003). Briefly, there were three experimental groups: rats that were given ES, rats that received IS that was yoked to the ES animal, and home cage control (HCC) rats that remained undisturbed in their home cages. All ES/IS treatments occurred between 0700 and 1000 h. ES and IS occurred in 14 11 17 cm3 Plexiglas wheel-turn boxes. A wheel consisting of two 14-cm diameter disks connected by 9-cm metal rods was mounted on one wall. ES and IS rats were treated as pairs, with the IS shock duration yoked to that of the ES animal. Each shock began simultaneously for the ES and IS subject, and terminated for both at the moment that the ES subject met the escape response requirement. The shock was terminated after 30 s if the escape subject did not perform
ARTICLE IN PRESS 378 the required response. The session consisted of 80 trials with an average intertrial interval (ITI) of 60 s and shock intensity was increased stepwise from 1.0 mA (0–30 min), to 1.3 mA (30–60 min) and then 1.6 mA (60–80 min). The rationale for this procedure is that at the low shock intensity, ES animals rapidly learn the contingency that wheel turning terminates tailshock, while higher shock intensity maintains the rate of behavioral responding throughout the session. Animals were sacrificed either 0 or 2 h post-shock. Hippocampus was dissected and gene expression measured by real-time RT-PCR. 2.2.2. Experiment 2: effect of glucocorticoid synthesis inhibition on IS-induced modulation of hippocampal FGF-2 and FGF-2-AS mRNA Animals were injected subcutaneously (1 ml/kg injection volume) either with temporary CORT synthesis inhibitors (metyrapone, 100 mg/kg and aminoglutethimide, 100 mg/kg) or vehicle 1.5 h prior to onset of IS. Metyrapone and aminoglutethimide were dissolved in 100% propylene glycol. Metyrapone, an inhibitor of 11-b hydroxylase, and aminoglutethimide, an inhibitor of cholesterol side-chain cleavage, were used in conjunction to ensure complete suppression of endogenous CORT levels (Plotsky and Sawchenko, 1987). Previously, we demonstrated that the dose and administration time point utilized here completely abrogates the stress-induced increase in CORT observed immediately after termination of IS (Der-Avakian et al., 2005). After pretreatment with metyrapone/aminoglutethimide (MA) or vehicle, animals were exposed to IS or served as HCC. ES was not employed given the results of exp. 1. Details of the present stressor protocol have been published previously (Johnson et al., 2002). Briefly, animals were placed in Plexiglas tubes (23.4 cm in length 7 cm in diameter) and exposed to 100 1.0 mA, 5 s tailshocks with a variable ITI ranging from 30 to 90 s (average ITI ¼ 60 s). All IS treatments occurred between 0700 and 1000 h. Animals were sacrificed immediately after termination of the stressor. HCC animals remained undisturbed in their home cages until sacrifice. Hippocampus was dissected and mRNA measured using real-time RT-PCR. 2.2.3. Experiment 3: effects of exogenous CORT on hippocampal FGF-2 and FGF-2-AS mRNA Animals were injected subcutaneously (2.5 mg/kg/ml) with either CORT or vehicle and returned to their home cages, where they remained undisturbed until sacrifice. CORT was dissolved in 100% propylene glycol. Animals were sacrificed 110 min post-injection, hippocampus dissected, and mRNA measured using real-time RT-PCR. The dose of CORT and time of sacrifice utilized here result in systemic CORT levels that mimic CORT levels induced by IS (Fleshner et al., 1995).
2.3. Tissue collection and processing Animals were given a lethal dose of sodium pentobarbital and transcardially perfused with heparinized (1 U/ml) .9% ice-cold saline for 3 min. Brain was rapidly extracted on ice and hippocampus dissected. Tissue was flash frozen in liquid nitrogen and stored at 80 1C.
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2.4. Real-time RT-PCR 2.4.1. RNA isolation and enrichment Total RNA was isolated from whole hippocampus utilizing a standard method of phenol:chloroform extraction (Chomczynski and Sacchi, 1987). Briefly, tissue was rapidly homogenized in 1 ml Trizol reagent (Invitrogen, Carlsbad, CA) using a Tissue Tearor homogenizer. After incubation at room temperature for 5 min, chloroform was added to supernatant, vortexed 2 min, and centrifuged (12,000g) for 10 min at 4 1C to achieve phase separation of nucleic acid. Isopropyl alcohol (.5 volumes of Trizol volume) was added to the aqueous phase to precipitate nucleic acid. Samples were briefly vortexed and incubated at room temperature for 10 min followed by centrifugation (12,000g) for 10 min at 4 1C. Nucleic acid precipitate was washed in 75% ethanol (1 ml) and centrifuged (7500g) for 5 min at 4 1C. UV spectrophotometric analysis of nucleic acid was performed at 260 nm to determine concentration. The 260:280 absorbance ratio was utilized to assess nucleic acid purity. Samples were Dnase-treated (DNA-free kit, Ambion, Austin, TX) to remove contaminating DNA and requantitated prior to cDNA synthesis. 2.4.2. cDNA synthesis Total RNA was reverse transcribed into cDNA using the SuperScript II First Strand Synthesis System for RT-PCR (Invitrogen). RNA (1 mg) was incubated for 5 min at 65 1C in a total reaction volume of 13 ml containing random hexamer primers (5 ng/ml) and dNTPs (1 mM). Samples were chilled on ice for at least 1 min. A cDNA synthesis buffer (6 ml) as described by the manufacturer was added to the reaction and incubated at 20 1C for 2 min. Reverse transcriptase (1 ml; 200 units SuperScript II) was added to the reaction and incubated at 25 1C for 10 min followed by 42 1C for 50 min. Reaction was terminated by heating to 70 1C for 15 min. cDNA was stored at 20 1C. 2.4.3. Primer specifications cDNA sequences were obtained from Genbank at the National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov). Primer sequences were designed to amplify FGF-2 (F: 50 -TATGAAGGAAGATGGACGGC-30 ; R: 50 TCGTTTCAGTGCCACATACC-30 ), FGF-2-AS (F: 50 -gttgttctcgg ctttgctcg-30 ; R: 50 -ATGTCCAGCTCGCCTTGTAG-30 ), full-length FGF-2-AS (F: 50 -GAACCGCACTTGTCAACG-30 ; R: 50 -ACCTGCAACCCCTACTTG-30 ), and glyceraldehyde-6-phosphate dehydrogenase (GAPDH) (F: 50 -GTTTGTGATGGGTGTGAACC30 ; R: 50 -TCTTCTGAGTGGCAGTGATG-30 ), which served as a housekeeping gene. It should be noted that 3 forms of FGF-2 of varying molecular masses are generated from alternate start codons (Prats et al., 1989). The FGF-2 primers used here amplify a region shared by all 3 forms of FGF-2. In addition, FGF-2-AS has 3 splice variants (Li and Murphy, 2000). The FGF-2-AS primer set falls within exon 1, which is shared by all 3 splice variants. The full-length FGF-2-AS primer set falls within exon 2, which is spliced out of the other 2 variants. Due to the technical limitations of realtime PCR and the detection chemistry used here, only the full-length splice variant was measured. Primer sequences were designed using the Qiagen Oligo Analysis & Plotting
ARTICLE IN PRESS Stress-induced glucocorticoids and FGF-2 Tool (oligos.qiagen.com/oligos/toolkit.php?) and tested for sequence specificity using the Basic Local Alignment Search Tool at NCBI (Altschul et al., 1997). Primers were obtained from Proligo (Boulder, CO). Primer specificity was verified by melt curve analysis (see Quantitative real-time PCR). 2.4.4. Quantitative real-time PCR PCR amplification of cDNA was performed using the Quantitect SYBR Green PCR Kit (Qiagen, Valencia, CA). cDNA (1 ml) was added to a reaction master mix (25 ml) containing 2.5 mM MgCl2, HotStar Taq DNA polymerase, SYBR Green I, dNTPs, fluorescein (10 nM) and gene-specific primers (500 nM each of forward and reverse primer). For each experimental sample, triplicate reactions were conducted in 96-well plates (BioRad, Hercules, CA). PCR cycling conditions consisted of a hot-start activation of HotStar Taq DNA polymerase (94 1C, 15 min) and 40 cycles of denaturation (95 1C, 15 s), annealing (55–58 1C, 30 s), and extension (72 1C, 30 s). A melt curve analysis was conducted to assess uniformity of product formation, primer dimer formation, and amplification of non-specific products. PCR product was denatured (95 1C, 1 min) and annealed (55 1C, 1 min) prior to melt curve analysis, which consisted of incrementally increasing reaction temperature (.5 1C/10 s) from 55 to 95 1C. The negative first derivative of the melt curve (fluorescence vs. temperature) plotted against temperature will yield a single peak (Tm of product) if primers are specific to the gene of interest. 2.4.5. Real-time detection and relative quantitation of PCR product Formation of PCR product was monitored in real time using the MyiQ Single-Color Real-Time PCR Detection System (BioRad). Fluorescence of SYBR Green I was captured at 72 1C. Threshold for detection of PCR product above background was set at 10 the standard deviation of mean background fluorescence for all reactions. Background fluorescence was determined from cycle 1 to 5 cycles prior to exponential amplification of product and subtracted from raw fluorescence of each reaction/cycle. Threshold for detection of PCR product fell within the exponential phase of amplification for each reaction. Threshold cycle (CT; number of cycles to reach threshold of detection) was determined for each reaction. 2.4.6. Relative quantitation of gene expression Relative gene expression was determined using the 2DDCt method (Livak and Schmittgen, 2001). Mean CT of triplicate measures was computed for each sample. Sample mean CT of the internal control (GAPDH) was subtracted from the sample mean CT of the respective gene of interest (DCT). The sample with the absolute highest mean DCT was selected as a calibrator and subtracted from the mean DCT of each experimental sample (DDCT). 2DDCt yields fold change in gene expression of the gene of interest normalized to the internal control gene expression and relative to the calibrator sample. Normalization to a housekeeping gene is valid only under experimental conditions where the housekeeping gene is not altered by the experimental treatments. In the present set of studies, GAPDH was not altered by the experimental treatments (data not shown).
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2.5. Statistics All data are presented as mean+SEM. Sample sizes are provided in figure captions. For experiments 1 and 2, statistical analyses consisted of ANOVA followed by Fisher’s PLSD post hoc comparisons. For experiment 3, t-tests were utilized. Threshold for statistical significance was set at a ¼ .05.
3. Results 3.1. Experiment 1: effect of ES and IS shock on hippocampal FGF-2 and FGF-2-AS mRNA 3.1.1. 2 h post-stress Based on prior studies showing that acute restraint stress upregulates FGF-2 within 2–3 h of stressor onset (Molteni et al., 2001; Fumagalli et al., 2005), the effects of ES and IS on FGF-2 expression were examined 2 h post-stress. Stress upregulated FGF-2 gene expression (Fig. 1A; F(2,13) ¼ 33.977, po.0001) compared to HCC. While both ES and IS increased FGF-2 expression compared to HCC (po.001 and po.0001, respectively), IS also resulted in a significant increase in FGF-2 compared to ES (po.01). FGF-2-AS was unaffected by either ES or IS (Fig. 1B; F(2,13) ¼ 2.339, p ¼ .1357) compared to HCC. To verify this result, the effect of stress on the full-length FGF-2-AS splice variant was assessed. Stress did not significantly alter the expression of the full-length splice variant (data not shown; F(2,13) ¼ 1.441, p ¼ .2722). Expression of FGF-2 and FGF-2-AS co-varies (BagumaNibasheka et al., 2005) suggesting that the ratio of these transcripts may be a relevant factor determining FGF-2 protein levels. Therefore, the ratio of FGF-2 to FGF-2-AS mRNA was computed and the effect of stress re-evaluated. As with FGF-2, stress significantly upregulated the ratio of FGF-2 to FGF-2-AS (Fig. 1C; F(2,13) ¼ 12.993, po.001). However, while both ES (po.001) and IS (po.001) increased FGF-2:FGF-2-AS expression compared to HCC, ES and IS did not significantly differ (p ¼ .79). 3.1.2. 0 h post-stress In light of evidence that changes in FGF-2-AS precede changes in FGF-2 expression (Baguma-Nibasheka et al., 2005), we examined the expression of these transcripts immediately after termination of stress. While termination of stress did not modulate FGF-2 expression (Fig. 2A; F(2,14) ¼ .203, p ¼ .82), FGF-2-AS expression was substantially reduced (Fig. 2B; F(2,14) ¼ 11.372, po.01). ES (po.01) and IS (po.001) similarly downregulated FGF-2-AS compared to HCC. The effects of ES and IS on FGF-2-AS were comparable (p ¼ .62). Analysis of the FGF-2 to FGF-2-AS expression ratio yielded a similar effect of stress (Fig. 2C; F(2,14) ¼ 34.937, po.0001), although effect sizes were considerably magnified. Of note, the magnitude of the effect of stress on the FGF-2 to FGF-2-AS expression ratio was comparable at 0 and 2 h post-stress (see Fig. 1C and 2C), though the effects are driven primarily by FGF-2 at 2 h and FGF-2-AS at 0 h. At 0 h post-stress, ES increased FGF-2:FGF-2AS 170% of HCC, while IS increased FGF-2:FGF-2-AS 182% of HCC. At 2 h post-stress, ES increased FGF-2:FGF-2-AS 175% of HCC, while IS increased FGF-2:FGF-2-AS 181% of HCC.
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Figure 1 (A) Effect of stress on FGF-2 gene expression 2 h poststress. Animals were exposed to ES (N ¼ 5), IS (N ¼ 6) or no stress (HCC; N ¼ 6) and hippocampal FGF-2 gene expression measured. *ES and IS significantly increased FGF-2 expression compared to HCC, po.001. #IS increased FGF-2 expression over ES, po.01. (B) Effect of stress on FGF-2-AS expression 2 h poststress. Animals were exposed to ES (N ¼ 5), IS (N ¼ 6) or no stress (HCC; N ¼ 6) and hippocampal FGF-2-AS gene expression measured. Neither ES nor IS significantly altered FGF-2-AS expression compared to HCC. (C) Effect of stress on FGF-2:FGF2-AS 2 h post-stress. Data presented in (A) and (B) were used to generate the ratio of FGF-2 to FGF-2-AS expression and reanalyzed. As in (A), ES and IS significantly increased FGF2:FGF-2-AS expression over HCC; however, ES and IS expression did not significantly differ. *po.001 compared to HCC.
3.2. Experiment 2: effect of glucocorticoid synthesis inhibition on IS-induced modulation of hippocampal FGF-2 and FGF-2-AS mRNA Given prior evidence that glucocorticoids increase FGF-2 expression, the present experiment tested whether glucocorticoids are necessary for the stress-induced alterations in both FGF-2 and FGF-2-AS expression. It should be noted here that exp. 2 employed only uncontrollable stress given the
Figure 2 (A) Effect of stress on FGF-2 expression 0 h poststress. Animals were exposed to ES (N ¼ 6), IS (N ¼ 6) or no stress (HCC; N ¼ 5) and hippocampal FGF-2 gene expression measured. Neither ES nor IS significantly altered FGF-2 expression compared to HCC. (B) Effect of stress on FGF-2-AS expression 0 h post-stress. Animals were exposed to ES (N ¼ 6), IS (N ¼ 6) or no stress (HCC; N ¼ 5) and hippocampal FGF-2-AS gene expression measured. *ES and IS significantly decreased FGF-2-AS expression compared to HCC, po.001. (C) Effect of stress on FGF-2:FGF-2-AS 0 h post-stress. Data presented in (A) and (B) were used to generate the ratio of FGF-2 to FGF-2-AS expression and reanalyzed. As in (A), ES and IS significantly increased FGF-2:FGF-2-AS expression over HCC, *po.0001. ES and IS expression did not significantly differ.
similar effects of ES and IS on gene expression in exp. 1. The uncontrollable stress protocol utilized in exp. 2 differs from exp. 1 in that animals are restrained in tubes and unable to control the offset of shock. Moreover, the duration of stress in exp. 2 occurred over 110 min, while in exp. 1 stress was applied over 80 min. Therefore, in exp. 2, 0 h post-IS is equivalent to 30 min post-stress in exp. 1. The stress protocol used in exp. 2 was selected to increase the likelihood of simultaneously detecting the effect of glucocorticoid synthesis inhibition on FGF-2 and FGF-2-AS. The IS protocol used in exp. 2 induces plasma glucocorticoid levels substantially higher than levels induced by the stress protocol used in exp. 1 (Fleshner et al., 1995; Maier
ARTICLE IN PRESS Stress-induced glucocorticoids and FGF-2
3.2.2. FGF-2-AS Inhibition of CORT synthesis abrogated the IS-induced suppression of FGF-2-AS expression (Fig. 3B; F(1,23) ¼ 6.956, po.01). As with exp. 1 at 0 h post-stress, IS reduced FGF-2-AS expression compared to HCC in the vehicle condition (po.001), whereas the suppressive effect of IS on FGF-2-AS was prevented by MA treatment (vehicle IS vs. MA IS, po.0001). The vehicle IS group significantly differed from the MA HCC (po.001) group. MA treatment effectively reversed the effects of IS on FGF-2-AS to levels comparable to MA-treated HCC (p ¼ .9). Vehicle and MA treatment in HCC exhibited similar effects on FGF-2-AS expression (p ¼ .39).
3.3. Experiment 3: effects of exogenous CORT on hippocampal FGF-2 and FGF-2-AS mRNA The results of exp. 2 demonstrated that glucocorticoids are necessary for the stress-induced alterations in FGF-2 and FGF-2-AS expression. To test the sufficiency of glucocorticoid modulation of both FGF-2 and FGF-2-AS expression, CORT was administered to naı¨ve animals at a dose that has been shown to result in systemic CORT levels comparable to the levels induced by IS (Fleshner et al., 1995). 3.3.1. FGF-2 Exogenous CORT upregulated FGF-2 expression compared to vehicle treatment (Fig. 4A; t(9) ¼ 7.007, po.0001). The magnitude of the CORT effect (136% of vehicle levels) was comparable to the effect of IS (156% of HCC) observed in the vehicle-treated HCC and IS animals of exp. 2 (see Section 3.2.1).
3.3.2. FGF-2-AS Exogenous CORT did not alter FGF-2-AS expression compared to HCC (Fig. 4B; t(10) ¼ .062, p ¼ .9522). To confirm this result, the effect of exogenous CORT on the full-length FGF-2-AS splice variant was assessed. CORT did not alter the
FGF-2 Relative Gene Expression
3.2.1. FGF-2 IS increased FGF-2 gene expression (po.001), similar to the effect of ES and IS in exp. 1. Inhibition of CORT synthesis using MA completely suppressed the effects of IS on FGF-2 expression (Fig. 3A; F(1,16) ¼ 17.865, po.001). As with exp. 1 at 2 h post-stress, IS increased FGF-2 expression compared to HCC in the vehicle condition (po.001). CORT synthesis inhibition in HCC had no effect relative to vehicle treatment in HCC (p ¼ .4). However, in animals exposed to IS, inhibition of CORT synthesis abrogated the IS-induced increase in FGF-2 expression (vehicle IS vs. MA IS, po.002). The vehicle IS group significantly differed from the MA HCC (po.001) group. MA suppressed the effect of IS on FGF-2 expression to levels comparable to HCC treated with MA (p ¼ .75).
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et al., 1986). Thus, if glucocorticoids modulate both FGF-2 and FGF-2-AS expression, the higher levels of glucocorticoids induced by the stress protocol in exp. 2 should increase the chances of detecting effects of metyrapone.
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Figure 3 (A) Effect of CORT synthesis inhibition on IS-induced increase in FGF-2 expression. Animals were injected with vehicle (N ¼ 5) or the CORT synthesis inhibitors metyrapone/ aminoglutethimide (MA; N ¼ 5) and served as HCC or injected with vehicle (N ¼ 5) or the CORT inhibitors MA (N ¼ 5) and exposed to IS. Immediately post-stress, FGF-2 gene expression was measured. MA completely blocked the IS-induced increase in FGF-2 expression comparable to expression levels in vehicletreated HCC and MA-treated HCC animals. *Vehicle IS vs. MA IS, po.002; vehicle IS vs. vehicle HCC, po.001; vehicle IS vs. MA HCC, po.001. (B) Effect of CORT synthesis inhibition on ISinduced suppression of FGF-2-AS expression. Animals were injected with vehicle (N ¼ 5) or the CORT synthesis inhibitors MA (N ¼ 7) and served as HCC or injected with vehicle (N ¼ 8) or the CORT inhibitors MA (N ¼ 7) and exposed to IS. Immediately post-stress, FGF-2-AS gene expression was measured. MA completely blocked the IS-induced suppression in FGF-2-AS expression to expression levels comparable to levels in vehicle-treated HCC and MA-treated HCC animals. *Vehicle IS vs. MA IS, po.0001; vehicle IS vs. vehicle HCC, po.001; vehicle IS vs. MA HCC, po.001.
expression of the full-length variant (data not shown; t (10) ¼ .349, p ¼ .73).
4. Discussion The translation of FGF-2 into protein is regulated by a unique molecular mechanism, whereby an AS transcript complementary to the 30 UTR of FGF-2 inhibits FGF-2 expression (Li and Murphy, 2000). The present results demonstrate that stress, independent of its controllability, downregulates this molecular mechanism. To our knowledge, this is the first demonstration that stress modulates
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Figure 4 (A) Effect of exogenous CORT on FGF-2 expression. Animals were injected with either vehicle (N ¼ 5) or CORT (N ¼ 6) and 110 min post-injection FGF-2 gene expression measured. CORT increased FGF-2 expression compared to vehicle controls, *po.0001. (B) Effect of exogenous CORT on FGF-2-AS expression. Animals were injected with either vehicle (N ¼ 6) or CORT (N ¼ 6) and 110 min post-injection FGF-2-AS gene expression measured. CORT did not significantly alter FGF2-AS expression compared to vehicle treatment.
FGF-2-AS. This downregulation of FGF-2-AS may be a mechanism involved in the stress-induced upregulation of FGF-2 observed in prior findings (Molteni et al., 2001; Fumagalli et al., 2005), which were corroborated in the present study. In addition, stress-induced modulation of FGF-2-AS preceded changes in FGF-2 expression, which is consistent with a prior study of the temporal relationship between the expression of these transcripts (BagumaNibasheka et al., 2005). Within a 2 h window post-stress, the effects of stress on FGF-2-AS dissipated to control levels, indicating that changes in FGF-2-AS expression are highly transient. Likewise, a prior study showed that transcriptional changes in FGF-2-AS are transitory (Baguma-Nibasheka et al., 2005). This same study also demonstrated that a narrow window may exist wherein changes in FGF-2-AS and FGF-2 expression overlap. In exp. 1, measurement of these transcripts 0 and 2 h post-stress did not reveal overlap in stress effects on gene expression. However, in exp. 2, FGF-2AS and FGF-2 expression were measured at a time point that corresponded to 30 min post-stress in exp. 1, thereby unmasking the concomitant effects of stress on FGF-2-AS and FGF-2. Taken together, these results highlight the importance in considering the gene expression of FGF-2 relative to FGF-2-AS when investigating FGF-2 translation control. It should be noted here that since the effects of
stress on FGF-2 and FGF-2-AS were restricted to hippocampus, the present results are obscure as to the specificity of these effects. Clearly, stress may differentially alter FGF-2 and FGF-2-AS expression depending on the brain region examined. In light of considerable evidence indicating that glucocorticoids upregulate FGF-2 expression both in vivo and in vitro, we tested whether glucocorticoids mediated the effects of stress on FGF-2 and FGF-2-AS. It should be noted that controllable and uncontrollable stress under the conditions used in the present experiment induce comparable elevations in circulating CORT (Maier et al., 1986) and similarly upregulated FGF-2 and downregulated FGF-2-AS expression, further implicating glucocorticoids in stressinduced modulation of FGF-2. Indeed, inhibition of CORT synthesis prevented both the stress-induced upregulation of FGF-2 and downregulation of FGF-2-AS suggesting that glucocorticoids are necessary for the stress-induced alterations in both FGF-2 and FGF-2-AS expression. To show that exogenous CORT reverses the effect of metyrapone on FGF-2 and FGF-2-AS would further substantiate the role of CORT in stress-induced regulation of FGF-2 and FGF-2-AS. To test the sufficiency of glucocorticoids in the regulation of FGF-2 and FGF-2-AS expression, the effects of exogenous CORT were assessed at a dose that is known to produce systemic concentrations across time similar to that produced by the stressors used here (Fleshner et al., 1995). Consistent with prior studies (Mocchetti et al., 1996; Colangelo et al., 1998; Hansson et al., 2000), exogenous CORT upregulated FGF-2 gene expression. However, CORT did not alter the expression of FGF-2-AS. The effects of CORT on FGF-2 suggest that levels of CORT that mimic stress-induced levels is sufficient to modulate FGF-2. However, conclusions regarding the lack of effect of this same level of CORT on FGF-2-AS are unclear given that exp. 3 was restricted to a single time point postCORT administration. We cannot exclude the possibility that CORT modulated FGF-2-AS at a prior time point and that the effect of CORT had simply dissipated. Also, the observation in exp. 1 that FGF-2-AS exhibits a transient expression pattern supports this alternate explanation of the absence of an effect of CORT on FGF-2-AS. Clearly, a full time course of the effects of exogenous CORT on FGF-2-AS expression is required to resolve this issue. Nevertheless, taken together, the present set of data suggests that stress-induced glucocorticoids are necessary for the effects of stress on FGF-2 translation control. The present study shows that stress reduces the expression of FGF-2-AS levels and that glucocorticoids mediate, in part, the effects of stress on FGF-2-AS. The effects of stressinduced glucocorticoids on both FGF-2 and FGF-2-AS gene expression may be necessary to induce an optimal ratio of FGF-2 to FGF-2-AS transcripts, thereby permitting FGF-2 translation to proceed. Stress-induced increases in FGF-2 may represent a compensatory mechanism for the potentially deleterious effects of glucocorticoids on the CNS. Excess levels of glucocorticoids as induced by acute and chronic stress inhibit neurogenesis (Gould, 1994) and induce atrophy of neuronal processes (Sapolsky, 1996). FGF-2 is important in adult neurogenesis and neuroprotection and therefore stress-induced FGF-2 may serve to ameliorate the damaging effects of glucocorticoids on the CNS. However, a neuroprotective role for FGF-2 during stress has not been
ARTICLE IN PRESS Stress-induced glucocorticoids and FGF-2 demonstrated. To further understand the role of FGF-2 during stress, future studies could examine whether inhibition of FGF-2 signaling exacerbates the effects of stress on the CNS.
Role of the funding source Funding from the National Institute on Drug Abuse was used to support post-doctoral training and research activity of Dr. Matthew G. Frank and Dr. Sondra T. Bland.
Conflict of interest statement We acknowledge that there were no actual or potential financial and other conflicts of interest related to the submitted manuscript.
Acknowledgements This work was supported by NIH post-doctoral fellowships to M.G.F. (DA15591) and S.T.B. (DA13006).
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M.G. Frank et al. expression in rat cortical astrocytes following dexamethasone treatment. J. Neurochem. 64, 2526–2533. Sapolsky, R.M., 1996. Stress, glucocorticoids, and damage to the nervous system: the current state of confusion. Stress 1, 1–19. Sapolsky, R.M., 1999. Glucocorticoids, stress, and their adverse neurological effects: relevance to aging. Exp. Gerontol. 34, 721–732. Weickert, C.S., Kittell, D.A., Saunders, R.C., Herman, M.M., Horlick, R.A., Kleinman, J.E., Hyde, T.M., 2005. Basic fibroblast growth factor and fibroblast growth factor receptor-1 in the human hippocampal formation. Neuroscience 131, 219–233. Woodward, W.R., Nishi, R., Meshul, C.K., Williams, T.E., Coulombe, M., Eckenstein, F.P., 1992. Nuclear and cytoplasmic localization of basic fibroblast growth factor in astrocytes and CA2 hippocampal neurons. J. Neurosci. 12, 142–152.
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Stress-induced glucocorticoids suppress the antisense molecular regulation of FGF-2 expression Matthew G. Frank, Andre Der-Avakian, Sondra T. Bland, Linda R. Watkins, Steven F. Maier Department of Psychology and Center for Neuroscience, Campus Box 345, University of Colorado, Boulder, CO 80309-0345, USA Received 18 April 2006; received in revised form 19 January 2007; accepted 1 February 2007
KEYWORDS Fibroblast growth factor; Hippocampus; Corticosterone
Summary Psychological stress can upregulate basic fibroblast growth factor (FGF-2) expression. Because glucocorticoids can also upregulate FGF-2 expression, the present studies investigated whether stress-induced glucocorticoids mediate the effects of stress on FGF-2. FGF-2 is regulated by an FGF-2 antisense (AS) molecular mechanism and so the present experiments also, for the first time, assessed the effects of stress on FGF-2-AS mRNA, as well as the mediating role of glucocorticoids. The effects of either escapable shock (ES) or yoked-inescapable tail shock (IS) on FGF-2 and FGF-2-AS were determined. To test whether glucocorticoids mediate the effect of stress on FGF-2 and FGF-2-AS, animals were pretreated with temporary corticosterone (CORT) synthesis inhibitors and exposed to IS. To test whether glucocorticoids are sufficient to modulate FGF-2 and FGF-2-AS mRNA, animals were injected with CORT and mRNA measured. ES and IS similarly downregulated FGF-2-AS mRNA at 0 h post-stress and upregulated FGF-2 mRNA 2 h post-stress. Inhibition of CORT synthesis abrogated the effect of IS on both FGF-2-AS and FGF-2 mRNA. Exogenous CORT mimicked the effects of ES and IS on FGF-2, but not FGF-2-AS mRNA. The present study demonstrates that glucocorticoids mediate the effects of stress on FGF-2 and FGF-2-AS. & 2007 Elsevier Ltd. All rights reserved.
1. Introduction Basic fibroblast growth factor (FGF-2) is widely and abundantly expressed throughout the CNS and plays an Corresponding author. Tel.: +1 303 919 8116;
fax: +1 303 492 2967. E-mail address: [email protected] (M.G. Frank). 0306-4530/$ - see front matter & 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.psyneuen.2007.02.001
important role in adult neurogenesis and neuroprotection (Reuss and von Bohlen und Halbach, 2003). Glucocorticoids and glucocorticoid analogues upregulate FGF-2 expression at both the protein (Chadi et al., 1993; Mocchetti et al., 1996) and mRNA level (Mocchetti et al., 1996; Colangelo et al., 1998; Hansson et al., 2000) throughout the CNS. In addition, reduction of basal glucocorticoid levels by adrenalectomy attenuates expression of FGF-2 (Follesa and Mocchetti, 1993; Chao and McEwen, 1994; Riva et al.,
ARTICLE IN PRESS Stress-induced glucocorticoids and FGF-2 1995a) indicating that glucocorticoids exert tonic control over FGF-2 expression. Glucocorticoids and glucocorticoid analogues also induce FGF-2 expression in astrocytes (Riva et al., 1995b; Niu et al., 1997; Magnaghi et al., 2000), which are considered the predominant source of FGF-2 in the CNS (Woodward et al., 1992; Gonzalez et al., 1995; Weickert et al., 2005). Glucocorticoid regulation of FGF-2 may be particularly relevant in conditions characterized by glucocorticoidmediated neurotoxicity. Excess glucocorticoid levels such as those induced under acute and chronic stress have numerous effects on the CNS, particularly the hippocampus, which is abundant in both mineralocorticoid and glucocorticoid receptors (McEwen, 1999). The effects of glucocorticoids can be deleterious to the CNS (Sapolsky, 1999) insofar as excess glucocorticoids disrupt learning, memory, and plasticity (McEwen and Sapolsky, 1995), inhibit neurogenesis (Gould, 1994), induce atrophy of neuronal processes (Sapolsky, 1996), and can be neurotoxic with prolonged exposure (Sapolsky, 1996). Similar to the effects of glucocorticoids, acute and chronic stress upregulate FGF-2 expression throughout the CNS (Molteni et al., 2001; Fumagalli et al., 2005). Early life stress both upregulates and downregulates basal FGF-2 expression in adults depending on the brain region examined (Fumagalli et al., 2005). While it is reasonable to assume that the effects of stress on FGF-2 expression are mediated by glucocorticoids, this assumption has not been experimentally tested. Of greater relevance is whether glucocorticoids and/or stress modulate a unique antisense (AS) molecular mechanism, which directly regulates translation of FGF-2. Unlike the genomic organization of most mammalian genes, the FGF-2 gene locus contains an RNA transcribed from the AS strand (FGF-2 antisense; FGF-2-AS), which is complementary to 464 nucleotides present in the 30 untranslated region of the FGF-2 gene (Knee et al., 1997). FGF-2-AS and FGF-2 exhibit inverse transcriptional expression (Li et al., 1996; Knee et al., 1997) suggesting that FGF2-AS may be involved in the post-transcriptional regulation of FGF-2 expression. In addition, the coordinated expression of FGF-2-AS and FGF-2 suggests that both genes may be regulated by common signaling pathways and molecules. FGF-2-AS is expressed in the rat CNS and inhibits FGF-2 translation in vitro (Li and Murphy, 2000). Further, in some cell types cellular FGF-2 protein levels are negatively correlated with FGF-2-AS mRNA levels demonstrating FGF2-AS translation control over FGF-2. Unlike FGF-2, FGF-2-AS exhibits rapid and transient expression patterns, which precede alterations in FGF-2 (Baguma-Nibasheka et al., 2005). Given the AS molecular regulation of FGF-2 expression, the present investigation examined whether glucocorticoid and stress effects on FGF-2 expression may be mediated, in part, through modulation of FGF-2-AS. In light of the inverse transcriptional expression of FGF-2 and FGF-2-AS, we initially tested whether stress downregulates FGF-2-AS mRNA, while upregulating FGF-2 mRNA. As a strong test of this prediction, we utilized a stress paradigm in which animals are exposed to escapable shock (ES) or yoked-inescapable tail shock (IS). Animals exposed to ES and IS simultaneously receive the same number, duration,
377 and intensity of shock, except that animals exposed to ES control the offset of each shock. Termination of shock by the ES animal automatically results in the offset of shock for the IS animal. Both ES and IS induce comparable systemic elevations in corticosterone (CORT) and adrenocorticotropin releasing hormone (Maier et al., 1986) under the exact conditions used here, as well as equivalent expression of neuropeptides, including corticotropin releasing hormone that drive HPA axis activation (Helmreich et al., 1999). Therefore, if glucocorticoids mediate the stress effects on FGF-2 and/or FGF-2-AS, ES and IS should induce similar changes in the expression of these genes even though they produce quite different effects on other hormones and transmitters (e.g. 5-HT; Maier and Watkins, 2005). If glucocorticoids mediate the effects of stress on FGF-2 and FGF-2-AS mRNA, inhibition of glucocorticoid synthesis should abrogate the effects of stress on expression of these genes. To test this notion, animals were pretreated with temporary CORT synthesis inhibitors, exposed to IS and FGF-2 and FGF2-AS mRNA measured. A final experiment was conducted to test whether glucocorticoids are sufficient to alter the expression of FGF-2 and FGF-2-AS mRNA. In this experiment, the effects of exogenous CORT on FGF-2 and FGF-2-AS mRNA were measured. The present investigation focused on hippocampal FGF-2 and FGF-2-AS mRNA given the particular vulnerability of this structure to the neurotoxic effects of excess glucocorticoids (Sapolsky, 1999).
2. Materials and methods 2.1. Animals Male Sprague–Dawley rats (60–90 d old; Harlan SpragueDawley, Inc., Indianapolis, IN, USA) were pair-housed with food and water available ad libitum. The colony was maintained at 25 1C on a 12 h light/dark cycle (lights on at 0700 h). All experimental procedures were conducted in accord with the University of Colorado Institutional Animal Care and Use Committee. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques.
2.2. Experimental design 2.2.1. Experiment 1: effect of ES and IS on hippocampal FGF-2 and FGF-2-AS mRNA Details of the stressor controllability protocol have been published previously (Bland et al., 2003). Briefly, there were three experimental groups: rats that were given ES, rats that received IS that was yoked to the ES animal, and home cage control (HCC) rats that remained undisturbed in their home cages. All ES/IS treatments occurred between 0700 and 1000 h. ES and IS occurred in 14 11 17 cm3 Plexiglas wheel-turn boxes. A wheel consisting of two 14-cm diameter disks connected by 9-cm metal rods was mounted on one wall. ES and IS rats were treated as pairs, with the IS shock duration yoked to that of the ES animal. Each shock began simultaneously for the ES and IS subject, and terminated for both at the moment that the ES subject met the escape response requirement. The shock was terminated after 30 s if the escape subject did not perform
ARTICLE IN PRESS 378 the required response. The session consisted of 80 trials with an average intertrial interval (ITI) of 60 s and shock intensity was increased stepwise from 1.0 mA (0–30 min), to 1.3 mA (30–60 min) and then 1.6 mA (60–80 min). The rationale for this procedure is that at the low shock intensity, ES animals rapidly learn the contingency that wheel turning terminates tailshock, while higher shock intensity maintains the rate of behavioral responding throughout the session. Animals were sacrificed either 0 or 2 h post-shock. Hippocampus was dissected and gene expression measured by real-time RT-PCR. 2.2.2. Experiment 2: effect of glucocorticoid synthesis inhibition on IS-induced modulation of hippocampal FGF-2 and FGF-2-AS mRNA Animals were injected subcutaneously (1 ml/kg injection volume) either with temporary CORT synthesis inhibitors (metyrapone, 100 mg/kg and aminoglutethimide, 100 mg/kg) or vehicle 1.5 h prior to onset of IS. Metyrapone and aminoglutethimide were dissolved in 100% propylene glycol. Metyrapone, an inhibitor of 11-b hydroxylase, and aminoglutethimide, an inhibitor of cholesterol side-chain cleavage, were used in conjunction to ensure complete suppression of endogenous CORT levels (Plotsky and Sawchenko, 1987). Previously, we demonstrated that the dose and administration time point utilized here completely abrogates the stress-induced increase in CORT observed immediately after termination of IS (Der-Avakian et al., 2005). After pretreatment with metyrapone/aminoglutethimide (MA) or vehicle, animals were exposed to IS or served as HCC. ES was not employed given the results of exp. 1. Details of the present stressor protocol have been published previously (Johnson et al., 2002). Briefly, animals were placed in Plexiglas tubes (23.4 cm in length 7 cm in diameter) and exposed to 100 1.0 mA, 5 s tailshocks with a variable ITI ranging from 30 to 90 s (average ITI ¼ 60 s). All IS treatments occurred between 0700 and 1000 h. Animals were sacrificed immediately after termination of the stressor. HCC animals remained undisturbed in their home cages until sacrifice. Hippocampus was dissected and mRNA measured using real-time RT-PCR. 2.2.3. Experiment 3: effects of exogenous CORT on hippocampal FGF-2 and FGF-2-AS mRNA Animals were injected subcutaneously (2.5 mg/kg/ml) with either CORT or vehicle and returned to their home cages, where they remained undisturbed until sacrifice. CORT was dissolved in 100% propylene glycol. Animals were sacrificed 110 min post-injection, hippocampus dissected, and mRNA measured using real-time RT-PCR. The dose of CORT and time of sacrifice utilized here result in systemic CORT levels that mimic CORT levels induced by IS (Fleshner et al., 1995).
2.3. Tissue collection and processing Animals were given a lethal dose of sodium pentobarbital and transcardially perfused with heparinized (1 U/ml) .9% ice-cold saline for 3 min. Brain was rapidly extracted on ice and hippocampus dissected. Tissue was flash frozen in liquid nitrogen and stored at 80 1C.
M.G. Frank et al.
2.4. Real-time RT-PCR 2.4.1. RNA isolation and enrichment Total RNA was isolated from whole hippocampus utilizing a standard method of phenol:chloroform extraction (Chomczynski and Sacchi, 1987). Briefly, tissue was rapidly homogenized in 1 ml Trizol reagent (Invitrogen, Carlsbad, CA) using a Tissue Tearor homogenizer. After incubation at room temperature for 5 min, chloroform was added to supernatant, vortexed 2 min, and centrifuged (12,000g) for 10 min at 4 1C to achieve phase separation of nucleic acid. Isopropyl alcohol (.5 volumes of Trizol volume) was added to the aqueous phase to precipitate nucleic acid. Samples were briefly vortexed and incubated at room temperature for 10 min followed by centrifugation (12,000g) for 10 min at 4 1C. Nucleic acid precipitate was washed in 75% ethanol (1 ml) and centrifuged (7500g) for 5 min at 4 1C. UV spectrophotometric analysis of nucleic acid was performed at 260 nm to determine concentration. The 260:280 absorbance ratio was utilized to assess nucleic acid purity. Samples were Dnase-treated (DNA-free kit, Ambion, Austin, TX) to remove contaminating DNA and requantitated prior to cDNA synthesis. 2.4.2. cDNA synthesis Total RNA was reverse transcribed into cDNA using the SuperScript II First Strand Synthesis System for RT-PCR (Invitrogen). RNA (1 mg) was incubated for 5 min at 65 1C in a total reaction volume of 13 ml containing random hexamer primers (5 ng/ml) and dNTPs (1 mM). Samples were chilled on ice for at least 1 min. A cDNA synthesis buffer (6 ml) as described by the manufacturer was added to the reaction and incubated at 20 1C for 2 min. Reverse transcriptase (1 ml; 200 units SuperScript II) was added to the reaction and incubated at 25 1C for 10 min followed by 42 1C for 50 min. Reaction was terminated by heating to 70 1C for 15 min. cDNA was stored at 20 1C. 2.4.3. Primer specifications cDNA sequences were obtained from Genbank at the National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov). Primer sequences were designed to amplify FGF-2 (F: 50 -TATGAAGGAAGATGGACGGC-30 ; R: 50 TCGTTTCAGTGCCACATACC-30 ), FGF-2-AS (F: 50 -gttgttctcgg ctttgctcg-30 ; R: 50 -ATGTCCAGCTCGCCTTGTAG-30 ), full-length FGF-2-AS (F: 50 -GAACCGCACTTGTCAACG-30 ; R: 50 -ACCTGCAACCCCTACTTG-30 ), and glyceraldehyde-6-phosphate dehydrogenase (GAPDH) (F: 50 -GTTTGTGATGGGTGTGAACC30 ; R: 50 -TCTTCTGAGTGGCAGTGATG-30 ), which served as a housekeeping gene. It should be noted that 3 forms of FGF-2 of varying molecular masses are generated from alternate start codons (Prats et al., 1989). The FGF-2 primers used here amplify a region shared by all 3 forms of FGF-2. In addition, FGF-2-AS has 3 splice variants (Li and Murphy, 2000). The FGF-2-AS primer set falls within exon 1, which is shared by all 3 splice variants. The full-length FGF-2-AS primer set falls within exon 2, which is spliced out of the other 2 variants. Due to the technical limitations of realtime PCR and the detection chemistry used here, only the full-length splice variant was measured. Primer sequences were designed using the Qiagen Oligo Analysis & Plotting
ARTICLE IN PRESS Stress-induced glucocorticoids and FGF-2 Tool (oligos.qiagen.com/oligos/toolkit.php?) and tested for sequence specificity using the Basic Local Alignment Search Tool at NCBI (Altschul et al., 1997). Primers were obtained from Proligo (Boulder, CO). Primer specificity was verified by melt curve analysis (see Quantitative real-time PCR). 2.4.4. Quantitative real-time PCR PCR amplification of cDNA was performed using the Quantitect SYBR Green PCR Kit (Qiagen, Valencia, CA). cDNA (1 ml) was added to a reaction master mix (25 ml) containing 2.5 mM MgCl2, HotStar Taq DNA polymerase, SYBR Green I, dNTPs, fluorescein (10 nM) and gene-specific primers (500 nM each of forward and reverse primer). For each experimental sample, triplicate reactions were conducted in 96-well plates (BioRad, Hercules, CA). PCR cycling conditions consisted of a hot-start activation of HotStar Taq DNA polymerase (94 1C, 15 min) and 40 cycles of denaturation (95 1C, 15 s), annealing (55–58 1C, 30 s), and extension (72 1C, 30 s). A melt curve analysis was conducted to assess uniformity of product formation, primer dimer formation, and amplification of non-specific products. PCR product was denatured (95 1C, 1 min) and annealed (55 1C, 1 min) prior to melt curve analysis, which consisted of incrementally increasing reaction temperature (.5 1C/10 s) from 55 to 95 1C. The negative first derivative of the melt curve (fluorescence vs. temperature) plotted against temperature will yield a single peak (Tm of product) if primers are specific to the gene of interest. 2.4.5. Real-time detection and relative quantitation of PCR product Formation of PCR product was monitored in real time using the MyiQ Single-Color Real-Time PCR Detection System (BioRad). Fluorescence of SYBR Green I was captured at 72 1C. Threshold for detection of PCR product above background was set at 10 the standard deviation of mean background fluorescence for all reactions. Background fluorescence was determined from cycle 1 to 5 cycles prior to exponential amplification of product and subtracted from raw fluorescence of each reaction/cycle. Threshold for detection of PCR product fell within the exponential phase of amplification for each reaction. Threshold cycle (CT; number of cycles to reach threshold of detection) was determined for each reaction. 2.4.6. Relative quantitation of gene expression Relative gene expression was determined using the 2DDCt method (Livak and Schmittgen, 2001). Mean CT of triplicate measures was computed for each sample. Sample mean CT of the internal control (GAPDH) was subtracted from the sample mean CT of the respective gene of interest (DCT). The sample with the absolute highest mean DCT was selected as a calibrator and subtracted from the mean DCT of each experimental sample (DDCT). 2DDCt yields fold change in gene expression of the gene of interest normalized to the internal control gene expression and relative to the calibrator sample. Normalization to a housekeeping gene is valid only under experimental conditions where the housekeeping gene is not altered by the experimental treatments. In the present set of studies, GAPDH was not altered by the experimental treatments (data not shown).
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2.5. Statistics All data are presented as mean+SEM. Sample sizes are provided in figure captions. For experiments 1 and 2, statistical analyses consisted of ANOVA followed by Fisher’s PLSD post hoc comparisons. For experiment 3, t-tests were utilized. Threshold for statistical significance was set at a ¼ .05.
3. Results 3.1. Experiment 1: effect of ES and IS shock on hippocampal FGF-2 and FGF-2-AS mRNA 3.1.1. 2 h post-stress Based on prior studies showing that acute restraint stress upregulates FGF-2 within 2–3 h of stressor onset (Molteni et al., 2001; Fumagalli et al., 2005), the effects of ES and IS on FGF-2 expression were examined 2 h post-stress. Stress upregulated FGF-2 gene expression (Fig. 1A; F(2,13) ¼ 33.977, po.0001) compared to HCC. While both ES and IS increased FGF-2 expression compared to HCC (po.001 and po.0001, respectively), IS also resulted in a significant increase in FGF-2 compared to ES (po.01). FGF-2-AS was unaffected by either ES or IS (Fig. 1B; F(2,13) ¼ 2.339, p ¼ .1357) compared to HCC. To verify this result, the effect of stress on the full-length FGF-2-AS splice variant was assessed. Stress did not significantly alter the expression of the full-length splice variant (data not shown; F(2,13) ¼ 1.441, p ¼ .2722). Expression of FGF-2 and FGF-2-AS co-varies (BagumaNibasheka et al., 2005) suggesting that the ratio of these transcripts may be a relevant factor determining FGF-2 protein levels. Therefore, the ratio of FGF-2 to FGF-2-AS mRNA was computed and the effect of stress re-evaluated. As with FGF-2, stress significantly upregulated the ratio of FGF-2 to FGF-2-AS (Fig. 1C; F(2,13) ¼ 12.993, po.001). However, while both ES (po.001) and IS (po.001) increased FGF-2:FGF-2-AS expression compared to HCC, ES and IS did not significantly differ (p ¼ .79). 3.1.2. 0 h post-stress In light of evidence that changes in FGF-2-AS precede changes in FGF-2 expression (Baguma-Nibasheka et al., 2005), we examined the expression of these transcripts immediately after termination of stress. While termination of stress did not modulate FGF-2 expression (Fig. 2A; F(2,14) ¼ .203, p ¼ .82), FGF-2-AS expression was substantially reduced (Fig. 2B; F(2,14) ¼ 11.372, po.01). ES (po.01) and IS (po.001) similarly downregulated FGF-2-AS compared to HCC. The effects of ES and IS on FGF-2-AS were comparable (p ¼ .62). Analysis of the FGF-2 to FGF-2-AS expression ratio yielded a similar effect of stress (Fig. 2C; F(2,14) ¼ 34.937, po.0001), although effect sizes were considerably magnified. Of note, the magnitude of the effect of stress on the FGF-2 to FGF-2-AS expression ratio was comparable at 0 and 2 h post-stress (see Fig. 1C and 2C), though the effects are driven primarily by FGF-2 at 2 h and FGF-2-AS at 0 h. At 0 h post-stress, ES increased FGF-2:FGF-2AS 170% of HCC, while IS increased FGF-2:FGF-2-AS 182% of HCC. At 2 h post-stress, ES increased FGF-2:FGF-2-AS 175% of HCC, while IS increased FGF-2:FGF-2-AS 181% of HCC.
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Figure 1 (A) Effect of stress on FGF-2 gene expression 2 h poststress. Animals were exposed to ES (N ¼ 5), IS (N ¼ 6) or no stress (HCC; N ¼ 6) and hippocampal FGF-2 gene expression measured. *ES and IS significantly increased FGF-2 expression compared to HCC, po.001. #IS increased FGF-2 expression over ES, po.01. (B) Effect of stress on FGF-2-AS expression 2 h poststress. Animals were exposed to ES (N ¼ 5), IS (N ¼ 6) or no stress (HCC; N ¼ 6) and hippocampal FGF-2-AS gene expression measured. Neither ES nor IS significantly altered FGF-2-AS expression compared to HCC. (C) Effect of stress on FGF-2:FGF2-AS 2 h post-stress. Data presented in (A) and (B) were used to generate the ratio of FGF-2 to FGF-2-AS expression and reanalyzed. As in (A), ES and IS significantly increased FGF2:FGF-2-AS expression over HCC; however, ES and IS expression did not significantly differ. *po.001 compared to HCC.
3.2. Experiment 2: effect of glucocorticoid synthesis inhibition on IS-induced modulation of hippocampal FGF-2 and FGF-2-AS mRNA Given prior evidence that glucocorticoids increase FGF-2 expression, the present experiment tested whether glucocorticoids are necessary for the stress-induced alterations in both FGF-2 and FGF-2-AS expression. It should be noted here that exp. 2 employed only uncontrollable stress given the
Figure 2 (A) Effect of stress on FGF-2 expression 0 h poststress. Animals were exposed to ES (N ¼ 6), IS (N ¼ 6) or no stress (HCC; N ¼ 5) and hippocampal FGF-2 gene expression measured. Neither ES nor IS significantly altered FGF-2 expression compared to HCC. (B) Effect of stress on FGF-2-AS expression 0 h post-stress. Animals were exposed to ES (N ¼ 6), IS (N ¼ 6) or no stress (HCC; N ¼ 5) and hippocampal FGF-2-AS gene expression measured. *ES and IS significantly decreased FGF-2-AS expression compared to HCC, po.001. (C) Effect of stress on FGF-2:FGF-2-AS 0 h post-stress. Data presented in (A) and (B) were used to generate the ratio of FGF-2 to FGF-2-AS expression and reanalyzed. As in (A), ES and IS significantly increased FGF-2:FGF-2-AS expression over HCC, *po.0001. ES and IS expression did not significantly differ.
similar effects of ES and IS on gene expression in exp. 1. The uncontrollable stress protocol utilized in exp. 2 differs from exp. 1 in that animals are restrained in tubes and unable to control the offset of shock. Moreover, the duration of stress in exp. 2 occurred over 110 min, while in exp. 1 stress was applied over 80 min. Therefore, in exp. 2, 0 h post-IS is equivalent to 30 min post-stress in exp. 1. The stress protocol used in exp. 2 was selected to increase the likelihood of simultaneously detecting the effect of glucocorticoid synthesis inhibition on FGF-2 and FGF-2-AS. The IS protocol used in exp. 2 induces plasma glucocorticoid levels substantially higher than levels induced by the stress protocol used in exp. 1 (Fleshner et al., 1995; Maier
ARTICLE IN PRESS Stress-induced glucocorticoids and FGF-2
3.2.2. FGF-2-AS Inhibition of CORT synthesis abrogated the IS-induced suppression of FGF-2-AS expression (Fig. 3B; F(1,23) ¼ 6.956, po.01). As with exp. 1 at 0 h post-stress, IS reduced FGF-2-AS expression compared to HCC in the vehicle condition (po.001), whereas the suppressive effect of IS on FGF-2-AS was prevented by MA treatment (vehicle IS vs. MA IS, po.0001). The vehicle IS group significantly differed from the MA HCC (po.001) group. MA treatment effectively reversed the effects of IS on FGF-2-AS to levels comparable to MA-treated HCC (p ¼ .9). Vehicle and MA treatment in HCC exhibited similar effects on FGF-2-AS expression (p ¼ .39).
3.3. Experiment 3: effects of exogenous CORT on hippocampal FGF-2 and FGF-2-AS mRNA The results of exp. 2 demonstrated that glucocorticoids are necessary for the stress-induced alterations in FGF-2 and FGF-2-AS expression. To test the sufficiency of glucocorticoid modulation of both FGF-2 and FGF-2-AS expression, CORT was administered to naı¨ve animals at a dose that has been shown to result in systemic CORT levels comparable to the levels induced by IS (Fleshner et al., 1995). 3.3.1. FGF-2 Exogenous CORT upregulated FGF-2 expression compared to vehicle treatment (Fig. 4A; t(9) ¼ 7.007, po.0001). The magnitude of the CORT effect (136% of vehicle levels) was comparable to the effect of IS (156% of HCC) observed in the vehicle-treated HCC and IS animals of exp. 2 (see Section 3.2.1).
3.3.2. FGF-2-AS Exogenous CORT did not alter FGF-2-AS expression compared to HCC (Fig. 4B; t(10) ¼ .062, p ¼ .9522). To confirm this result, the effect of exogenous CORT on the full-length FGF-2-AS splice variant was assessed. CORT did not alter the
FGF-2 Relative Gene Expression
3.2.1. FGF-2 IS increased FGF-2 gene expression (po.001), similar to the effect of ES and IS in exp. 1. Inhibition of CORT synthesis using MA completely suppressed the effects of IS on FGF-2 expression (Fig. 3A; F(1,16) ¼ 17.865, po.001). As with exp. 1 at 2 h post-stress, IS increased FGF-2 expression compared to HCC in the vehicle condition (po.001). CORT synthesis inhibition in HCC had no effect relative to vehicle treatment in HCC (p ¼ .4). However, in animals exposed to IS, inhibition of CORT synthesis abrogated the IS-induced increase in FGF-2 expression (vehicle IS vs. MA IS, po.002). The vehicle IS group significantly differed from the MA HCC (po.001) group. MA suppressed the effect of IS on FGF-2 expression to levels comparable to HCC treated with MA (p ¼ .75).
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et al., 1986). Thus, if glucocorticoids modulate both FGF-2 and FGF-2-AS expression, the higher levels of glucocorticoids induced by the stress protocol in exp. 2 should increase the chances of detecting effects of metyrapone.
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Figure 3 (A) Effect of CORT synthesis inhibition on IS-induced increase in FGF-2 expression. Animals were injected with vehicle (N ¼ 5) or the CORT synthesis inhibitors metyrapone/ aminoglutethimide (MA; N ¼ 5) and served as HCC or injected with vehicle (N ¼ 5) or the CORT inhibitors MA (N ¼ 5) and exposed to IS. Immediately post-stress, FGF-2 gene expression was measured. MA completely blocked the IS-induced increase in FGF-2 expression comparable to expression levels in vehicletreated HCC and MA-treated HCC animals. *Vehicle IS vs. MA IS, po.002; vehicle IS vs. vehicle HCC, po.001; vehicle IS vs. MA HCC, po.001. (B) Effect of CORT synthesis inhibition on ISinduced suppression of FGF-2-AS expression. Animals were injected with vehicle (N ¼ 5) or the CORT synthesis inhibitors MA (N ¼ 7) and served as HCC or injected with vehicle (N ¼ 8) or the CORT inhibitors MA (N ¼ 7) and exposed to IS. Immediately post-stress, FGF-2-AS gene expression was measured. MA completely blocked the IS-induced suppression in FGF-2-AS expression to expression levels comparable to levels in vehicle-treated HCC and MA-treated HCC animals. *Vehicle IS vs. MA IS, po.0001; vehicle IS vs. vehicle HCC, po.001; vehicle IS vs. MA HCC, po.001.
expression of the full-length variant (data not shown; t (10) ¼ .349, p ¼ .73).
4. Discussion The translation of FGF-2 into protein is regulated by a unique molecular mechanism, whereby an AS transcript complementary to the 30 UTR of FGF-2 inhibits FGF-2 expression (Li and Murphy, 2000). The present results demonstrate that stress, independent of its controllability, downregulates this molecular mechanism. To our knowledge, this is the first demonstration that stress modulates
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Figure 4 (A) Effect of exogenous CORT on FGF-2 expression. Animals were injected with either vehicle (N ¼ 5) or CORT (N ¼ 6) and 110 min post-injection FGF-2 gene expression measured. CORT increased FGF-2 expression compared to vehicle controls, *po.0001. (B) Effect of exogenous CORT on FGF-2-AS expression. Animals were injected with either vehicle (N ¼ 6) or CORT (N ¼ 6) and 110 min post-injection FGF-2-AS gene expression measured. CORT did not significantly alter FGF2-AS expression compared to vehicle treatment.
FGF-2-AS. This downregulation of FGF-2-AS may be a mechanism involved in the stress-induced upregulation of FGF-2 observed in prior findings (Molteni et al., 2001; Fumagalli et al., 2005), which were corroborated in the present study. In addition, stress-induced modulation of FGF-2-AS preceded changes in FGF-2 expression, which is consistent with a prior study of the temporal relationship between the expression of these transcripts (BagumaNibasheka et al., 2005). Within a 2 h window post-stress, the effects of stress on FGF-2-AS dissipated to control levels, indicating that changes in FGF-2-AS expression are highly transient. Likewise, a prior study showed that transcriptional changes in FGF-2-AS are transitory (Baguma-Nibasheka et al., 2005). This same study also demonstrated that a narrow window may exist wherein changes in FGF-2-AS and FGF-2 expression overlap. In exp. 1, measurement of these transcripts 0 and 2 h post-stress did not reveal overlap in stress effects on gene expression. However, in exp. 2, FGF-2AS and FGF-2 expression were measured at a time point that corresponded to 30 min post-stress in exp. 1, thereby unmasking the concomitant effects of stress on FGF-2-AS and FGF-2. Taken together, these results highlight the importance in considering the gene expression of FGF-2 relative to FGF-2-AS when investigating FGF-2 translation control. It should be noted here that since the effects of
stress on FGF-2 and FGF-2-AS were restricted to hippocampus, the present results are obscure as to the specificity of these effects. Clearly, stress may differentially alter FGF-2 and FGF-2-AS expression depending on the brain region examined. In light of considerable evidence indicating that glucocorticoids upregulate FGF-2 expression both in vivo and in vitro, we tested whether glucocorticoids mediated the effects of stress on FGF-2 and FGF-2-AS. It should be noted that controllable and uncontrollable stress under the conditions used in the present experiment induce comparable elevations in circulating CORT (Maier et al., 1986) and similarly upregulated FGF-2 and downregulated FGF-2-AS expression, further implicating glucocorticoids in stressinduced modulation of FGF-2. Indeed, inhibition of CORT synthesis prevented both the stress-induced upregulation of FGF-2 and downregulation of FGF-2-AS suggesting that glucocorticoids are necessary for the stress-induced alterations in both FGF-2 and FGF-2-AS expression. To show that exogenous CORT reverses the effect of metyrapone on FGF-2 and FGF-2-AS would further substantiate the role of CORT in stress-induced regulation of FGF-2 and FGF-2-AS. To test the sufficiency of glucocorticoids in the regulation of FGF-2 and FGF-2-AS expression, the effects of exogenous CORT were assessed at a dose that is known to produce systemic concentrations across time similar to that produced by the stressors used here (Fleshner et al., 1995). Consistent with prior studies (Mocchetti et al., 1996; Colangelo et al., 1998; Hansson et al., 2000), exogenous CORT upregulated FGF-2 gene expression. However, CORT did not alter the expression of FGF-2-AS. The effects of CORT on FGF-2 suggest that levels of CORT that mimic stress-induced levels is sufficient to modulate FGF-2. However, conclusions regarding the lack of effect of this same level of CORT on FGF-2-AS are unclear given that exp. 3 was restricted to a single time point postCORT administration. We cannot exclude the possibility that CORT modulated FGF-2-AS at a prior time point and that the effect of CORT had simply dissipated. Also, the observation in exp. 1 that FGF-2-AS exhibits a transient expression pattern supports this alternate explanation of the absence of an effect of CORT on FGF-2-AS. Clearly, a full time course of the effects of exogenous CORT on FGF-2-AS expression is required to resolve this issue. Nevertheless, taken together, the present set of data suggests that stress-induced glucocorticoids are necessary for the effects of stress on FGF-2 translation control. The present study shows that stress reduces the expression of FGF-2-AS levels and that glucocorticoids mediate, in part, the effects of stress on FGF-2-AS. The effects of stressinduced glucocorticoids on both FGF-2 and FGF-2-AS gene expression may be necessary to induce an optimal ratio of FGF-2 to FGF-2-AS transcripts, thereby permitting FGF-2 translation to proceed. Stress-induced increases in FGF-2 may represent a compensatory mechanism for the potentially deleterious effects of glucocorticoids on the CNS. Excess levels of glucocorticoids as induced by acute and chronic stress inhibit neurogenesis (Gould, 1994) and induce atrophy of neuronal processes (Sapolsky, 1996). FGF-2 is important in adult neurogenesis and neuroprotection and therefore stress-induced FGF-2 may serve to ameliorate the damaging effects of glucocorticoids on the CNS. However, a neuroprotective role for FGF-2 during stress has not been
ARTICLE IN PRESS Stress-induced glucocorticoids and FGF-2 demonstrated. To further understand the role of FGF-2 during stress, future studies could examine whether inhibition of FGF-2 signaling exacerbates the effects of stress on the CNS.
Role of the funding source Funding from the National Institute on Drug Abuse was used to support post-doctoral training and research activity of Dr. Matthew G. Frank and Dr. Sondra T. Bland.
Conflict of interest statement We acknowledge that there were no actual or potential financial and other conflicts of interest related to the submitted manuscript.
Acknowledgements This work was supported by NIH post-doctoral fellowships to M.G.F. (DA15591) and S.T.B. (DA13006).
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ARTICLE IN PRESS 384 and oxytocin after pharmacological adrenalectomy. Endocrinology 120, 1361–1369. Prats, H., Kaghad, M., Prats, A.C., Klagsbrun, M., Lelias, J.M., Liauzun, P., Chalon, P., Tauber, J.P., Amalric, F., Smith, J.A., et al., 1989. High molecular mass forms of basic fibroblast growth factor are initiated by alternative CUG codons. Proc. Natl. Acad. Sci. USA 86, 1836–1840. Reuss, B., von Bohlen und Halbach, O., 2003. Fibroblast growth factors and their receptors in the central nervous system. Cell Tissue Res. 313, 139–157. Riva, M.A., Fumagalli, F., Blom, J.M., Donati, E., Racagni, G., 1995a. Adrenalectomy reduces FGF-1 and FGF-2 gene expression in specific rat brain regions and differently affects their induction by seizures. Brain Res. Mol. Brain Res. 34, 190–196. Riva, M.A., Fumagalli, F., Racagni, G., 1995b. Opposite regulation of basic fibroblast growth factor and nerve growth factor gene
M.G. Frank et al. expression in rat cortical astrocytes following dexamethasone treatment. J. Neurochem. 64, 2526–2533. Sapolsky, R.M., 1996. Stress, glucocorticoids, and damage to the nervous system: the current state of confusion. Stress 1, 1–19. Sapolsky, R.M., 1999. Glucocorticoids, stress, and their adverse neurological effects: relevance to aging. Exp. Gerontol. 34, 721–732. Weickert, C.S., Kittell, D.A., Saunders, R.C., Herman, M.M., Horlick, R.A., Kleinman, J.E., Hyde, T.M., 2005. Basic fibroblast growth factor and fibroblast growth factor receptor-1 in the human hippocampal formation. Neuroscience 131, 219–233. Woodward, W.R., Nishi, R., Meshul, C.K., Williams, T.E., Coulombe, M., Eckenstein, F.P., 1992. Nuclear and cytoplasmic localization of basic fibroblast growth factor in astrocytes and CA2 hippocampal neurons. J. Neurosci. 12, 142–152.