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*12-O-TETRADECANOYLPHORBOL-13-ACETATE
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Endocrinology Vol. 140, No. 9 4095-4103
Copyright © 1999 by The Endocrine Society

ARTICLES

Transcriptional Regulation of Corticotropin-Releasing Hormone-Binding Protein Gene Expression in Astrocyte Cultures1

Shanna J. McClennen and Audrey F. Seasholtz

Department of Biological Chemistry (S.J.M., A.F.S.) and the Mental Health Research Institute (A.F.S.), The University of Michigan, Ann Arbor, Michigan 48109

Address all correspondence and requests for reprints to: Audrey F. Seasholtz, Ph.D., Mental Health Research Institute, 205 Zina Pitcher Place, Ann Arbor, Michigan 48109-0720. E-mail: aseashol{at}umich.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The molecular mechanisms involved in regulation of CRH-binding protein (CRH-BP) gene expression were examined using primary rat astrocyte cultures. The cells were treated with various regulators, and CRH-BP messenger RNA (mRNA) levels were determined using ribonuclease protection assays. Forskolin (Fsk, 10 µM) or 12-O-tetradecanoyl-phorbol 13-acetate (TPA, 100 nM) increases CRH-BP mRNA levels up to 30 times control level, and together they act synergistically to increase CRH-BP gene expression up to 100 times control levels. CRH can also positively regulate CRH-BP gene expression to 6.1 times control levels. All of these increases in steady-state CRH-BP mRNA levels can be repressed by dexamethasone, a synthetic glucocorticoid. To determine whether these changes in steady-state CRH-BP mRNA levels are caused by altered transcription or RNA stability, heteronuclear (hn) CRH-BP species were examined using ribonuclease protection assays. CRH-BP hnRNA transcripts can be detected transiently after the addition of Fsk or TPA, and dexamethasone can repress Fsk- or TPA-induced CRH-BP hnRNA levels in this assay. These results demonstrate that CRH, glucocorticoids, and the protein kinase A and protein kinase C signaling pathways are involved in regulation of CRH-BP gene expression in astrocyte cultures, and that this regulation is caused, at least in part, by altered transcription of the gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CRH IS A 41-AMINO acid neuropeptide with multiple roles in the mammalian stress response. It has been most extensively characterized in its role as an endocrine mediator in the hypothalamus-pituitary-adrenal axis; yet, it has also been shown to be widely distributed in the central nervous system, where it functions as a neurotransmitter/neuromodulator (1). At CRH target sites, CRH binds to specific seven-transmembrane spanning Gs protein-coupled receptors, increasing intracellular cAMP levels through activation of adenylate cyclase (2, 3, 4, 5). The type 1 CRH-receptor (CRH-R1) is expressed in pituitary and a number of brain regions, including brain stem, cerebellum, and cerebral cortex (6, 7). The type 2 CRH-receptor (CRH-R2) is present in two alternatively spliced isoforms in rodents. CRH-R2{alpha} is expressed most highly in lateral septum, the ventromedial nucleus of the hypothalamus, and amygdala. CRH-R2ß is most abundantly expressed in peripheral tissues, including heart and skeletal muscle (4, 8, 9, 10, 11, 12).

An additional protein, the CRH-binding protein (CRH-BP), has been shown to bind CRH with high affinity (13, 14). CRH-BP is a 37-kDa glycoprotein that colocalizes with CRH at several sites in the brain, including the central nucleus of the amygdala, bed nucleus of the stria terminalis, olfactory bulb, and lateral septal nucleus. It is also expressed in a subset of anterior pituitary corticotrophs, where CRH-R1 is also expressed (15). CRH-BP binds CRH with an affinity higher than that of CRH-R1 [Ki = 0.4 and 1.7 nM, respectively (13, 16, 17)], and has been shown to block the ACTH-releasing activity of CRH in primary pituitary cultures and in cultured AtT-20 cells (16, 17).

Both in vitro and in vivo studies have begun to elucidate the molecular mechanisms involved in regulation of CRH-BP gene expression. Transfection experiments with CRH-BP-reporter constructs demonstrate positive regulation of the CRH-BP promoter by cAMP and by CRH in cells expressing CRH-R1 (18). Experiments with primary rat astrocyte cultures have also demonstrated positive regulation of endogenous CRH-BP steady-state messenger RNA (mRNA) levels by cAMP and increased secretion of CRH-BP in response to forskolin (Fsk) or phorbol myristate acetate (PMA) (19, 20). Studies using an immortalized amygdalar neuronal cell line demonstrate positive regulation of steady-state CRH-BP mRNA levels by Fsk, PMA, and dexamethasone (Dex) (21). Our in vivo studies examining CRH-BP gene expression in rat pituitaries show that restraint stress increases CRH-BP mRNA levels, whereas adrenalectomy decreases pituitary CRH-BP mRNA levels (22). Together, these results demonstrate that the expression and secretion of CRH-BP is highly regulated by multiple second-messenger pathways.

In this study, we have examined the regulation of endogenous CRH-BP gene expression in primary rat astrocyte cultures using ribonuclease (RNase) protection assays. Our results demonstrate that extracellular signals such as CRH, and intracellular signals from the adrenal steroid hormone, protein kinase A (PKA), and protein kinase C (PKC) pathways, mediate dramatic changes in CRH-BP gene expression in these cells. Our studies also demonstrate that the changes in CRH-BP mRNA levels in primary astrocyte cultures are caused, at least in part, by altered transcription of the CRH-BP gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Fsk was purchased from Calbiochem (San Diego, CA). 12-O-Tetradecanoyl-phorbol 13-acetate (TPA, also known as PMA), Dex, trypsin, deoxyribonuclease (DNase) I, and trypsin inhibitor were purchased from Sigma Chemical Co. (St. Louis, MO). Ovine CRH was purchased from American Peptide Co. (Sunnyvale, CA). DMEM with D-valine, antibiotic/antimycotic, and Trizol Reagent were purchased from Life Technologies (Gaithersburg, MD). FCS was purchased from HyClone Laboratories, Inc. (Logan, UT). The antibody to glial fibrillary acidic protein (GFAP) was purchased from INCSTAR Corp. (Stillwater, MN), and the antibody for neural specific class III ß-Tubulin (TUJ-1) was purchased from Berkeley Antibody Co. (Richmond, CA). The Cy-3 conjugated rabbit antimouse secondary antibody was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Nineteen-day-pregnant Sprague Dawley rats were purchased from Charles River Laboratories, Inc. (Wilmington, MA). All animal procedures were conducted following National Institutes of Health guidelines for proper animal care and were approved by the University of Michigan Committee on Use and Care of Animals.

Astrocyte cultures
Primary astrocyte cell cultures were prepared using a method modified from Ruzicka et al. (23). All solutions were filter sterilized, and all work was carried out in a sterile environment. Briefly, 1- to 2-day-old rat pups were killed, and their brains were isolated. The brain stem and cerebellum were removed, and the forebrain and midbrain were placed in 10 ml solution A (0.91 mM Na2HPO4, 116.4 mM NaCl, 26.2 mM NaHCO3, 1.5 mM MgSO4, 14 mM D-glucose, 5.3 mM KCl, and 0.3% BSA) per 7 brains in a 10-cm2 tissue culture dish. Two hundred microliters of 1.5% trypsin (dissolved in water) was added to the 10 ml solution A, and the brains were incubated on a rotating platform shaker at 37 C for 30 min. Tissue was then transferred to a 50-ml conical tube containing 10 ml solution A with 70 µg DNase I (10 mg/ml stock prepared in water) and inverted 20 times. These tubes were then incubated on ice until the tissue settled. The supernatant was removed, and 4 ml solution B (solution A except final concentration of MgSO4 is 3.0 mM) with 80 µg DNase I and 4 mg Type I-S trypsin inhibitor (10 mg/ml stock prepared in water) was added to the tissue. Tissue was gently triturated 10 times with a 10-ml sterile disposable pipet. The undisrupted tissue was allowed to settle. The supernatant was then layered carefully onto 4 ml solution C (solution A with a final concentration of 3.0 mM MgSO4 and 4.0% BSA) and centrifuged at 100 x g for 10 min. The cell pellet was placed on ice. Another 4 ml solution B with DNase and trypsin inhibitor was added to the remaining undisrupted tissue, and trituration and centrifugation were repeated as above. The cell pellets were resuspended in 10 ml growth medium (see below) for determination of cell density using a hemacytometer.

Cells were seeded at 5 x 105 per 10-cm2 tissue culture dish in 5 ml growth medium (DMEM with D-valine) supplemented with 33 mM D-glucose, 1x antibiotic/antimycotic, and 10% FCS. One day after plating, the medium was aspirated and replaced with 10 ml cold growth medium. For maintenance, every third day, 5 ml medium was removed and replaced with 5 ml cold, fresh medium. Astrocytes were maintained in culture for 8–10 days before harvesting for RNA extractions. Astrocyte cultures were treated with various regulators, as described in the text. For all drug treatments, 5 ml medium was removed (from the total of 10 ml) and twice the desired final concentration was delivered to the cells in 5 ml growth medium for the final drug concentration, as reported in the text. This procedure ensured that astrocyte-conditioned medium was still present during the drug treatment. Regulator stocks were prepared as follows: Fsk, 10 mM in 100% ethanol; TPA, 1.6 mM in 100% ethanol; Dex, 2 mM in 100% ethanol; CRH, 0.1 mM in 1 mg/ml ascorbic acid, 10 mg/ml BSA, and 9 mg/ml sodium chloride.

At the time of drug treatments, the cultures were approximately 95% astrocytic in nature (Ref. 23 ; and McClennen, data not shown). We used immunocytochemistry for GFAP to confirm the astrocytic content of the cultures and for the neural specific class III ß-Tubulin (TUJ-1) to determine the neuronal content of the cultures. The procedure for GFAP staining is detailed by Ruzicka et al. (23). The immunocytochemistry, using TUJ-1 antibody, was conducted in the same manner, except that visualization of ß-Tubulin was carried out with fluorescence microscopy using a Cy-3-conjugated rabbit antimouse secondary antibody.

RNA isolation
Total RNA was isolated from the astrocyte cultures with Trizol reagent. After drug treatments, medium was removed from the plates, and 2 ml Trizol was aliquoted per dish, and the isolation was carried out according to the manufacturer’s instructions. Generally, Trizol suspension from 5 plates of cells was pooled and transferred to Falcon 2059 tubes and extracted with 2 ml chloroform. Tubes were tightly capped, shaken, and allowed to settle for 5 min, then centrifuged at 12,000 x g at 4 C for 15 min. The aqueous phase was transferred to a new 2059 tube and precipitated with 5 ml isopropanol at -20 C for an hour. Samples were centrifuged at 12,000 x g at 4 C for 15 min, and resulting RNA pellets were resuspended in 300 µl sterile water and reprecipitated with 6 µl 5 M NaCl and 600 µl 100% ethanol overnight. Samples were resuspended in 20 µl sterile water and used for subsequent experiments. Concentrations of RNA samples were determined by optical density at 260 nm.

RNase protection assays
RNase protection assays were carried out as described by McClennen et al. (22). The rat cyclophilin transcript was used as an internal positive control in all experiments. To determine steady-state levels of CRH-BP mRNA in these cultures after treatment with various regulators, the 565-bp PstI fragment of CRH-BP was used as the riboprobe template (22). This template is linearized with ScaI to produce a 252-base riboprobe, which protects 232 bases of exon 7 of the CRH-BP mature transcript. To compare levels of heteronuclear (hn) RNA vs. mature mRNA transcript, a 278-bp SacI/KpnI fragment of the rCRH-BP gene (from +66 bp in exon I (SacI site) to +172 bp of intron I (KpnI site) (18)) was cloned into pGEM-3Z vector (Promega Corp., Madison, WI) to produce plasmid pBPEx/In. This plasmid was linearized with EcoRI and transcribed with SP6 RNA polymerase (Epicentre Technologies, Madison, WI) to produce a 318-base complementary RNA (cRNA) template. This template (Ex/In) produced two major protected bands, at 278 bp and 106 bp, which represent the hnCRH-BP species and mature CRH-BP spliced transcript, respectively.

Data analysis
Gels were exposed to a Phosphorimager screen (Molecular Dynamics, Inc., Sunnyvale, CA) and Biomax MS film and intensifying screens (Eastman Kodak Co., Rochester, NY). Phosphorimager analysis was carried out using ImageQuant software (Molecular Dynamics, Inc.), and all quantitations were determined to be within the linear range of the Phosphorimager. RNase protection assays with the cyclophilin cRNA probe generate two protected fragments, of 84 and 85 bases, most likely caused by breathing of the hybrid; both bands were included in the quantitation. CRH-BP image densities were divided by cyclophilin densities to normalize for variations in RNA concentrations and recovery. The normalized values are presented as CRH-BP/cyclophilin mRNA ratio, relative to control, except in Fig. 4Go, where the data are presented as CRH-BP/cyclophilin mRNA ratio. All results are expressed as the mean ± SEM. The significance of differences was assessed by ANOVA test with Fisher’s least-significant difference (LSD) post hoc analysis using Statview software (Abacus Concepts, Berkeley, CA).



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  		Figure 4. CRH-BP gene expression is regulated in part by increased hnRNA and mRNA expression. A, Rat CRH-BP gene fragment used to generate the riboprobe spanning exon 1 and intron 1. The 318 base cRNA probe spans 106 bases of exon I, 172 bases of intron I, and contains 40 bases of linker sequence. The sizes of the protected hybrids are shown. The translation start codon is denoted by ATG. B shows a representative autoradiograph of an RNase protection assay using the gene-specific CRH-BP riboprobe. Time courses from 5–360 min of 10 µM Fsk or 100 nM TPA treatment shows the changes in CRH-BP RNA species with time. The top panel is a darker exposure than the rest of the figure to facilitate visualization of the nonabundant hnRNA species (278 base protected hybrid, arrow). The center and bottom panels are overnight Phosphorimager exposures of the gel and represent the exon-specific hybrid (labeled CRH-BP mRNA, 106 base major protected hybrid, top arrow; and minor hybrid, bottom arrow) and the internal control, cyclophilin (84–85 base protected hybrid, arrow), respectively. C is the quantitation of the representative experiment shown in B. These data are CRH-BP/cyclophilin ratios that have not been expressed relative to control due to the absence of detectable heteronuclear transcript under control conditions. The normalized levels of hnRNA species (speckled bars) are plotted on the left-hand y-axis and the normalized levels of mature CRH-BP mRNA species (solid bars) are plotted on the right-hand y-axis.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Primary rat astrocyte cultures express CRH-BP, making them a useful model system for studying regulation of CRH-BP gene expression. The primary astrocyte cultures used were previously determined to be 95% or more astrocytic in nature (23). Our cultures were derived from only the forebrain and midbrain regions, and we confirmed the astrocytic content of the cultures using immunocytochemistry for GFAP (data not shown).

Fsk and TPA positively regulate CRH-BP mRNA levels in rat astrocyte cultures
To examine the effects of intracellular second messengers on CRH-BP steady-state mRNA expression in these cultures, cells were treated for 6 h with 10 µM Fsk (an adenylate cyclase activator), 100 nM TPA (a PKC activator), or a combination of the two regulators. RNA was harvested, and RNase protection assays (22) were carried out using a 252-base CRH-BP riboprobe [which protects nucleotides 1039–1271 of the rat CRH-BP complementary DNA (cDNA) (18)]. This riboprobe was used in the experiments shown in Figs. 1–3GoGoGo. The effects of Fsk and TPA on CRH-BP steady-state mRNA expression are depicted in Fig. 1AGo. The low basal level of CRH-BP expression is shown in the control lane, which represents RNA isolated from untreated astrocytes. There is a dramatic increase in CRH-BP steady-state mRNA levels in astrocytes after Fsk, TPA, or Fsk/TPA treatment, as shown by the significant increases in protected hybrids in the top panel of Fig. 1AGo. The protected hybrid of the internal control, rat cyclophilin, is depicted in the bottom panel of this figure. To properly visualize the control CRH-BP hybrid, the rest of the hybrids are overexposed in the autoradiograph. However, quantitations were performed within the sensitivity range of the Phosphorimager. A figure comparing the migration pattern of the full-length CRH-BP and cyclophilin probes compared with the protected hybrids can be found in McClennen et al., 1998 (22).



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  		Figure 1. Fsk and TPA positively regulate CRH-BP steady-state mRNA levels in primary astrocyte cultures. A, Representative autoradiograph from an RNase protection assay depicting 6 h treatments of cells with Fsk (10 µM), TPA (100 nM), or both Fsk and TPA. Control is defined as CRH-BP expression in untreated astrocyte cultures. CRH-BP protected hybrids are in the top set of panels and cyclophilin internal control hybrids are in the bottom set of panels. B, Dose-response curve of CRH-BP gene expression after 6 h treatment with Fsk (black bars) or TPA (open bars). All concentrations represent an n = 3 or more. Error bars for 1.0 nM TPA are insignificant on this scale. *, P < 0.05, compared with control. C, Time course of CRH-BP gene expression with 10 µM Fsk (black bars), 100 nM TPA (open bars), or 10 µM Fsk/100 nM TPA (gray bars) treatment. Data for B and C are presented as CRH-BP/cyclophilin density ratio relative to control. *, P < 0.05, compared with control. Values for 1, 3, 6, and 48 h time points represent mean ± SEM for n = 3 or more while the 18 and 24 h time points represent the values from one or two independent experiments. Error bars for Fsk and TPA 48 h time points are insignificant on this scale.

 


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  		Figure 2. Dex negatively regulates Fsk- and TPA-induced CRH-BP mRNA levels. The inset in A is a representative RNase protection assay autoradiograph showing the effect of Dex on levels of CRH-BP mRNA after Fsk and/or TPA treatment. The bands in the inset are over-exposed to facilitate visualization, but all quantitations were done within the linear range of the Phosphorimager. The histogram presents the quantitation of several independent experiments. F = 10 µM Fsk (black bars), T = 100 nM TPA (open bars), FT = Fsk/TPA (gray bars), D = 1 µM Dex (hatched bars) and all drug treatments were for 6 h. *, P < 0.05, compared with control; #, P < 0.05, compared with Fsk, TPA, or Fsk/TPA-treated sample. Values for all experiments represent mean ± SEM for n = 3 or more. Error bars for F/D, T/D, and FT/D are insignificant on this scale. B shows the dose-response curve of CRH-BP gene expression after 6 h treatments with increasing concentrations of Dex (hatched) in the presence of Fsk (10 µM, black bars) or TPA (100 nM, open bars). *, P < 0.05, compared with Fsk or TPA treated sample; n = 3 or more for all data except Fsk/Dex 0.1 and 1.0 nM where n = 2.

 


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  		Figure 3. CRH positively regulates CRH-BP mRNA levels and Dex negatively regulates CRH-induced CRH-BP mRNA levels. The inset is a representative autoradiograph from an RNase protection assay showing the CRH and CRH/Dex effects on CRH-BP mRNA levels after a 6-h treatment. The histogram depicts the quantitation of several RNase protection assays showing the time course of CRH treatment in the absence or presence of Dex on CRH-BP gene expression. Cells were treated with 20 nM CRH (open bars) or 20 nM CRH/1 µM Dex (hatched bars) for 6, 18, or 48 h. *, P < 0.05, compared with control; #, P < 0.05, compared with 6 h CRH treatment.

 
The doses of Fsk or TPA required for maximal CRH-BP gene expression in these cells were determined by treating astrocyte cultures with increasing concentrations of either drug. The results are shown in Fig. 1BGo. The dose that achieved the maximal Fsk response was 10 µM. For TPA response, the maximal CRH-BP mRNA induction was obtained at 100 nM. For all subsequent experiments, 10 µM Fsk and 100 nM TPA were used individually and in combination, as described in the text.

The time course of steady-state CRH-BP mRNA expression was also examined. Cells were treated with 10 µM Fsk, 100 nM TPA, or both regulators for various times: 1, 3, 6, 18, 24, or 48 h before harvest. The results of these experiments are summarized in Fig. 1CGo. Fsk increased steady-state CRH-BP mRNA levels detectably at 3 h (15.9 ± 6.1 times untreated levels; n = 3; P = 0.15) to a maximum of 32.4 ± 10.3 times the level of control astrocytes at 6 h of drug treatment [n = 6, P = 0.0009 (compared with control)]. The levels decreased to 2.9 times control levels by 48 h after treatment. TPA also increased steady-state CRH-BP mRNA levels to 28.5 ± 7.9 times control levels after 6 h of drug treatment [n = 4, P = 0.0008 (compared with control)]. By 48 h post treatment, mRNA expression had returned to 1.7 times control levels (n = 6). Interestingly, Fsk and TPA act synergistically on CRH-BP mRNA expression and increase expression in these cells up to 100 ± 12.6 times control levels at 6 h [n = 4, P < 0.0001 (compared with control)], with a slight sustained increase (9.6 ± 3.5) over basal at 48 h after treatment.

Dex negatively regulates Fsk- or TPA-induced CRH-BP mRNA levels in astrocyte cultures
Cells were also treated with the synthetic glucocorticoid, Dex, either alone or in combination with 10 µM Fsk and 100 nM TPA. Preliminary studies examined the effect of Dex on basal expression of CRH-BP at 6 and 48 h. No significant increase in CRH-BP mRNA levels was observed. However, the low basal levels of CRH-BP transcripts made it difficult to determine whether CRH-BP levels were unaltered or decreased by the addition of Dex. We therefore focused on examining the effects of Dex on CRH-BP gene expression in combination with Fsk and TPA. Dex could significantly repress the increase in CRH-BP mRNA induced by Fsk, TPA, or the combination of Fsk/TPA, as shown in Fig. 2Go, A and B. The inset in Fig. 2AGo shows a representative RNase protection autoradiograph, with CRH-BP steady-state mRNA hybrids in the top panel and cyclophilin hybrids in the bottom panel. The quantitation of several experiments is shown in the histogram in Fig. 2AGo. Dex (1 µM, 6-h treatment) decreased Fsk-induced expression of CRH-BP from 32.4 ± 11.2 times control to 4.9 ± 0.8 times control [n = 4, P = 0.0027 (compared with Fsk treatment)]. TPA-induced CRH-BP expression was reduced from 30.5 ± 4.7 to 7.9 ± 1.0 times control [n = 4, P = 0.0113 (compared with TPA treatment)]. Similarly, Fsk/TPA-induced CRH-BP mRNA levels were decreased from 94.1 ± 13.2 to 18.7 ± 3.1 times control [n = 3, P < 0.0001 (compared with Fsk/TPA treatment)] by cotreatment with 1 µM Dex for 6 h.

To determine the dose dependence of Dex for repression of Fsk- or TPA-induced CRH-BP gene expression, various concentrations of Dex, from 0.1–100 nM, were administered with constant doses of Fsk (10 µM) or TPA (100 nM) for 6 h. Maximal inhibition of CRH-BP gene expression was observed at 10 nM Dex (Fig. 2BGo). A 6-h coincubation with 10 nM Dex reduced Fsk-induced levels of CRH-BP mRNA from 33.7 ± 8.5 times control level to 8.7 ± 3.3 times control level (n = 5, P = 0.0015). A 6-h coincubation with 10 nM Dex reduced TPA-induced levels of CRH-BP mRNA from 25.6 ± 2.4 times control levels to 8.8 ± 1.0 times control levels (n = 4, P = 0.019). A maximal repression at 10 nM Dex is consistent with a GR-mediated effect (24).

CRH positively regulates CRH-BP mRNA levels and Dex negatively regulates CRH-induced CRH-BP mRNA levels
Previous studies have demonstrated the expression of CRH receptors in astrocyte cultures (25). Because CRH receptors are coupled to a stimulatory G protein, thereby increasing intracellular cAMP levels, and Fsk has been shown to increase CRH-BP mRNA expression, CRH would be expected to increase endogenous CRH-BP mRNA levels. The cells were treated with 20 nM CRH for 6, 18, or 48 h. Maximal induction of steady-state CRH-BP mRNA was observed at 6 h, as depicted in Fig. 3Go. Gene expression at 6 h was 6.1 ± 2.9 times control levels [n = 5, P = 0.026 (compared with control)], but CRH-BP expression had returned to control levels by 18 h. To determine whether this increase in CRH could be repressed by Dex, cells were treated with 20 nM CRH and 1 µM Dex simultaneously for 6, 18, and 48 h. The only significant decrease in CRH-induced CRH-BP gene expression occurred at 6 h, where CRH-BP expression was decreased to 1.3 ± 0.7 times basal levels [n = 4, P = 0.043 (compared with CRH at 6 h)]. The inset in Fig. 3Go is a representative autoradiograph showing the CRH induction and Dex repression of CRH-induced CRH-BP mRNA levels, compared with control, after 6 h of treatment.

The regulation of CRH-BP gene expression is caused, in part, by increased transcription
RNase protection assays are powerful in their ability to detect low levels of nonabundant transcripts. However, as normally used, the assay reflects changes in steady-state mRNA levels that could represent changes in transcription or mRNA stability. To begin to examine whether the increases in steady-state CRH-BP mRNA levels after Fsk and TPA treatment were caused by increased gene transcription or increased mRNA stability, a riboprobe was designed that is specific for the exon I/intron I junction of the CRH-BP gene, as depicted in Fig. 4AGo. The 318-base cRNA probe spans 106 bases of exon I and 172 bases of intron I, and it contains 40 bases of linker sequence. Increases in CRH-BP transcription would show increasing levels of hnRNA transcripts (a 278-base hybrid including both exon I and intron I), whereas increases in mRNA stability would show accumulation of the 106-base mRNA hybrid in the absence of the 278-base hnRNA hybrid during the time course.

Astrocyte cultures were treated with Fsk or TPA for intervals from 5–360 min, and RNA was harvested for RNase protection assays. Figure 4BGo shows a representative RNase protection assay showing the changes in RNA species over time. Fsk-treated samples are on the left and TPA-treated samples are on the right. By 30 min after initiation of drug treatment, there is a visible increase in Fsk-induced CRH-BP hnRNA, which increases until 60 min of treatment and then disappears. The decrease in hnRNA at 180 min corresponds with the dramatic increase in mature CRH-BP transcripts shown in the middle panel, suggesting that splicing of the intron sequence has occurred. For TPA-treated samples, the CRH-BP hnRNA transcripts reach a peak by 45–60 min after addition of TPA and disappear as the mature transcript increases at 3 and 6 h of drug treatment. The exon-specific fragment of the riboprobe protects a doublet with the 106-base protected hybrid as the major species. Both bands were used in the quantitation. Although the hybrids shown in the middle panel are specific only for exon I, this time course of increased gene expression corresponds directly with the results observed when using the 252-base riboprobe (protects nucleotides 1039–1271 of the rat CRH-BP cDNA), which was used in all previous experiments (data not shown). Figure 4CGo is the quantitation from the experiment shown in Fig. 4BGo, demonstrating the change in RNA species from hnRNA to mRNA during the time course examined. The experiment was repeated three times with consistent results. Data are presented as absolute CRH-BP/cyclophilin ratios (not relative to control) because of the differences in abundance of the transcripts and absence of detectable hnRNA species in the control lanes.

The same time course of CRH-BP hnRNA vs. mature mRNA species was conducted with Fsk/Dex- and TPA/Dex-treated astrocytes. The Fsk vs. Fsk/Dex profile and quantitation are shown in Fig. 5Go. The time course profiles for the two treatments are similar, except that the Fsk/Dex-treated samples have decreased intensity of both the CRH-BP hnRNA and CRH-BP mRNA hybrids. The results for TPA vs. TPA/Dex were comparable (data not shown). These results demonstrate that the repression of Fsk- or TPA-induced CRH-BP expression by Dex is consistent with a decrease in gene transcription. However, although changes in hnRNA levels are usually thought to reflect altered transcription, similar results could be obtained by changes in hnRNA stability.



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  		Figure 5. Dex decreases CRH-BP hnRNA and mRNA expression. A is a representative autoradiograph of RNase protection assays using the exon/intron specific riboprobe. Cells were treated with 10 µM Fsk or 10 µM Fsk/100 nM Dex for the time course shown in the figure. The top panel is a darker Phosphorimager exposure to facilitate visualization of the nonabundant hnRNA band. B is the quantitation for the representative autoradiograph shown in A. Data are presented as in Fig. 4CGo. This experiment was repeated with consistent results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Studies examining the regulation of endogenous CRH-BP gene expression have been limited to date. To begin to dissect the mechanisms involved in CRH-BP gene regulation, we have characterized the transcriptional regulation of CRH-BP gene expression in rat primary astrocyte cultures. These cells express low, but detectable, levels of CRH-BP mRNA in the untreated state, with dramatic increases in CRH-BP expression after treatment with a variety of drugs that activate known intracellular signaling pathways.

Astrocyte cultures were first treated with Fsk, a well-characterized activator of adenylate cyclase, which increases cAMP levels and signaling through the PKA pathway. A dramatic increase in both CRH-BP hnRNA and CRH-BP steady-state mRNA levels was observed with Fsk treatment, consistent with increases in CRH-BP transcription. Previous transfection studies in our lab showed an increase in rat CRH-BP promoter activity after Fsk treatment. This increase was mediated, in large part, via the cAMP-responsive element (CRE) at -127 bp upstream of the rat CRH-BP transcription start site (18). The CREB/ATF family of transcription factors binds to this sequence to mediate increased transcription. It is most likely that the CRE sequence in the CRH-BP promoter and the family of CREB/ATF transcription factors are also responsible for the increased transcription of CRH-BP in Fsk-treated astrocytes. Our data are also consistent with other studies completed in primary rat astrocyte cultures, which showed increased steady-state CRH-BP mRNA levels with Fsk/IBMX treatment, as detected by in situ hybridization experiments (19), as well as increased secretion of CRH-BP in response to Fsk (20).

On a more physiological level, this study is the first to demonstrate that CRH increases endogenous CRH-BP gene expression. Astrocytes are known to have CRH receptors (25); and because CRH receptors are coupled to a stimulatory G protein and increase intracellular cAMP levels, it is not surprising that CRH and Fsk both mediate increases in CRH-BP gene expression. The difference in the levels of induction by Fsk and CRH is most likely attributable to the limiting number of CRH receptors present on the surface of the astrocytes. Transfection experiments with CRH-BP reporter constructs demonstrated CRH-mediated increases in CRH-BP promoter activity through CRH-R1 (18). The CRH effect was mediated, in large part, through the CRE in the CRH-BP promoter. Together, these results suggest that the increase in CRH-BP transcription, observed in astrocyte cultures after Fsk or CRH treatment, is mediated via the CRE in the CRH-BP promoter. The positive regulation of CRH-BP gene expression by CRH could also have implications for feedback control on CRH activity in astrocytes.

Regulation of CRH-BP gene expression by TPA, a known PKC activator, was also examined. In response to TPA, CRH-BP hnRNA and steady-state mRNA levels were increased to a level similar to that observed with Fsk. It is possible that this transcriptional effect is mediated via the putative AP-1 sites that have been identified in the promoter of the rat CRH-BP gene (18). Consistent with our data, a study done in primary astrocyte cultures showed an increase in secretion of CRH-BP in response to TPA (20).

These data suggest that both the PKA and PKC signaling pathways contribute to increased CRH-BP gene expression in astrocyte cultures. Similar results have been demonstrated in an immortalized amygdalar neuronal cell line. Studies in this neuronal cell line show increases in CRH-BP steady-state mRNA levels of 75% and 35% in response to Fsk and TPA, respectively, but do not examine the effects of Fsk and TPA combined (21). In our astrocyte cultures, a synergistic effect is observed when both Fsk and TPA are used to treat the cells (up to 100 times control levels of steady-state CRH-BP mRNA are observed). The synergistic increases in CRH-BP expression seen with Fsk/TPA in these astrocyte cultures suggest that the effects of these regulators may be mediated via concerted action of transcription factors at nearby elements in the CRH-BP promoter, which act cooperatively to further activate transcription of the CRH-BP gene. It should be noted, however, that in previous studies performed with primary astrocyte cultures (19), the increases in CRH-BP protein secretion in response to Fsk and TPA do not seem to be synergistic.

Our results further demonstrate that glucocorticoids repress CRH-BP transcription in astrocyte cultures when the cells are treated simultaneously with Dex and Fsk, TPA, or CRH. Consistent with our data, Maciejewski et al. (20) show decreased levels of CRH-BP secretion when primary astrocyte cultures are treated with Dex and TPA. In contrast, Mulchahey et al. (21) show significant increases in steady-state CRH-BP mRNA levels with Dex treatment in their immortalized amygdalar cell line. Our group has also shown dramatic decreases in steady-state CRH-BP mRNA by adrenalectomy in rat pituitary (22), suggesting positive glucocorticoid regulation of pituitary CRH-BP gene expression. Together, these data demonstrate that CRH-BP regulation by glucocorticoids is cell-type-specific. The glucocorticoid regulation of a variety of other genes, including the CRH gene, has also been shown to be cell-type-specific (24).

There are several mechanisms by which glucocorticoids are known to alter transcription. The ligand-activated glucocorticoid receptor (GR) can bind to glucocorticoid response elements (GREs), either positive or negative, in the promoter region of the glucocorticoid-responsive gene to mediate increased or decreased transcription of the gene. Alternatively, the activated GR can interact via protein-protein interactions with other transcription factors (such as AP-1, NF{kappa}B, and CREB) to mediate repression of gene transcription (26). An examination of the first 600 bases upstream of the transcription start site in the CRH-BP promoter shows no putative GREs, but GRE sequences may be present further upstream or in intronic sequences. However, the rat CRH-BP promoter contains CREB, AP-1, and NF{kappa}B binding sites, suggesting that the negative glucocorticoid regulation of CRH-BP gene expression seen in astrocyte cultures may be mediated via GR interaction with one or more of these transcription factors. CREB and GR have previously been proposed to interact directly or indirectly in the negative glucocorticoid regulation of CRH-reporter activity in AtT-20 cells (24).

The presence of endogenously expressed CRH-BP in primary astrocyte cultures suggests an interesting new role for CRH-BP. Primary astrocyte cells are one of only a few cell types expressing both CRH-BP and CRH receptor. It is known that glial cells are present in significant numbers around the termini of both pre- and postsynaptic neurons. One potential role for CRH-BP in astrocytes is to control the amount of free CRH that is present at the synapse. When a signal stimulates CRH release from the presynaptic neuron, it is possible that excess CRH not bound by CRH receptors on the postsynaptic neuron will bind to the CRH receptors on the glial cells. CRH binding will stimulate transcription of the CRH-BP gene and CRH-BP release from the astrocytes. The secreted CRH-BP then binds and sequesters excess CRH to decrease the neuronal CRH signal. The fate of the CRH-BP/CRH complex, once binding has occurred, is not known. It is possible that the interaction is transient and that CRH is released from CRH-BP in its bioactive state at a later time, although it seems more likely that the CRH-BP/CRH complex targets CRH for degradation (27). Finally, it should be added that the CRH-BP also binds urocortin, a 40-amino acid CRH-like peptide (28). Thus, the CRH-BP may be important for binding not only CRH but also urocortin and other potential CRH-like ligands.

The results presented in this paper are the first to clearly demonstrate increased transcription of the endogenous CRH-BP gene by activation of the PKA and PKC signaling pathways. In addition, we have shown that CRH increases CRH-BP mRNA levels in astrocyte cultures, suggesting intricate feedback mechanisms. Finally, we demonstrate negative transcriptional regulation of CRH-BP gene expression by glucocorticoids in astrocyte cultures, suggesting differential glucocorticoid regulation of CRH-BP gene expression in different cell types. Together, these results demonstrate that extracellular signals, such as CRH and intracellular signals from the adrenal steroid hormone, PKA, and PKC signaling pathways, are all involved in regulation of the CRH-BP gene expression in astrocyte cultures. These same extracellular and intracellular signaling pathways may play important regulatory roles in CRH-BP gene expression in the brain and pituitary. Increased understanding of the regulation of the CRH-BP may provide new insights into the functions of the CRH-BP and its potential role in disorders associated with aberrant regulation or activity of CRH.


   Acknowledgments
 
The authors would like to thank Dr. David Turner for providing us with the TUJ-1 antibody and Dr. John Kasckow for sharing results before publication.


   Footnotes
 
1 This work is supported by National Institutes of Health Grant DK-42730 (to A.F.S) and a Young Investigator Award (to A.F.S.) from National Alliance for Research on Schizophrenia and Depression. Back

Received January 6, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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