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Endocrinology Vol. 140, No. 1 251-259
Copyright © 1999 by The Endocrine Society

ARTICLES

Coordinate and Divergent Regulation of Corticotropin-Releasing Factor (CRF) and CRF-Binding Protein Expression in an Immortalized Amygdalar Neuronal Cell Line1

J. J. Mulchahey, A. Regmi, S. Sheriff, A. Balasubramaniam and J. W. Kasckow2

Department of Psychiatry (J.J.M., A.R., J.W.K.) and Department of Surgery (S.S., A.B.), University of Cincinnati College of Medicine, and University of Cincinnati Neuroscience Program (A.B., J.W.K.), Cincinnati, Ohio 45267; and Cincinnati Veteran’s Affairs Medical Center (J.W.K.), Psychiatry Service, Cincinnati, Ohio 45220

Address all correspondence and requests for reprints to: Jeff Mulchahey, Ph.D., Department of Psychiatry, University of Cincinnati, College of Medicine, 231 Bethesda Avenue, P.O. Box 670559, Cincinnati, Ohio 45267-0559. E-mail: mulchajj{at}email.uc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CRF is a 41-amino acid neuropeptide best known for its hypophysiotropic actions. CRF is widely distributed in the central nervous system in areas beyond the hypothalamus. CRF-binding protein (CRF-BP) regulates the bioavailability of CRF, and knowledge of the regulation of CRF-BP synthesis is an integral component of understanding the actions of CRF. To better study the regulation of CRF and CRF-BP, primary amygdalar cultures were immortalized by transfection with the SV 40 large T antigen. A clonal line that expresses CRF immunoreactivity and messenger RNA was selected. The production of CRF peptide and message by this line is regulated in a manner indistinguishable from primary cultures. We also observed that the immortalized cells express CRF-BP immunoreactivity and messenger RNA. The expression of both CRF and CRF-BP is positively regulated by forskolin and interleukin-6. Unlike CRF, the expression of CRF-BP message and peptide was increased by phorbol 12-myristate 13-acetate or dexamethasone. These results demonstrate that the synthesis of CRF and CRF-BP in this clonal cell line may be regulated in parallel by some agents but not by others. These data also suggest that dexamethasone may decrease the biological availability of CRF in the amygdala by increasing the expression of CRF-BP, rather than by decreasing CRF expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CRF IS A 41-amino acid neuropeptide, the structure and function of which were first elucidated in the context of the hypophysiotropic regulation of ACTH secretion from the anterior pituitary (1, 2, 3, 4). The hypothalamo-pituitary-adrenal axis is activated by stress, and it mediates the metabolic responses to stress (5, 6, 7). However, it seems that extrahypothalamic structures, the amygdala in particular, play a significant role in mediating behavioral manifestations of stress (8, 9). CRF is localized in high concentrations in the central nucleus of the amygdala (8, 9). Central administration of exogenous CRF mimics (5), and CRF antagonists ameliorate (10), the behavioral effects of stress, suggesting that the relationship between dysregulation of CRF and behavioral effects is a causal one. Furthermore, local delivery of CRF or CRF antagonists to the amygdala, which presumably restricts their site of action to the amygdala, produces behavioral effects similar to those seen with intracerebroventricular delivery (10, 12). This suggests that the behaviorally relevant site of action for CRF is likely to be the amygdala.

The biological actions of CRF may be attenuated by CRF-binding protein (CRF-BP; Refs. 13, 14, 15). This 37-kDa protein was first isolated from human plasma by CRF affinity chromatography (13) and was subsequently sequenced and cloned (14). CRF-BP has been localized to corticotropic cells of the anterior pituitary, where it is thought to play a paracrine role in the regulation of ACTH secretion by binding to CRF and inhibiting its ACTH-releasing actions (15). CRF-BP is widely distributed in the brain, where it is believed to modulate activation of CRF receptors by limiting the availability of CRF. The distribution of CRF-BP has been mapped, using immunohistochemistry and in situ hybridization, and has been detected in the amygdala (15). Despite extensive mapping studies in several species, relatively little is known about the cellular regulation of CRF-BP expression. It seems that activators of both protein kinase A (PKA) and C (PKC) pathways will increase the expression of CRF-BP in mixed neuronal, as well as pure astrocyte cultures (16, 17). Although the amygdala is one of several brain regions expressing both CRF and CRF-BP, and the two proteins have been colocalized in the central nucleus of the amygdala at the cellular level (8, 9, 15), we are unaware of studies exploring the coordinated regulation of these two species in a single tissue or cell type.

We have recently characterized some of the responses of amygdalar CRF neurons to several stimuli. Using primary cultures of dispersed amygdalar neurons, we observed that, like hypothalamic CRF neurons, CRF in amygdalar neurons is positively regulated by cytokines and forskolin (18). Unlike hypothalamic CRF neurons (19, 20), however, primary amygdalar CRF neurons seem to be refractory to the negative feedback effects of glucocorticoids (18). The use of primary cultures of amygdalar neurons presents several challenges. These include the limited amount of tissue available from embryonic donor aminals, the protracted amount of time required in culture before experimental use, and the heterogeneous nature of primary cultures. As a result, primary neuronal cultures are of limited utility in studies of the regulation of amygdalar CRF expression. We therefore set out to establish an immortalized amygdalar CRF neuronal cell line that facilitates such studies. This report describes the generation of such a cell line and extends our observations of the regulation of CRF expression in the amygdala to include regulation of CRF-BP expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Immortalization of amygdalar cells
Cultures of amygdalar cells were established with day 19 Sprague Dawley rat embryos as tissue donors. This procedure was approved by the University of Cincinnati institutional review board and has been used by our group for studies of primary neuronal cultures. This procedure is described in detail in Kasckow et al. (18). Briefly, the amygdala was dissected from the embryonic brain and then dissociated in trypsin and deoxynuclease I. Cells were maintained in culture in DMEM/Ham F-12 (1:1) supplemented with 14 mM glucose, 15 mM NaHCO3, 5 mM HEPES, 0.05 U/ml penicillin-steptomycin, and 7.5% FBS (Atlanta Biologicals, Norcross, GA). Cells were grown on gelatin- and polyornithine-coated plastic culture dishes for 3 days before viral transformation.

Virus producer cell line {Psi}CRE/pZIPTEX (provided by Dr. J. Jacobberger, Case Western Reserve, Cleveland, OH) was propagated at 1 x 106 cells per 100-mm culture dish in the same medium used for the amygdalar cultures. Infection of the primary cultures was performed as described previously (21), with minor modifications. Viral culture supernatant was mixed with polybrene (final concentration of 4 µg/ml) and 2 x 106 amygdalar cells. This mixture was incubated with gentle agitation for 6 h at 37 C. At the time of infection, the virus producer line had been propagated for 3 days. Thirty hours after infection, the cells were treated with 0.8 µg/ml geneticin (G418, Sigma Chemical Co., St. Louis, MO) to select for transformed cells. Surviving colonies were cloned by limiting dilution in 96-well culture dishes. Wells initially containing single cells were propagated and replated in 6-well plates. Clones were selected for subsequent use, on the basis of CRF peptide release into the culture supernatant, which was monitored by RIA (described below).

Cells were passaged by removing adherent cells from the substrate with 0.025% trypsin and were split at a ratio of 10:1. The immortalized cells displayed a doubling time of approximately 2 days. Cells from generations 5–7 were used in our initial characterization. Cells from generations 19–22 were also used to determine the persistence of the phenotype and to characterize their responses to various stimuli.

Stimulation experiments
A single clone was selected for further study, and cells from this clone were plated at a density of 2 x 106 cells per well in 6-well plates (35-mm wells; Corning Costar, Cambridge, MA). Cells to be used in CRF gene expression studies were plated into 100-mm tissue culture plates (Falcon, Becton, Dickinson and Co., Cockeysville, MD). Cells were washed in serum-free media (ß-pit Julip containing 0.1% BSA; Sigma Chemical Co.), and test substances were added as described in Kasckow et al. (18). Our initial experiments sought to compare the responses of the immortalized cells with our results from primary amygdalar cultures. Cells were incubated with the following test substances and concentrations: forskolin (3, 10, 30 µM; Sigma Chemical Co.), phorbol 12 myristate 13-acetate (TPA; 1, 20, 50 nM; Sigma Chemical Co.), interleukin-6 (IL-6; 10, 50, 100 pM; Promega Corp., Woods Hollow, WI), and dex (10, 50, 100 nM; Sigma Chemical Co.). Exposure times of 6, 12, and 24 h were examined by Northern analysis for CRF message expression (total RNA extraction and Northern procedure, described below), and times of 12 and 24 h were examined by RIA for CRF peptide production. Culture medium was removed and frozen at -20 C before CRF RIA. Intracellular CRF content was measured after cell lysis in 0.1% NP-40 in 0.75 ml of incubation medium; samples were frozen and assayed, as for culture supernatants.

CRF and CRF-BP messenger RNA (mRNA) detection by Northern hybridization
Total RNA was isolated using the PUREscript RNA isolation kit (Gentra Systems, Minneapolis, MN), following the manufacturer’s instructions. Twenty micrograms of total RNA per lane was electrophoresed in an agarose-formaldehyde gel (1.2%/2.2 M) for 2 h at 80 V. RNA was transferred to a Hybond-N nylon membrane overnight at 0.25 mA at 2 C and was fixed to the membrane using a Stratalinker 1800 UV cross-linker and was prehybridized in ExpressHyb hybridization solution (CLONTECH Laboratories, Inc., Palo Alto, CA) for 30 min at 65 C. The membrane was hybridized with 1 x 106 cpm/ml of labeled probe in ExpressHyb solution for 1 h at 65 C. A 32P-labeled DNA probe was generated, which contains the rat CRF exon, using a PrimeIt-II random oligonucleotide priming kit (Stratagene, La Jolla, CA) and a pGEM3Zbam761 construct containing a 761-bp BamHI portion of the rat CRF exon II (provided by Dr. A. Seasholtz, University of Michigan, Ann Arbor, MI). The CRF-BP probe was generated similarly using a 500-bp PstI fragment from a pBluescript SK construct provided by Dr. W. Vale (Salk Institute, La Jolla, CA). Membranes probed for CRF were washed at 65 C for 2 x 15 min in 0.5% SDS in 10 mM disodium phosphate monhydrate (buffer 1), followed by 5 min at 65 C in buffer 1 containing 0.05% SDS. Membranes probed for CRF-BP were washed at 65 C for 2 x 5 min in buffer 1. After washing, the membrane was exposed to Xomatic film (Kodak, Rochester, NY) for 24–48 h and then developed. A Foto/Eclipse Imager (Fotodyne, Inc.; Hartland, WI), interfaced with a Power Macintosh 8100/110 using NIH ImageQuant analysis software, was used for image capture and analysis of hybridization signals. CRF and CRF-BP expression studies were performed in independent experi-ments.

CRF immunocytochemistry
Cells were grown in 8-well Lab-Tek II Chamber Slide Systems (Nalge Nunc International, Naperville, IL) and fixed, using 4% paraformaldehyde (pH 7.4), as described previously (18). Cells were incubated overnight at room temperature in a 1:2000 dilution of rabbit polyclonal anti-CRF antiserum (rc70, Dr. W. Vale) in PBS containing 0.2% Triton X-100. The slides were then incubated with a Vectastain ABC antirabbit reagent kit (Vector Laboratories, Inc., Burlingame, CA) for 2 h at room temperature, following the manufacturer’s instructions. The chromagenic substrate was diaminobenzidine (Vector) applied for 20 min at room temperature. The cells were also incubated in a mouse anti-GFAP preparation (1:100; Novocastra Laboratories, Newcastle upon Tyne, UK) followed by a Vectastain ABC antimouse reagent kit (Vector) and diaminobenzidine. Alternately, the cells were also incubated in 2 µg/ml antineuron specific microtubule-associated protein-2 (MAP2) monoclonal antibody (Boerhinger Mannheim, Indianapolis, IN) overnight at 4 C. The slides were then incubated with a Histomouse-SP kit (Zymed Laboratories, San Francisco, CA) according to the manufacturer’s instructions. The chromagenic substrate was aminoethylcarbazole, applied for 3 min. Slides were washed, coverslipped, and photographed using an Ortholux microscope from Leitz (Rockleigh, NJ) and a Wild MPS52 camera from Leica Corp. (Heerbrugg, Switzerland).

CRF-BP determination by Western blot
Cells were lysed in 25 mM Tris buffer (pH 8.8) containing 192 mM glycine, 0.1% SDS, and 0.5 mM phenylmethylsulfonyl fluoride. Total protein (25 ug) was electrophoresed in 10% acrylamide at 100 V for 3 h. Protein was transferred to a nitrocellulose membrane (Amersham, Buckinghamshire, UK) overnight at 2 C and 25 V. The membrane was incubated with rabbit antihuman CRF-BP (1:10,000; provided by Dr. W. Vale) overnight at 4 C. After washing, the membrane was incubated with goat antirabbit antibody (1:1,000; Vector). The membrane was washed at room temperature, and the immunoreactive protein bands were detected using the enhanced chemiluminescence ECL kit (Amersham). The membrane was exposed to Xomatic film (Kodak) for 2–60 sec and then developed. A Foto/Eclipse Imager (Fotodyne, Inc.), interfaced with a Power Macintosh 8100/110 using NIH ImageQuant analysis software, was used for densitometric analysis of band intensity. Rainbow markers (Gibco BRL, Grand Island, NY) were used to estimate protein size. Recombinant CRF-BP (25 pg; provided by Dr. W. Vale) served as a positive control.

CRF RIA
CRF RIA was performed using reagents and protocols from IgG Corp (Nashville, TN). The primary antiserum was rabbit anti-human CRF, diluted 1:100 in buffer (63 mM Na2HPO4 (pH 7.4); 13 mM EDTA, 3 mM sodium azide, 0.1% Triton X-100, and 250 kIU/ml aprotinin). One hundred microliters of culture supernatant or cell lysate were incubated with primary antiserum for 3 days at 4 C. One hundred microliters of [125I]-0Tyr-CRF (DuPont NEN; Wilmington, DE), containing 1000 cpm, was then added, and the samples were incubated for 2 days at 4 C. After this, goat antirabbit {gamma} globulin was added; and after 4 h, the samples were pelleted by centrifugation. Pellets were counted using a Packard Multi Prias 4 {gamma} counter (Packard Instruments, Downers Grove, IL). The CRF RIA exhibited an ED90 of 1.66 ± 0.13 pg/100 µl, with an interassay coefficient of variation of 2.91%.

Statistical analysis
Data are expressed in this report as mean ± SE of three independent experiments, unless indicated otherwise. Data were analyzed using ANOVA, followed by Duncan’s new multiple-range test. Differences were considered to be statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunohistochemistry
Our initial selection criteria for immortalized amygdalar CRF neurons was that a clone express both CRF peptide and neuron-specific MAP2. Figure 1Go illustrates the results of immunostaining for the clone, identified as AR-5, ultimately selected for further study. Figure 1AGo illustrates that these cells contain immunoreactive CRF, whereas Fig. 1CGo demonstrates that the cells are also MAP2 positive. Cells stained with anti-CRF antiserum displayed a diffuse brown reaction product throughout the cytoplasm with nuclear sparing. Figure 1Go, B and D, displays specificity controls for the immunostaining, in that, omission of the anti-CRF or anti-MAP2 antiserum, respectively, eliminated the immunohistochemical reaction product. Immunostaining for GFAP was negative (data not shown).



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  		Figure 1. Detection of CRF-like and MAP2-like immunoreactivities by immunohistochemistry. These brightfield photomicrographs illustrate the morphology of immortalized amygdalar neurons in culture. A, Cells immunostained for CRF with a diffuse reaction product distributed throughout the cytoplasm; B, a negative control for CRF immunostaining, in which nonimmune rabbit serum has been substituted for anti-CRF serum; C, a cohort culture immunostained for MAP2; D, a corresponding negative control for the mouse immunoreagents. Scale bar = 50 µm.

 
CRF transcript size
We next determined whether the AR-5 clone expresses a CRF message of the appropriate size. The CRF gene transcript size appeared to be a single product of a size (1.3 kb; data not shown) consistent with that reported for CRF in other rat neuronal systems (22).

Effects of forskolin, IL-6, TPA or dex on CRF gene expression
We compared the forskolin and IL-6 concentration- and time course- responses of immortalized amygdalar neurons to those we determined for primary cultures of amygdalar neurons. The results for forskolin stimulation of CRF expression are shown in Fig. 2AGo. The insert displays representative Northern analyzes of CRF expression after 6 h treatment with various concentrations of forskolin. Forskolin induced concentration dependent increases in CRF message expression over the range of concentrations tested (i.e. 0 to 30 µM). These were also probed for actin (data not shown). The actin blots show no change in actin signal as a result of forskolin or any subsequent challenge and were used to correct the CRF blot for gel loading. The main panel shows a graphic summary of densitometric analysis of this result summed over three such experiments, as well as the results of similar determinations at 12 and 24 h of treatment. Thirty µM forskolin induced significant increases in CRF message expression after 6 and 12 h of treatment while 10 µM forskolin also induced a significant increase in CRF expression at 12 h. It appears on the basis of these experiments that the maximum response to forskolin occurs at a concentration of 30 µM and after 12 h of treatment.



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  		Figure 2. Regulation of CRF gene expression by forskolin and IL-6. A (insert), Typical autoradiograms of total RNA, probed for CRF after challenge for 6 h with various concentrations of forskolin. The main panel A is a graphic representation of experimental results summed across three independent experiments, and it displays the concentration- and time-dependence of forskolin (FOR)-stimulated CRF gene expression in immortalized amygdalar neurons. The data are expressed as percent of untreated control, after correction for gel loading by normalization to actin. (*, P < 0.05 vs. control). B, This figure is similar to A, in that the insert illustrates a representative Northern analysis of total RNA, probed for CRF after challenge for 6 h with various concentrations of IL-6. The main panel displays the concentration- and time-dependence of IL-6-stimulated CRF gene expression in immortalized amygdalar neurons. The data are expressed as percent of untreated control, after normalization to actin. The insert is representative of three such experiments, whereas the graph is a summary across the three independent experiments (*, P < 0.05 vs. control).

 
The results of similar experiments probing the effects of IL-6 treatment on CRF gene expression are presented in Fig. 2BGo. The insert displays a representative Northern analysis of CRF expression after 6 h treatment with various concentrations of IL-6. IL-6 induced concentration dependent increases in CRF message expression over a concentration range of 0 to 100 pM. The main panel shows a graphic summary of this result summed over three such experiments, as well as the results of determinations at 12 and 24 h of treatment. One hundred pM IL-6 induced significant increases in CRF gene expression at 6 and 12 h. No significant effects were observed at lower concentrations of IL-6 or other times of incubation. These experiments suggest that the maximal effects of IL-6 to increase CRF gene expression occur after 12 h treatment with 100 pM IL-6. Both TPA and dex were tested for their ability to affect CRF mRNA expression by AR-5 cells and neither compound was effective at any concentration (1 to 50 pM and 1 to 100 nM, respectively) or time (6–24 h) examined (data not shown).

Effects of forskolin, IL-6, TPA and dex on CRF peptide production
We have demonstrated that immortalized amygdalar neurons produce immunoreactive CRF in addition to expressing the CRF message. We then analyzed culture supernatants and cell lysates for the levels of immunoreactive CRF in the presence and absence of forskolin or IL-6. We also examined the effects of TPA and dex for comparison with nontransformed amygdalar cells in primary culture. The results of these studies using the CRF RIA are summarized in Fig. 3Go. Panel A shows CRF detected in the culture supernatant. Treatment of immortalized amygdalar neurons with 30 µM forskolin resulted in a significant increase in immunoreactive CRF detected in the media at 12 and 24 h. Similarly, treatment of these cells with 100 pM IL-6 also resulted in significant increases in media CRF at 12 and 24 h. Treatment of these cells with TPA or dex was without significant effect on the amount of CRF detected in the media.



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  		Figure 3. CRF peptide production by immortalized amygdalar neurons. The upper graph displays CRF peptide detected by RIA in culture supernatants after 12 and 24 h of challenge with forskolin (FOR; 30 µM), IL-6 (100 pM), dex (100 nM), or TPA (50 nM). The lower graph represents data obtained from cell lysates for intracellular CRF in the same experiments. Both figures were obtained from the same experiment and are representative of three independent experiments. The rationale for the times and concentrations is described in the accompanying text. *, P < 0.05 vs. control.

 
The amount of CRF detected in cell lysates after treatment with forskolin, IL-6, dex or TPA are illustrated in Panel 3B. The effects of these treatments on CRF content of the cells is similar to that observed for CRF detected in the culture supernatants. Both forskolin (30 µM) and IL-6 (100 pM) caused significant increases in the amount of CRF detected in cell lysates after 12 and 24 h of treatment while both dex and TPA were without significant effect on lysate CRF levels. We also examined earlier (3, 6 and 9 h) time points besides 12 and 24 h for changes in culture supernatant and cell lysate CRF. However, CRF was not consistently detectable at these earlier time points (i.e. 3, 6, or 9 h) and thus these data are are not shown.

Effects of forskolin or IL-6 in combination with dex on CRF expression
Figure 4Go illustrates the CRF responses of immortalized amygdalar neurons to forskolin or IL-6, alone or in combination with dex. Panel A displays the effects of these agents, at the concentrations used previously, on CRF mRNA expression after 12 h of treatment. Both forskolin and IL-6 caused significant increases over control CRF message expression by 40% and 86%, respectively. When these treatments were combined with 100 nM dex, the increases were 74% and 73%, respectively. Both pairs of increases represent statistically significant increases over control. However, none of the stimulated values (i.e. forskolin ± dex and IL-6 ± dex) are significantly different from one another, indicating that the dex treatment did not alter the ability of forskolin or IL-6 to increase CRF gene expression by these cells.



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  		Figure 4. Effects of dex, with forskolin or IL-6, on CRF expression. The upper graph displays a typical Northern analysis of CRF gene expression after treatment of these cells with dex (100 nM) alone or in combination with forskolin (30 µM) or IL-6 (100 pM). These data are expressed as percent of untreated control, after normalization to actin (not shown). The lower graph displays the CRF peptide content of additional cultures treated in the same manner. Both panels show effects after 12 h of treatment within the same experiment and are representative of three independent experiments. *, P < 0.05 vs. control.

 
Panel B of Fig. 4Go illustrates the effect of these combined treatments on CRF peptide content after 12 h of treatment This time point was selected on the basis of results described above. As was seen for CRF mRNA, forskolin or IL-6 treatment caused a significant increase over control in CRF content. Addition of 100 nM dex did not alter the forskolin or IL-6 effect on CRF content. Consistent with the results observed for CRF message expression, all of the stimulated peptide values are statistically similar, indicating that cotreatment with dex failed to alter the production of CRF peptide.

Expression of CRF-BP by AR-5 cells
We next sought to determine if AR-5 immortalized amygdalar neurons express CRF-BP. Lane A of Fig. 5Go displays a Northern blot analysis of total RNA in which a single species of the expected 1.85 kb size (as described in 14) is detected by the CRF-BP probe. Lane B displays a positive control of the Western blot in which the anti-CRF-BP antiserum detects recombinant CRF-BP (25 pg) at the appropriate size of 37 kDa. Lane C displays the Western analysis of total protein (25 µg) prepared from AR-5 cells. An immunoreactive protein of a size similar to the recombinant CRF-BP control is detected by the anti-CRF-BP antiserum in AR-5 extracts. These results suggest that AR-5 cells express both CRF-BP mRNA and immunoreactive CRF-BP.



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  		Figure 5. CRF-BP Northern and Western analyses. Lane A, Foto/Eclipse Imager Northern blot of total AR-5 cell RNA probed for CRF-BP. The hybridization signal consisted of a single species with an apparent size of 1.85 kb, which is consistent with other reports of the size of the rat CRF-BP mRNA. B and C, Western analysis of recombinant CRF-BP (B) and AR-5 cell total protein (C), probed with a polyclonal anti-CRF-BP antiserum. The antiserum revealed an immunoreactive species at 37 kDa in both lanes.

 
Regulation of CRF-BP expression by AR-5 cells
We questioned whether the expression of CRF-BP by AR-5 cells was responsive to the same agents used to examine CRF regulation. Figure 6AGo demonstrates that treatment with forskolin at concentrations of 10 and 30 µm resulted in significant increases in CRF-BP mRNA expression; the responses at 10 and 30 µm were not statistically different. Figure 6BGo demonstrates that treatment with IL-6 at concentrations of 100 and 500 pM also resulted in significant increases in CRF-BP gene expression; in addition, the responses at 100 and 500 pM were not significantly different. These data together with those presented above indicate that forskolin and IL-6 both increase the expression of CRF and CRF-BP mRNA in a concentration dependent manner. These and subsequent experiments in this section were performed using 12 h treatment; preliminary observations (not shown) indicated that maximal responses occurred at this time.



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  		Figure 6. Regulation of CRF-BP gene expression by forskolin, IL6, TPA, and dex. Inserts, Representative autoradiograms of total RNA, probed for CRF-BP, after treatment with various concentrations of forskolin, IL-6, TPA, or dex (A, B, C, and D, respectively). The main panels are graphic representations of experimental results summed across three independent experiments, and they display the concentration-dependence of forskolin-, IL-6, TPA-, or dex- stimulated CRF-BP gene expression in immortalized amygdalar neurons. The data are expressed as percent of untreated control, after normalization to actin. *, P < 0.05 vs. zero concentration control.

 
Figure 6CGo demonstrates that treatment of AR-5 cells with TPA resulted in a concentration-dependent increases in CRF-BP mRNA expression. This effect reached statistical significance at 50 pM. Figure 6DGo demonstrates that treatment of these cells with dex also resulted in a concentration dependent increase in CRF-BP gene expression which achieved statistical significance at 100 nM. These results in combination with those presented earlier indicate that while neither TPA nor dex affect the expression of CRF, both of these agents stimulate the expression of CRF-BP mRNA.

We extended our observations of CRF-BP mRNA expression to the protein level by performing semiquantitative Western analysis of total cellular protein after treatment with forskolin, TPA, IL-6 or dex. The results of these experiments are presented in Fig. 7Go. Treatment of AR-5 cells with each of these agents at the concentrations used above which increased CRF-BP mRNA expression also increased the expression of immunoreactive CRF-BP when compared with unstimulated control cells. These experiments examining CRF-BP expression were performed after 12 h treatment.



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  		Figure 7. Regulation of CRF-BP protein expression by various agents. Semiquantitative Western analysis reveals that treatment of AR-5 cells with forskolin (30 µM), IL-6 (100 pM), dex (100 nM), or TPA (50 pM) increased CRF-BP protein expression, relative to untreated control cells (*, P < 0.05 vs. zero concentration control). Insert, Representative Western blot chemiluminescence image.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report in this paper the generation of an immortalized CRF producing neuronal cell line which we have named AR-5. This AR-5 cell line results from retroviral-mediated transfer of the SV 40 large T antigen into embryonic amygdalar cells. Immortalized neuronal cell lines have been produced from various brain regions by several investigators using similar techniques (23). Whyte et al. (24) developed clonal differentiated neuronal cell lines of hypothalamic GnRH-producing neurons by targeting SV 40 large T antigen-mediated tumorigenesis with the GnRH promoter. Gao and Hatten (25) developed cell lines from the external germinal layer of the cerebellum by retroviral infection with the SV 40 large T antigen. Additionally, Eves et al. (23) used the SV 40 large T antigen to produce immortalized hippocampal cell lines expressing neuronal markers.

Our use of SV 40 large T antigen transformation has resulted in an immortalized cell line with a polygonal appearance. The presence of the neuron-specific MAP2 epitope and the absence of the glial marker GFAP in these cells argue in favor of a neuronal origin of the cell line. Significantly, these cells express immunoreactive CRF, as detected by RIA and immunohistochemistry. Consistent with this expression of CRF peptide, these cells also express the CRF gene product, as determined by Northern analysis. The size of the transcript detected (1.3 kb) is identical in size to that detected in primary cultures of amygdalar or hypothalamic neurons (22).

The expression of CRF message and peptide seems to be regulated in these immortalized cells. Forskolin, a pharmacological activator of PKA, increases CRF message and peptide production in a time- and concentration-dependent fashion. The time course of increases in CRF peptide expression, which first showed significant increases at 12 h and was still elevated at 24 h, lagged the increases in CRF message, which had returned to unstimulated levels by 24 h. This delay is not unexpected, because changes in peptide expression generally lag behind changes in message expression. Furthermore, we examined levels of mature CRF message, as opposed to heteronuclear CRF. The latter is a more sensitive indicator of changes in CRF message but presents challenges in detection, because it is present in much lower abundance.

In addition, our studies do not distinguish between increases in message transcription or message stability. We hypothesize that the mechanism by which forskolin stimulates CRF gene expression is by PKA-mediated phosphorylation of cAMP response element binding protein (CREB). The CRF gene contains a cAMP response element in its 5' region (26), and a similar mechanism is present in the hypothalamic CRF system (27, 28). However, we are unaware of reports implicating CREB in amygdalar CRF responses, nor have we yet performed similar experiments in these immortalized cells. The time courses of CRF message responses to forskolin and IL-6 were similar. Unlike the effects observed with forskolin, the increase in peptide expression lagged behind the increase in message expression, although the time course of the increase in peptide expression was similar to that observed with forskolin treatment.

Our results with AR-5 cells are congruent with the results we have reported for primary cell cultures of amygdala. Primary amygdalar cultures increase CRF expression in response to forskolin and IL-6. The time course of CRF responses is similar in the two systems. The relative magnitude of the responses of CRF message and peptide are slightly lower in immortalized AR-5 cells, compared with primary cultures. For example, CRF mRNA increased by 110% with forskolin in immortalized cells vs. an increase of 200% in primary amygdalar cultures. CRF mRNA increased 165% with IL-6 in immortalized cells, whereas it increased 200% in primary amygdalar cultures. Activation of PKC does not increase CRF expression in the immortalized cells. This is consistent with our observations in primary cultures. Both primary and immortalized amygdalar cells are also similar to the placenta, in this regard, but both differ from the hypothalamus, where PKC activation increases CRF expression. The immortalized and primary amygdalar cells are also similar, in that CRF in these two systems is refractory to dex. Though the negative feedback effects of glucocorticoids are well known for the hypothalamic CRF system (19, 20, 29), studies using adrenalectomized rats do not support such a role for glucocorticoids in the regulation of CRF in the amygdala (30). However, positive and negative regulation of amygdalar neurons, by direct administration of glucocorticoids to rats, has been reported (29). Furthermore, glucocorticoids have been reported to increase CRF mRNA levels in primary placental cultures (31). A precedent thus exists for a failure of dex to suppress CRF expression, depending on the CRF system being investigated.

Taken together, the results stated, thus far, indicate that the immortalized amygdalar neuronal cells are similar to primary amygdalar cultures cells, in terms of their CRF responses to known regulators of amygdalar CRF. These results suggest that our immortalized cells represent a model that is a valid alternative to primary cultures of the amygdala in experiments investigating the regulation of CRF in that structure. If so, then the immortalized cell line offers important advantages over primary cultures, in terms of offering a virtually unlimited supply of cells.

We questioned whether the lack of a dex effect was the result of basal CRF secretion that could not be suppressed. Immortalized cells were challenged with IL-6 or forskolin, in the presence and absence of dex, to determine whether dex might suppress the increases in CRF expression observed with these stimulators alone. The amounts of CRF message and peptide detected when dex was combined with IL-6 or forskolin were not statistically different from the amounts detected with these stimulators alone. We conclude, therefore, that the immortalized cells are refractory to the effects of dex to inhibit CRF production, both in basal and stimulated states. This is consistent with our report that dex is ineffective at altering basal CRF production in primary amygdalar cultures (18), although both observations contrast with reports for hypothalamic CRF systems, where dex inhibits both basal and stimulated CRF production (19, 20, 29). This differential effect of glucocorticoids on CRF regulation in various brain regions may be caused by the differential expression of glucocorticoid receptors in the various experimental systems; this is an area requiring experimental investigation in our cells. A second important conclusion is that these experiments were performed with cells from generations 19–21. This indicates that the phenotype of regulated CRF production persists in these cells, to at least generation 21, the last generation of cells we have examined. Quantitative differences seem to exist in the magnitude of CRF mRNA responses across experiments (compare Figs. 2AGo and 4AGo). We ascribe these to interexperiment differences. We have not observed a consistent difference in CRF mRNA responses as a function of culture confluence or passage number.

CRF-BP colocalizes with CRF in the amygdala (15), and CRF-BP is believed to modulate the biological activity of CRF (31). Because the AR-5 cell line seems to be an appropriate model of at least one CRF-producing cell type found in the amygdala, we questioned whether AR-5 cells also produce CRF-BP. The AR-5 cells were found to express an RNA species that is the appropriate size (1.85 kDa; Ref. 14). These cells also produce a protein of the same apparent molecular size as CRF-BP. In light of these data, it is reasonable to conclude that the AR-5 cell line produces CRF-BP. The exact role of CRF-BP in the central nervous system has not been established. CRF-BP has been reported to occur in both a secreted/soluble form and in a membrane-associated form (13, 32). Our experiments examined only cell content (intracellular or membrane associated) and not culture supernatants (secreted or soluble form). This emphasis on cell-associated CRF-BP is consistent with current models of central CRF-BP in which the molecule is membrane bound and functions as a perisynaptic sink for CRF to modulate the synaptic activity of CRF (32). We also note that, although we have confirmed the presence of CRF-BP mRNA and an immunoreactive protein, we have not explored the ability of this protein from AR-5 cells to bind CRF.

The regulation of CRF-BP expression has not been extensively studied. Activation of the PKA pathway has been shown to increase CRF-BP expression in cultured astrocytes and mixed neuronal cultures (16, 17), and structural analysis of the promoter has revealed a cAMP response like-element centered around base -127 (33). Our observations, that forskolin increases CRF-BP mRNA and protein expression in AR-5 cells, are consistent with these reports. Consistent with Maciejewski et al. (17), who demonstrated that PKC activators increase CRF-BP secretion in astrocytes, we have demonstrated that the PKC activator TPA will increase CRF-BP mRNA and the cell-associated form of the protein. Because we are unaware of reports implicating the IL-6-dependent JAK/Stat pathways in the regulation of CRF-BP expression, our observation that IL-6 will increase CRF-BP expression in AR-5 cells is novel. The mechanisms underlying these effects are not presently known.

The observation that some stimuli that increase CRF expression can also increase the expression of CRF-BP suggests that the net result of this interaction is a change in CRF bioactivity that is different than that predicted on the basis of changes in CRF alone. Although our experiments were not explicitly designed to compare the relative magnitude of changes in CRF and CRF-BP expression, it is interesting to note that 30 µM forskolin induced an approximate doubling in CRF (Fig. 3AGo), but this same treatment induced an increase of approximately 50% in CRF-BP (Fig. 7Go). A more intriguing observation, particularly in light of the discrepant reports of dex effects on CRF in the amygdala, is that treatment of AR-5 cells with dex (100 nM) did not alter CRF expression (Fig. 3Go, A and B), while CRF-BP expression was approximately doubled by this treatment (Fig. 7Go). This finding provides a potential model for a mechanism regulating CRF bioactivity, in which CRF bioactivity is reduced not by a reduction in CRF but instead by an increase in CRF-BP.

In conclusion, we have produced a clonal line of immortalized amygdalar cells that express a neuronal marker. These cells also express CRF, and the expression of this neuropeptide is regulated in a manner consistent with that observed with primary cultures of amygdalar neurons. This cell line offers the typical advantages of a cell line, namely, a limitless model of an otherwise scarce cell type. These cells also produce CRF-BP. We have exploited these cells to learn that CRF and CRF-BP are regulated in a similar manner by some agents and in a divergent manner by others. The homogeneous nature of this clonal line should facilitate investigations of the molecular processes involved in the regulation of CRF and CRF-BP in the amygdala. These cells seem to represent a fertile model for additional investigation in this area.


   Footnotes
 
1 Portions of this report were presented in abstract form at the 1997 and 1998 annual meetings of The Endocrine Society. Back

2 Supported by a National Alliance for Research on Schizophrenia and Depression (NARSAD) Young Investigator Award. Back

Received May 4, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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