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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
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Introduction |
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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.
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Materials and Methods |
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Virus producer cell line 
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 
globulin was added; and after 4 h, the
samples were pelleted by centrifugation. Pellets were counted using a
Packard Multi Prias 4 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.
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Results |
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illustrates the results of immunostaining for the clone, identified as
AR-5, ultimately selected for further study. Figure 1A illustrates that
these cells contain immunoreactive CRF, whereas Fig. 1C 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 1, 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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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. 2A. 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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. 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. 3. 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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Effects of forskolin or IL-6 in combination with dex on CRF
expression
Figure 4 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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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. 5 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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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 6B 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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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 6D
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. 7. 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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Discussion |
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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. 2A and 4A). 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. 3A), but this same treatment induced an increase of
approximately 50% in CRF-BP (Fig. 7). 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. 3, A and B), while
CRF-BP expression was approximately doubled by this treatment (Fig. 7).
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.
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Footnotes |
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2 Supported by a National Alliance for Research on Schizophrenia and
Depression (NARSAD) Young Investigator Award.
Received May 4, 1998.
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