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Endocrinology Vol. 144, No. 4 1292-1300
Copyright © 2003 by The Endocrine Society

ARTICLE

Cyclic Adenosine 3',5'-Monophosphate Regulation of Corticotropin-Releasing Hormone Promoter Activity in AtT-20 Cells and in a Transformed Hypothalamic Cell Line

Maria Nikodemova, John Kasckow, Hanguan Liu, Vincent Manganiello and Greti Aguilera

Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development (M.N., G.A.), and Pulmonary/Critical Care Medicine Branch, National Heart, Lung, and Blood Institute (V.M.), National Institutes of Health, Bethesda, Maryland 20892; and Department of Psychiatry, University of Cincinnati (J.K.), Cincinnati, Ohio 45267

Address all correspondence and requests for reprints to: Dr. Greti Aguilera, Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 10N 262, 10 Center Drive, MSC 1862, Bethesda, Maryland 20892-1862. E-mail: greti{at}helix.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The regulation of CRH promoter activity by cAMP was studied in two cell lines, the pituitary corticotroph cell line AtT-20 and the immortalized hypothalamic cell line 4B, which expresses CRH and vasopressin. In 4B cells transfected with a CRH promoter-luciferase construct, the adenylyl cyclase stimulator, forskolin, increased luciferase activity in parallel with increases in intracellular cAMP. In 4B cells, however, the phosphodiesterase inhibitor, isobutylmethylxanthine, potentiated forskolin-stimulated cAMP without affecting further increases in luciferase activity. In AtT-20 cells, forskolin plus isobutylmethylxanthine elevated cAMP only slightly, but increased luciferase activity to levels similar to those observed in 4B cells. AtT-20 cells were also unresponsive to 8-bromo-cAMP, due in part to higher phosphodiesterase (PDE) activities. Although both cells contained PDE1, -3, and -4, inhibition of either PDE4 or PDE1 potentiated luciferase activity stimulated by submaximal forskolin concentrations in 4B cells, while only simultaneous inhibition of PDE3 and PDE4 was effective in AtT-20 cells. The data show that minor elevations in intracellular cAMP are sufficient for full stimulation of CRH promoter activity regardless of the cell line. Furthermore, poor CRH promoter activation in AtT-20 cells appears to result from deficient cAMP production and rapid cAMP degradation by PDE.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CRH IS SYNTHESIZED in the parvocellular subdivision of the paraventricular hypothalamic nucleus (PVN) and is a key regulator of the hypothalamus-pituitary-adrenal axis. CRH regulates the expression of the proopiomelanocortin gene and secretion of corticotropin (ACTH) in the pituitary (1). Maintenance of appropriate levels of CRH expression in the PVN as well as secretion of the peptide into the pituitary portal circulation are critical for maintaining the responsiveness of the hypothalamus-pituitary-adrenal axis during stress (2, 3). In addition to the PVN, CRH is expressed in other areas of the central nervous system and peripheral organs, including adrenal medulla, lymphocytes, pancreas, skin, gonads, uterus, and placenta (4, 5, 6, 7, 8, 9, 10). The regulation of CRH gene expression appears to differ in various tissues expressing CRH. For example, although glucocorticoids have been recognized as having inhibitory effects on CRH expression in the PVN (11, 12, 13, 14), they have stimulatory effects in the placenta (15, 16).

Studies in a number of systems indicate that cAMP-dependent signaling activates CRH gene expression. A functional cAMP-responsive element (CRE) has been identified in both rat and human CRH promoters, 221 bp upstream from the putative transcription start point (17, 18). It has been shown that activation of the protein kinase A (PKA) pathway with forskolin leads to binding of CRE-binding protein to the CRE in the CRH promoter in human placental JEG-3 cells (19). Moreover, stimulation of PKA in F9 cells, which contain low levels of CRE-binding protein, does not result in CRH promoter stimulation (20). Despite the wealth of information derived from studies in CRH-expressing extrahypothalamic cells or cell lines that do not express CRH, little is known about the signaling pathways involved in the control of CRH transcription in parvocellular PVN neurons. A major impediment to studying the mechanism regulating CRH transcription in parvocellular neurons has been the lack of availability of a representative cell line. Recently, a rat fetal hypothalamic cell line, 4B, has been developed from primary hypothalamic culture by retroviral transformation using a construct expressing the large T antigen. 4B cells show neuronal phenotype as determined by the expression of the marker microtubule-associated protein 2. These cells express CRH mRNA and display CRH-like immunoreactivity, which coelutes with a CRH standard by reverse phase HPLC analysis. In addition, 4B cells express type-1 CRH receptor mRNA and have functional CRH receptors, as shown by binding assays and the ability of CRH to stimulate cAMP production and PKA activation (21).

In our study we compared the responses of CRH promoter activity after PKA activation in two cell lines. These include the pituitary corticotroph cell line, AtT-20, commonly used in CRH promoter studies, and the CRH-expressing hypothalamic cell line, 4B.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Constructs
A 613-bp restriction fragment containing the CRH promoter (–498 to +115 bp relative to the proximal transcription start point) was obtained by XbaI/KpnI digestion of the CRH gene clone prCRHBglII (provided by Dr. Audrey Seasholtz, Ann Arbor, MI). This DNA fragment was used to produce the CRH promoter-driven luciferase reporter construct, pCRH-Luc, by a two-step cloning method. First, the DNA fragment was cloned into the XbaI/KpnI sites of pcDNA3.1(-) (Invitrogen, San Diego, CA) and then it was subcloned into the NheI-HindIII sites of pGL3Basic (Promega Corp., Madison, WI).

Additionally, an 803-bp restriction fragment of the vasopressin (VP) promoter was digested with BamHI and HindIII from pXP1-arginine vasopressin (AVP) 803 (provided by Drs. Y. Iwasaki and J. A. Majzoub, Children’s Hospital, Boston, MA) and subcloned into BglII and HindIII cloning sites of pGL3Basic to create pVP-Luc.

Cell cultures and transfection
AtT-20 cells were purchased from American Type Culture Collection (Manassas, VA) and cultured in DMEM supplemented with 15% fetal bovine serum and 100 U penicillin and 100 µg streptomycin/ml. The hypothalamic cell line 4B was cultured in DMEM supplemented with 10% horse serum, 10% fetal bovine serum, and 100 U penicillin and 100 µg streptomycin/ml.

Cells were plated in 24-well plates at a density of 20 x 103 (4B) or 50 x 103 (AtT-20) per well and were cultured at 37 C under 5% CO2/95% air. After 24 h of culture, cells were transfected with 0.3 µg DNA (pCRH-Luc or the control vector, pGL3-Basic) using Lipofectamine Plus (Life Technologies, Inc., Gaithersburg, MD) in Opti-MEM medium (Life Technologies, Inc.) for 3 h. After this they were switched to the initial serum-containing medium. To test the specificity of the effects of cAMP on CRH promoter activity in the two cell lines, in some experiments cells were transfected with 0.3 µg pVP-Luc. Eighteen hours after transfection, cells were incubated in DMEM supplemented with 0.1% BSA with various concentrations of the adenylyl cyclase stimulator, forskolin, in the presence or absence of 1 mM isobutylmethylxanthine (IBMX) or with 8-bromo-cAMP (8Br-cAMP), as indicated in Results and the figure legends. Cells were then lysed using 100 µl 1x passive lysis buffer (Promega Corp.) for 15 min. Twenty microliters of cell extract were processed for the luciferase assay using kit reagents (Promega Corp.).

RT-PCR for CRH and vasopressin
Total RNA from 4B and AtT-20 cells was isolated using RNAzol B (Tel-Test, Friendswood, TX) following the manufacturer’s protocol. Polyadenylated [poly(A)+] RNA was isolated from the total RNA, using oligo(deoxythymidine) PolyATract mRNA Isolation Systems (Promega Corp.). Rat hypothalamic total RNA was used as a positive control. For CRH, 500 ng total RNA were used for RT-PCR amplification using the Superscript One-Step RT-PCR Platinum Taq system (Life Technologies, Inc.) and rat CRH primers: forward, 5'-CTCTCTGGATCTCACCTTCCAC-3'; and reverse, 5'-CTAAATGCAGAATCGTTTTGGC-3'. Single-stranded cDNA was synthesized at 52 C for 30 min, followed by 35 PCR cycles consisting of 1 min at 94 C, 40 sec at 56 C, and 40 sec at 72 C, followed by a 10-min extension at 72 C. RT-PCR for VP was performed as described previously (22) using the primers: forward, 5'-CGCAGTGCCCACCTATGCTCGCCA-3'; and reverse, 5'-TCGGCCACGCAGCTCTCATCGCTG-3'. Single-stranded cDNA was synthesized at 52 C for 30 min, followed by 35 PCR cycles consisting of 1 min at 94 C, 1 min at 65 C, and 40 sec at 72 C, followed by a 10-min extension at 72 C. The PCR products were separated and visualized in a 2% Tris-acetate-EDTA-agarose gel containing ethidium bromide and were sized using PCR markers (Promega Corp.).

Measurement of intracellular cAMP
AtT-20 and 4B cells were plated at a density of 5 x 104 or 3 x 104 cells/well, respectively, in 24-well plates. Twenty-four hours later, cells were incubated with 10 µM forskolin in the presence and absence of 1 mM IBMX for 15, 30, 60, and 180 min or 24 h. After treatment, cells were washed twice with PBS, and intracellular cAMP was extracted after the addition of 250 µl 0.01 M HCl, followed by three cycles of freezing and thawing. cAMP production was determined using a RIA kit from Perkin-Elmer (Norwalk, CT).

Measurement of phosphodiesterase (PDE) activities
AtT-20 or 4B cells were homogenized in a buffer consisting of 50 mM HEPES, 5 mM MgCl2, 0.1 mM EGTA, 5 mM benazamidine, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1% Triton X-100 (pH 7.4). The homogenate was centrifuged at 1000 x g for 5 min, and the supernatant was assayed for PDE activity.

Total cAMP PDE activity was assayed for 30 min at 30 C in 50 mM HEPES (pH 7.4), 5 mM MgCl2, 0.1 mM EGTA, and 1 µM [3H]cAMP (50,000 dpm) with or without the PDE inhibitor IBMX (300 µM). PDE1 activity was assessed in the presence or absence of 1 µM calmodulin and 4 mM Ca2+ (EGTA was omitted in the medium). PDE3 and PDE4 activities were determined by using the following selective inhibitors: 1 µM cilostamide (PDE3 inhibitor) or 5 µM rolipram (PDE4 inhibitor). PDE3 and PDE4 activities were calculated by subtracting the activity in the presence of cilostamide or rolipram, respectively, from total PDE activity.

To determine the role of PDE1, PDE3, and PDE4 in potentiating forskolin-stimulated CRH promoter activity, AtT-20 and 4B cells transfected with pCRH-Luc were incubated with forskolin after preincubation with or without specific PDE inhibitors. The PDE inhibitors, 80 µM 8-methoxy-IBMX, 1 µM cilostamide, and 5 µM rolipram, were added 15 min before incubation for 6 h with submaximal stimulatory concentrations of forskolin (10 µM for AtT-20 cells and 0.5 µM for 4B cells). The use of these low stimulatory concentrations of forskolin was necessary to detect potentiation of this activity by the different PDE inhibitors, as in 4B cells full promoter activation was observed with 10 µM forskolin. After incubation, cells were lysed for measurement of luciferase activity as described above.

Data analysis
Differences between groups were tested by one-way ANOVA, followed by pairwise multiple comparison (Tukey’s test) and multiple comparison vs. control (Dunn’s method), using the software package SigmaStat (SPSS, Inc., Chicago, IL). When appropriate, two-way ANOVA was performed, followed by Bonferroni’s t test and Tukey’s test. Data are presented as the mean ± SEM from at least three independent experiments. The difference between experimental conditions was considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CRH and VP expression in 4B cells
RT-PCR on total RNA extracted from 4B cells revealed a 150-bp band corresponding to the expected size for the CRH DNA fragment, which was identical to the band obtained with rat hypothalamic RNA (Fig. 1AGo). This confirmed that the 4B hypothalamic cell line expresses endogenous CRH mRNA (21). In addition, as shown in Fig. 1BGo, RT-PCR on poly(A)+ RNA isolated from 4B cells revealed a 365-bp band corresponding to the VP band obtained with rat hypothalamic RNA.



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  		Figure 1. RT-PCR for CRH and VP mRNAs in 4-B cells. A, RT-PCR analysis of CRH mRNA in 4B cells performed on 500 ng total RNA (lane 2). The RT step was omitted in lane 1 to exclude the genomic DNA contamination. B, RT-PCR analysis of VP mRNA using 200 ng of poly(A)+ RNA from rat hypothalamus (lane 1) or 4B cells (lanes 2).

 
Effects of forskolin and IBMX on CRH promoter activity and cAMP production
Forskolin had no significant effect on luciferase activity in AtT20 or 4B cells transfected with the promoterless vector, pGL3Basic. In 4B cells transiently transfected with the CRH promoter construct, incubation with 10 µM forskolin led to marked increases in luciferase activity (12.6 ± 1.5-, 12.3 ± 2.76-, and 7.6 ± 1.85-fold of basal CRH promoter activity after 3, 6, and 24 h, respectively; Fig. 2AGo). Unexpectedly, in AtT-20 cells, incubation with forskolin resulted in only a minor increase in luciferase activity (1.81 ± 0.42-, 1.87 ± 0.36-, and 0.75 ± 0.14-fold induction after 3, 6, and 24 h, respectively; Fig. 2BGo).



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  		Figure 2. Effect of forskolin on CRH promoter activity. 4B cells (A) and AtT-20 cells (B) were transiently transfected with pCRH-Luc. Luciferase activity was measured after treatment with 10 µM forskolin with or without 1 mM IBMX. In 4B cells (A), two-way ANOVA followed by Bonferroni’s t test revealed a significant effect of both forskolin and forskolin plus IBMX on CRH promoter activity (P < 0.001; n = 3), but no effect of IBMX on forskolin-stimulated activity (P = 1.0; n = 3). In AtT-20 cells (B), there was no effect of forskolin alone, but there was a significant effect of forskolin in combination with IBMX (P < 0.001; n = 3)

 
Analysis of intracellular cAMP levels in 4B cells after forskolin treatment indicated marked increases (99 ± 28.5-, 48 ± 14.5-, 19 ± 5.4-, and 2.9 ± 0.6-fold at 15, 30, 60, and 180 min, respectively; Fig. 3AGo). By 24 h cAMP levels returned to baseline (not shown). In contrast, in AtT-20 cells forskolin failed to significantly increase intracellular cAMP (Fig. 3BGo). Treatment of the cells with forskolin in the presence of 1 mM IBMX further increased cAMP levels in 4B cells (271 ± 84-, 200 ± 70-, 172 ± 39-, and 34 ± 2.8-fold at 15, 30, 60, and 180 min, respectively). Despite the increase in cAMP production, IBMX was not associated with further elevations in luciferase activity. In AtT-20 cells, IBMX caused a minor 1.6 ± 0.1-fold potentiation of intracellular cAMP. However, this small elevation in cAMP was associated with increased CRH promoter activity comparable to that seen in 4B cells (15 ± 4.1-, 20 ± 3.2-, and 8 ± 1.2-fold at 3, 6, and 24 h, respectively).



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  		Figure 3. Time course of the effect of forskolin on intracellular cAMP. 4B and AtT-20 cells were incubated with 10 µM forskolin with and without 1 mM IBMX for the indicated times. cAMP production was determined by RIA in acid extracts of the cells. Two-way ANOVA followed by Bonferroni’s t test revealed a significant effect of forskolin in 4B cells (P < 0.001; n = 5–8 for different time points), whereas in AtT-20 cells it had no effect (P < 0.06; n = 3). There was a significant difference between forskolin and forskolin plus IBMX (P < 0.001) in both cell lines.

 
We then determined the concentration-response effect with forskolin in 4B cells. Cells were transfected with pCRH-Luc and treated with increasing forskolin concentrations (1–20 µM). This resulted in a concentration-dependent increase in luciferase activity, with an EC50 of 1.2 µM and a maximal stimulatory concentration of 3 µM forskolin (Fig. 4AGo). The concentration-response curve for intracellular cAMP accumulation after forskolin stimulation (Fig. 4BGo) increased progressively up to a concentration of 20 µM forskolin. As shown in the inset, low concentrations of forskolin from 1–5 µM lead to small increases in cAMP, but the changes were statistically significant only when compared with basal values by t test.



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  		Figure 4. Concentration-dependent effects of forskolin on luciferase activity (A) and cAMP (B) production in 4B cells. A, Cells were transiently transfected with pCRH-Luc. Eighteen hours post transfection, cells were incubated with various concentrations of forskolin (1–20 µM) for 6 h, and luciferase activity was measured. Each concentration of forskolin used significantly increased luciferase activity as determined by one-way ANOVA, followed by Tukey’s test (P < 0.01). There was no significant difference between 3 µM and higher concentrations of forskolin. B, 4B cells were incubated with increasing concentrations of forskolin (1–20 µM) for 15 min. cAMP levels determined by RIA were significantly increased with the 7-, 10-, and 20-µM concentrations of forskolin as determined by one-way ANOVA, followed by Dunn’s test for multiple comparisons (P < 0.05). Responses to 1–5 µM cAMP shown in the inset were statistically significant only when compared with basal values by t test. #, P < 0.001.

 
Effects of forskolin and IBMX on VP promoter
To determine the promoter specificity of the effects of cAMP, the responses to forskolin and the PDE inhibitor, IBMX, were assessed in cells transfected with the VP promoter construct, pVP-Luc. As shown in Fig. 5AGo, in 4B cells 10 µM forskolin increased VP promoter activity by 4-fold. Similar to the findings with the CRH promoter in this cell line, the effect of forskolin was not potentiated by the PDE inhibitor, IBMX (data not shown). However, the EC50 for forskolin stimulation of VP promoter activity was 5.5 µM, which was more than 4 times higher than that obtained for stimulation of the CRH promoter. Consistent with a higher cAMP requirement for stimulation of the VP promoter, forskolin had no effect on stimulation of VP promoter in AtT-20 cells, which showed minimal elevations of cAMP even in the presence of IBMX (Figs. 5BGo and 3BGo).



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  		Figure 5. The effect of forskolin on VP promoter activity. 4B and AtT-20 cells were transiently transfected with pVP800. A, Eighteen hours after transfection, 4B cells were stimulated with various concentrations of forskolin (0–20 µM) for 6 h, and luciferase activity was measured. All concentrations used significantly increased luciferase activity (P < 0.05, by one-way-ANOVA followed by Bonferroni’s t test). There was no significant difference in the effects of 7, 10, and 20 µM forskolin on VP promoter. B, AtT-20 cells were stimulated with 10 µM forskolin with or without 1 mM IBMX for 6 h. There was no significant effect of either treatment on VP promoter activity.

 
Effect of 8Br-cAMP on CRH promoter
The blunted cAMP responses to forskolin in AtT-20 cells in the presence of PDE inhibitors suggested a lower cAMP production in this cell line. To bypass the action of adenylyl cyclase, cells transfected with pCRH-Luc were incubated with increasing concentrations of 8Br-cAMP (25–1000 µM). In two experiments, 4B cells displayed clear dose-dependent increases in luciferase activity after 6-h incubation with 8Br-cAMP. AtT-20 cells showed a significant response only with the highest 8Br-cAMP concentration, 1 mM (Fig. 6Go). This suggests that there are alterations in cAMP signaling at a site downstream of adenylyl cyclase.



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  		Figure 6. Concentration response for the effect of 8Br-cAMP on CRH promoter activity in 4B and AtT-20 cells. Cells were transiently transfected with pCRH-Luc, and luciferase activity was measured 6 h after stimulation with increasing concentrations of 8Br-cAMP (25–1000 µM). One-way ANOVA revealed significant dose-dependent increases in CRH promoter activity (*, P < 0.001 vs. basal, for 4B cells; #, P < 0.02 vs. basal for AtT-20).

 
PDE activity in 4B and AtT-20 cells
As activation of the CRH promoter by forskolin in the presence of the PDE inhibitor IBMX was similar in both cell lines, the inability of AtT-20 cells to respond to 8Br-cAMP suggests that there was an increase in PDE activity in this cell line. To test this hypothesis, we measured PDE activities in the two cell lines. Total PDE activity (excluding calmodulin-dependent PDE) was 7-fold higher in AtT-20 cells than in 4B cells. In both cell lines, the addition of the broad spectrum PDE inhibitor, IBMX, markedly inhibited PDE activity (82% and 84% in 4B and AtT-20, respectively; Fig. 7AGo). As shown in Fig. 7BGo, all PDE subtypes measured were higher in AtT-20 cells than in 4B cells. The predominant subtype in AtT-20 cells was PDE1, and its activity was 10-fold higher than that in 4B cells. PDE3 and PDE4 activities were also higher in AtT-20 cells. PDE3 and PDE4 levels were about 38% and 46%, respectively, of the values observed for PDE1. In 4B cells, PDE4 activity was similar to that of PDE1, whereas PDE3 was very low.



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  		Figure 7. PDE activities in 4B and AtT-20 cells. A, Total PDE (excluding PDE1) with and without 300 µM IBMX was assayed as described in Materials and Methods. Total PDE was significantly higher in AtT-20 than in 4B cells (*, P < 0.001, by one-way ANOVA followed by Bonferroni’s t test; n = 3). IBMX significantly inhibited PDE activity in both cell lines (#, P < 0.001). B, PDE subtypes in 4B and AtT-20 cells. PDE1 activity was determined in the presence of 1 µM calmodulin and 4 mM Ca2+. PDE3 and PDE4 activities were determined in the presence of the selective inhibitors, cilostamide and rolipram, respectively. Two-way ANOVA followed by Tukey’s test, revealed significantly higher levels of all PDEs determined in AtT-20 cells than in 4B (*, P < 0.03; n = 3). In AtT-20 cells, PDE1 was significantly higher than PDE3 and PDE4 (#, P < 0.006). There was no difference between PDE3 and PDE4 in AtT-20 cells. In 4B cells, PDE3 was significantly lower than PDE1 and PDE4 (@, P < 0.01). There was no significant difference between PDE1 and PDE4 in 4B cells.

 
Effects of PDE inhibitors on forskolin-stimulated CRH promoter
To determine the roles of different phosphodiesterase subtypes, which are responsible for modulating cAMP-dependent CRH promoter activity, we tested the abilities of different PDE subtype inhibitors to potentiate CRH promoter activation by submaximal stimulatory concentrations of forskolin. In 4B cells, the PDE4 inhibitor rolipram was able to potentiate forskolin-stimulated luciferase activity. On the other hand, the PDE3 inhibitor cilostamide had no effect. The PDE1-selective inhibitor 8-methyl-IBMX potentiated forskolin-stimulated luciferase activity in 4B cells, and this effect appeared to be additive to that of rolipram (Fig. 8AGo). In contrast, in AtT-20 cells there was no effect of either rolipram or cilostamide alone, but the combination of the two inhibitors markedly potentiated forskolin-stimulated luciferase activity (Fig. 8BGo). Furthermore, in AtT-20 cells, the selective PDE1 inhibitor, 8-methyl–IBMX, was ineffective and demonstrated no additive effect when combined with rolipram.



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  		Figure 8. The effect of subtype-specific PDE inhibitors on luciferase activity in 4B (A) and AtT-20 (B) cells. Cells were transiently transfected with pCRH-Luc; 18 h later, cells were preincubated with the selective PDE inhibitors for 15 min, followed by 6-h stimulation with submaximal stimulatory concentrations of forskolin (0.5 and 10 µM for 4B and AtT-20 cells, respectively) and the respective PDE inhibitors (PDE1, 80 µM 8-methoxy-IBMX; PDE3, 1 µM cilostamide; PDE4, 5 µM rolipram). F, Forskolin; R, rolipram; C, cilostamide; mI, 8-methoxy-IBMX. In 4B cells, one-way ANOVA showed a significant increase in forskolin-stimulated luciferase activity with rolipram, 8-methoxy-IBMX, and their combination (*, P < 0.004 for R+C and R+mI vs. forskolin; n = 3 or n = 2). The effect of rolipram and 8-methoxy-IBMX was additive, as this treatment further increased luciferase activity comparing to rolipram or 8-methoxy-IBMX alone (#, P < 0.04). In AtT-20 cells, only the combination of rolipram and cilostamide had a significant effect on forskolin-stimulated luciferase activity (*, P < 0.001 for R+C and R+mI vs. forskolin; n = 3 or 2), whereas rolipram or cilostamide alone did not have a significant effect.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although previous studies in nonhypothalamic cell lines have indicated that cAMP/PKA-dependent pathways play an important role in activating CRH expression (17, 18, 19), little information is available on the signaling mechanisms controlling CRH transcription in parvocellular neurons. As a major aim of this study was to study the regulation of CRH expression by cAMP in a cell line representative of parvocellular CRH neurons, we used the recently described hypothalamic cell line 4B, previously shown to express CRH and functional response to CRH (21). In addition to confirming that 4B cells express CRH, the present study showed that they coexpress vasopressin. These cells have also been shown to express glucocorticoid receptors (21), which is consistent with a parvocellular neuron phenotype. These characteristics of 4B cells suggest that this hypothalamic cell line may prove to be a useful model for studying the regulation of hypothalamic CRH.

In these experiments we used the 4B cell line to study CRH promoter activation by cAMP and compared the results with those observed in AtT-20 cells, a cell line previously used to study the mechanisms of CRH expression (17, 23). It is clear from the present data that elevations of intracellular cAMP were able to activate the 498-bp CRH promoter fragment used in the study equally in both cell lines regardless of the ability of the cell line to express endogenous CRH. However, it is possible that the use of different promoter constructs may reveal tissue-specific differences in cAMP regulation of CRH promoter activity. In this regard, experiments in transgenic mice have revealed expression in inappropriate regions of the brain of the promoter fragment used in the present study and have shown that tissue specificity of expression was improved with inclusion of an 8.5-kb upstream fragment (24).

It is noteworthy that minimal elevations in intracellular cAMP were sufficient for maximal activation of CRH promoter activity in both cell lines. This was evident from the higher sensitivity of the concentration responses of forskolin-stimulated CRH promoter activity than those for stimulation of cAMP production as well as from the full CRH promoter stimulation in the presence of minor increases in cAMP production in AtT-20 cells. A dissociation between cAMP production and cellular responses has been reported in other studies, in which the concentrations of stimulatory hormones required for cAMP production are at least 1 order of magnitude higher than those needed for hormone secretion (25, 26, 27). Studies in testicular Leydig cells stimulated by gonadotropins or in adrenocortical cells stimulated by ACTH have indicated that small increases in intracellular cAMP production, undetectable by RIA, are able to dissociate the catalytic subunit of PKA (28, 29, 30). Although both cell lines in the present study exhibited marked differences in intracellular cAMP responses to forskolin, it is clear from these studies that small elevations in cAMP production are sufficient for stimulation of CRH gene expression. To determine whether this requirement for low increases in cAMP for CRH promoter activation is promoter specific, we examined the forskolin concentration dependence for activation of the VP promoter. It is interesting to note that the increase in cAMP after forskolin stimulation in AtT-20 cells was not sufficient to stimulate VP promoter activity. This suggests that the levels of cAMP needed for activation of the VP promoter are higher than those required for CRH promoter activation. This was confirmed by the finding that the EC50 for forskolin stimulation of the VP promoter was more than 4-fold higher than that for stimulation of the CRH promoter in 4B cells. The mechanism responsible for this differential regulation of CRH and VP promoters by cAMP is likely to involve different affinities of transcription factors for the CRE. Although cAMP regulation of the CRH promoter is mediated by a consensus CRE, regulation of the VP promoter appears to involve half-CRE sequences (31). The different sensitivities of the CRH and VP promoters to cAMP signaling could contribute to the differential regulation by stress of these genes, which are expressed within the same parvocellular neuron (1, 32).

Intracellular levels of cAMP are dependent upon the types and levels of adenylyl cyclase and PDE isoforms expressed in a particular cell type. The predominant subtype of adenylyl cyclase present in AtT-20 cells is adenylyl cyclase IX, a subtype susceptible to inhibition by the calcium-dependent Ser/Thr phosphatase, calcineurin (33). It has been shown that elevations in cytosolic calcium inhibit CRH or ß-adrenergic stimulation of cAMP production in a calcineurin-dependent manner (34), and this mechanism may mediate the low levels of intracellular cAMP accumulation observed in the present study. In addition, other factors, such as defective adenylyl cyclase-G protein coupling, may contribute to the low cAMP responses of AtT-20 cells. For example, it has been shown that the affinity of adenylyl cyclase for forskolin is greatly reduced in the absence of Gs{alpha} (35). The fact that other studies (17, 23) reporting effects of cAMP on CRH promoter activation in AtT-20 cells have been performed in the presence of a PDE inhibitor could be consistent with the low cAMP responses observed in this study. Assuming that a deficient cAMP production is responsible for the low CRH promoter activation in AtT-20 cells, we expected that 8Br-cAMP would stimulate promoter activity equally in the two cell lines. However, responses to the cAMP analog were equally blunted in AtT-20 cells. Previous studies have indicated that 8Br-cAMP can stimulate CRH promoter activity (23, 36) and the {alpha}1C subunit of the L-type calcium channel in AtT-20 cell at concentrations 5-fold higher than that those used in our studies (37). This insensitivity to 8Br-cAMP suggested that AtT-20 cells degrade cAMP more efficiently than 4B cells. This possibility was confirmed by data indicating that AtT-20 cells express much higher PDE activity levels than 4B cells. Thus, it is clear from these experiments that cAMP signaling is attenuated in the strain of AtT-20 cells used in these experiments, and that this is due to both deficient cAMP production as well as increases in cAMP degradation by PDE.

The use of subtype-specific PDE inhibitors in these studies made it possible to define PDE subtypes involved in the modulation of cAMP signaling in both 4B and AtT-20 cells. Of the 11 subtypes of PDE known, 3 subtypes evaluated, PDE1, -3, and -4, were present in both cell types. It is of interest that these subtypes could be actually up-regulated by forskolin and/or calmodulin. After stimulation of adenylyl cyclase, increased cAMP and activation of PKA can result in phosphorylation/activation of certain PDE3 and PDE4 isoforms, resulting in feedback regulation of cAMP. Although it is not clear whether activation of PDE1 by calmodulin requires increases in cytosolic calcium, it is possible that increases in cytosolic calcium in these cells result in activation of calmodulin-dependent PDE (37).

Similar to the findings of studies using pituitary corticotrophs (38), the predominant PDE subtypes expressed in 4B cells included the calcium/calmodulin-dependent PDE1 and the cAMP-selective PDE4. As shown by the potentiating effect of rolipram (PDE4 inhibitor) and 8-methyl-IBMX (PDE1 inhibitor) on forskolin-stimulated luciferase activity, both PDE subtypes appear to be biologically relevant in these cells. In AtT-20 cells, PDE1 was also the predominant subtype expressed, but in marked contrast to 4B cells, PDE1 inhibition did not potentiate forskolin-stimulated luciferase. This suggests that this PDE subtype fails to be activated in AtT-20 cells or that it resides in a different spatial or functional compartment or microdomain in these cells (37). As even in the presence of a PDE inhibitor, IBMX, forskolin caused only minor elevations in cAMP in AtT-20 cells, it is possible that the low cAMP levels produced were insufficient to act as a substrate for PDE1 (38). In another report using pituitary corticotrophs, Ang and Antoni (39) found that a PDE1 inhibitor did not alter cAMP responses to CRH, but did enhance responses after treatment with both CRH and VP. Also, in contrast to 4B cells, AtT-20 cells contain levels of PDE3 as high as PDE4, and both subtypes are important in blunting the effects of cAMP, as shown by the need for the combination of the two inhibitors to potentiate forskolin-stimulated luciferase activity.

In summary, the data show that the ability of cAMP to activate a 498-bp CRH promoter fragment is similar in two cell lines regardless of their capacity to express endogenous CRH. Although minor elevations in intracellular cAMP are sufficient for full stimulation of CRH promoter activity, higher increases are required for activation of the VP promoter, suggesting that different sensitivity to cAMP contributes to the differential regulation of both peptides in parvocellular neurons. In addition, AtT-20 cells show an altered cAMP signaling due to both deficient cAMP production and increased cAMP degradation by elevated PDE levels.


   Footnotes
 
Abbreviations: 8Br-cAMP, 8-Bromo-cAMP; CRE, cAMP-responsive element; IBMX, isobutylmethylxanthine; PDE, phosphodiesterase; PKA, protein kinase A; poly(A)+, polyadenylated; PVN, paraventricular hypothalamic nucleus; VP, vasopressin.

Received September 20, 2002.

Accepted for publication December 31, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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