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Endocrinology Vol. 142, No. 7 3041-3050
Copyright © 2001 by The Endocrine Society

ARTICLES

Cyclic Adenosine 3',5'-Monophosphate Inhibits Insulin-Like Growth Factor I Gene Expression in Rat Glioma Cell Lines: Evidence for Regulation of Transcription and Messenger Ribonucleic Acid Stability1

Lai Wang and Martin L. Adamo

Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900

Address all correspondence and requests for reprints to: Dr. Martin L. Adamo, Department of Biochemistry, Mail Code 7760, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78229-3900. E-mail: adamo{at}biochem.uthscsa.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
cAMP inhibits growth and stimulates differentiation in glioma cells. We examined the effect of cAMP on insulin-like growth factor I (IGF-I) gene expression in the C6 cell line, a rat glioma cell line previously reported to grow in response to autocrine IGF-I. cAMP potently inhibited IGF-I messenger RNA (mRNA) and peptide secretion in C6 cells, associated with an attenuation of DNA synthesis. Exogenous IGF-I peptide at least partially prevented the inhibition of DNA synthesis, suggesting that the reduction in IGF-I biosynthesis may contribute to the inhibitory effect of cAMP on C6 cell growth. cAMP also inhibited IGF-I mRNA in rat RG2 glioma cells, but not in three other nonglioma tumor cell lines. The nuclear IGF-I pre-mRNA level and the half-life of mature IGF-I mRNA were both reduced by cAMP in C6 cells, suggesting effects on gene transcription and mRNA stability. However, cAMP had no effect on the activities of IGF-I exon 1 promoter-luciferase constructs. Protein synthesis inhibition partially reduced the inhibition of IGF-I mRNA by cAMP. Inhibition of cAMP-activated protein kinase A activity by H89 did not alter the inhibition of IGF-I gene expression in response to cAMP, suggesting that protein kinase A does not mediate the cAMP inhibitory effect on IGF-I gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MALIGNANT GLIOMAS represent as many as 50% of all brain tumors and are associated with a high incidence of morbidity and mortality. There are low levels of intracellular adenylate cyclase activity and cAMP in brain tumors compared with those in normal brain tissue (1), suggesting that the relative inability of brain tumors to generate cAMP may be important for the progression of brain tumors. In support of this idea are the observations that increasing intracellular cAMP levels can suppress the mitogenic responses of human astrocytoma cells to growth factors (2) and that cAMP inhibits new DNA synthesis of rat C6 glioma cells (3). In addition to the inhibitory effect on cell growth, cAMP has been reported to induce differentiation in glioma cells, including C6 cells (4, 5). Therefore, modulation of cAMP-dependent signaling pathways may represent a possible approach for treating malignant gliomas. However, the mechanisms by which cAMP regulates glioma growth are not yet well understood. Moreover, to our knowledge, there is only one report about the effect of cAMP on autocrine growth factor production in gliomas (6), which may influence their tumorigenesis and metastasis.

Both insulin-like growth factor I (IGF-I) and IGF-II mRNAs and peptides have been identified in gliomas (7, 8, 9, 10). There are reports showing that IGF-I mRNA and peptide levels are higher in gliomas than in normal tissue (7, 9), suggesting that IGF-I may contribute to the tumorigenicity of the gliomas by acting as an autocrine growth factor. Rat C6 glioma cells have been extensively used as a glioma cell model in studies related to tumor cell biology (11). IGF-I has been suggested to be an important autocrine mitogen in C6 cells (12, 13). Inhibition of IGF-I gene expression using antisense technology leads to the loss of tumorigenicity when C6 cells are implanted in animals (14). Furthermore, inhibition of IGF-I receptor gene expression using antisense oligonucleotides, stably transfected antisense plasmid or a triple helix strategy inhibits the growth of C6 cells both in vivo and in vitro, induces apoptosis, and decreases tumor progression (15, 16, 17, 18). Mutant forms of the IGF-I receptor also decrease proliferation and induce apoptosis of C6 cells (19, 20).

Modulation of IGF-I gene expression by cAMP has been observed in a variety of cell culture models (21, 22, 23, 24, 25, 26, 27, 28). Signaling mechanisms by which these effects occur are largely uncharacterized, with the exception of the recently characterized stimulation of IGF-I transcripts by cAMP in osteoblasts, which is mediated by CCAAT/enhancer-binding protein {delta} (C/EBP{delta}) (29). The regulation of IGF-I gene expression in response to cAMP in gliomas has never been reported. In this study we used C6 cells as a glioma cell model to study the influence of cAMP on IGF-I gene expression and cell growth.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Rat C6 glioma cells, human SK-N-MC neuroblastoma cells, human OVCAR-3 ovarian carcinoma cells, and rat GH3 pituitary adenoma cells were obtained from American Type Culture Collection (Manassas, VA) and were cultured as previously described (30, 31). Rat RG2 glioma cells were also obtained from American Type Culture Collection and were cultured in DMEM with 4.5 g/liter glucose, 4 mM glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% FBS. All cell lines were grown to confluence, followed by 24-h incubation in appropriate medium with 1% FBS for C6 cells, GH3 cells, SK-N-MC cells, and OVCAR-3 cells or with 0.1% BSA for RG2 cells in place of 10% FBS. Cells were treated with 8-(4-chloropenylthio)-cAMP (8-CPT-cAMP; Sigma, St. Louis, MO), a cell-permeable cAMP analog, isoproterenol (Sigma); or forskolin (Sigma) in appropriate medium. Cells were then harvested at the indicated times for total RNA extraction. For mRNA stability studies, C6 cells were preincubated for 3 h with or without 100 µM 8-CPT-cAMP before the addition of 75 µM 5,6-dichloro-1ß-D-ribofuranosylbenzimidazole (DRB; Sigma), which is a RNA polymerase II inhibitor. In some experiments C6 cells were treated with 8-CPT-cAMP and DRB simultaneously. After 12 h of incubation fresh DRB was added into the conditioned medium. C6 cells were harvested after 0, 3, 6, 12, and 24 h of DRB treatment. For treatment with cycloheximide (Sigma) or H89 (Calbiochem, San Diego, CA), cells were cultured as described in figure legends.

Total RNA extraction and RNase protection assays (RPAs)
Total RNA was prepared using the Ultraspec reagent (Tel-Test, Inc., Friendswood, TX). RNA concentrations were determined using the absorbance at 260 nm. Antisense RNA probes were labeled and synthesized using either the MaxiScript kit from Ambion, Inc. (Austin, TX), or the protocol described previously (32) with reagents from Promega Corp. (Madison, WI) and Ambion, Inc. [{alpha}32p]-UTP (800 Ci/mmol) used for labeling was obtained from NEN Life Science Products (Boston, MA). Solution hybridization/ribonuclease protection assays were conducted using either the RPA II kit from Ambion, Inc., or the protocol described previously (32) with reagents supplied by Ambion, Inc. The intron 1-specific antisense RNA probe used to measure rat IGF-I pre-mRNA was described previously (33), as was the insulin receptor antisense RNA probe used to detect rat insulin receptor pre-mRNA (34). A protected band of about 500 bases was obtained in the RPA, reflecting IGF-I pre-mRNA and a protected band of about 680 bases was obtained in the RPA, reflecting insulin receptor pre-mRNA. All of the other antisense RNA probes used in this study to measure levels of rat IGF-I, human IGF-I, rat IGF-I receptor, rat ß-actin, and human ß-actin mature mRNA were described previously (31). Rat IGF-I and human IGF-I antisense probes were constructed differently (31), in that the rat probe contains contiguous exon 2, exon 3, and a portion of exon 4, whereas the human probe is based on the complementary DNA to exon 1 containing mRNA. Thus, the rat exon 1 containing mRNA protects a shorter fragment of probe than does rat exon 2 containing mRNA, whereas the reverse is true when using the human probe and IGF-I mRNA from human cells.

IGF-I RIA
The IGF-I RIAs were performed by Dr. Clifford J. Rosen and Julie Burgess at the Maine Center for Osteoporosis Research and Education Laboratory (Bangor, ME) using a protocol described previously (35), which was a modification of another protocol (36).

Nuclear RNA preparation
C6 cells were treated with or without 100 µM 8-CPT-cAMP for 24 h. Nuclei were prepared as described previously (37). In brief, cells were washed with PBS and harvested in lysis buffer [10 mM Tris-Cl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40]. Lysates were examined under the microscope to confirm the presence of free nuclei. Nuclei were pelleted at 500 x g, and nuclear RNA was extracted from nuclear pellets using the Ultraspec reagent.

Transient transfection assay
Before transient transfection, C6 cells were grown to confluence, followed by 24-h incubation in Ham’s F-12 medium with 1% FBS. Transient transfection was performed using 2 µg pGL2-Basic DNA or equal molar amounts of pGL2-Control or IGF-I promoter/luciferase fusion constructs, as previously described (38), with the Lipofectamine Plus system in Opti-MEM medium (Life Technologies, Inc., Gaithersburg, MD). Three hours after transfection, Opti-MEM medium was replaced with Ham’s F-12 medium containing 1% FBS with or without 100 µM 8-CPT-cAMP. Twenty-four hours later, cellular lysates were prepared and were assayed for luciferase activity and protein concentration as described previously (31, 38).

Protein kinase A (PKA) activation assay
The PKA activation assay has been described previously (39). In brief, cells were collected and lysed in 100 µl HP buffer consisting of 10 mM potassium phosphate (pH 6.8), 1 mM ß-mercaptoethanol, 10 µg/ml leupeptin, 10 mM magnesium acetate, 10 µM ATP containing 5 x 105 cpm [{gamma}-32P]ATP (6,000 Ci/mmol, NEN Life Science Products), and 300 µg/ml Kemptide substrate (Sigma). A parallel plate was harvested at each time point in HP buffer without Kemptide. Reaction mixtures were incubated for 5 min at 30 C and then spotted onto Whatman P-81 paper (Whatman, Maidstone, UK). The filters were washed in 75 mM phosphoric acid twice for 1 min each time. 32P incorporation was determined by liquid scintillation counting. The level of 32P incorporation of the samples without Kemptide was subtracted from that of the samples with Kemptide at each time point to obtain specific Kemptide phosphorylation.

DNA synthesis
C6 cells were plated in 48-well cell culture clusters (Corning, Inc. Corning, NY) at 5 x 104 cells/well in complete Ham’s F-12 medium and were cultured for 24 h, followed by another 24-h incubation in Ham’s F-12 medium with 1% FBS in place of 10% FBS. Then cells were treated with 10 or 100 µM 8-CPT-cAMP or 1 µM isoproterenol in the presence or absence of IGF-I peptide (Austral Biologicals, San Ramon, CA) in Ham’s F-12 medium containing 1% FBS. Twenty-four hours later, 1 µCi [methyl-3H]thymidine (NEN Life Science Products, Boston, MA) was added to each well. After 4-h incubation, C6 cells were washed three times with PBS and incubated with 250 µl 10% trichloroacetic acid for 15 min on ice. The trichloroacetic acid precipitates were solubilized by adding 200 µl 0.1 M NaOH and were placed on ice for 10 min, followed by washing the well with 200 µl 0.1 M HCl. The amount of [3H]thymidine incorporated was measured by scintillation counting.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
cAMP inhibits IGF-I gene expression in C6 and RG2 glioma cells
Confluent C6 cells were treated with isoproterenol, a ß-adrenergic receptor agonist. As shown in Fig. 1Go, A and B, the IGF-I mRNA level was potently inhibited by isoproterenol in a dose-dependent manner after 24 h of treatment. IGF-I mRNA was reduced by 90% when cells were treated with isoproterenol at a dose of 0.01 µM or more. In contrast, the ß-actin mRNA level was relatively constant (Fig. 1AGo). The down-regulation of IGF-I gene expression in response to 1 µM isoproterenol began to appear as early as 1 h of treatment and was maximal at 12 h (Fig. 1Go, C and D). During the 48-h incubation, isoproterenol did not change the level of the ß-actin transcripts (Fig. 1CGo). These data suggest that the effect of isoproterenol on IGF-I gene expression in C6 cells is rapid and potent. Consistent with these results, a synthetic cAMP analog, 8-CPT-cAMP, also down-regulated IGF-I mRNA in a dose-dependent manner after 24 h of treatment. 8-CPT-cAMP (100 µM) almost extinguished IGF-I mRNA (Fig. 2Go). In contrast, IGF-I receptor and ß-actin mRNA levels were relatively constant (Fig. 2Go). Moreover, forskolin, an adenylate cyclase activator, also inhibited IGF-I mRNA expression in C6 cells (data not shown). IGF-I peptide levels in the conditioned medium measured by RIA were reduced from 2.91 ± 0.28 to 1.06 ± 0.19 ng/ml (P < 0.01) after 24 h of 100 µM 8-CPT-cAMP treatment (data are the mean ± SEM for five separate experiments).



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  		Figure 1. Isoproterenol effect on IGF-I and ß-actin mRNA levels. A and B, C6 cells were harvested after 24 h of treatment with the indicated concentration of isoproterenol. C and D, C6 cells were treated with (+) or without (-) 1 µM isoproterenol and harvested at the indicated time points. The autoradiographs of RPAs are shown in A and C. B and D, Quantification of the IGF-I mRNA level, expressed as a percentage of that in untreated cells. Each data point is the mean ± SEM for three separate experiments, each performed on a single plate of cells.

 


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  		Figure 2. cAMP effect on IGF-I, IGF-I receptor, and ß-actin mRNA levels. Confluent C6 cells were treated with 0, 10, 30, or 100 µM 8-CPT-cAMP for 24 h. Cells were then harvested for total RNA extraction. The autoradiographs of RPAs are shown in A. B, Quantified mRNA levels, expressed as a percentage of those in untreated cells. Each data point is the mean ± SEM for two separate experiments, each performed on a single plate of cells.

 
Another rat glioma cell line, RG2, expressed mainly exon 1-containing transcripts, but unlike C6 cells, RG2 cells also expressed very low levels of exon 2-containing transcripts (Fig. 3AGo). 8-CPT-cAMP (100 µM) and forskolin (10 µM) inhibited exon 1-containing mRNA by 65% and 61%, respectively, in RG2 cells (Fig. 3Go). However, 1 µM isoproterenol did not alter the IGF-I mRNA level (Fig. 3Go). The level of exon 2-containing mRNA was too low to permit accurate determination of the percent inhibition by cAMP.



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  		Figure 3. cAMP effect on IGF-I and ß-actin mRNA levels in RG2 cells. Confluent RG2 cells were treated without (control) or with 100 µM 8-CPT-cAMP, 1 µM isoproterenol, or 10 µM forskolin. Cells were then harvested 24 h later for total RNA extraction. Exon 1 represents IGF-I mRNA transcribed from the exon 1 promoter. Exon 2 represents IGF-I mRNA transcribed from the exon 2 promoter. The autoradiographs of RPAs of IGF-I mRNA and ß-actin mRNA are shown in A. Exon 1 containing mRNA was quantified and is shown in B as a percentage of that in untreated cells. Each data point is the mean ± SEM for four separate experiments, each performed on a single plate of cells.

 
The effect of cAMP on IGF-I gene expression is not general to all tumor cells
To determine whether the regulation of IGF-I gene expression in response to cAMP is general to tumor cells, three nonglioma tumor cell lines, human SK-N-MC neuroblastoma cells, human OVCAR-3 ovarian carcinoma cells, and rat GH3 pituitary adenoma cells, were studied. In SK-N-MC cells, neither isoproterenol nor 8-CPT-cAMP altered IGF-I mRNA (Fig. 4Go, A and B). In OVCAR-3 cells, both 100 µM 8-CPT-cAMP and 1 µM isoproterenol stimulated IGF-I mRNA by approximately 2-fold (Fig. 4Go, C and D). In GH3 cells, IGF-I mRNA was stimulated by approximately 30% after 100 µM 8-cAMP treatment, whereas isoproterenol did not alter the IGF-I mRNA level (Fig. 4Go, E and F). These data suggest that the inhibition of IGF-I mRNA by cAMP treatment is probably not general to all tumor cells, but may be glioma cell specific.



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  		Figure 4. cAMP effect on IGF-I and ß- actin mRNA levels in SK-N-MC cells, OVCAR-3 cells, and GH3 cells. Cells were treated without (control) or with 100 µM 8-CPT-cAMP or 1 µM isoproterenol. Cells were then harvested 24 h later for total RNA extraction. Exon 1 represents IGF-I mRNA transcribed from the exon 1 promoter. Exon 2 represents IGF-I mRNA transcribed from the exon 2 promoter. The results from SK-N-MC cells are shown in A and B. The results from OVCAR-3 cells are shown in C and D. The results from GH3 cells are shown in E and F. The autoradiographs of RPAs are shown in A, C, and E. B, Quantified IGF-I mRNA level as a percentage of the exon 1-containing mRNA in untreated cells. D and F, Quantified IGF-I mRNA level as a percentage of the exon 2-containing mRNA in untreated cells. Each data point is the mean ± SEM for three separate experiments, each performed on a single plate of cells.

 
cAMP reduces the abundance of IGF-I pre-mRNA
Measuring the pre-mRNA level in nuclear RNA preparations has been used to determine changes in IGF-I gene transcription, and results are consistent with those obtained from the nuclear run-on assay (40). Nuclear RNA from C6 cells was prepared, and the IGF-I pre-mRNA level was measured by RPAs using a probe containing portions of exon 1 and intron 1. A 96% reduction of IGF-I pre-mRNA was observed when C6 cells were treated with 100 µM 8-CPT-cAMP (Fig. 5Go), suggesting that cAMP potently inhibits IGF-I gene transcription. In contrast, the insulin receptor pre-mRNA level was not significantly altered by cAMP treatment (Fig. 5Go).



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  		Figure 5. cAMP effect on IGF-I and insulin receptor pre-mRNA levels. Cells were treated without (control) or with 100 µM 8-CPT-cAMP for 24 h, followed by extraction of nuclear RNA. The autoradiographs of RPAs of IGF-I pre-mRNA and insulin receptor pre-mRNA are shown in A. Quantified results are shown in B. Each data point is the mean ± SEM for four separate experiments, each performed on a single plate of cells.

 
Transient transfection experiments were performed with rat IGF-I exon 1 promoter/luciferase fusion constructs to map potential cAMP-response element(s) in C6 cells. However, the promoter activities of the constructs containing 1500, 1000, or 500 bp of the 5'-flanking region and 319 bp of the exon 1 sequence were not altered by cAMP treatment in C6 cells (Fig. 6Go). These data suggest that the IGF-I promoter region from -1500 to +319 (+1 is designated the most 5' exon 1 start site) does not contain cAMP-response element(s) in C6 cells.



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  		Figure 6. cAMP effect on IGF-I exon 1 promoter activity. C6 cells were transfected with IGF-I exon 1 luciferase fusion constructs and then treated without (-) or with (+) 100 µM 8-CPT-cAMP. The luciferase activities were determined after 24 h, normalized to protein concentration, and expressed as fold increase over pGL2-Basic. The promoter activity of the pGL2-control is shown at 0.01x to fit in the same graph. Each data point is the mean ± SEM for three separate experiments, each performed on a single plate of cells.

 
IGF-I mRNA half-life was reduced by cAMP treatment
To determine whether IGF-I mRNA stability was altered by cAMP treatment, C6 cells were treated with the RNA polymerase II inhibitor, DRB. As shown in Fig. 7Go, A and B, IGF-I mRNA decreased faster in the cAMP-treated cells compared with the untreated cells. The IGF-I mRNA half-life was calculated by fitting the standard exponential decay equation. The IGF-I mRNA half-life was 15.2 h in the absence of cAMP and 7 h in the presence of 100 µM 8-CPT-cAMP, i.e. cAMP caused a 54% reduction in IGF-I mRNA half-life. When cells were treated with cAMP and DRB simultaneously, similar results were obtained (data not shown).



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  		Figure 7. cAMP effect on IGF-I mRNA stability and requirement of protein synthesis for the response of IGF-I mRNA to cAMP treatment. For the mRNA stability study (A and B), C6 cells were treated with (+) or without (-) 100 µM 8-CPT-cAMP 3 h before the addition of 75 µM DRB. Total RNA was extracted at the indicated time points after the addition of DRB. For the protein synthesis inhibitor study (C and D), confluent C6 cells were treated with (+) or without (-) 100 µM 8-CPT-cAMP in the presence (+) or absence (-) of 1 µg/ml cycloheximide. Twenty-four hours later, total RNA was extracted. The autoradiographs of RPAs are shown in A and C. B and D, Quantified IGF-I mRNA level as a percentage of that in untreated cells (control). For the mRNA stability study, each data point is the mean ± SEM for two separate experiments, each performed on a single plate of cells. For the protein synthesis inhibitor study, each data point is the mean ± SEM for five separate experiments, each performed on a single plate of cells.

 
Protein synthesis is required for the full inhibitory effect of cAMP on IGF-I gene expression
To determine whether on-going protein synthesis is required for the inhibition of IGF-I in response to cAMP, cycloheximide, a protein synthesis inhibitor, was used to treat C6 cells. Cycloheximide reduced IGF-I mRNA by 18% in the absence of cAMP (Fig. 7Go, C and D). cAMP reduced IGF-I mRNA by 94% in the absence of cycloheximide, but decreased IGF-I mRNA by 72% in the presence of cycloheximide (Fig. 7Go, C and D). These results indicate that on-going protein synthesis is required for the full inhibition of IGF-I mRNA by cAMP.

The influence of on-going protein synthesis on IGF-I gene transcription and mRNA stability was also determined. Cycloheximide potently inhibited the IGF-I pre-mRNA level by 85% in the absence of cAMP (Fig. 8Go, A and B), suggesting that on-going protein synthesis is required for basal IGF-I gene transcription. Although the reduction of IGF-I pre-mRNA in response to cAMP was largely decreased by cycloheximide, there was still a 60% decrease in the IGF-I pre-mRNA level in response to cAMP in the presence of cycloheximide (Fig. 8Go, A and B). Therefore, the effect of cAMP on IGF-I transcription requires, but does not fully depend upon, new protein synthesis. When transcriptionally arrested C6 cells were treated with cycloheximide, the inhibitory effect of cAMP on IGF-I mRNA stability was completely eliminated (Fig. 8Go, C and D). Therefore, on-going protein synthesis is essential for cAMP to reduce the IGF-I mRNA half-life. In the absence of cAMP, cycloheximide had no effect on the IGF-I mRNA level in transcriptionally arrested cells (Fig. 8Go, C and D), suggesting that under these conditions, cycloheximide does not affect IGF-I mRNA stability.



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  		Figure 8. The influence of on-going protein synthesis on IGF-I gene transcription and mRNA stability. A and B, C6 cells were treated with (+) or without (-) 100 µM 8-CPT-cAMP in the presence (+) or absence (-) of 1 µg/ml cycloheximide. Twenty-four hours later, cells were harvested for nuclear RNA. C and D, C6 cells were treated with 75 µM DRB in the presence (+) or absence (-) of 100 µM 8-CPT-cAMP without (-) or with (+) 1 µg/ml cycloheximide. Twenty-four hours later, cells were harvested for total RNA. The autoradiographs of RPAs are shown in A and C. The quantified IGF-I mRNA level is shown in B and D as a percentage of that in untreated cells (control). For the pre-mRNA study, each data point is the mean ± SEM for three separate experiments, each performed on a single plate of cells. For the mRNA stability study, each data point is the mean ± SEM for four separate experiments, each performed on a single plate of cells.

 
cAMP inhibits IGF-I mRNA in a PKA-independent manner
As shown in Fig. 9AGo, cAMP potently stimulated PKA activity after 30 min of treatment. This stimulation was less, but still significant, after 3 and 24 h of cAMP treatment. Pretreatment with H89, a PKA inhibitor, prevented PKA activation by cAMP at all three time points. Total RNA was prepared from parallel plates. The IGF-I mRNA level was inhibited by cAMP to the same extent in the presence of H89 as in the absence of H89 (Fig. 9Go, B and C), suggesting that PKA does not mediate the inhibitory effect of cAMP on IGF-I gene expression in C6 cells.



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  		Figure 9. cAMP inhibits IGF-I gene expression in a PKA-independent manner. Confluent C6 cells were pretreated with (+) or without (-) 5 µM H89 for 3 h and were then treated with (+) or without (-) 100 µM 8-CPT-cAMP. A, Cells were harvested after 30 min, 3 h, and 24 h for PKA activity assays as described in Materials and Methods. As cells after 30 min of cAMP treatment exhibited highest PKA activity, this value was normalized as 100% in each experiment. Parallel plates were harvested 24 h later for total RNA. The autoradiograph of RPA is shown in B, and the quantified IGF-I mRNA level is shown in C as a percentage of that in untreated cells (control). Each data point is the mean ± SEM for three separate experiments, each performed on a single plate of cells.

 
Exogenous IGF-I can prevent the inhibitory effect of cAMP on C6 cell growth
[3H]Thymidine incorporation assays were performed in the presence or absence of 8-CPT-cAMP or isoproterenol and with or without exogenous IGF-I. 8-CPT-cAMP (10 µM), 100 µM 8-CPT-cAMP, and 1 µM isoproterenol inhibited DNA synthesis by 41%, 88%, and 65%, respectively (Fig. 10Go). Exogenous IGF-I peptide at 10 nM totally overcame the attenuation of DNA synthesis caused by isoproterenol and 10 µM 8-CPT-cAMP, and it partially prevented the profound inhibition of DNA synthesis caused by 100 µM 8-CPT-cAMP (Fig. 10Go). The effects of cAMP and IGF-I on C6 cell growth were confirmed by measuring the change in cell number. As shown in Fig. 11Go, there was no significant difference in the growth curves of C6 cells cultured in 1% FBS or 1% FBS plus 100 nM IGF-I. In contrast, cAMP inhibited C6 cell proliferation after 2–3 days, and exogenous IGF-I partially prevented the decrease in cell number caused by cAMP (Fig. 11Go).



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  		Figure 10. cAMP effect on DNA synthesis. C6 cells were treated without (control) or with 10 or 100 µM 8-CPT-cAMP or 1 µM isoproterenol and the indicated concentration of IGF-I peptide. After 24 h, cells were labeled with [3H]thymidine for another 4 h. The [3H]thymidine incorporation rate is shown as percentage of that in untreated cells (control, 0 nM IGF-I). Each data point is the mean ± SEM for three separate experiments, each performed on duplicate wells of cells.

 


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  		Figure 11. cAMP effect on cell number. C6 cells were plated at a density of 0.5 million cells/60-mm plate. Cells were grown in Ham’s F-12 medium with 1% FBS in the presence or absence of 100 µM 8-CPT-cAMP and with or without 100 nM IGF-I. Fresh 8-CPT-cAMP and IGF-I were added every 24 h. Cell numbers were counted on 3 consecutive days. Each data point is the mean ± SEM for two separate experiments, each performed on a single plate of cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The effect of cAMP on IGF-I gene expression has been extensively studied in various cell lines. In most studies, including those using testicular cells, osteoblasts, hepatocytes, ovarian granulosa cells, thyroid cells, and fetal dermal fibroblasts, IGF-I mRNA levels are increased in response to cAMP, or in response to hormones whose second messenger is cAMP (21, 22, 23, 24, 27, 28). There are only two reports showing that IGF-I mRNA is inhibited in response to cAMP: in Leydig cells and in mouse macrophages (25, 26). As shown in the present study, the inhibition of IGF-I mRNA by cAMP in C6 cells is much more potent than in those two previous reports. A high dose of 8-CPT-cAMP almost completely extinguished IGF-I mRNA in C6 cells. Furthermore, the effect of cAMP is quite rapid, suggesting that the reduction of IGF-I gene expression is one of the early responses of C6 cells after cAMP treatment. To our knowledge, this is the first report showing the regulation of IGF-I gene expression by cAMP in tumor cells. Our results indicate that this inhibitory effect of cAMP on IGF-I gene expression is also tumor cell type specific. Similar to C6 cells, both the cAMP analog and forskolin inhibited IGF-I gene expression in another rat glioma cell line, RG2. Although isoproterenol reduced IGF-I mRNA in C6 cells, it was ineffective in RG2 cells. We do not know the reason for this result. Possibilities include a lower level of ß-adrenergic receptors on RG2 cells and/or decreased coupling of those receptors to adenylate cyclase. In contrast, cAMP either slightly induced or had no effect on IGF-I gene expression in human SK-N-MC neuroblastoma cells, human OVCAR-3 ovarian carcinoma cells, and rat GH3 pituitary adenoma cells. A tumor cell type-specific expression of signaling intermediate(s) and/or factors that regulate transcription, processing, and/or stability of IGF-I mRNA may play a role in this regulation.

To determine the mechanism by which cAMP regulates IGF-I gene expression in C6 cells, we characterized the influence of cAMP on both gene transcription and mRNA stability. Our data suggest that cAMP inhibits IGF-I gene transcription and reduces IGF-I mRNA stability in C6 cells. In Leydig cells, cAMP slightly inhibited IGF-I gene transcription (25). In contrast to the observations in C6 cells, IGF-I mRNA stability was not altered by cAMP in Leydig cells. Apparently, different mechanisms are used by cAMP to inhibit IGF-I gene expression in C6 and Leydig cells. In another report (26), Fournier et al. showed that in macrophages, cAMP or PGE2 reduced IGF-I mRNA half-life from 15 h to 6 h, which is similar to the reduction of IGF-I mRNA half-life caused by cAMP in C6 cells, i.e. from 15.2 h to 7 h. However, there is no evidence in that report showing a change in the gene transcription rate. Moreover, in macrophages, although IGF-I mRNA expression was down-regulated by cAMP, IGF-I peptide biosynthesis was elevated after cAMP or PGE2 treatment (26). An enhancement of the translation efficiency of the remaining mRNA after cAMP treatment was suggested. In this study we report that the IGF-I peptide level in the C6 cell-conditioned medium was reduced after addition of cAMP, which correlates with decreased IGF-I mRNA levels.

The results of experiments using cycloheximide suggest that on-going protein synthesis is required for the full inhibitory effect of cAMP on the IGF-I mRNA level in C6 cells. In contrast, in transcriptionally arrested C6 cells, inhibition of protein synthesis completely prevented the ability of cAMP to inhibit IGF-I mRNA levels, suggesting that the reduction in IGF-I mRNA stability caused by cAMP in C6 cells is totally dependent on new protein synthesis. In addition, it is possible that cAMP alters the transcription of some genes that may be involved in the degradation of IGF-I mRNA. To test this possibility, DRB was added to C6 cell cultures at the same time as cAMP to block any cAMP-induced transcription. However, even in these transcriptionally arrested cultures, cAMP caused a similar reduction in IGF-I mRNA stability as that observed when C6 cells were preincubated with cAMP before transcriptional arrest. Thus, we would hypothesize that cAMP induces the synthesis of the protein(s), but not the transcription of the gene(s), important in IGF-I mRNA degradation. Our data also suggest that the effect of cAMP on IGF-I gene transcription requires, but does not fully depend upon, new protein synthesis. Treatment with cycloheximide caused a potent reduction of the abundance of IGF-I pre-mRNA in the absence of cAMP, whereas it had a small effect on IGF-I mature mRNA levels. This is probably due to the fact that cycloheximide itself has no effect on IGF-I mRNA stability and that IGF-I mRNA has a long half-life, i.e. 15.2 h. Therefore, even when the IGF-I pre-mRNA level was potently decreased by cycloheximide, without altering mRNA stability, there will still be a large portion of preexisting IGF-I mRNA after 24 h of treatment with cycloheximide.

In the present study we were unable to identify any cAMP responsiveness in 1.8 kb of the IGF-I exon 1 proximal promoter region. In control experiments using fetal rat osteoblasts, the luciferase activity of the IGF-I exon 1 promoter construct -1500/+319 was stimulated by forskolin, whereas the luciferase activity of another construct extending from -133 to +75 was not altered by forskolin (data not shown). These results are consistent with the report by McCarthy et al. (41). In osteoblasts, treatment with a cAMP-elevating reagent, prostaglandin E2 (PGE2), causes the nuclear translocation of CCAAT/enhancer-binding protein {delta} (C/EBP{delta}) protein in a PKA-dependent manner, which trans-activates the IGF-I exon 1 promoter by binding to a site located approximately 192 nucleotides downstream of the first exon 1 transcription start site (29). It is possible that in C6 cells, the functional suppressive cAMP response element(s) is not located in this region. Alternatively, chromatin structure may be important for suppression of IGF-I transcription by cAMP in C6 cells, and this cannot be assessed by transient transfection assays. Interestingly, when IGF-I exon 1 promoter constructs were assessed in dermal fibroblasts in which IGF-I exon 1-containing mRNA was induced by a cAMP-elevating reagent, PTH-related protein (PTHrP), no cAMP response was detected on the IGF-I exon 1 promoter (28).

As PKA was activated by increasing the intracellular cAMP level, we asked whether PKA mediates the inhibition of IGF-I gene expression in response to cAMP. However, when the PKA-specific inhibitor H89 was used in the combination with cAMP, the IGF-I mRNA level was still inhibited to the same extent by cAMP as in the absence of H89. The PKA activity assays demonstrated that cAMP activated PKA rapidly and that the effect was sustained for at least 24 h. Moreover, H89 did prevent PKA activation by cAMP. These data suggest that cAMP may act in a non-PKA-dependent fashion to alter IGF-I gene expression in C6 cells. Similar to our observation, another report showed that cAMP induces glial fibrillary acidic protein expression in a PKA-independent manner in C6 cells (42). Recently, two groups independently reported that cAMP can directly activate several guanine nucleotide exchange factors (Epac) to alter rap-1 activity (43, 44). In addition, stimulation of extracellular signal- regulated kinases in melanocytes and stimulation of the phosphatidylinositol 3-kinase/Akt pathway in thyroid cells by cAMP are demonstrated to be independent of PKA (45, 46).

An autocrine IGF-I loop has been suggested to be important for C6 cell growth, survival, and tumorigenesis (12, 13, 14, 15, 16, 17, 18, 19, 20). The results of the present study showed that exogenous IGF-I can at least partially overcome the inhibition of C6 cell growth caused by cAMP. These results support the hypothesis that the reduction of endogenous IGF-I biosynthesis may play a role in the inhibitory effect of cAMP on C6 cell growth. At the low dose of 8-CPT-cAMP or isoproterenol, IGF-I totally prevented the inhibition of DNA synthesis, whereas only a partial rescue was observed when a higher dose of 8-CPT-cAMP was used. In addition to IGF-I, a family of IGF-binding proteins (IGFBPs) may be involved in the inhibition of C6 cell growth in response to cAMP. We observed that cAMP markedly changed the profile of IGFBP gene expression and protein secretion in C6 cells (Wang, L., et al., manuscript submitted). Depending on the particular IGFBP, how they are modified, and the specific cell lines, IGFBPs can inhibit or potentiate IGF-I action (47). IGFBPs can have IGF-I-independent stimulatory or inhibitory effects on cell growth as well (47). Therefore, the changes in IGFBP gene expression may contribute to the inability of exogenous IGF-I to fully overcome the inhibition of DNA synthesis caused by cAMP. Furthermore, the possibility that other factors or signaling molecules besides IGF-I and IGFBPs are required for the full effect of cAMP on C6 cell growth cannot be excluded.

In summary, cAMP inhibits IGF-I biosynthesis in rat C6 and RG2 glioma cells. This effect is rapid, potent, and cell type specific. The reduction in IGF-I gene expression in C6 cells occurs at both the transcriptional and mRNA stability levels. The addition of exogenous IGF-I can at least partially overcome the inhibitory effect of cAMP on C6 cell growth. Over the past few years, modulation of cAMP-regulated signal transduction has attracted increasing attention in antiglioma therapy (48). Understanding how cAMP acts in glioma cells may provide a rational mechanistic basis for developing new therapeutic targets.


   Acknowledgments
 
We thank Dr. Clifford J. Rosen and Julie Burgess at the Maine Center for Osteoporosis Research and Education Laboratory (Bangor, ME) for performing IGF-I RIAs. We thank Dr. John C. Lee (UTHSCSA) for providing fetal rat osteoblasts. We also thank Xiuye Ma for her excellent technical support.


   Footnotes
 
1 This work was supported by Grant DK-47357 from the NIDDK, NIH; Grant AQ-1385 from the Robert A. Welch Foundation; and Grant 07 from the Children’s Cancer Research Center of University of Texas Health Science Center. A portion of these studies was presented in Abstract 1004 at the 82nd Annual Meeting of The Endocrine Society, Toronto, Canada, 2000. Back

Received November 21, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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