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Endocrinology Vol. 142, No. 9 3917-3925
Copyright © 2001 by The Endocrine Society

ARTICLES

Differential Regulation of IGF-Binding Protein Gene Expression by cAMP in Rat C6 Glioma Cells

Lai Wang, Xiuye Ma, Lee-Chuan C. Yeh 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
 
We previously reported that cAMP inhibits autocrine IGF-I gene expression in rat C6 glioma cells. In this study we examined the influence of cAMP on IGF-binding protein gene expression in C6 cells. cAMP potently inhibited IGF-binding protein-3 mRNA and, to a lesser extent, IGF-binding protein-4 mRNA and transiently stimulated IGF-binding protein-5 mRNA. The changes in secreted IGF-binding proteins whose molecular weights were consistent with IGF-binding protein-3 and -5 correlated with those of mRNA levels. cAMP decreased the IGF-binding protein-3 mRNA half-life, but did not alter IGF-binding protein-4 and -5 mRNA half-lives. An IGF-binding protein-5 promoter/luciferase fusion construct containing 888 bp of 5'-flanking sequence and the first 114 bp of exon 1 sequence was stimulated by cAMP after 24 h by approximately 2-fold in transient transfection assays. 5'- or 3'-deletion to -33 or +10 (the transcription start site was designated as +1), respectively, did not alter the increase caused by cAMP. Site-directed mutagenesis of the region from -14 to -5 led to a loss of the ability of the IGF-binding protein-5 promoter to respond to cAMP. H89, a cell-permeable protein kinase A inhibitor, did not alter the regulation of IGF-binding protein mRNAs in response to cAMP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLIOMAS ARE TUMORS that originate from the glial cells of brain tissue. Patients with glioblastoma (the most common and the most malignant form) have a very poor prognosis, with average survival of less than 1 yr (1). Although a number of treatment strategies have been attempted, the overall survival of patient with malignant gliomas has not been significantly improved (2). Over the past few years, modulation of cAMP-dependent signal transduction has attracted increasing attention in antiglioma therapy (3). cAMP has been shown to inhibit growth and induce differentiation of glioma cells (4, 5, 6, 7). However, the mechanisms by which cAMP regulates glioma growth are not yet well understood. Rat C6 glioma cells have been extensively used as a glioma cell model in studies related to tumor cell biology (8). An autocrine IGF-I loop has been suggested to be important for C6 cell growth, survival and tumorigenesis (9, 10, 11, 12). Recently, we reported that cAMP inhibits IGF-I gene expression in C6 cells (13). Addition of exogenous IGF-I at least partially overcame the inhibition of cell growth by cAMP, suggesting that the reduction of endogenous IGF-I biosynthesis may contribute to the inhibitory effect of cAMP on C6 cell growth.

The biological actions of IGF-I are modulated by a family of IGF-binding proteins. There are six high affinity IGF-binding proteins, which inhibit or potentiate IGF-I action, depending on the particular IGF-binding protein (IGFBP), how they are modified, and the specific cells or tissues (14). Moreover, IGFBPs can have IGF-I independent stimulatory and inhibitory effects on cell growth as well (14, 15). Expression of IGFBP-3 and IGFBP-4 mRNA and protein has been demonstrated in C6 cells, whereas IGFBP-2 production in C6 cells is very low compared with that in primary astrocytes (16, 17).

Modulation of IGFBP gene expression by cAMP has been observed in a variety of cell culture models (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35). The stimulation of IGFBP-5 gene expression in dermal fibroblasts (36) and osteoblasts (37, 38) involves promoter-dependent and/or promoter-independent mechanisms. In kidney cells and mammary epithelial cells, cAMP has been clearly shown to increase IGFBP-3 by increasing its gene transcription and mRNA stability (34, 35). In contrast, nothing is known about the mechanisms of cAMP inhibition of IGFBP-3 gene expression or of the mechanisms by which cAMP regulates IGFBP-4 mRNA. Moreover, the influence of cAMP on IGFBP gene expression in glioma cells has not been previously reported. In this study we found that cAMP inhibited IGFBP-3 as well as IGFBP-4 gene expression, but stimulated IGFBP-5 gene expression in a protein kinase A (PKA)-independent manner in C6 cells. We showed that the inhibition of IGFBP-3 gene expression involves at least in part a reduction in mRNA stability, whereas the stimulation of IGFBP-5 gene expression is associated with increased promoter activity, suggesting an effect on transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Rat C6 glioma cells were obtained from the American Type Culture Collection (Manassas, VA) and were cultured as previously described (39). Cells were grown to confluence, followed by 24-h incubation in Ham’s F-12 with 1% FBS (Life Technologies, Inc., Gaithersburg, MD). Cells were then treated with 8-(4-chloropenylthio)-cAMP (8-CPT-cAMP; Sigma, St. Louis, MO) or forskolin (Sigma) in fresh Ham’s F-12 with 1% FBS and harvested at the indicated time for total RNA extraction. For mRNA stability studies, 75 µM of the RNA polymerase II inhibitor 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB; Sigma) was used to treat C6 cells in the presence or absence of 100 µM 8-CPT-cAMP. After 12-h incubation, fresh DRB was added to the conditioned medium. C6 cells were harvested after 0, 3, 6, 12, and 24 h of DRB treatment. In some experiments, C6 cells were preincubated with 8-CPT-cAMP for 3 h before the addition of DRB. For treatment with cycloheximide (Sigma) or H89 (Calbiochem, San Diego, CA), cells were cultured as described in the figure legends.

Total RNA extraction and ribonuclease protection assays (RPAs)
Total RNA was prepared using the Ultraspec reagent (TelTest, 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 a protocol described previously (40) with reagents from Promega Corp. (Madison, WI) and Ambion, Inc. Solution hybridization/RPAs were conducted using either the RPA II kit from Ambion, Inc., or a protocol described previously (40) with reagents supplied by Ambion, Inc. All of the antisense RNA probes used in this study were described previously (41).

Plasmids and site-directed mutagenesis
All IGFBP-5 promoter constructs were cloned upstream of the firefly luciferase structural gene in the pGL2-Basic vector (Promega Corp., Madison, WI). A vector consisting of the SV40 promoter/enhancer directing luciferase transcription (pGL2-Control, Promega Corp.) was used as a positive control. IGFBP-5 promoter constructs containing between 888 and 33 bp of 5'-flanking sequence and the first 114 bp of exon 1 fused to luciferase have been described previously (42). The -71/+80, -71/+40, and -71/+10 constructs (the transcription start site was designated as +1) were generated by PCR amplification of these regions using a sense primer with a KpnI restriction site and an antisense primer with a HindIII restriction site. Primers were synthesized at the Center for Advanced DNA Technologies (University of Texas Health Science Center at San Antonio). The PCR products were ligated into the same sites of pGL2-Basic and were confirmed by sequence analysis.

Mutant constructs, M1, M2, M3, M4, and M5, were generated to mutate the sequence between -23 to -7, -6 to -1, +1 to +10, -24 to -15, and -14 to -5, respectively. Mutagenesis was performed by PCR amplification of the region between -71 and +10 using an antisense mutagenic primer containing the respective mutant sequence and a wild-type sense primer. For each antisense mutagenic primer, the respective sequence was mutated following the rule G->A, C->T, T->G, A->C. The identities of the mutations in the -71/+10 constructs were confirmed by DNA sequence analysis.

Western ligand blot assays
The levels of IGFBPs were determined from conditioned medium using the Western ligand blot (WLB) assay protocol described previously (41). Conditioned medium (CM) was collected and centrifuged to remove dead cells and debris. The protease inhibitors aprotinin, leupeptin, and pepstatin at final concentrations of 6.5, 10, and 0.69 µg/ml, respectively, were added to the CM. Trichloroacetic acid was added to a final concentration of 5%, and the proteins were allowed to precipitate overnight at 4 C. Samples were centrifuged, and the pellets were dissolved in Laemmli buffer [100 mM Tris-HCl (pH 6.8), 2% SDS, 20% glycerol, and 0.04% bromophenol blue]. Proteins were separated by 10% SDS-PAGE and transferred electrophoretically to nitrocellulose membrane (Millipore Corp., Bedford, MA). The membrane was blocked in 3% Nonidet P-40 in WLB saline [10 mM Tris-HCl (pH 7.4), 8.8 g/liter NaCl, and 0.5 g/liter sodium azide] for 30 min. The membrane was incubated in 1% BSA solution in WLB saline for 2 h, followed by incubation in 0.1% Tween 20 solution in WLB saline for 10 min and then incubated overnight in a solution containing 0.1% Tween 20, 1% BSA, and 2.5 x 105 cpm [125I]IGF-I (2215 Ci/mmol; NEN Life Science Products, Boston, MA) in WLB saline. The membrane was washed twice in 0.1% Tween 20 in WLB saline for 15 min, washed once for 15 min in WLB saline, and then washed twice more for 5 min each time in WLB saline. The membrane was air-dried and then exposed to film overnight at -80 C.

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 IGFBP-5 promoter/luciferase fusion constructs with the Lipofectamine Plus system in Opti-MEM medium (Life Technologies, Inc.). 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 (43).

Statistical analysis
Statistical differences between means were determined using one-way ANOVA in the SIMSTAT 3 package (Normand Peladeau, Provalis Research, Montréal, Canada).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Influence of cAMP on IGFBP gene expression
Our previous study has shown that cAMP rapidly and potently inhibits IGF-I gene expression in rat C6 cells (13). As IGF-I gene expression and action are closely associated with that of IGFBPs, we examined whether cAMP alters endogenous IGFBP gene expression in C6 cells. A synthetic cAMP analog, 8-CPT-cAMP, was used to treat C6 cells. No expression of IGFBP-1, -2, and -6 mRNAs was detected by RPAs in either 8-CPT-cAMP-treated or untreated C6 cells (data not shown). 8-CPT-cAMP regulated IGFBP-3, -4, and -5 mRNAs in a dose-dependent manner (Fig. 1Go). After 24 h of 100 µM 8-CPT-cAMP treatment, IGFBP-3 mRNA expression was maximally reduced by 10-fold compared with that in untreated cells (Fig. 1Go). IGFBP-4 mRNA was reduced by 2-fold, whereas IGFBP-5 mRNA was stimulated by 2-fold after 24 h of incubation with 100 µM 8-CPT-cAMP (Fig. 1Go). In contrast, the ß-actin mRNA level was not altered by 8-CPT-cAMP (Fig. 1Go). The assay for ß-actin mRNA has been shown previously (13) and is also shown here, as the assay was performed on the same RNA that was generated in our prior study (13). Similar changes in IGFBP-3, -4, and -5 mRNA levels were observed when C6 cells were treated with forskolin, an adenylate cyclase activator (data not shown), which suggests that the effects of the cell-permeable cAMP analog were due to increased intracellular levels of cAMP. The inhibitory effect of 8-CPT-cAMP on IGFBP-3 mRNA was significant after only 3 h of treatment (P < 0.01; Fig. 2Go). In contrast, the inhibitory effect of 8-CPT-cAMP on IGFBP-4 mRNA was slower, not reaching significance until 24 h (Fig. 2Go). Interestingly, the effect on IGFBP-5 mRNA was transient, with a maximal 2.9-fold stimulation at 12 h, followed by a decline at 48 h to the same level as that in untreated cells (Fig. 2Go).



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  		Figure 1. The effects of cAMP on IGFBP-3, IGFBP-4, IGFBP-5, and ß-actin mRNA levels. Confluent C6 cells were treated with 0, 10, 30, or 100 µM 8-CPT-cAMP for 24 h, followed by preparation of total RNA and RPAs of IGFBP and ß-actin mRNAs. The autoradiographs of representative RPAs are shown in A. B, Quantified mRNA levels, expressed as percentage of those in untreated cells. Each data point is the mean + SEM for three separate experiments, each performed on a single plate of cells. **, The experimental value of the cAMP-treated sample is significantly different from that of the untreated sample (P < 0.01).

 


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  		Figure 2. Time-course of the effect of cAMP on IGFBP mRNA levels. C6 cells were treated with (+) or without (-) 100 µM 8-CPT-cAMP, followed by total RNA preparation at the indicated time points and RPA of IGFBP mRNAs. The autoradiographs of representative RPAs are shown in A. B, Quantified IGFBP mRNA levels, expressed as a percentage of those in untreated cells harvested at the same time points. Each data point is the mean + SEM for three separate experiments, each performed on a single plate of cells. * and **, The experimental value of the cAMP-treated sample is significantly different from that of the untreated sample (P < 0.05 and P < 0.01, respectively).

 
WLB assays were performed to determine the IGFBP protein levels in the CM. C6 cells secreted high levels of IGFBPs with Mr of 40 and 31 kDa (Fig. 3AGo) and a very low level of IGFBP with a Mr of 24 kDa (not observable in this blot). The 40- and 24-kDa IGFBP have been previously identified to be IGFBP-3 and IGFBP-4, respectively (16, 17). The identity of the 31-kDa IGFBP was deduced to be IGFBP-5 based on its molecular mass (14), the detection of IGFBP-5 mRNA, and the lack of detectable levels of IGFBP-1, -2, or -6 mRNAs. In the CM of untreated cells, IGFBP-3 and IGFBP-5 protein levels were increased with incubation time, which is probably due to accumulation of secreted IGFBPs (Fig. 3AGo). When IGFBP protein levels in the CM of 8-CPT-cAMP-treated cells were normalized to those of untreated cells at the same time point, there was a reduction in IGFBP-3 protein level by cAMP at 24 and 48 h, to 59% and 33% of levels in untreated cells, respectively (Fig. 3BGo). In contrast, the IGFBP-5 protein level was transiently stimulated by 8-CPT-cAMP, with the highest stimulation of 2.8-fold at 12 h (Fig. 3BGo). Thus, the changes in IGFBP-3 and IGFBP-5 protein levels were consistent with those of IGFBP-3 and IGFBP-5 mRNA levels. The low level of IGFBP-4 protein precluded accurate quantification of changes in untreated or cAMP-treated cells.



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  		Figure 3. Time-course of the effect of cAMP on secreted IGFBP protein levels in C6 CM. C6 cells were treated with (+) or without (-) 100 µM 8-CPT-cAMP, and CM were collected at the indicated time points. An equal volume of CM was used for each assay. IGFBP protein levels were assessed by [125I]IGF-I ligand blot analysis as described in Materials and Methods. The autoradiograph from a representative blot is shown in A. B, Quantified IGFBP protein levels, expressed as a percentage of those in untreated cells harvested at the same time points. Each data point is the mean + SEM for three separate experiments, each performed on a single plate of cells. * and **, The experimental value of the cAMP-treated sample is significantly different from that of the untreated sample (P < 0.05 and P < 0.01, respectively).

 
IGFBP-3 mRNA stability was reduced by cAMP treatment
To determine whether IGFBP mRNA stability was altered by cAMP treatment, C6 cells were treated with the RNA polymerase II inhibitor, DRB. As shown in Fig. 4Go, cAMP accelerated the degradation of IGFBP-3 mRNA in transcriptionally arrested C6 cells. In contrast, 8-CPT-cAMP did not alter the decay curves of IGFBP-4 and IGFBP-5 mRNA (data not shown). The IGFBP mRNA half-life was calculated by fitting the standard exponential decay equation. Results are summarized in Table 1Go. The IGFBP-3 mRNA half-life was 15.7 h in the absence of 8-CPT-cAMP and 11 h in the presence of 100 µM 8-CPT-cAMP, i.e. cAMP caused a 30% reduction in IGF-I mRNA half-life. IGFBP-4 and IGFBP-5 mRNA half-lives were not significantly changed by cAMP treatment, which suggests that cAMP does not alter IGFBP-4 and IGFBP-5 mRNA stability. When cells were preincubated with 8-CPT-cAMP for 3 h before the addition of DRB, similar results were obtained (data not shown).



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  		Figure 4. The effect of cAMP on IGFBP-3 mRNA stability. C6 cells were treated with (+) or without (-) 100 µM 8-CPT-cAMP in the presence of 75 µM DRB. Total RNA was extracted at the indicated time points and assayed for IGFBP-3 mRNA by RPA. The autoradiograph of a representative RPA is shown in A. B, Quantified IGFBP-3 mRNA level, expressed as percentage of that in untreated cells at the same time point. Each data point is the mean + SEM for four separate experiments, each performed on a single plate of cells.

 

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  		Table 1. Half-life of IGFBP-3, -4, and -5 mRNA in C6 cells

 
Requirement of protein synthesis for cAMP effects on IGFBP gene expression
To determine whether on-going protein synthesis is required for the regulation of IGFBP gene expression by cAMP, cycloheximide, a protein synthesis inhibitor, was used in C6 cells. Cycloheximide alone caused a 40% decrease in the IGFBP-3 mRNA level. A 100-µM concentration of 8-CPT-cAMP reduced IGFBP-3 mRNA by 20-fold in the absence of cycloheximide, but decreased IGFBP-3 mRNA by only 3.6-fold in the presence of cycloheximide (Fig. 5Go, A and B), suggesting that the inhibitory effect of cAMP on IGFBP-3 gene expression requires, but does not fully depend upon, new protein synthesis. As the IGFBP-3 mRNA half-life was reduced by cAMP, we also determined whether this effect on mRNA stability requires on-going protein synthesis. In transcriptionally arrested C6 cells, 100 µM 8-CPT-cAMP decreased IGFBP-3 mRNA level by 2.5-fold (Fig. 5Go, C and D). However, cycloheximide totally abolished this inhibition (Fig. 5Go, C and D). Thus, new protein synthesis is essential for cAMP to destabilize IGFBP-3 mRNA. Moreover, cycloheximide alone potently stimulated IGFBP-3 mRNA in transcriptionally arrested C6 cells (Fig. 5Go, C and D). This suggests that the degradation of IGFBP-3 mRNA depends on on-going protein synthesis even in the absence of cAMP.



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  		Figure 5. The influence of on-going protein synthesis on the abundance and stability of IGFBP-3 mRNA in response to cAMP treatment. For A and B, confluent C6 cells were treated with (+) or without (-) 100 µM 8-CPT-cAMP in the presence (+) or absence (-) of 1 µg/ml cycloheximide. For C and D, C6 cells were treated with (+) 75 µM DRB in the presence (+) or absence (-) of 100 µM 8-CPT-cAMP and/or 1 µg/ml cycloheximide. Twenty-four hours later, total RNA was extracted, followed by RPA of IGFBP-3 mRNA. The autoradiographs of representative RPAs are shown in A and C. B, Quantified IGFBP-3 mRNA level 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. D, Quantified IGFBP-3 mRNA level as a percentage of that in the cells treated with DRB alone. Each data point is the mean ± SEM for five separate experiments, each performed on a single plate of cells.

 
Cycloheximide significantly reduced IGFBP-4 mRNA and totally abolished the 2-fold reduction of IGFBP-4 mRNA caused by 8-CPT-cAMP (Fig. 6Go, A and B), suggesting an essential role for on-going protein synthesis in cAMP-inhibited IGFBP-4 gene expression. The effect of cycloheximide on IGFBP-5 mRNA was more complex. In the absence of 8-CPT-cAMP, cycloheximide caused a 5.8-fold reduction of the IGFBP-5 mRNA level, whereas in the presence of 8-CPT-cAMP, cycloheximide induced IGFBP-5 gene expression by 1.5-fold (Fig. 6Go, C and D). Therefore, on-going protein synthesis may be essential for IGFBP-5 basal gene expression, but not cAMP-induced IGFBP-5 gene expression. Rather, cycloheximide may actually sensitize IGFBP-5 gene expression to cAMP treatment.



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  		Figure 6. The requirement of on-going protein synthesis for the responses of IGFBP-4 and IGFBP-5 mRNAs to cAMP treatment. 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 and then assayed by RPAs for IGFBP-4 and IGFBP-5 mRNAs. The autoradiographs of representative RPAs are shown in A and C. B and D represent quantified IGFBP-4 and IGFBP-5 mRNA levels, expressed as a percentage of those in untreated cells. Each data point is the mean + SEM for three separate experiments, each performed on a single plate of cells.

 
cAMP stimulated IGFBP-5 promoter activity
As cAMP did not alter IGFBP-5 mRNA stability, we characterized potential transcriptional mechanisms, using IGFBP-5 promoter constructs fused to a luciferase reporter gene in transient transfections assays, in C6 cells. We first investigated IGFBP-5 basal promoter activity in C6 cells. An IGFBP-5 promoter construct containing 888 bp of 5'-flanking region and the first 114 bp of exon 1 sequence stimulated luciferase activity by 13-fold over pGL2-Basic (Fig. 7Go). Deletion of the sequence from -888 to -390 increased IGFBP-5 promoter by 3-fold (the transcription start site is designated +1; Fig. 7Go), suggesting that a silencer may be located in the region between -888 and -390. Further 5'-deletion to -71, -50, and -33 led to progressive reduction of promoter activity (Fig. 7Go). However, the IGFBP-5 promoter region between -33/+114 still significantly stimulated luciferase activity by 2.8-fold over pGL2-Basic (P < 0.01; Fig. 7Go), suggesting that this 147-bp sequence contains a minimal IGFBP-5 promoter in C6 cells. 3'-Deletions of constructs containing 71 bp of 5'-flanking sequence to +80, +40, and +10 had a minor effect on IGFBP-5 promoter activity (Fig. 7Go). These deletion analyses suggest that the minimal IGFBP-5 promoter resides between -33 and +10 in C6 cells.



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  		Figure 7. IGFBP-5 basal promoter activity in C6 cells. Various IGFBP-5 promoter-luciferase fusion plasmids (depicted in the left panel) were transfected into C6 cells using Lipofectamine Plus as described in Materials and Methods. {square}, 5'-Flanking region; {blacksquare}, exon 1 sequence. Luciferase activities were measured on cell lysates 24 h later, normalized to protein concentration, and expressed as the fold increase over pGL2-Basic. The luciferase activity of pGL2-Control is shown at 0.1x to fit in the same graph. Each data point is the mean + SEM for the number of separate experiments, indicated as n, each performed on a single plate of cells.

 
IGFBP-5 promoter-luciferase fusion constructs were then used to identify potential cAMP response element(s) in C6 cells. Twenty-four hours of treatment with 100 µM 8-CPT-cAMP increased the activities of all promoter constructs by 1.5- to 2.1-fold (Fig. 8Go), which is consistent with the 2-fold increase in IGFBP-5 mRNA level at 24 h. Therefore, the 43 bp of promoter region between -33 and +10 contains a cAMP response element in C6 cells. Three mutation constructs, designated M1, M2, and M3, were generated to mutate this 43-bp promoter region between -23 to -7, -6 to -1, and +1 to +10, respectively (Fig. 9AGo). These mutations left the putative TATA box at -32 intact. Mutations M1, M2, and M3 decreased IGFBP-5 promoter activity by 7.4-, 3.3-, and 2.3-fold, respectively, compared with the wild-type construct (-71/+10; Fig. 9BGo), suggesting that the mutated sequences contribute to basal promoter activity. Constructs M2 and M3 both remained sensitive to stimulation of luciferase activity by 8-CPT-cAMP, whereas the promoter activity of construct M1 was not significantly altered by cAMP treatment (P > 0.05; Fig. 9BGo). To further locate the precise site of the cAMP response element(s) in the IGFBP-5 promoter in C6 cells, we further mutated the regions between -24 to -15 and -14 to -5, and mutants were designated M4 and M5, respectively (Fig. 9AGo). The basal promoter activities of M4 and M5 were decreased by 52% and 29%, respectively, compared with that of the wild-type construct (Fig. 9BGo). The response to cAMP was abolished in M5, whereas the response was not altered in M4 (Fig. 9BGo). These data suggest that a cAMP response element(s) may be located between -14 and -5 in the IGFBP-5 promoter region in C6 cells.



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  		Figure 8. The effect of cAMP on IGFBP-5 promoter activity in C6 cells. Various IGFBP-5 promoter-luciferase fusion plasmids (depicted in the left panel) were transfected into C6 cells. Cells were then treated with or without 100 µM 8-CPT-cAMP for 24 h, followed by luciferase assays. Luciferase activities were normalized to protein concentration and luciferase activity of pGL2-Basic. The fold stimulation by cAMP was calculated by dividing promoter activity in the presence of cAMP over that in the absence of cAMP and presented as a bar graph. Each data point is the mean + SEM for the number of experiments indicated as n, each performed on a single plate of cells. * and **, The experimental value of the cAMP-treated sample is significantly different from that of the untreated sample (P < 0.05 and P < 0.01, respectively).

 


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  		Figure 9. The effect of mutations on IGFBP-5 promoter activity in response to cAMP. A, Sequence of the IGFBP-5 promoter region from -33 to +10, and shows the locations of mutants M1 through M5. {square}, 5'-Flanking region; {blacksquare} exon 1 sequence. The promoter activities of mutant and wild-type constructs were normalized to protein concentration and expressed as the fold increase over pGL2-Basic, shown in B. Each data point is the mean + SEM for the number of experiments indicated as n, each performed on a single plate of cells. The mean value of the fold stimulation by cAMP was calculated and presented under the bar graph. **, The experimental value of the cAMP-treated sample is significantly different from that of the untreated sample (P < 0.01).

 
cAMP regulates IGFBP gene expression in a PKA-independent manner
Previously, we showed that cAMP potently stimulated PKA activity in C6 cells after 30 min and that the stimulation was sustained for at least 24 h (13). Pretreatment with H89, a PKA inhibitor, prevented PKA activation. Now we asked whether the regulation of IGFBP gene expression by cAMP is mediated by PKA. As shown in Fig. 10Go, IGFBP-3, -4, and -5 mRNAs were regulated by 8-CPT-cAMP to the same extent in the presence of H89 as in the absence of H89. Therefore, it is unlikely that activation of PKA by cAMP contributes to the alteration in IGFBP gene expression by cAMP that we have observed in C6 cells.



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  		Figure 10. cAMP regulates IGFBP 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. The autoradiographs of representative RPAs are shown in A and the quantified IGFBP-3, -4, and -5 mRNA levels are shown in B, C, and D as a percentage of those in untreated cells. Each data point is the mean + SEM for three separate experiments, each performed on a single plate of cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We previously showed that cAMP rapidly and potently inhibits IGF-I gene expression in rat C6 glioma cells, in association with the inhibition of cell growth by cAMP (13). In this study we determined the influence of cAMP on IGFBP gene expression in C6 cells. We detected only IGFBP-3, -4, and -5 mRNAs in C6 cells using RPAs. Our results also suggested that IGFBP-3 and IGFBP-5 are the major IGFBPs present in the conditioned medium of C6 cells, with a very low level of IGFBP-4 in CM. Although previous studies did not report the expression of IGFBP-5 in C6 cells, Bradshaw et al. detected an IGFBP with a Mr of 28 or 34 kDa in the CM of C6 cells (44, 45), which is similar to the size of 31-kDa IGFBP detected in our results. Based on its molecular mass and the detection of IGFBP-5 mRNA by RPA in C6 cells, we deduced that this 31-kDa IGFBP is IGFBP-5. We do not know why Bradshaw et al. did not detect IGFBP-5 mRNA in C6 cells using Northern blot analysis (45). This may be due to the difference of the sensitivity between Northern blot and RPA analyses and/or, alternatively, the different expression levels of IGFBP-5 mRNA in different clones of C6 cells used by the two laboratories.

It has been reported that cAMP stimulates IGFBP-5 mRNA and protein secretion in dermal fibroblasts (20), osteoblasts (25), and Schwann cells (31), whereas cAMP inhibits IGFBP-5 mRNA and protein secretion in thyroid cells (29). Recently, it was shown that cAMP can also stimulate IGFBP-5 protein secretion in mesangial cells (30) and rat L6 myoblasts (33). In C6 cells, cAMP transiently stimulated IGFBP-5 gene expression and protein secretion, and the stimulation was diminished to control levels after 48 h of treatment. This transient regulation is distinct from the sustained stimulation of IGFBP-5 gene expression in osteoblasts (37) and the sustained inhibition of IGF-I (13), IGFBP-3, and IGFBP-4 gene expression in C6 cells. It is possible that there are both stimulatory and inhibitory effects of cAMP on IGFBP-5 gene expression in C6 cells, and the stimulatory effect is more dominant in the first 24 h, or alternatively, that the stimulatory effect of cAMP on IGFBP-5 gene expression is only transient in C6 cells.

The fact that IGFBP-5 promoter activity is stimulated by cAMP to a similar extent as mRNA, whereas IGFBP-5 mRNA stability is not altered by cAMP in C6 cells indicates that the stimulation of IGFBP-5 gene expression by cAMP may be solely a transcriptional event. Our results suggest that the IGFBP-5 promoter region between -14 and -5 may contain a cAMP response element(s). This region is distinct from the locations of IGFBP-5 promoter cAMP response element(s) reported in osteoblasts (38, 46) or in dermal fibroblasts (36) (Fig. 11Go). In dermal fibroblasts, an activator protein-2 site located at -45, i.e. just upstream of TATA box, was suggested to contribute to the stimulation of IGFBP-5 promoter activity by cAMP (36) (Fig. 11Go). In osteoblasts, there is disagreement about the location of the cAMP response element in the IGFBP-5 promoter region and also about whether any regulation of IGFBP mRNA occurs at the level of mRNA stability. Pash and Canalis (38) reported that there was no change in IGFBP-5 mRNA stability after treatment with PGE2, which increases intracellular cAMP levels, and that there are two PGE2-responsive regions located upstream of -330 in the IGFBP-5 promoter (Fig. 11Go). In contrast, McCarthy et al. reported that mechanisms of both transcription and mRNA stability are involved in the stimulation of IGFBP-5 gene expression by cAMP in osteoblasts (37). Moreover, the same group recently showed that a C/EBP site at -68, an E box at -58, and a nuclear factor-1 site at -54 may all play roles in the response of the IGFBP-5 promoter to PGE2 in osteoblasts (46) (Fig. 11Go). Clearly, a different mechanism is used by cAMP to stimulate IGFBP-5 promoter activity in C6 cells compared with osteoblasts and dermal fibroblasts. The cAMP-responsive sequence that we have located between -14 to -5 in C6 cells, 5'-CGACCAGAGC-3', does not correspond to the consensus sequences of reported transcription factors (47). Therefore, a novel transcription factor(s) may be regulated by cAMP to stimulate IGFBP-5 promoter. Identification of this transcription factor(s) will be required to understand the mechanism by which cAMP stimulates the IGFBP-5 promoter in C6 cells.



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  		Figure 11. cAMP-responsive regions in the IGFBP-5 proximal promoter in various cell types. Numbers are relative to transcription start site +1.

 
In the present study we showed that cAMP rapidly and potently inhibited IGFBP-3 gene expression. The IGFBP-3 protein level in CM followed the profile of the change in mRNA level. Similarly, it was found that cAMP inhibits IGFBP-3 mRNA and protein secretion in Sertoli cells (21), thyroid cells (29), and ovarian granulosa cells (22). In contrast, in all other reported studies, IGFBP-3 gene expression and/or protein secretion were stimulated by cAMP, including in kidney cells (34), articular chondrocytes (28), osteoblasts (25), dermal fibroblasts (20), mesangial cells (30), mammary epithelial cells (35), breast cancer cell lines (19, 27), and HEC-1B endometrial carcinoma cells (19). We showed that in C6 cells at least part of the mechanism by which cAMP inhibited IGFBP-3 gene expression involved a decrease in IGFBP-3 mRNA stability. However, the 30% decrease in IGFBP-3 mRNA half-life is not sufficient to account for the 10- to 20-fold reduction in IGFBP-3 mRNA abundance. Thus, it is likely that transcriptional regulation is also involved in the inhibition of IGFBP-3 gene expression by cAMP.

The results of experiments using cycloheximide suggest that the effect of cAMP on IGFBP-3 gene expression requires, but does not fully depend upon, new protein synthesis. In contrast, in transcriptionally arrested C6 cells, inhibition of protein synthesis completely prevented the ability of cAMP to decrease the IGFBP-3 mRNA level, suggesting that the reduction in IGFBP-3 mRNA stability is totally dependent on new protein synthesis. Similar results were observed for IGF-I (13), suggesting that cAMP induces the translation of some labile protein(s) important for both IGF-I and IGFBP-3 mRNA degradation. Moreover, as shown in Fig. 5Go, cycloheximide increased IGFBP-3 mRNA level in the presence of DRB, whereas cycloheximide decreased the IGFBP-3 mRNA level in the absence of DRB. Thus, inhibition of protein synthesis could increase IGFBP-3 mRNA stability, but decrease IGFBP-3 mRNA abundance, which indicates that inhibition of protein synthesis may inhibit basal IGFBP-3 gene transcription.

Compared with IGFBP-3 mRNA, the reduction in IGFBP-4 mRNA is less pronounced and delayed. cAMP also inhibits IGFBP-4 mRNA in thyroid cells (29). However, stimulation of IGFBP-4 gene expression and/or protein secretion by cAMP is more commonly observed, including in dermal fibroblasts (20), osteoblasts (25), articular chondrocytes (32), ovarian granulosa cells (24), bone marrow stromal cells (26), mesangial cells (30), L6 myoblasts (33), BPE-1 endothelial cells (23), and TE-85 osteosarcoma cells (18). We have shown that in C6 cells, the inhibition of IGFBP-4 mRNA does not occur at the level of mRNA stability. Therefore, a transcriptional mechanism may be used by cAMP to inhibit IGFBP-4 gene expression. Moreover, the study using cycloheximide suggests that new protein synthesis is essential for the inhibition of IGFBP-4 mRNA by cAMP.

Similar to IGF-I (13), the regulation of IGFBP gene expression by cAMP is also PKA independent in C6 cells. PKA-independent action of cAMP has attracted increasing attention in the past few years. Two groups recently independently identified several rap guanine nucleotide exchange factors (Epac) that can be activated directly by binding to cAMP (48, 49). In addition, stimulation of ERKs in melanocytes and stimulation of the PI3K/Akt pathway in thyroid cells by cAMP are also PKA independent (50, 51). Thus, cAMP may regulate IGF-I and IGFBP gene expression by altering these pathways or signaling molecules independently of PKA in C6 cells.

In the previous study we showed that exogenous IGF-I did not fully overcome the inhibition of C6 cell growth caused by a high dose of 8-CPT-cAMP. That result suggests that other factor(s), in addition to endogenous IGF-I gene expression, is required for cAMP to exert its full inhibitory effect on C6 cell growth. One of the possibilities is that changes in levels of IGFBPs contribute to the growth inhibitory effect of cAMP on C6 cells. IGFBP-4 is universally reported to inhibit IGF-I-stimulated growth, whereas IGFBP-3 and IGFBP-5 both have growth stimulatory and growth inhibitory actions (14). To understand the physiological functions of these binding proteins in C6 cells will be one of the future goals of this study.


   Acknowledgments
 
We thank Dr. John C. Lee for helpful discussions and for assistance in providing the -888/+114, -390/+114, -71/+114, -50/+114, and -33/+114 rat IGFBP-5 promoter constructs, and Drs. Shimasaki and Ling for supplying initial rat IGFBP cDNA constructs.


   Footnotes
 
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 at San Antonio (to M.L.A.).

Abbreviations: CM, Conditioned medium; 8-CPT-cAMP, 8-(4-chloropenylthio)-cAMP; DRB, 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole; IGFBP, IGF-binding protein; PKA, protein kinase A; RPA, ribonuclease protection assay; WLB, Western ligand blot.

Received March 6, 2001.

Accepted for publication May 3, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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