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Endocrinology Vol. 140, No. 7 3073-3081
Copyright © 1999 by The Endocrine Society

ARTICLES

Growth Hormone-Mediated Regulation of Insulin-Like Growth Factor I Promoter Activity in C6 Glioma Cells1

Carlos Benbassat, Lungile N. N. Shoba, Marsha Newman, Martin L. Adamo, Stuart J. Frank and William L. Lowe, Jr.

Department of Medicine (C.B., L.N.N.S., M.N., W.L.L.), Veterans’ Affairs Chicago Healthcare System, Lakeside Division, and Northwestern University Medical School, Chicago, Illinois 60611; Department of Biochemistry (M.L.A.), University of Texas Health Science Center, San Antonio, Texas 78284-7760; Department of Medicine (S.J.F.), University of Alabama at Birmingham and Birmingham Veterans’ Affairs Medical Center, Alabama 35294

Address all correspondence and requests for reprints to: William L. Lowe, Jr., M.D., Center for Endocrinology, Metabolism, and Molecular Medicine, Tarry 15–703, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: wlowe{at}nwu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The molecular mechanisms by which GH regulates insulin-like growth factor (IGF-I) gene expression remain obscure. One difficulty has been the lack of established GH-responsive cell lines that express the IGF-I gene. To develop such a cell line, we used rat C6 glioma cells which, as determined by RNase protection assay, express the IGF-I gene but not the GH receptor gene. To confer GH responsiveness, C6 cells were cotransfected with vectors that express the GH receptor (pRc/CMV WTrGHR) and Jak2 (pRc/CMV Jak2). GH responsiveness was demonstrated using luciferase reporter genes containing either the Sis-inducible element from the c-fos gene (pTK81-SIE-Luc) or 6 copies of the GH-responsive GAS-like element (GLE) from the rat spi2.1 gene (pSpi-GLE-Luc). The SIE is activated by binding of STAT1 and 3, whereas the GLE binds STAT5. In cells cotransfected with pRc/CMV WTrGHR, pRc/CMV Jak2, and either pTK81-SIE-Luc or pSpi GLE-Luc, treatment with 500 ng/ml GH for 24 h stimulated a 3.1- and 1.7-fold increase in luciferase activity, respectively. These data suggest that in C6 cells cotransfected with pRc/CMV WTrGHR and pRc/CMV Jak2, GH activates STAT1, 3, and 5. To determine whether GH-responsive IGF-I promoter activity could be demonstrated, C6 cells were cotransfected with pRc/CMV WTrGHR, pRc/CMV Jak2, and an IGF-I-luciferase fusion gene that contained a fragment of the rat IGF-I gene that extended from -412 in the 5'-flanking region of exon 1 to the Met-22 in exon 3. GH stimulated a modest, but reproducible, 1.7-fold increase in luciferase activity in these cells, suggesting that a GH-responsive element is present in this region of the IGF-I gene. To better localize the GH-responsive element, cells were cotransfected with pRc/CMV WTrGHR, pRc/CMV Jak2 plus one of several IGF-I-luciferase fusion genes containing either fragments of one of the two promoters in the IGF-I gene or a fragment of intron 2 that includes a GH-responsive DNase I hypersensitivity site. For all constructs, treatment with GH for 24 h did not stimulate a significant increase in luciferase activity, suggesting that GH-responsive sequences are not located in these specific regions of the IGF-I gene or that GH-directed transcription of the IGF-I gene is mediated via several different regions of the IGF-I gene and the effect of any one of these regions in isolation was not sufficiently robust to be detected in this model system. In summary, transient expression of the GH receptor and Jak2 in C6 cells creates a GH-responsive system that activates STAT1, 3, and 5. Moreover, a fragment of the IGF-I gene that contains exons 1 and 2, a fragment of exon 3, and introns 1 and 2 is GH responsive using this model system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factor-I (IGF-I) is a peptide that has both mitogenic and metabolic effects and is expressed in a wide range of tissues (1, 2, 3). Among its actions, IGF-I mediates many of the growth-promoting effects of GH, which, in turn, is one of the primary regulators of IGF-I gene expression (1, 2, 3, 4). The effect of GH on IGF-I gene expression is mediated at the level of gene transcription (5, 6). Although this effect of GH on IGF-I gene transcription is well described, the molecular mechanisms responsible for it are unknown.

Despite a relatively simple protein structure, the IGF-I gene and its messenger RNA (mRNA) are complicated (7). In humans and rats, IGF-I is a single copy gene that is approximately 70 kb in size and contains six exons that are differentially spliced, leading to different forms of IGF-I mRNAs. Sequences 5' to and within exon 1 regulate expression of IGF-I mRNAs that contain exon 1 and are referred to as the major promoter or promoter 1 (8, 9, 10, 11, 12, 13, 14, 15). A second promoter, promoter 2, is present in sequences 5' to exon 2 and regulates expression of IGF-I mRNAs that contain exon 2 (8, 11, 15). Promoter 1 of the IGF-I gene is unique in that it lacks typical proximal transcriptional control elements, including a TATAA and CAAT box (9, 14). Consistent with this, multiple transcription initiation sites have been described for mRNAs that contain exon 1 (14, 16). Previous studies using transient transfection assays have demonstrated that basal activity of promoter 1 could be localized to a discrete region that spanned the most 5' transcription initiation site and included sequences in exon 1 (10, 12). Promoter 2 also lacks a TATAA box, and transcription initiates from two clusters in exon 2 located 1.8 kb from the 3'-end of exon 1 (14, 16, 17, 18, 19). Recently, a CACCC box that is 53 bp 5' to the more proximal transcription initiation site has been shown to be important for basal promoter 2 activity (20). A variety of protein binding sites, as demonstrated using DNase I footprinting and gel shift analyses, have been described in promoters 1 and 2, but none of these sites binds to protein in a GH-responsive fashion (10, 15, 20, 21, 22, 23).

Cloning of the GH receptor gene and identification of Jak2 as a GH receptor-associated tyrosine kinase have facilitated the understanding of how GH acts at a molecular level (24). Yet, there are few clues as to the location of GH-responsive elements in the IGF-I gene. GH-inducible luciferase activity in brain cells isolated from a transgenic mouse bearing an IGF-I-luciferase transgene that contained an 11.3-kb fragment of the IGF-I gene has been reported (25), but otherwise a GH-inducible IGF-I promoter construct and further localization of GH-responsive sequences have not been reported. Interestingly, some studies have shown greater GH responsiveness of IGF-I mRNAs that contain exon 2 compared with exon 1, suggesting that sequences in the region of exon 2 may be important for GH-induced IGF-I gene transcription (26, 27). This is consistent with the presence of a GH-inducible DNase I hypersensitivity site in intron 2 (5). DNase I hypersensitivity sites often accompany alterations in chromatin structure that occur at sites of transcriptional activity. This DNase I hypersensitivity site has been mapped to a 350-bp region of intron 2, but GH-inducible protein binding to this region could not be demonstrated (28). Otherwise, inspection of the sequence of the IGF-I gene has not revealed obvious GH response elements based upon the sequences of known GH response elements from other genes.

The lack of progress in defining the mechanisms of GH-mediated IGF-I gene transcription is not due solely to the complexity of the IGF-I gene. GH-responsive cell lines that transcribe the IGF-I gene have been difficult to find. In the present study, we have developed a cell culture model system using a rat C6 glioma cell line that transiently expresses the GH receptor and Jak2 genes and have demonstrated its responsiveness to GH stimulation. Such a cell culture system can be used to examine the regulation of IGF-I gene expression by GH.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Rat C6 glioma cells were maintained in 75-cm2 flasks in F12/DMEM supplemented with 5% FBS, penicillin (50 U/ml), streptomycin (50 U/ml), 2 mM L-glutamine, and 10 mM sodium bicarbonate at 37 C in 5% CO2 as described previously (29). Upon reaching confluence, the cells were replated at a 1:5 dilution.

RNA extraction
RNA was prepared using the guanidine thiocyanate cesium chloride method as described previously (29, 30). RNA was quantified by measuring absorbance at 260 nm. The accuracy of quantification and integrity of the RNA were confirmed by size-separating the RNA from different samples by denaturing agarose gel electrophoresis as described previously (30).

Hybridization probes
For the quantification of IGF-I mRNA levels by solution hybridization/RNase protection assays, a 322-bp rat IGF-I complementary DNA (cDNA) was subcloned into a pGEM-2 vector (Promega Biotech, Madison, WI), and the plasmid DNA was linearized with EcoRI to allow for transcription of antisense IGF-I RNAs (27). To quantify GH receptor mRNA levels, a 900-bp BglII fragment of the rat GHR cDNA was subcloned into the vector pT7T3 (kindly provided by Dr. L. Mathews). For transcription of antisense GH receptor RNAs, the plasmid DNA was linearized with BamHI, which results in transcription of GH receptor antisense RNAs 439 bases in length.

Solution hybridization/RNase protection assay
GH receptor and IGF-I mRNA levels were quantified using a solution hybridization/RNase protection assay as described previously (27, 30). Briefly, 32P-labeled antisense RNAs were transcribed from linearized plasmid DNA and incubated with either 10 µg of rat liver total RNA or 100 µg of C6 cell total RNA at 45 C in 75% formamide/0.4 M NaCl. After a 16-h incubation, the samples were digested with RNases A and T1. The protected double-stranded hybrids were collected by ethanol precipitation and electrophoresed on an 8% polyacrylamide/8 M urea denaturing gel. All assays were performed in duplicate.

Western blot analyses
For Western blot analyses, cells were lysed in 50 mM Tris, pH 8.0, 1 mM EDTA, 0.25 M sucrose, 17.4 µg/ml 0.1 M phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, 10 µg/ml leupeptin, 10 µg/ml antipain, and 10 µg/ml chymostatin. Lysates were clarified by centrifuging at 4 C at 15,000 x g for 5 min, and protein concentrations were determined using the Coomassie Blue protein assay (Bio-Rad Laboratories, Inc., Richmond, CA) reagent. Protein samples were then mixed 1:1 with Laemmli sample buffer with 2-mercaptoethanol (5%) and heated at 95 C for 4 min. SDS-PAGE was performed on a 7.5% acrylamide slab gel. Pre-stained molecular weight markers (Bio-Rad Laboratories, Inc.) were used as standards. Electrophoretic transfer of proteins to polyvinylidene difluoride membranes (Immobilon-P, 0.45 µm, Millipore Corp., Bedford, MA) was accomplished with the semi dry Trans-Blot transfer system from Bio-Rad Laboratories, Inc. in a transfer buffer consisting of 25 mM Tris-HCl, 192 mM glycine and 20% methanol for 1 h at 150 mAmps. Membranes were blocked for 90 min at room temperature in 20 mM Tris, pH 7.6, 137 mM sodium chloride, 0.1% Tween-20 (TBST) with 4% nonfat dried milk. Membranes were incubated overnight in TBST at 4 C with primary antibody. Primary antibodies were as follows. Anti-STAT1, anti-STAT3, and anti-PI-3 kinase mouse monoclonal antibodies were obtained from Transduction Laboratories, Inc. and used at dilutions of 1:5,000, 1:15,000, and 1:2,500, respectively. Anti-IRS-1 mouse polyclonal antibody was obtained from Transduction Laboratories, Inc. and used at a dilution of 1:250. Anti-STAT5a and 5b rabbit polyclonal antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and used at a dilution of 1:2,500. Blots were then incubated with either antimouse (dilution of 1:7,500, Promega Corp., Madison, WI) or antirabbit (dilution of 1:5,000, Promega Corp.) IgG horseradish peroxidase conjugated antibodies for 90 min at room temperature. Following three washes in TBST, immunoreactive bands were detected using the enhanced chemiluminescence (ECL) detection system from Amersham Corp. (Arlington, IL), according to the manufacturer’s instructions.

Plasmid constructions
Generation of pRc/CMV WTrGHR, pRc/CMV GHR1–275, and pRc/CMV Jak2 has been described previously (31, 32). Briefly, the wild-type rabbit GH receptor (WTrGHR), a rabbit GH receptor cDNA truncated at residue 275 (GHR1–275), or a murine Jak2 cDNA were subcloned into the pRC/cytomegalovirus (CMV) expression plasmid (Invitrogen, San Diego, CA). The cloning of pTK81-SRE-Luc, which contained a single copy of the 28-bp fragment of the serum response element (SRE) from the human c-fos gene cloned 5' to a minimal thymidine kinase promoter and luciferase gene has been described previously (33). The plasmid pTK81-SIE-Luc was constructed by cloning a single copy of the 21-bp fragment of the sis-inducible element (SIE) from the c-fos gene into the SmaI site of the parent vector pTK81-Luc, which contains a minimal thymidine kinase promoter and the luciferase gene. The sequence of the SIE was a previously identified mutant of the SIE with increased binding activity (34) and was as follows: 5'-GAGCATTTCCCGTAAATCCCT-3'. The vector p-Fos-Luc was constructed as described previously (35). Briefly, it contained the region -361 to +157 of the human c-fos gene, which had been subcloned 5' to the coding region of the luciferase gene in the vector pA3Luc. The vector pSpi-GLE-Luc was constructed by cloning 3 copies of the sequence 5'-ATGTTCTGAGAAATCgtacATGTTCTGAGAAATCgtac-3' in the antisense orientation 5' to the luciferase gene in the KpnI site in the vector pTK81-Luc. This sequence contained two copies (indicated in capital letters) of a previously described GH response element from the rat spi 2.1 gene (36, 37). The pSpi 2.1-Luc construct was kindly provided by Dr. Nils Billestrup (38). pSpi 2.1-Luc contained two copies of the sequence -147 to -103 of the rat spi 2.1 gene cloned 5' to the luciferase gene in the vector pGL2 Promoter.

The cloning of pGL2IGF-18, pGL2-IGF-412, pGL2IGF-1690, pGL2IGF-P2–485, and pGL2IGF-P2–1500 has been described previously (10, 13, 15). For construction of pGL2IGF-18-Intron 2 and pGL3 Promoter-Intron 2, a 402-bp fragment of intron 2 that has been shown previously to contain a GH-inducible DNase I hypersensitivity site was amplified using PCR and a previously characterized rat genomic clone that contains intron 2 as template DNA (13). The resulting amplified fragment was purified, and its sequence was confirmed by DNA sequencing using an PE Applied Biosystems 373A DNA Sequencer. The amplified fragment was then subcloned 5' to a minimal IGF-I promoter in the vector pGL2IGF-18 or the SV40 promoter in the vector pGL3 Promoter (Promega Corp.). The construction of pGL3IGF-mini was accomplished using a previously described rat genomic clone that contained 412 bp of 5'-flanking region for exon 1 (promoter 1), exon 1, intron 1 (which contains promoter 2), exon 2, intron 2, exon 3, and a fragment of intron 3 (13). A map of the genomic clone and a schematic diagram of pGL3IGF-mini are shown in Fig. 1Go. PCR was used to amplify a 1.3-kb fragment of the IGF-I gene that extended from the EcoRI site in intron 2 to the ATG at Met-22 in exon 3. The sense and antisense oligonucleotides used for PCR were as follows: 5'-AACCATCGGCTAGCTTGTGCCAA-3' and 5'-GTGAAGACGCCATGGTGTGTATC-3'. The underlined bases in the antisense oligonucleotide represent changes from the native IGF-I sequence that result in the introduction of an NcoI site surrounding the Met-22 ATG. The sequence of the amplified fragment was confirmed by DNA sequence analysis. Following restriction of the amplified fragment with EcoRI and NcoI, it was subcloned into pSVK3 (Amersham Pharmacia Biotech, Piscataway, NJ) that had been restricted with NcoI and EcoRI. Following restriction of the genomic clone with SacI and EcoRI, the 1.1-kb SacI-EcoRI fragment was purified and subcloned 5' to the EcoRI-NcoI fragment in pSVK3. The SacI-NcoI fragment was then released by restriction with SacI and NcoI and subcloned 5' to the luciferase gene in pGL3 Basic. Subsequently, the 3.1-kb SacI-SacI fragment from the genomic clone was purified and subcloned 5' to the SacI-NcoI fragment in pGL3 Basic. DNA sequencing was used to determine the orientation of the subcloned SacI-SacI fragment. Clones with the SacI-SacI fragment in both the forward (pGL3IGF-mini) and reverse orientation (pGL3IGF-mini-rev) were isolated.



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  		Figure 1. Schematic diagram of a rat IGF-I genomic clone and the IGF-I-luciferase fusion genes pGL3IGF-mini and pGL3IGF-mini-rev. A previously isolated rat IGF-I genomic clone that contained 412 bp of the 5'-flanking region of exon 1 (relative to the most 5' transcription start site for exon 1), exon 1, intron 1, exon 2, intron 2, exon 3, and a portion of intron 3 was used to construct two IGF-I-luciferase fusion genes, pGL3IGF-mini and pGL3IGF-mini-rev. The open, numbered boxes represent the indicated exons, whereas P1 and P2 indicate the locations of promoter 1 and 2, respectively. The cross-hatched box in intron 2 represents the approximate location of the GH-responsive DNase 1 hypersensitivity site. The Met-22 ATG that is present in exon 3 is indicated, as are in-frame ATGs present in exons 1 and 2. Whether these ATGs in exons 1 and 2 are used in vivo is not known.

 
Transient transfection and luciferase assays
For transient transfection assays, cells were plated onto 12-well plates at a density of 1 x 105 cells per well. Plasmid DNA that was purified by cesium chloride centrifugation was transfected into cells using the cationic lipid Lipofectin according to the manufacturer’s instructions as described previously (10, 13). Cells were incubated initially for 1 h in Opti-mem and then for an additional 18 h in normal growth medium in the continued presence of cationic lipid and plasmid DNA. Cells were then washed twice with PBS and treated with or without recombinant human GH (a kind gift from Eli Lilly & Co., Indianapolis, IN) in serum-free F12/DMEM with 0.25% BSA for the indicated periods of time. Cells were then harvested, and luciferase assays were performed as described previously (10). Light emission was measured with an AutoLumat LB953 luminometer (E. G. & G. Berthold, Bad Wildbad, Germany). The luciferase activity present in each sample was normalized using the protein content of the sample, as determined using the method of Lowry et al. (39). All luciferase assays were performed in triplicate.

Statistical analyses
Values are reported as the mean ± SEM. P values were calculated using the Mann-Whitney Rank Sum Test or Kruskal-Wallis one-way ANOVA on Ranks with the Dunnett’s Pairwise Multiple Comparison Procedure, as appropriate, using SigmaStat 2.0 software (Jandel Corp., San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of the IGF-I and GH receptor genes and GH-activated signaling molecules in C6 glioma cells
An initial series of studies was performed to characterize IGF-I and GH receptor expression in rat C6 glioma cells. Consistent with findings reported previously (29), IGF-I mRNA was present in C6 cells, indicating that the IGF-I gene is transcribed (Fig. 2AGo). The antisense IGF-I RNA used in the RNase protection assay is able to discriminate between IGF-I mRNAs that contain exon 1 or exon 2. As has been described previously, IGF-I mRNAs that contained both exon 1 and exon 2 were present in rat liver, whereas C6 cells expressed only IGF-I mRNAs that contain exon 1 (27, 29). In contrast to expression of the IGF-I gene, GH receptor mRNA was undetectable in C6 cells, whereas the mRNAs encoding the GH receptor and binding protein were easily detectable in liver (Fig. 2BGo).



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  		Figure 2. Expression of IGF-I and GH receptor mRNA in rat C6 glioma cells. Total RNA prepared from either rat C6 glioma cells (lane a, panels A and B) or rat liver (lane b, panels A and B) was hybridized to either 32P-labeled IGF-I (panel A) or GH receptor (GHR) (panel B) antisense RNAs and subjected to solution hybridization/RNase protection analysis. The protected bands representing IGF-I mRNAs containing either exon 1 or 2 and mRNAs encoding either the GH receptor or GH binding protein are indicated by the arrows. The integrity of the RNA used in the above assays is demonstrated in panel C, which is a photograph of an ethidium bromide stained denaturing agarose gel in which 5 µg of either the C6 glioma cell (lane a) or rat liver RNA (lane b) were size-separated by denaturing agarose gel electrophoresis. The 28S and 18S ribosomal RNA bands are indicated by the arrows.

 
Having established that C6 cells transcribe the IGF-I gene but do not express GH receptors, studies were performed to document expression of other signaling molecules that are activated by GH. The tyrosine kinase that is associated with the GH receptor and appears to be necessary for most of the signals transmitted via the receptor is Jak2 (24). Western blot analyses demonstrated low levels of Jak2 in C6 cells (Fig. 3AGo). Among the early signaling molecules activated in response to GH binding to its receptor are STAT1, 3, 5a, and 5b (24), and each of these signaling molecules is present in C6 cells, as demonstrated by Western blot analysis (Fig. 3Go, B–E). Western blot analyses were also performed to examine the expression of PI-3 kinase and IRS-1, two other molecules that contribute to GH-mediated signal transduction. PI-3 kinase was present in C6 cells, but IRS-1 was undetectable (data not shown).



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  		Figure 3. Expression of the Stats and Jak2 in rat C6 glioma cells. Total cellular protein was prepared from C6 cells as described in Materials and Methods. Protein from C6 cells (lane c, panel A; lane b, panels B–E) or control extracts (lanes a and b, panel A; lane a, panels B–E) was size-separated using 7.5% SDS-PAGE, blotted onto polyvinylidene difluoride membranes, and probed with antibodies directed against the indicated signaling molecules. Antibody binding was detected using an ECL detection system. Control extracts in panel A were prepared from fetal rat liver (lane a) and Cos 7 cells (lane b), whereas the control extract for panels B–E was adult rat liver. For each panel, the specific protein being detected is indicated by the arrow.

 
Generation of GH-responsive C6 cells
Given the absence of GH receptors in C6 cells, cells were transfected with an expression vector that expressed the rabbit GH receptor (pRc/CMV WTrGHR) to confer GH responsivity on C6 cells. In pilot studies to determine if GH responsivity had been conferred, the cells were cotransfected with a reporter gene that contained a 21-bp fragment of the SIE from the c-fos gene cloned 5' to a minimal thymidine kinase promoter and luciferase reporter gene (pTK81-SIE-Luc). Previous studies have documented GH-induced binding of STAT1 and 3 to the SIE (24, 40, 41, 42). In cells cotransfected with pRc/CMV WTrGHR and pTK81-SIE-Luc, treatment with 500 ng/ml of GH for 24 h stimulated only a 1.3 ± 0.1-fold increase in luciferase activity (mean ± SEM, n = 4). In contrast, in cells cotransfected with the above two vectors in addition to a vector that expressed murine Jak2 (pRc/CMV Jak2), treatment with 500 ng/ml of GH for 24 h stimulated a 2.2 ± 0.2-fold (mean ± SEM, n = 4; P < 0.05 compared with SIE activity in cells not transfected with pRc/CMV Jak2) increase in luciferase activity. When pTK81-Luc was used as the reporter gene in cells that had been cotransfected with pRc/CMV WTrGHR and pRc/CMV Jak2, luciferase activity increased only 1.2 ± 0.1-fold (mean ± SEM, n = 5) in response to GH, suggesting that the effect of GH on luciferase activity was not mediated via effects on the thymidine kinase promoter. The above data suggested that GH responsivity could be conferred upon C6 cells but that, despite the low levels of JAK2 present in C6 cells, increased expression of Jak2 was required.

Having determined a means of conferring GH responsivity on C6 cells, the cells were cotransfected with pRc/CMV WTrGHR and pRc/CMV Jak2 in addition to one of several reporter genes. Previous studies have documented GH responsivity of the c-fos gene (43, 44), and, in C6 cells, treatment with 500 ng/ml of GH for 24 h increased c-fos promoter activity 1.9-fold (Fig. 4Go). In addition to the GH-responsive SIE, the c-fos promoter contains an SRE that also mediates the effect of GH on c-fos promoter activity (43, 44). Multiple experiments demonstrated that GH treatment for 24 h increased pTK81-SIE-Luc activity 3.1-fold compared with the activity in cells not treated with GH (Fig. 4Go). To determine whether GH-induced SRE activity could also be demonstrated in C6 cells, cells were cotransfected with pRc/CMV WTrGHR, pRc/CMV Jak2, and pTK81-SRE-Luc, which contained a 28-bp fragment of the SRE. Interestingly, treatment with GH for 24 h had no effect (1.2 ± 0.1-fold, n = 5) on luciferase activity when compared with the activity in cells not treated with GH. As a positive control, C6 cells that had been transfected with these same plasmids were treated with 1 µM of the phorbol ester phorbol 12-myristate 13-acetate (PMA). PMA stimulated a 3.2-fold (n = 2) increase in SRE activity compared with the activity in cells not treated with PMA, demonstrating that the SRE could be activated in these cells. A final series of experiments was performed to examine the effect of GH on the activity of a GH response element from the rat spi 2.1 gene, which binds to STAT5 (36, 45, 46). Initial experiments were performed with pSpi 2.1-Luc, which contained two copies of the sequence extending from -147 to -103 in the rat spi 2.1 gene cloned 5' to the luciferase gene in the vector pGL2 Promoter. Treatment of cells transfected with pRc/CMV WTrGHR, pRc/CMV Jak2, and pSpi 2.1-Luc with 500 ng/ml GH stimulated a 1.8 ± 0.2-fold (mean ± SEM, n = 8) increase in luciferase activity compared with the activity in cells not treated with GH (data not shown). Because the GH-responsive element in pSpi 2.1-Luc has been shown to bind to C/EBP molecules in addition to STAT5 (37), additional studies were performed using a second vector, pSpi-GLE-Luc, that contained six copies of the 15-bp interferon-{gamma}-activated sequence-like element (GLE) that is present in the rat Spi 2.1 promoter and binds to Stat5 (36). Similar to the findings with pSpi 2.1-Luc, GH stimulated a 1.7-fold increase in luciferase activity in cells transfected with pRc/CMV WTrGHR, pRc/CMV Jak2, and pSpi-GLE-Luc (Fig. 4Go).



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  		Figure 4. GH-induced reporter gene expression. Rat C6 glioma cells were transfected with either pRc/CMV WTrGHR (wild-type GHR) or pRc/CMV GHR1–275 (mutant GHR) in addition to pRc/CMV Jak2 and the indicated reporter gene as described in Materials and Methods. Following transfection, the cells were treated for 24 h without or with 500 ng/ml GH in serum-free F12/DMEM + 0.25% BSA. The cells were then harvested, and luciferase activity was determined. The values represent the relative luciferase activity compared with the activity in cells maintained in serum-free F12/DMEM + 0.25% BSA in the absence of GH, which was defined as 1.0. For cells transfected with pRc/CMV WTrGHR, the values are the mean ± SEM of either 6 (pFos), 17 (pTK81-SIE), or 6 (pSpi-GLE-Luc) independent experiments performed in triplicate. For cells transfected with pRc/CMV GHR1–275, the values are the mean ± SEM of either 4 (pFos), 3 (pTK81-SIE), or 6 (pSpi-GLE-Luc) independent experiments performed in triplicate. *, P < 0.01 compared with the level in cells transfected with pRc/CMV GHR1–275.

 
As a control, studies also were performed using a vector that expressed a truncated GH receptor (pRc/CMV GHR1–275) that is unable to activate Jak2. For these studies, C6 cells were cotransfected with pRc/CMV GHR1–275, pRc/CMV Jak2, and one of the reporter genes. In cells transfected with pFos-Luc, pTK81-SIE-Luc, or pSpi-GLE-Luc, luciferase activity was identical to the activity in cells not treated with GH (Fig. 4Go). Similar results were obtained using cells transfected with pSpi 2.1-Luc in which luciferase activity was decreased by 20% in GH-treated compared with untreated cells (data not shown). These data demonstrate that the effect of GH on reporter gene activity is dependent upon the presence of a functional GH receptor.

To determine the sensitivity of cells transfected with pRc/CMV WTrGHR and pRc/CMV Jak2 to GH treatment, dose-response studies were performed using pTK81-SIE-Luc as a reporter gene (Fig. 5Go). The effect of GH on luciferase activity was dose dependent, and a maximal effect was achieved with 10 ng/ml GH. Studies performed using doses of GH as a high as 500 ng/ml demonstrated that higher doses of GH had no further effect on luciferase activity (data not shown).



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  		Figure 5. Dose-response effect of GH on SIE activity. Rat C6 glioma cells were transfected with pRc/CMV WTrGHR, pRc/CMV Jak2, and pTK81-SIE as described in Materials and Methods. Following transfection, the cells were treated for 24 h with the indicated dose of GH in serum-free F12/DMEM + 0.25% BSA. The cells were then harvested, and luciferase activity was determined. The values represent the relative luciferase activity compared with the activity in cells maintained in serum-free F12/DMEM + 0.25% BSA, which was defined as 1.0, and are the mean ± SEM of three independent experiments performed in triplicate. *, P < 0.05 compared with the activity in cells maintained in serum-free F12/DMEM + 0.25% BSA.

 
Effect of GH on IGF-I promoter activity in GH-responsive C6 cells
The rat IGF-I gene contains two alternative promoters, promoter 1 (P1) and promoter 2 (P2), which control expression of IGF-I mRNAs that contain either exon 1 or exon 2, respectively (4). At present, it is unclear whether one, both, or neither of these promoters is GH responsive. To begin to define the molecular mechanisms for GH-responsive IGF-I gene expression, a fragment of the rat IGF-I gene that contained 412 bp of the region flanking the most 5' transcription initiation site in exon 1, exon 1, intron 1, exon 2, intron 2, and the first 18 bp of exon 3 was cloned 5' to the luciferase gene in the promoterless plasmid pGL3 Basic to create the IGF-I-luciferase fusion gene pGL3IGF-mini (see Fig. 1Go for schematic diagram of pGL3IGF-mini). This IGF-I-luciferase fusion gene contains a fragment of promoter 1 and the entirety of promoter 2 and intron 2, which is the location of a GH-inducible DNase I hypersensitivity site. Initial studies were performed to confirm that this fragment of the IGF-I gene had promoter activity in C6 cells. For these studies, either pGL3IGF-mini, pGL3IGF-mini-rev (in which the portion of the IGF-I promoter construct between its 5' end and the SacI site in intron 2 is present in the reverse orientation), or pGL3 Basic was transfected into C6 cells. In extracts from cells transfected with pGL3IGF-mini, luciferase activity was increased 7.4 ± 1.7-fold (mean ± SEM, n = 16) compared with the activity in cells transfected with pGL3 Basic (P < 0.05). This increase in luciferase activity confirms that this fragment of the IGF-I gene functions as a promoter. Interestingly, transfection of pGL3IGF-mini-rev also increased luciferase activity (2.1 ± 0.3-fold, mean ± SEM, n = 16) compared with pGL3 Basic (P < 0.05), although its activity was markedly reduced compared with pGL3IGF-mini.

Having confirmed that this fragment of the IGF-I gene has promoter activity in the context of a luciferase fusion gene, its responsiveness to GH was next examined. For these studies, C6 cells were cotransfected with pRc/CMV WTrGHR and pRc/CMV Jak2 in addition to either pGL3IGF-mini, pGL3IGF-mini-rev, or pGL3 Basic and treated for 24 h with 50 ng/ml of GH (Fig. 6Go). As can be seen in cells transfected with pGL3IGF-mini, GH treatment stimulated a small but highly reproducible 1.7-fold increase in luciferase activity. In contrast, GH treatment had a minimal effect on luciferase activity in cells transfected with pGL3IGF-mini-rev and no effect on luciferase activity in cells transfected with pGL3 Basic. These data suggest that a GH-responsive element is present in the region of the IGF-I gene represented in pGL3IGF-mini.



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  		Figure 6. Effect of GH on IGF-I promoter activity. pRc/CMV WTrGHR, pRc/CMV Jak2, and either pGL3 Basic, pGL3IGF-mini, or pGL3IGF-mini-rev were transfected into rat C6 glioma cells as described in Materials and Methods. Following transfection, the cells were treated for 24 h without or with 50 ng/ml GH in serum-free F12/DMEM + 0.25% BSA. The cells were then harvested, and luciferase activity was determined. The values represent the relative luciferase activity compared with the activity in cells maintained in serum-free F12/DMEM + 0.25% BSA, which was defined as 1.0, and are the mean ± SEM of eight independent experiments performed in triplicate. *, P < 0.001 compared with the activity in cells transfected with pGL3IGF-mini and maintained in serum-free F12/DMEM + 0.25% BSA. +, P < 0.001 compared with the activity in cells transfected with pGL3 and treated with 50 ng/ml GH. **, P = 0.01 compared with the activity in cells transfected with pGL3IGF-mini-rev and maintained in serum-free F12/DMEM + 0.25% BSA.

 
To determine whether sequences in promoter 1 or 2 or the region of intron 2 that contains the GH-inducible DNase I hypersensitivity site were responsible for the inducibility of pGL3IGF-mini activity by GH, a series of transient transfection assays using IGF-I-luciferase genes that contained these different fragments of the rat IGF-I gene were performed. Previous studies have documented that rat IGF-I-luciferase fusion genes that contain fragments of either promoter 1 or promoter 2 are active in C6 cells (10, 13, 20). For these studies, C6 cells were cotransfected with pRc/CMV WTrGHR and pRc/CMV Jak2 in addition to one of a variety of IGF-I-luciferase fusion genes and then treated for 24 h with 500 ng/ml GH (Fig. 7Go). Vectors pGL2IGF-18, pGL2IGF-412, and pGL2IGF-1690 contained fragments of promoter 1 that included 302 bp of exon 1 and either 18, 412, or 1690 bp of 5'-flanking region, respectively. The vectors pGL2IGF-P2–485 and pGL2IGF-P2–1500 contained fragments of promoter 2 that included 44 bp of exon 2 and either 485 or 1500 bp, respectively, of the region of intron 1 that is 5' to exon 2. As can be seen, GH had little to no effect on activity of either promoter 1 or promoter 2. Of note, use of pGL2IGF-1690 also determined whether GH-responsive elements were present in the 1278 bp of promoter 1 5' to pGL3IGF-mini. To determine whether longer treatment periods were required to observe an effect of GH, C6 cells were cotransfected with pRc/CMV WTrGHR, pRc/CMV Jak2 and pGL2IGF-1690 and treated with GH for either 36 or 48 h. Similar to the above results, a GH-induced increase in luciferase activity was not observed (data not shown).



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  		Figure 7. Effect of GH on the activity of IGF-I-luciferase fusion genes. pRc/CMV WTrGHR, pRc/CMV Jak2, and the indicated IGF-I-luciferase fusion gene were transfected into rat C6 glioma cells as described in Materials and Methods. Following transfection, the cells were treated for 24 h without or with 500 ng/ml GH in serum-free F12/DMEM + 0.25% BSA. The cells were then harvested, and luciferase activity was determined. The values represent the relative luciferase activity compared with the activity in cells maintained in serum-free F12/DMEM + 0.25% BSA, which was defined as 1.0, and are the mean ± SEM of four (pGL2IGF-P2–1500), five (pGL2IGF-18, pGL2IGF-P2–485, pGL2IGF-18-Intron 2, and pGL3 Promoter-Intron 2), six (pGL2IGF-412), seven (pGL2), or eight (pGL2IGF-1690) independent experiments performed in triplicate.

 
As noted, a GH-inducible DNase I hypersensitivity site has been localized in intron 2 of the rat IGF-I gene (5). DNase I hypersensitivity sites often accompany alterations in chromatin structure that occur at sites of transcriptional activity, suggesting that the effect of GH may be mediated via DNA sequences in intron 2. To determine whether this DNase I hypersensitivity site mediated the effect of GH on IGF-I promoter activity, a 402-bp fragment of intron 2 that contains the GH-inducible DNase I hypersensitivity site was cloned 5' to either an SV40 promoter or a minimal IGF-I promoter (pGL2IGF-18). In cells cotransfected with pRc/CMV WTrGHR, pRc/CMV Jak2, and one of the above reporter genes, GH-inducible luciferase activity was not observed (Fig. 7Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although significant progress has been made in defining the promoters that regulate IGF-I gene expression and the signal transduction pathways that are activated by GH, the molecular mechanisms by which GH regulates IGF-I gene expression remain obscure. Previous in vivo studies have demonstrated that GH treatment activates IGF-I gene expression within 15 min and that protein synthesis is not required for GH-induced expression of IGF-I mRNA (47, 48). Although multiple sites of protein binding to the rat IGF-I gene have been demonstrated, including sites within promoter 1, promoter 2, and the GH-induced DNase I hypersensitivity site in intron 2 (10, 20, 22, 23, 28), GH-inducible protein binding to these sites could not be demonstrated (22, 23, 28). To begin to define the molecular mechanisms by which GH regulates IGF-I gene expression, we have developed a GH-responsive cell line that expresses the IGF-I gene. Rat C6 glioma cells have been shown previously to transcribe the IGF-I gene (29, 49), but, as demonstrated in these studies, they do not express the GH receptor. To confer GH responsivity, the C6 cells were cotransfected with vectors that express the GH receptor and Jak2, the tyrosine kinase that mediates most of the effects of the GH receptor (24). Using this model system, we have now demonstrated that a fragment of the IGF-I gene that extends from -412 in the region flanking exon 1 to the Met-22 ATG in exon 3 is GH-responsive in the context of an IGF-I-luciferase fusion gene (pGL3IGF-mini), suggesting that at least some of the sequences that mediate the effect of GH on IGF-I gene expression are present in this region. This is the first demonstration of a GH-inducible IGF-I promoter construct using a transient transfection assay system. The transient transfection system was also used to address the issue of the sequences that mediate the effect of GH on IGF-I gene expression. Interestingly, none of the IGF-I-luciferase fusion genes that contained fragments of promoter 1, promoter 2, or the GH-responsive DNase I hypersensitivity site in intron 2 demonstrated a consistent response to GH. Taken together with the data obtained using pGL3IGF-mini, these data suggest several possibilities. A straightforward possibility is that GH-responsive sequences are not present in any of these fusion genes, although these constructs contained most of the sequence present in pGL3IGF-mini, except for large portions of intron 2. A second possibility is that the effect of GH on IGF-I gene transcription is mediated via several different regions (e.g. sequences in promoter 1, promoter 2, and/or intron 2), such that the effect of any one of these in isolation in a fusion gene was insufficient to detect GH responsiveness using this model system. Finally, GH responsiveness of the DNase I hypersensitivity site in intron 2 might be position dependent.

As noted, a recently published study demonstrated that GH induced a 4.9-fold increase in the expression of an 11.3-kb IGF-I-luciferase transgene in mouse brain cells in primary culture (25). This transgene had a similar 3'-end to pGL3IGF-mini but contained 5.3-kb of sequence 5' to exon 1. There are several potential explanations for the more robust response achieved in those studies compared with the present study. For one, additional sequences not present in our IGF-I fusion gene were present, and these sequences may have enhanced the response to GH. Secondly, the differences could represent differences in the responsiveness of stably integrated DNA compared with transiently transfected DNA. Finally, the magnitude of the response could be cell type-specific due to differences in the signal transduction pathways activated by GH in the two cell types. Specific signaling molecules required for a robust response to GH stimulation may be absent or poorly activated in C6 glioma cells. To date, the signal transduction pathways that mediate the effect of GH on IGF-I gene expression have not been defined, but improved definition of GH-activated signal transduction pathways and studies using mice with null mutations of specific genes have provided some insight into the relevant pathways.

Subsequent to GH binding to its receptor and activating Jak2, a variety of signal transduction pathways are activated (24). Among the proteins that are phosphorylated/activated in response to GH are the mitogen-activated protein kinases (MAPKs), ERK-1 and -2, insulin receptor substrate-1 and -2 (IRS-1 and -2), phosphatidlyinositol-3 kinase (PI-3 kinase), and the transcription factors, STAT1, 3, and 5. Mouse models have facilitated identification of pathways that are important for GH-mediated IGF-I gene expression. Disruption of the STAT5b gene results in male mice with decreased body weight gain that first becomes evident at 3 weeks of age (50, 51). STAT5 is activated by the pulsatile pattern of GH stimulation in male mice, and the decrease in body weight gain in STAT5b-/- mice is accompanied by a 2-fold elevation in plasma GH levels and a 50% reduction in plasma IGF-I levels. Female STAT5b-/- mice demonstrated a minimal decrease in body weight gain with normal plasma GH levels but an approximately 50% reduction in plasma IGF-I levels. STAT5a-/- mice are not growth-retarded (51, 52), but both male and female STAT5a/b-/- mice are growth-retarded. Moreover, the growth retardation of male STAT5a/b-/- is more severe than that of male STAT5b-/- mice (51). Taken together, the above data suggest that STAT5a and STAT5b contribute to GH-mediated expression of the IGF-I gene. In the present studies, STAT5 was activated by GH in the GH-responsive C6 cells, as demonstrated using a reporter gene that contained six copies of a STAT5 binding site from the rat spi 2.1 gene. Interestingly, the modest GH-induced increase in the activity of this reporter gene was essentially the same as that of pGL3IGF-mini.

Because activation of the SIE by GH appears to be mediated by binding of STAT1 and 3, the studies using the reporter gene pTK81-SIE-Luc suggest that GH induced STAT1 and -3 activation in the C6 cells, (24, 40, 41, 42). Whether STAT1 and/or 3 are important for GH-mediated IGF-I gene expression is not known, although mice with a null mutation of the STAT1 gene are not small (53, 54). A null mutation of the STAT3 gene is an embryonic lethal (55), so its potential role in GH-mediated IGF-I gene expression remains unclear. Interestingly, GH had no effect on SRE activity in the C6 cells, despite the ability of phorbol esters to activate the SRE. Previous studies have demonstrated GH-inducible SRE activity that is dependent upon binding of both ternary complex factor and serum response factor to the SRE (56). Moreover, GH has been shown to stimulate phosphorylation and activation of a ternary complex factor, Elk-1, presumably via activation of the ERKs (56, 57). The pathway from the GH receptor to ERK activation, in turn, appears to involve assembly of a SHC-Grb2-SOS complex that activates Ras and subsequently Raf (58, 59). The results of the present study suggest that GH is unable to activate this pathway in C6 cells. Whether this pathway contributes to the induction of IGF-I promoter activity by GH is not known, but it may provide one explanation for the modest effect of GH on IGF-I promoter activity in these cells.

Mice with a null mutation of the hepatocyte nuclear factor-1{alpha} (HNF-1{alpha}) gene, a transcription factor that is expressed primarily in liver, but also in kidney, intestine, stomach, and pancreas, also demonstrate changes of GH resistance (60). At 12 weeks of age, both male and female Hnf-1{alpha}-/- mice were approximately 40% of the body weight of wild-type and heterozygote mice. Moreover, serum GH levels were approximately 3.5- and 10-fold elevated in male and female Hnf-1{alpha}-/- mice, respectively, whereas serum IGF-I levels were approximatley 25 and 50% of the levels in wild-type mice in female and male Hnf-1{alpha}-/- mice, respectively. These data suggest that HNF-1{alpha} is important for GH-mediated IGF-I gene expression, which is consistent with an earlier study demonstrating that overexpression of HNF-1{alpha} in Hep 3B cells transactivates the IGF-I gene via a site in promoter 1 of the human IGF-I gene (61). Given the tissue distribution of HNF-1{alpha}, it is unlikely that it is expressed in C6 cells, which is consistent with the inability of GH to stimulate the activity of the IGF-I-luciferase fusion genes that contained the HNF-1{alpha} binding site (pGL2IGF-1690, pGL2IGF-412, and pGL2IGF-18).

In summary, we have now defined an IGF-I-luciferase fusion gene that is GH responsive. Studies are in progress to increase its sensitivity to GH. With that accomplished, the model system described in these studies will be useful for dissecting the signal transduction pathways and elucidating the sequences within the IGF-I gene that mediate the effect of GH on IGF-I gene expression.


   Acknowledgments
 
The authors would like to thank Dr. Lawrence Mathews for providing the GH receptor cDNA.


   Footnotes
 
1 This work was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs and NIH Grants DK-46935 (to S.J.F.) and DK-47357 (to M.L.A.). Back

Received August 17, 1998.


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 Introduction
 Materials and Methods
 Results
 Discussion
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S. J. Frank, X. Wang, K. He, N. Yang, P. Fang, R. G. Rosenfeld, V. Hwa, T. R. Chaudhuri, L. Deng, and K. R. Zinn
In Vivo Imaging of Hepatic Growth Hormone Signaling
Mol. Endocrinol., November 1, 2006; 20(11): 2819 - 2830.
[Abstract] [Full Text] [PDF]

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 Endocr. Rev.Home page
V. C. Russo, P. D. Gluckman, E. L. Feldman, and G. A. Werther
The Insulin-Like Growth Factor System and Its Pleiotropic Functions in Brain
Endocr. Rev., December 1, 2005; 26(7): 916 - 943.
[Abstract] [Full Text] [PDF]

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 EndocrinologyHome page
K. He, K. Loesch, J. W. Cowan, X. Li, L. Deng, X. Wang, J. Jiang, and S. J. Frank
Janus Kinase 2 Enhances the Stability of the Mature Growth Hormone Receptor
Endocrinology, November 1, 2005; 146(11): 4755 - 4765.
[Abstract] [Full Text] [PDF]

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 J Biomol ScreenHome page
L. Rosengren, H. Simko, L. Aryan, P. Axelsson-Lendin, J. Chmielewska, A. Mode, and V. Parrow
Antisense and Sense RNA Probe Hybridization to Immobilized Crude Cellular Lysates: A Tool to Screen Growth Hormone Antagonists
J Biomol Screen, April 1, 2005; 10(3): 260 - 269.
[Abstract] [PDF]

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 J. Biol. Chem.Home page
Y. Wang and H. Jiang
Identification of a Distal STAT5-binding DNA Region That May Mediate Growth Hormone Regulation of Insulin-like Growth Factor-I Gene Expression
J. Biol. Chem., March 25, 2005; 280(12): 10955 - 10963.
[Abstract] [Full Text] [PDF]

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 Mol. Endocrinol.Home page
Y. Huan