Transcriptional regulation of IGF-I expression in skeletal muscle

G. E. McCall,1 D. L. Allen,2 F. Haddad,1 and K. M. Baldwin1

1University of California, Irvine, California 92697-4560; and 2University of Colorado, Boulder, Colorado 80304

Submitted 4 February 2003 ; accepted in final form 22 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study investigated the role of transcription in the regulation of insulin-like growth factor (IGF)-I expression in skeletal muscle. RT-PCR was used to determine endogenous expression of IGF-I pre-mRNA and mRNA in control (Con) and functionally overloaded (FO) rat plantaris. The transcriptional activities of five different-length IGF-I promoter fragments controlling transcription of a firefly luciferase (FLuc) reporter gene were tested in vitro by transfection of myoblasts or in vivo during FO by direct gene transfer into the plantaris. Increased endogenous IGF-I gene transcription during 7 days of plantaris FO was evidenced by an ~140-160% increase (P < 0.0001) in IGF-I pre-mRNA (a transcriptional marker). IGF-I mRNA expression also increased by ~90% (P < 0.0001), and it was correlated (R = 0.93; P < 0.0001) with the pre-mRNA increases. The three longest IGF-I exon 1 promoters induced reporter gene expression in proliferating C2C12 and L6E9 myoblasts. In differentiated L6E9 myotubes, promoter activity increased approximately two- to threefold over myoblasts. Overexpression of calcineurin and MyoD increased the activity of the -852/+192 promoter in C2C12 myotubes by ~5- and ~18-fold, respectively. However, FO did not induce these exogenous promoter fragments. Nevertheless, the present findings are consistent with the hypothesis that the IGF-I gene is transcriptionally regulated during muscle hypertrophy in vivo as evidenced by the induction of the endogenous IGF-I pre-mRNA during plantaris FO. The exon 1 promoter region of the IGF-I gene is sufficient to direct inducible expression in vitro; however, an in vivo response to FO may require elements outside the -852/+346 region of the exon 1 IGF-I promoter or features inherent to the endogenous IGF-I gene.

muscle fiber; hypertrophy; functional overload; transcription factor; myogenic regulatory factor; pre-messenger ribonucleic acid; myotube


THE ROLE OF LOCALLY PRODUCED insulin-like growth factor (IGF)-I protein in the hypertrophy of mechanically loaded skeletal muscle and in the prevention of atrophy in aging muscle has received considerable interest as a fundamental factor that can modulate changes in muscle mass (for reviews see Refs. 4, 5, and 18). The time course for increased skeletal muscle IGF-I mRNA expression during functional overload (FO) of the rat plantaris is consistent with the hypothesis that the increase in IGF-I protein involves pretranslational processes (6). In addition, several different IGF-I isoforms are created by posttranscriptional processing of the IGF-I primary transcripts by alternative splicing, including an isoform expressed in skeletal muscle, which has been designated as mechano growth factor (MGF) (16). MGF is postulated to be the major IGF-I gene product mediating load- and stretch-induced adaptations in skeletal muscle (36). However, despite evidence that these locally derived IGF-I isoforms are fundamental in the plasticity of skeletal muscle, no study has verified whether an increase in IGF-I gene transcription occurs during hypertrophy. Also, little is known concerning the regulatory elements of the IGF-I promoter region that are responsive to altered mechanical loading in skeletal muscle. Defining the regulatory mechanisms controlling IGF-I gene expression in skeletal muscle could facilitate the development of pharmacological and/or other strategies to enhance muscle IGF-I expression to counter the muscle atrophy that accompanies aging and other states of reduced neuro-muscular activity and/or loading.

The human and rat IGF-I genes (Fig. 1A) consist of six exons, with distinct promoter regions regulating transcription initiation for both exons 1 and 2 (17). Exons 1 and/or 2 encode untranslated alternate leader sequences, whereas exons 3 and 4 encode the mature peptide domains A, B, C, and D. Exon 4 also encodes a small portion of an extension (E) domain that serves as a signaling peptide, whereas exons 5 and 6 each encode alternate E domains (35). Circulating IGF-I in adult rats is primarily synthesized in the liver, with hepatic transcription initiated by both exon 1 and 2 promoters (30). In contrast, local IGF-I transcription appears to be regulated by the exon 1 promoter in most other tissues, including skeletal muscle (17, 30).



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Fig. 1. Schematic of insulin-like growth factor (IGF)-I gene and promoter-reporter constructs. A: illustration of the IGF-I gene, which includes 6 exons (boxes) and 5 introns (lines connecting exons). Promoter transcription start sites (TSS) on exons 1 and 2 are depicted by arrows; the height and weight of the arrowhead indicate the strength of the TSS. The 5.5-kb fragment of DNA encompassing the IGF-I minigene construct is indicated by a bracket. The positions of promoter TSS and exon/intron sizes are not to scale. B: IGF-I exon 1 promoter-firefly luciferase (FLuc) reporter constructs used for in vitro transfection and in vivo injection. Arrows indicate TSS, with TSS 1 designated as +1 using the nomenclature of Adamo et al. (2). Numbers indicate the locations of deletion constructs and are relative to TSS 1.

 

The structure of the proximal exon 1 promoter (Fig. 1) reveals the complexity of the rat IGF-I gene, which lacks typical promoter regulatory elements such as TATA boxes, CCAAT boxes, and GC-rich regions (17, 34). Adding further to this complexity, previous in vitro studies have identified two major and two minor exon 1 transcription start sites (TSS) in the ~0.36-kb promoter region located within exon 1 (34). TSS 3 was identified as the primary TSS in several types of nonmuscle cells in vitro, because deletion of this site caused an ~1.5- to 9-fold decrease in promoter activity (34). Moreover, ribonuclease protection assays indicate that TSS 3 also appears to be the main TSS in skeletal muscle (17).

Given the absence of any previous information showing that transcription controls the elevated IGF-I mRNA and protein during muscle overload, the first aim of the present study was to test the hypothesis that IGF-I induction during muscle overload is transcriptionally regulated. We hypothesized that muscle IGF-I pre-mRNA (the primary product of transcription) levels would increase in 7-day FO rat plantaris muscles, because this time point (7 days) was previously reported as the peak elevation for muscle IGF-I mRNA and protein (6). The second aim was to characterize the promoter region(s) regulating transcription of IGF-I in skeletal muscle. To achieve this aim, we investigated the activity of different-length exon 1 promoter fragments in muscle cells both in vitro and in vivo with reporter gene assay technology.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
FO experiments. FO of the left plantaris was accomplished in female Sprague-Dawley rats (~121 ± 10 g body wt; n = 3-10 rats/group) by surgical ablation of the gastrocnemius and soleus muscles (10). The nonoperated right plantaris served as the control (Con). IGF-I exon 1 promoter activity was tested by injection of promoter-reporter plasmids into the FO and Con plantaris muscles with a 29-gauge needle attached to a 0.5-ml insulin syringe. Plasmids were injected in Con and FO plantaris immediately before the FO ablation surgery. For the Con plantaris, a skin incision was made to access the plantaris muscle. Plasmid mixtures consisted of the rat IGF-I exon 1 test promoter (molar equivalent to 10 µg of -852/+346 IGF-I pGL3 promoter construct) and a fixed amount (3.29 µg) of the human skeletal {alpha}-actin reference promoter construct (HSA2000-pRL) in 20 µl of sterile phosphate-buffered saline (PBS). To further enhance plasmid uptake after injection, the plantaris was electroporated (4 pulses at 200 V/cm, 50 ms each, 1 Hz, followed by 4 identical pulses in reverse polarity) with two gold-plated electrodes set 4 mm apart and placed on the injection site (23). After 7 days of FO, rats were euthanized with pentobarbital sodium (100 mg/kg) and plantaris muscles were dissected, trimmed of excessive connective tissue, quick frozen, and stored at -80°C until analysis. All procedures involving rats were approved by the University's internal review board.

Plasmid constructs. The initial IGF-I promoter sequence (see Fig. 1B) was a gift from Dr. Peter Rotwein (Oregon Health Sciences University, Portland, OR) and consisted of the rat exon 1 IGF-I promoter encompassing -852 to +346 bp [+1 is the first TSS as identified by Adamo et al. (3)]. This promoter sequence was linked to a firefly luciferase (FLuc) reporter gene in the pGL3 basic vector (Promega) with standard cloning techniques. Deletion fragments of the 5' and/or 3' ends were produced from this longest fragment by using high-fidelity PCR to generate specific promoter fragments that were subcloned into the pGL3 basic plasmid at unique restriction enzyme sites in the multicloning site upstream of the FLuc reporter gene (Fig. 1B). In addition, a 5.5-kb IGF-I minigene (11) [obtained from Dr. Martin Adamo (University of Texas Health Science Center, San Antonio) and courtesy of Dr. William Lowe (Northwestern University Medical School, Chicago, IL)] containing 412 bp of the region upstream from TSS 1 and exons 1 and 2 and a portion of exon 3 plus introns 1 and 2 inserted into pGL3 also was investigated (Fig. 1A). The -852/+346 bp, -852/+192 bp, -250/+192 bp, -250/+64 bp, and minigene IGF-I promoter pGL3 constructs were individually tested in a series of separate experiments. For in vivo experiments, a coinjected reference promoter consisted of the human skeletal {alpha}-actin promoter (a kind gift from Dr. Steven Swoap, Williams College, Williamstown, MA) extending from -2000 to +250 bp relative to the TSS (26) and linked to the Renilla luciferase (RLuc) reporter gene in the pRL vector (Promega). The IGF-I promoter activity in Con and FO plantaris was expressed relative to the activity of a promoterless pGL3 basic plasmid.

In vivo reporter expression assays. Each plantaris was homogenized in 1.5 ml of ice-cold passive lysis buffer (Promega) supplemented with protease inhibitors [0.2 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 5 µg/ml aprotinin, and 5 µg/ml leupeptin] made in nuclease-free deionized water. A 250-µl aliquot of this homogenate was immediately mixed with 750 µl of TRI-LS reagent (Molecular Research Center) and stored at -80°C for RNA extraction. The remaining homogenate was centrifuged at 10,000 g for 10 min at 4°C. The supernatant was decanted and used to measure reporter gene activity with the Promega Dual Luciferase Assay kit, which is designed for detection of both FLuc and RLuc activities from a single sample. Five microliters of supernatant was used in the assay to measure light output integrated over 10 s with a luminometer (Analytical Luminescence) and expressed as relative light units (RLUs). Background activity was determined from noninjected normal muscle extracts and was subtracted from the activity of plasmid-injected muscles.

Pre-mRNA and mRNA analyses. Total RNA was extracted from 250 µl of total muscle homogenate with TRI-LS reagent according to the supplied procedure (Molecular Research Center). The RNA pellet was suspended in nuclease-free water and treated with DNase I according to the supplier's (Invitrogen) recommendation to remove any genomic DNA contamination. After DNase I treatment, RNA concentration was determined by ultraviolet light (UV) absorbance at 260 nm. One microgram of RNA was reverse transcribed in twenty microliters of total volume with Superscript II (Invitrogen) and a primer mix consisting of oligo(dT) (100 ng/µl) and random decamers (200 ng/µl) according to the supplier's (Invitrogen) recommendation. PCR amplification to detect IGF-I pre-mRNA used 1 µl of cDNA amplified for 30 cycles. Two separate sets of IGF-I pre-mRNA PCR primers ~55 kb apart were designed to distinguish between transcripts either near transcription initiation (exon/intron 1 primers) or as elongation proceeded (exon/intron 3 primers). Primers designated as I1 produced a 363-bp PCR product with the following primers: a forward primer based on exon 1 sequence, TGCGCAATCGAAATAAAGTCCTCA, and a reverse primer complementary to the sequence from intron 1 of the IGF-I gene, AGCTCCCACAGAACCGCACATT. Primers designated as I3 produced a 295-bp PCR product with the following primers: a forward primer based on exon 3 sequence, ACAGACGGGCATTGTGGATGA, and a reverse primer complementary to sequence from intron 3 of the IGF-I gene, TTGGAAGGGTGGACCCATAAGC. Furthermore, additional primers were designed to evaluate initiation at TSS 2 and TSS 3 as identified by Adamo et al. (1). For TSS 2, the forward primer was TGTTCCCCCAGCTGTTTCCTGTCT and resided between TSS 2 and TSS 3 on exon 1, with the reverse primer complementary to the sequence from exon 3, CACTCATCCACAATGCCCGTCTGT, to produce a 539-bp PCR product. In combination with this same exon 3 reverse primer, another exon 1 forward primer distal to TSS 3, GCCTGCGCAATCGAAATAAAGTCC, was used to detect a 417-bp mRNA transcript from combined TSS 2 and TSS 3.

PCR reactions to determine IGF-I mRNA used 0.1 µl of cDNA amplified for 25 cycles to produce a 202-bp product with the following primers: a forward primer derived from exon 3 sequence, GCATTGTGGATGAGTGTTGC, and a reverse primer complementary to exon 5 sequence, GGCTCCTCCTACATTCTGTA. The PCR reaction mixture consisted of 1x PCR buffer, 0.5 µl of 10 mM dNTP, 0.75 µl of 50 mM MgCl2, 3 µl of 5' + 3' IGF-I primers at 5 pmol/µl, 0.15 µl of Biolase DNA polymerase (Bioline), and water to a final volume of 25 µl. Amplification was performed by using a Stratagene thermocycler with an initial denaturization step of 3 min at 96°C followed by 25 or 30 cycles of 1 min at 96°C, 45 s at 58°C, and 45 s at 72°C. The final elongation step was 3 min at 72°C. All samples were run in duplicate, and PCR products were separated on 2% agarose gels and stained with ethidium bromide. Pictures were taken in UV conditions with Polaroid 55 film, negatives were scanned (Molecular Dynamics densitometer), and band intensity was measured by volume integration with Image Quant 5.0 software (Molecular Dynamics). All PCR primers were designed with sequence data from rat GenBank accession no. NW044015, with the IGF-I gene located between positions 1,109,034 and 1,183,518.

Myoblast cell culture: cell culture and transfections. Mouse C2C12 myoblasts and rat L6E9 myoblasts were obtained from American Type Culture Collection (Rockville, MD). Primary mouse muscle fibroblasts were isolated from adult mouse skeletal muscle with standard techniques. Briefly, adult C57 male mice were killed by cervical dislocation and the thigh and calf muscles were isolated, minced, and placed in ice-cold Dulbecco's modified Eagle's medium (DMEM; GIBCO). The muscle fragments were digested with 0.01% trypsin and 0.5% collagenase type IV for 3 x 10 min. The fragments were triturated by pipetting up and down 20-30 times and then filtered through a 40-µm mesh (Becton Dickinson). The resulting suspension was centrifuged for 5 min at 500 g, and the pellet was resuspended in fresh growth medium consisting of DMEM plus 10% fetal bovine serum (FBS; Hyclone) and preplated on uncoated 10-cm dishes for 30 min at 37°C. These cells, which were enriched for fibroblasts, were grown to near confluence and then passaged two or three times to allow fibroblasts to proliferate. At this stage >99% of the cells were nonmyogenic as determined by staining for the muscle-specific intermediate filament protein desmin (data not shown).

Transfections for all three cell types were carried out under identical conditions. An aliquot of cells was thawed, grown to ~80% confluence, and then passaged (1/4 for C2C12 cells, 1/3 for L6E9 cells, and 1/2.5 for fibroblasts) onto 0.5% gelatin-coated 24-well tissue culture plates. The following day (~80% confluence) the cells were transfected with Lipofectamine 2000 reagent (Invitrogen) per the manufacturer's instructions. Briefly, 2.0 µl of Lipofectamine 2000 and 0.8 µg of total DNA per well were diluted in 50 µl each of DMEM without penicillin-streptomycin, allowed to complex for 20 min, and added to the wells. In addition to the IGF-I promoter constructs, other cultures were transfected with an embryonic myosin heavy chain (MHC) promoter-reporter construct as a positive control. The following day, C2C12 and L6E9 myoblasts and 1-day transfected primary fibroblasts were harvested by removing the medium, rinsing with PBS, and scraping them into 100 µl of 1x passive lysis buffer (Promega). At this point, C2C12 and L6E9 myoblasts were placed in differentiation medium consisting of DMEM plus 1% horse serum (GIBCO), differentiated for 2 days, and harvested as described for myoblasts. For cotransfections, equal amounts of IGF-I promoter construct plasmid DNA and constructs overexpressing specific signaling molecules or transcription factors were added to the cells. The following genes were used for cotransfections, and overexpression was driven by a cytomegalovirus (CMV) promoter unless noted otherwise: MyoD, which was driven by the RSV promoter; serum response factor (SRF); myocyte enhancer factor-2C (MEF-2C); a constitutively active nuclear form of the nuclear factor-activator of T cells (NF-AT) of transcription 3 gene (NFAT3); GATA-4; MEKK1; constitutively active calcineurin; focal adhesion kinase (FAK); and FAK-related nonkinase (FRNK), a dominant-negative inhibitor of FAK activity. These constructs were described previously (8). The FAK and FRNK constructs were kindly provided by Dr. David Schlaepfer (Scripps Research Institute, San Diego, CA); the MEKK1 was purchased from Stratagene; all others were kindly provided by Dr. Eric Olson (University of Texas-Dallas Southwestern Medical Center). All cell culture experiments were replicated three or four times in quadruplicate wells. Ten microliters of cell homogenate was used to assay for FLuc activity with the Promega luciferase kit. In all experiments, a promoterless pGL3 basic construct was also transfected into an equal amount of wells and all IGF-I luciferase values were normalized to pGL3 basic luciferase values to generate the fold difference between the IGF-I promoter-containing constructs and the promoterless pGL3 basic construct. To control for potential effects of the cotransfection, such as promoter interference, a CMV-{beta}-galactosidase promoter-reporter plasmid construct also was cotransfected with the IGF-I promoter construct. These control experiments found no adverse effects of CMV-{beta}-galactosidase cotransfection on the IGF-I promoter activity, which was approximately one- to twofold and approximately fivefold above the promoterless pGL3 in myoblasts and myotubes, respectively, and thus essentially identical to the IGF-I promoter-only transfection condition (see Fig. 4).



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Fig. 4. Cotransfection of C2C12 (A) and L6E9 (B) myotubes with the -850/+192 IGF-I promoter constructs and other plasmid constructs overexpressing common muscle signaling and transcription factors. Data are calculated as fold activity relative to mean promoterless (basic) pGL3 (gray line). Values are means ± SE. Each bar represents the mean of 3-4 experiments, with 4 replicate wells/experiment. *Significantly greater than myotubes transfected with only -852/+192 IGF-I promoter. SRF, serum response factor; NF-AT, nuclear factor-activator of T cells; MEF, myocyte enhancer factor; FAK, focal adhesion kinase; FRNK, FAK-related nonkinase; Ca iono, calcium ionophore.

 

Statistical analysis. IGF-I pre-mRNA and mRNA levels were each compared between Con and FO groups by paired t-tests. In addition, the percent increases of FO plantaris from Con plantaris were calculated with the respective mean Con value and compared by a paired t-test. The fold activation of the IGF-I promoter (i.e., FLuc activity) and the ratio of IGF-I to {alpha}-actin (i.e., FLuc:RLuc activities) were calculated relative to the mean values from the promoterless (basic) pGL3 experiment and compared among the different IGF-I promoter-reporter construct experiments with ANOVA and Fisher's post hoc test. For cell culture, the fold activity of the IGF-I promoter constructs relative to promoterless pGL3 were compared by ANOVA and Fisher's post hoc test. P < 0.05 was considered significant. Data are reported as means ± SE. Statistical analyses were performed with Stat-view (SPSS) or Prism3 (GraphPad) software.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IGF-I pre-mRNA and mRNA during FO. Seven days of unilateral plantaris FO induced by surgical removal of the synergist gastrocnemius muscle resulted in a significant (P <= 0.05) increase in mean plantaris muscle wet weight (mean increase 32 ± 9%) in all experiments. FO in plantaris was associated with ~140% (I1 primers) to ~160% (I3 primers) increases (P < 0.0001) in IGF-I pre-mRNA expression and an ~90% increase (P < 0.0001) in IGF-I mRNA expression as determined by semiquantitative RT-PCR (Fig. 2A). The increase in IGF-I pre-mRNA (I3 primers) was significantly greater than the increase in IGF-I mRNA (P = 0.0003). Moreover, the relationship between pre-mRNA (I3 primers) and mRNA across the Con and FO groups was highly correlated (R = 0.93, P < 0.0001; Fig. 2B), indicating that increased IGF-I mRNA expression resulted from increased pre-mRNA production rather than an increase in its stability. The similar increases and significant correlation (R = 0.83, P < 0.0001) between IGF-I pre-mRNA detected by the I1 and I3 PCR primers indicate that the increase in pre-mRNA in FO plantaris is due to transcription initiation rather than elongation. Thus the induction of IGF-I mRNA in response to FO is consistent with a transcriptional regulation. With regard to which TSS of exon 1 is active in muscle and responsive to FO, RT-PCR analysis with primers differentiating between TSS 2 and TSS 3 showed that TSS 3 is 15-20 times more active than TSS 2. Both TSS 2 and TSS 3 mRNA products increased in FO plantaris (data not shown).



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Fig. 2. IGF-I pre-mRNA and mRNA analysis in control and overload plantaris. A: plantaris IGF-I pre-mRNA and mRNA each increase significantly (*P < 0.0001; paired t-tests) from control (Con) during functional overload (FO), and the % increase in IGF-I pre-mRNA (I3 primers) is significantly greater ({dagger}P = 0.0003; paired t-test) than the % increase in mRNA. Values are means ± SE. B: positive correlation (Pearson R = 0.93; P < 0.0001) between plantaris IGF-I pre-mRNA (I3 primers) and IGF-I mRNA levels in Con and FO muscles. OD, optical density.

 

Promoter activity in cultured myocytes and muscle fibroblasts. The shortest IGF-I promoter construct (-250/+64), which lacked the major TSS 3, was not significantly different from the promoterless pGL3 in C2C12 and L6E9 myoblasts and myotubes (Fig. 3, A and B). However, the three longer IGF-I promoter constructs all showed significantly greater (P < 0.0001) activity than the promoterless pGL3 plasmid in myoblasts and myotubes. The activities of these three longer promoter constructs were approximately one- to threefold greater than the promoterless pGL3 in mouse C2C12 cells (Fig. 3A) and approximately twofold greater in rat L6E9 myoblasts (Fig. 3B). Although IGF-I promoter activity was not significantly different between C2C12 myoblasts and myotubes, in L6E9 cells IGF-I promoter activity was approximately four- to sixfold above promoterless pGL3 in L6E9 myotubes compared with approximately twofold greater in myoblasts (Fig. 3B). In comparison, the embryonic MHC promoter activities relative to promoterless pGL3 were ~50- and 230-fold higher in proliferating myoblasts and ~525- and ~2,180-fold higher in differentiated myotubes in L6E9 and C2C12 cultures, respectively.



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Fig. 3. IGF-I promoter-reporter activity in C2C12 (A) and L6E9 (B) myoblasts and muscle-derived mouse fibroblasts (C) expressed as fold activity relative to mean promoterless (basic) pGL3 (gray line). Values are means ± SE. Each bar represents the mean of 3-4 experiments, with 4 replicate wells/experiment. *Significantly greater than promoterless pGL3 (P < 0.05).

 

Because the identity of IGF-I-expressing cells in skeletal muscle has not been definitively established, we also examined IGF-I promoter activity in muscle-derived fibroblasts. In fibroblasts derived from mouse skeletal muscles, promoter activity over promoterless pGL3 at 1 day after transfection was higher only for the -250/+192 IGF-I construct (Fig. 3C). However, at 3 days after transfection, promoter activity was approximately two- to fourfold higher (P <= 0.005) than promoterless pGL3 for the -852/+346, -250/+192, and minigene constructs (Fig. 3C).

We also tested whether the activity of the -852/+192 IGF-I promoter construct in myotubes could be further induced by cotransfection with overexpression constructs containing a variety of transcription factors and signaling molecules associated with muscle growth and differentiation. None of the cotransfected constructs had an effect on IGF-I promoter activity in L6E9 myotubes (Fig. 4B). However, cotransfection of C2C12 myotubes with either the myogenic regulatory factor MyoD or a constitutively active form of the calcium signaling phosphatase calcineurin resulted in ~5- and ~18-fold (P < 0.0001) increases in -852/+192 IGF-I promoter construct, respectively (Fig. 4A). These data demonstrate that in C2C12 myotubes, IGF-I promoter activity can be further augmented by overexpression of factors associated with muscle growth and differentiation.

In vivo promoter activity of FO plantaris. Across all experiments in both the Con and FO groups, the FLuc reporter of IGF-I promoter activity was very low relative to promoterless (basic) pGL3 (Fig. 5A). The lowest promoter activity was generally observed for the shortest construct (-192/+64), which lacked the major TSS 3, suggesting that this site is a major TSS for skeletal muscle in vivo. Only the IGF-I minigene showed higher (P = 0.01) basal FLuc activity than basic (promoterless) pGL3 (Fig. 5A). When expressed relative to the coinjected {alpha}-actin promoter activity, the IGF-I promoter activities of both the IGF-I minigene and the longest exon 1 fragment, -852/+346, were greater than the basic pGL3 (Fig. 5B). Moreover, the ratio of IGF-I to {alpha}-actin promoter activities was similar between FO and Con muscles for each of the constructs tested (Fig. 5B). Thus, although the reporter activities of the two longer IGF-I promoter fragments were above a promoterless pGL3, their activity was not induced further in response to FO.



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Fig. 5. IGF-I promoter-reporter activity in Con and FO plantaris muscles 7 days after injection with promoter-reporter constructs. Values are means ± SE. Fold activity relative to promoterless basic pGL3 for FLuc, the reporter for the IGF-I promoter (A) and the ratio of IGF-I-to-{alpha}-actin reporter activity (B) are shown. *Con and FO promoter activity are significantly greater than Con basic pGL3 (P < 0.05).

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Numerous studies have demonstrated a fundamental role for increased IGF-I gene expression in skeletal muscles in response to altered loading states, thereby indicating an autocrine/paracrine role for this growth factor in the regulation of muscle plasticity (for recent reviews see Refs. 5 and 16). In addition, there is considerable evidence that IGF-I primary transcripts can be alternatively spliced to give rise to a load- and/or stretch-sensitive isoform of IGF-I in skeletal muscle, which has been named mechano growth factor (MGF) (22, 35). Although a previous study demonstrated an increase in muscle IGF-I mRNA in response to FO (6), it was not clear whether this increased RNA expression involved transcriptional processes. In the present study, an increase in endogenous IGF-I gene transcription in overloaded rat plantaris was confirmed by the ~140-160% increase in the IGF-I pre-mRNA, which is the initial RNA product of gene transcription. Moreover, the increase in pre-mRNA detected by I1 primers strongly indicates that FO induced transcription initiation of the IGF-I gene. Prior studies confirmed that hepatic IGF-I pre-mRNA levels reflect transcriptional activity, as evidenced by a positive correlation between pre-mRNA levels with data quantified from nuclear run-on assays during fasting conditions (19), development (20), and growth hormone treatment (12). In the present study, the strong positive correlation (R = 0.93, P < 0.0001) between the levels of IGF-I pre-mRNA and mRNA in Con and FO muscles demonstrates that the alterations in IGF-I pre-mRNA and mRNA levels are tightly linked. In this regard, the present results confirm that the increase in local IGF-I protein expression in overloaded skeletal muscle occurs, at least in part, by inducing transcription of IGF-I. Given that MGF is produced by alternative splicing of the pre-mRNA, an increase in IGF-I transcription also provides more substrate for MGF-I production, further indicating that IGF-I transcription is essential for expression of this local stretch-induced growth factor. Therefore, the results of the present study indicate that IGF-I gene transcription is a key mechanism for increasing these two IGF-I protein isoforms in overloaded muscles.

Very little information is available concerning the regulatory elements controlling IGF-I gene expression in skeletal muscle or other tissues in vivo. Previous attempts to identify the regulatory features of the rat IGF-I gene promoter regions revealed the uniqueness and complexity of its structure. Two promoter regions have been identified, each of which precedes/includes exon 1 and 2 (1). Initiation of transcription at these distinct promoter regions gives rise to IGF-I mRNAs with alternate untranslated leader sequences. The exon 1 promoter controls expression in liver and in many other tissues that produce IGF-I, including skeletal and cardiac muscle (30). IGF-I expression controlled by exon 2 promoter activity occurs in relatively fewer types of tissue, but this promoter is particularly active in liver and kidney (30). The exon 1 promoter includes four TSS located within ~0.36 kb of exon 1 (2). TSS 3 appears to be the major site of transcription initiation in most tissues, including rat skeletal muscles that were analyzed by ribonuclease protection assay techniques (17, 30). The results of the present study are consistent with TSS 3 as the most active TSS in skeletal muscle, with TSS 2 transcripts expressed at much lower levels. However, TSS 2 and TSS 3 showed comparable increases in response to muscle overload.

We tested the activity of four IGF-I exon 1 promoter fragments that encompassed up to 1.2 kb of the regions previously shown to modulate in vitro reporter activity in various nonmuscle cells (2). In addition, we tested an IGF-I minigene that encompassed 5.5 kb of the IGF-I gene beginning at -412 bp proximal to exon 1 and continuing through the initial portion of exon 3. To gain insight into the muscle-specific regulation of these IGF-I promoter-reporter plasmids, cell culture experiments were undertaken with myocyte cell lines derived from mice (C2C12) and rats (L6E9). In both muscle cell lines, the tested IGF-I promoter activity was highest for the three constructs that included the major TSS 3 located at approximately +135 bp downstream from TSS 1 (33). Activity of the -250/+64 construct, which included only TSS 1 and 2, exhibited reporter activity similar to the basic/promoterless pGL3 control. Thus our results are consistent with TSS 3 functioning as the major TSS for cultured myoblasts. Moreover, IGF-I promoter activity increased on differentiation in the L6E9, but not C2C12, cells (Fig. 3, A and B). However, it was surprising that promoter activity did not increase further in the C2C12 myotubes because Yoshiko et al. (37) recently reported an IGF-I-dependent regulation of differentiation in C2C12 cells. The IGF-I exon 1 promoter constructs we tested apparently do not contain the regulatory region(s) of the IGF-I promoter capable of increasing the expression of the reporter gene despite the reported presence of IGF-I mRNA in proliferating C2C12 myoblasts and the further induction of IGF-I mRNA during differentiation of C2C12 myotubes (37).

To gain insight into IGF-I promoter activity in other cell types associated with skeletal muscle, primary cultures of muscle-derived fibroblasts also were transfected with the IGF-I plasmid constructs. The IGF-I promoter activity observed in fibroblasts provides several key observations. First, it shows that nonmuscle cells might also contribute to IGF-I mRNA and protein levels measured from whole muscle homogenates. Second, the findings demonstrate that the IGF-I promoter constructs are active in cells derived from primary cultures derived from skeletal muscle despite the absence of reporter activity in the in vivo muscle transfection experiments. Third, they indicate the weakness of this promoter region in yet another in vitro cell system, perhaps because critical regulatory elements are missing that are required to induce a more robust transcriptional activity across the diverse range of cell types that have been studied to date. However, we also cannot rule out the possibility that muscle-derived fibroblasts merely do not exhibit a high level of IGF-I expression.

The relatively moderate level of reporter activity in the cell culture experiments demonstrates that the IGF-I exon 1 promoter region is a weak promoter compared with other inducible muscle-specific genes. For example, Allen and coworkers showed that the MHC isoform IIa promoter activity in C2C12 myotubes can be induced ~10-20-fold by treatment with calcium ionophores (7) and up to 200-fold by cotransfection of constitutively active calcineurin (8). These same investigators also reported that overexpression of MyoD induced an ~10-fold increase in the promoter activity of the MHC IIb isoform in C2C12 myotubes (8). The approximately one- to sixfold increase in IGF-I promoter activity we observed in myocytes and muscle-derived fibroblasts under various conditions is, however, generally comparable to the activities previously reported for similar IGF-I constructs in Chinese hamster ovary (34), osteoblasts (31), and glial, pituitary, and ovarian tumor cells (34). Rotwein and colleagues (17, 21) also reported IGF-I exon 1 promoter activities similar to that of the present study for an IGF-I-producing human neuroblastoma cell line (SK-N-MC). However, a more recent study by Rotwein's group (24), as well as a study by Wang et al. (33), reported a more robust 16- to 70-fold IGF-I promoter activity in this same cell line. The reason for discrepancies between their earlier and more recent results in this neuroblastoma cell line is unclear. Nevertheless, the results across the majority of in vitro studies using transient gene transfection techniques in multiple cell types do not demonstrate high levels of activity for the regions of the IGF-I exon 1 promoter studied to date, and the present results are consistent with these observations.

Several previous in vitro studies identified putative control elements in a variety of nonmuscle cell types. Potential regulatory elements within the IGF-I exon 1 promoter region include binding sites for Sp1 (38), CCAAT/enhancer-binding protein (32), GATA (33), and response elements for prostaglandin E2 (27) and cAMP (31). In the present study, cotransfecting C2C12 myotubes with plasmid vectors that overexpress common muscle signaling molecules or transcription factors showed that IGF-I promoter activity was further induced in C2C12 myotubes by either MyoD or constitutively active calcineurin (Fig. 4A). Interestingly, MyoD and calcineurin overexpression had no effect on IGF-I promoter activity in L6E9 myotubes. Each of these molecules has been investigated for its role in muscle hypertrophy and/or phenotype plasticity (6, 28). MyoD is a member of a family of DNA-binding transcription factors called myogenic regulatory factors (MRFs) that regulate muscle-specific gene transcription (8) and can control the myogenic determination and/or differentiation of myoblasts during development (9). Inspection of the IGF-I -852/+192 exon 1 fragment indicates five regions that contain the putative consensus binding sequence (CANNTG) for MRFs known as E-boxes. The results of the present study indicate that calcineurin can also induce IGF-I gene expression in C2C12 myotubes. Calcineurin is a calcium-activated phosphatase that has been shown to mediate the effects of IGF-I signaling on hypertrophy of C2C12 (29) and L6E9 myotubes (25). Thus, in the case of C2C12 myotubes, IGF-I and calcineurin may be operating in a positive feedback loop with one another such that these factors cooperatively enhance their respective roles during myotube hypertrophy. Calcineurin affects transcriptional activity via dephosphorylation of NF-AT, which can regulate transcription by binding to NF-AT response elements in target genes (28). Indeed, sequence analyses of the IGF-I -852/+192 exon 1 fragment indicates that five putative NF-AT response elements (GGAAA) reside within this region. These observations, to our knowledge, are the first insights into which regulatory factor(s) might be involved in the induction of IGF-I gene transcription in skeletal muscle.

We also tested the activity of these exon 1 IGF-I promoters in vivo by injecting the promoter-reporter plasmids into plantaris muscles subjected to chronic muscle overload. Only the longest IGF-I exon I promoter (-852/+346) and minigene constructs exhibited significant reporter activity above the promoterless control in our experimental paradigm, which significantly induces transcription of the endogenous IGF-I gene. The inability of these IGF-I promoter regions to drive more robust expression in vivo was not due to low levels of plasmid uptake, because a coinjected skeletal muscle {alpha}-actin promoter expressing the RLuc gene showed very high activity (~400-fold higher than background) and increased by ~50% in FO plantaris. In addition, Giger et al. (15) used the direct gene transfer approach to study regulation of the type I MHC promoter in overloaded skeletal muscle and reported an ~100-130% increase in the type I MHC promoter activity in FO plantaris.

One possible reason for the failure of exogenous IGF-I promoter fragments to drive measurable reporter activity is that they could lack essential regulatory element(s) required for high levels of promoter activity. These elements could be located as far as several kilobases upstream or downstream of a TSS. The likelihood that control elements exist beyond the more well-characterized exon 1 promoter regions tested in the present study is supported by the inability of a previous study to identify prostaglandin A2 regulatory elements known to repress IGF-I transcription in glioma cells (13). In that study, Bui et al. (13) concluded that the prostaglandin A2 repressive element(s) extend beyond the exon 1 promoter region between -1711 to +328. The fact that even the IGF-I minigene construct was not induced by FO in the present study provides additional information that regulation of IGF-I transcription during increased muscle loading in vivo requires an even more extensive promoter region of this gene. Alternatively, the inactivity of the exogenous IGF-I promoters in muscle after direct gene transfer might be due to the fact that naked plasmid DNA lacks chromatin structural components that could be important in the recruitment of transcriptional activators necessary to drive its transcription. The endogenous gene is a constitutive component of the chromatin, which is a highly complex structure involving multiple protein-DNA dynamic interactions (14). The remodeling of chromatin structure alters the accessibility of genomic DNA to specific activators or repressors, general transcription factors, and RNA polymerase (14). This issue could be addressed if the IGF-I promoter-reporter constructs were integrated into the chromosomes such as occurs in the generation of transgenic animals.

In summary, muscle IGF-I gene expression was induced by chronic overload as evidenced by increased IGF-I pre-mRNA in the FO plantaris. The exon 1 IGF-I promoter constructs we tested showed moderate activity in myoblasts in cell culture and were inducible by overexpression of the muscle transcription factor MyoD and the calcium signaling molecule calcineurin. However, these IGF-I promoter constructs did not initiate in vivo load-stimulated reporter gene expression in overloaded muscles despite endogenous IGF-I gene transcription. This different behavior of the IGF-I promoter in vivo vs. in vitro indicates that presently unidentified regions and/or structural features of the IGF-I gene and chromosome might be required for in vivo transcription, further illustrating the complexity and uniqueness of in vivo IGF-I gene regulation.


    DISCLOSURES
 
This work was supported by National Space and Biomedical Research Institute Grant NCC9-58-A and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-30346 (to K. M. Baldwin).


    ACKNOWLEDGMENTS
 
We thank Dr. Peter Rotwein for providing the IGF-I exon-1 promoter, Drs. William Lowe and Martin Adamo for the IGF-I minigene, and Dr. Steven Swoap for the {alpha}-actin promoter. In addition, we thank Dr. Leslie Leinwand for assistance with the in vitro experiments.


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. M. Baldwin, Univ. of California, Irvine, Physiology and Biophysics, 346-D Med Sci I, Irvine, CA 92697-4560 (E-mail: kmbaldwi{at}uci.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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