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J. Biol. Chem., Vol. 277, Issue 52, 50535-50542, December 27, 2002

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Insulin-like Growth Factor-1 Increases Skeletal Muscle Dihydropyridine Receptor _1S Transcriptional Activity by Acting on the cAMP-response Element-binding Protein Element of the Promoter Region^*

Zhenlin Zheng, Zhong-Min Wang, and Osvaldo Delbono^§¶

From the  Department of Physiology and Pharmacology, ^§ Department of Internal Medicine, Gerontology, and ^¶ Neuroscience Program, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157

Received for publication, October 15, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Previous work from our laboratory has shown that insulin-like growth factor 1 (IGF-1) increases the expression of the skeletal muscle dihydropyridine receptor (DHPR) ₁ subunit by regulating DHPR _1S nuclear transcription. In this study, we investigated the mechanism by which IGF-1 enhances expression of the DHPR _1S gene. To this end, the promoter region of the mouse DHPR _1S gene was recently cloned and sequenced and various promoter deletion-luciferase reporter constructs were used. These constructs were transfected into C2C12 cells and IGF-1 effects were measured by recording luciferase activity. IGF-1 significantly enhanced DHPR _1S transcription in those constructs carrying cAMP-response element-binding protein (CREB) binding site but not in CREB core binding site mutants. Gel mobility shift assay using a double stranded oligonucleotide for the CREB site in the promoter region, and competition experiments with excess unlabeled or mutated promoter oligonucleotide, and unlabeled consensus CREB oligonucleotide demonstrated that IGF-1 induces CREB binding to the DHPR _1S promoter. IGF-1-mediated enhancement in charge movement was prevented by incubating the cells with antisense but not with sense oligonucleotides against CREB. These results support the conclusion that IGF-1 regulates DHPR _1S transcription in muscle cells by acting on the CREB element of the promoter.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study we investigated the mechanisms by which insulin-like growth factor 1 (IGF-1)¹ regulates the expression of the skeletal muscle L-type Ca²⁺ channel or dihydropyridine-sensitive receptor DHPR _1S. IGF-1 is a peptide structurally related to proinsulin and has a primary role in promoting skeletal muscle differentiation and growth (1). We have shown that IGF-1 regulates the ion permeation function of the dihydropyridine (DHP)-sensitive L-type Ca²⁺ channel in skeletal muscle (2, 3). DHPR and ryanodine receptor and sarcoplasmic reticulum Ca²⁺ content are directly involved in regulating the amplitude of the muscle fiber Ca²⁺ influx (see Ref. 4). Prior studies from our laboratory have shown that the age-related decrease in the number of DHPR and ryanodine receptor 1 isoforms can be prevented by overexpression of IGF-1 in skeletal muscle (5). We have also shown that IGF-1 enhances skeletal muscle charge movement, [³H]PN200-110 binding sites, and DHPR _1S message expression in single muscle fibers from adult rats (6). Whether IGF-1 regulates DHPR _1S expression by acting on specific consensus sequences of the DHPR _1S 5'-flanking region is not known. To address this issue, a combination of molecular and electrophysiological techniques was used in the present study.

The DHPR _1S (known also as Ca_v1.1 ₁1.1, _1S, or CaCh1) is encoded in the human chromosome 1q31-32 by the CACNA1S gene and expressed exclusively in skeletal muscle (7, 8). The DHPR consists of five subunits (₁, ₂, , , and ), ₁ being the subunit that senses changes in membrane voltage, forms the Ca²⁺ conduction pore, binds to dihydropyridines, and interacts with the sarcoplasmic reticulum Ca²⁺ release channel or ryanodine receptor 1 isoform to release Ca²⁺ from the organelle into the myoplasm in response to membrane depolarization (9-11). The DHPR is located at the infoldings of the sarcolemma, named T-tubule, and plays a critical role in excitation-contraction coupling (4). Because of the pivotal role of the DHPR _1S subunit in excitation-contraction coupling, its expression is of crucial importance for skeletal muscle contraction. DHPR _1S cDNA restores excitation-contraction coupling in dysgenic mice (12, 13). DHPR _1S subunit expression is subject to regulation by a series of factors, including aging (5, 14), development (15), calcium (16), trophic factors (14, 17, 18), activity (19), and muscle denervation (20-22). Recently, we have demonstrated that IGF-1 and age regulate DHPR _1S gene transcription in murine skeletal muscle (23). All these factors result in changes in DHPR _1S subunit abundance. Despite the diverse modulation of channel expression, the molecular mechanisms underlying this process are not known. The recent characterization of the DHPR _1S 5'-flanking region allows for a better understanding of channel transcription and abundance (24). In that study, we have identified the consensus sequence for three transcription factors involved in the regulation of the DHPR _1S expression in muscle cells. Deletion experiments in the core of the consensus sequence for these transcription factors and antisense procedures support that GATA-2, CREB, and SOX-5 play a significant role in the DHPR _1S transcription and DHPR _1S subunit functional expression in differentiated skeletal muscle cells. Whether these transcription factors mediate IGF-1-induced DHPR _1S expression enhancement is not known.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- The mouse C2C12 muscle cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA), cultured in standard conditions, and maintained in growth medium (Dulbecco's modified Eagle's medium, supplemented with 20% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin). Dulbecco's modified Eagle's medium supplemented with 2% horse serum, 100 units/ml penicillin, and 100 µg/ml streptomycin was used as the differentiation medium.

Charge Movement Recordings-- For charge movement recordings C2C12 cells were plated on glass coverslips and mounted in a small flow-through Lucite chamber positioned on a microscope stage. Myotubes were continuously perfused with the external solution (see below) using a push-pull syringe pump (WPI, Saratoga, FL). Cells were voltage-clamped in the whole cell configuration of the patch clamp (25) using an Axopatch-200B amplifier (Axon Instruments, Foster City, CA). Micropipettes were pulled from borosilicate glasses (Boralex) using a Flaming Brown micropipette puller (P97, Sutter Instrument Co., Novato, CA) to obtain electrode resistance ranging from 2 to 4 M. The composition of the internal solution (pipette) was (mM): 140 Cs-aspartate, 5 Mg-aspartate₂, 10 Cs₂EGTA, 10 HEPES, pH was adjusted to 7.4 with CsOH. The high concentration of Mg²⁺ in the pipette solution helped to maintain the preparation stable for longer time. The external solution contained (mM): 145 tetraethylammonium hydroxide-Br, 10 CaCl₂, 10 HEPES, and 0.001 tetrodotoxin (26). Solution pH was adjusted to 7.4 with CsOH. This solution was used for forming gigaohm seals. For charge movements recording, calcium current was blocked with a solution containing (mM): 145 tetraethylammonium hydroxide-Br, 2 CaCl₂, 0.5 Cd²⁺, 0.3 La³⁺, 10 HEPES, and 0.001-0.003 tetrodotoxin (27). The maximum integral of the charge movement (see below) was used for the statistical analysis.

Whole cell currents were acquired and filtered at 5 kHz with pClamp 6.04 software (Axon Instruments). A Digidata 1200 interface (Axon Instruments) was used for A-D conversion. Membrane current during a voltage pulse, P, was initially corrected by analog subtraction of linear components. The remaining linear components were digitally subtracted on-line using hyperpolarizing control pulses of one-quarter test pulse amplitude (P/4 procedure) (20). The four control pulses were applied before the test pulse. Charge movements were evoked by 25-ms depolarizing voltage steps from the holding potential (80 mV) to command potentials ranging from 70 to 70 mV. Intramembrane charge movements were calculated as the integral of the current in response to depolarizing pulses (charge on, Q_on) and were expressed per membrane capacitance (coulombs per farad). The complete blockade of the inward calcium current was verified by the Q_on-Q_off linear relationship. Membrane capacitance was calculated as the integral of the transient current in response to a brief hyperpolarizing pulse from 80 mV (holding potential) to 90 mV.

Transfection of C2C12 Cell with Antisense Oligonucleotides-- Sense and antisense oligonucleotides were synthesized and phosphorothioated in 3 nucleotides at both ends by IDT (Coralville, IA). The following sense and antisense oligonucleotides were used: sense for CREB, 5'-GAATCTGGAGCAGAC-3' and antisense for CREB, 5'-GTCTGCTCCAGATTC-3' (24, 28-30).

The oligonucleotides were transfected into cultured C2C12 myotubes using FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer. After transfection, the dishes were gently rocked and then incubated at a 10% CO₂ atmosphere at 37 °C, for 48 h before recordings.

Construction of _1S Subunit Promoter-Luciferase Fusion Plasmid-- The full sequence or partial fragments of the DHPR _1S promoter (24) (GenBank^TM accession number for the 1.2-kb 5'-flanking region is AF343753) were fused with the luciferase reporter gene as explained previously (24). Sequential deletion constructs were generated by PCR cloning using specific primers. The correct orientation of the constructs and sequence was confirmed by DNA sequencing (ABI Prism Cycle Sequencing, PerkinElmer Life Sciences).

Transfection of C2C12 with _1S Promoter-Luciferase Fusion Plasmid and Dual Luciferase Assay-- All of the plasmids used in the present work were purified with QIAfilter (Qiagen Inc., Valencia, CA). Cells were plated at a density of 2 × 10⁴ cells, and grown in growth medium on 35-mm dishes till reaching 70% confluence. For cell transfection, 1 µg of each plasmid and 200 ng of the control vector pRL-TK (Promega) were mixed with 2 µl of FuGENE 6 (Roche Molecular Biochemicals) for 20 min at room temperature and added to the medium directly. Cells were induced to differentiate by changing to differentiation medium and cultured for 3 days. Cell lysis was prepared using a passive lysis buffer for the dual luciferase reporter assay (Promega). Luciferase and renilla activity were measured using a luminometer (Turner 20E, Sunnyvale, CA) and expressed as arbitrary units. Values for the luciferase assay were normalized to renilla luciferase activity to minimize differences in transfection efficiency for each experiment.

Site-directed Mutagenesis of Luc/P-146 Construct-- The Luc/P-146 construct was mutated using the GeneEditor in vitro site-directed mutagenesis system (Promega). The specific primers used for deletion of core nucleotides in transcription factor binding sites in this system were: for CREB-del, 5'-TCCAGTCCAGCCGGATCCCCATCTGCCCC-3'; for MZF1-del, 5'-CCTCGGGGGCAGATGTGTCACCGGCTGGAC-3'; for GATA-2-del, 5'-AGCCGGTGACATCCCTGCCCCCGAGGAGGC-3'. After alkaline denaturation of the Luc/P-146 construct, the DNA template was hybridized with phosphorylated primers and the bottom or top strand selection primer. The mutant strand was synthesized by T4 DNA polymerase and T4 DNA ligase. The mutant reactions were transformed into BMH 71-18 mutS competent cells, which were selected by GeneEditor antibiotic and ampicillin once plated in LB agar. The mutant clones were transformed into JM109 competent cells and confirmed by DNA sequence.

RNase Protection Assay-- RNase protection assays (RPA) were used to measure DHPR _1S mRNA concentrations in C2C12 cells. In vitro transcription probe labeling was performed with SP6 RNA polymerase (Maxiscript Kit, Ambion Inc., Austin, TX) and [³²P]UTP (ICN Pharmaceuticals Inc., Costa Mesa, CA). The reaction was gel purified and the labeled probe (2 × 10⁴ cpm) was hybridized at 56 °C overnight with 25 µg of total RNA from mouse skeletal muscle. After RNase digestion, the protected fragment was separated on a denaturing polyacrylamide gel and exposed to x-ray film. The radiolabeled RNA century marker (Ambion) was loaded on the same gel as molecular weight marker.

RPA was performed with total RNA extracted from C2C12 cells. The DNA template for detecting DHPR _1S mRNA (accession number L06234: 1461-1600 bp) in skeletal muscle and C2C12 cells were prepared by reverse transcriptase-PCR, cloned into pCRII vector, and linearized with BsrDI (New England Biolabs Inc., Beverly, MA). DHPR _1S probe (140 bp plus 82-bp vector fragment) was labeled with [³²P]UTP by in vitro transcription. The probe for 28 S rRNA consists of 115 bp plus the 67-bp vector fragment. Both the pTRI-RNA-28S antisense control template and the RNA century marker template set (Ambion Inc.) were also labeled by the in vitro transcription method using different dilutions of [³²P]UTP.

Nuclear Protein Extraction and Gel Mobility Shift Assay-- Nuclear proteins from myoblast and myotubes of C2C12 cells were extracted as described (23). Briefly, the cells were collected and washed in Tris-buffered saline. The cell pellets were resuspended in cold buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride) by gentle pipetting. After swelling on ice for 15 min, 10% Nonidet P-40 solution was added and vortexed for 10 s. After centrifugation, the nuclear pellets were suspended in cold buffer C (20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride) and rocked at 4 °C for 15 min. The supernatants were frozen in aliquots at 70 °C. Protein concentration was determined with Coomassie Plus Protein Reagent (Pierce Chemical Co.).

Sense and antisense DNA oligonucleotides (from 122 to 95) were synthesized and purified by PAGE (Integrated DNA Technologies, Coralville, IA). Both single strand oligos were annealed to form a double stranded DNA (D8 probe), which was end-labeled by T4 polynucleotide kinase (Promega, WI) and [-³²P]ATP (7000 mCi/mmol; ICN) and used as a probe for the gel shift assay. The nuclear proteins (5 µg) were incubated at room temperature for 10 min in a total 20 µl of reaction mixture containing 10 mM Tris-HCl, pH 7.5, 1 mM MgCl₂, 0.5 mM EDTA, 0.5 mM dithiothreitol, 5% glycerol, and 50 µg/ml polydeoxyinosinic-deoxycytidylic acid strand DNA at room temperature for 10 min, labeled probe was added and incubated for 20 min. DNA-protein complexes were resolved on 10% native polyacrylamide gel electrophoresis and visualized in x-ray film. In competition analysis, nuclear extracts were incubated for 10 min with unlabeled double-stranded oligonucleotides prior to the addition of the radiolabeled probe. The following double-stranded oligonucleotides were used as probes or competitors in gel mobility shift assays (only the sequence of the top strand is shown): D8 probe, 5'-GATGGGGATGTCACCGGCTGGACTGGAA-3', D8-MUT oligo, 5'-GATGGGGATGTCTGCGGCTGGACTGGAA-3', CREB consensus oligonucleotide, 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3' (Promega, Madison, WI).

Immunoprecipitation and Western Blot Analysis for the DHPR ₁ Subunit-- C2C12 cells were washed with phosphate-buffered saline, treated with 1% digitonin buffer (1% digitonin, 185 mM KCl, 1.5 mM CaCl₂, 10 mM HEPES, pH 7.4) on ice, and centrifuged at 10,000 × g for 10 min at 4 °C. Protein concentration was measured using the BCA protein assay (Pierce). The lysate (500 µg of total cellular protein) was precleared by adding 0.5 µg of the appropriate control IgG (normal goat IgG) together with 20 µl of the resuspended volume of the appropriate agarose conjugate (Protein G-agarose). After centrifugation at 2,500 rpm (1,000 × g), the supernatant was transferred to a fresh tube on ice. Goat anti-DHPR _1S N-19 primary antibody (1 µg) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added and incubated. Only goat IgG was added to the control tube. The resuspended volume of the Protein G-agarose (20 µl) was added to each tube and incubated at 4 °C on a rotating device overnight. After centrifugation (2,500 rpm, 1,000 × g) the pellets were washed with phosphate-buffered saline and resuspended in 20 µl of electrophoresis sample buffer. All samples were boiled for 2-3 min and analyzed in 10% SDS-PAGE gel. Rainbow Molecular Weight Markers (Amersham Biosciences) were loaded. Proteins were transferred from the gel to the nitrocellulose membrane. Nonspecific binding was blocked by incubating the nitrocellulose membrane in 5% milk, Tris buffered-saline Tween for 30-60 min at room temperature. Incubation in the primary antibody (1:100 diluted in blot buffer) was done for 1 h at room temperature and washed three times for 5 min each with Tris-buffered saline, 0.05% Tween 20. The membrane was incubated with anti-goat IgG conjugated with horseradish peroxidase, washed, and finally incubated in ECL reagent (Pierce) and visualized in x-ray films.

Statistical Analysis-- Data have been analyzed using Student's t test or analysis of variance. A value of p < 0.05 was considered significant. Data are expressed as mean ± S.E. with the number of observations (n).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We investigated the effects of IGF-1 on DHPR ₁ subunit protein, DHPR _1S mRNA, and the role of IGF-1 in the basal expression of the DHPR ₁ subunit, to identify the element(s) involved in the IGF-1-mediated regulation of DHPR _1S expression. We also defined the transcription factor(s) critical for IGF-1 effects on DHPR _1S promoter-luciferase fusion plasmids, and examined the functional effects of antisense oligonucleotides for this transcription factor on charge movement.

IGF-1 Increases DHPR _1S Expression in Muscle Cells-- We have previously demonstrated that IGF-1 enhances DHPR _1S protein expression (6) by increasing nuclear transcriptional activity in skeletal muscle (31). To study the specific regulatory elements involved in IGF-1-mediated enhancement of DHPR _1S, we examined first the effects of IGF-1 on the levels of expression of the channel subunit in C2C12 cells. To this end, a combination of immunoprecipitation and Western blot was used to improve the signal of the DHPR ₁ subunit. Repeated determinations (n = 4) revealed that the DHPR ₁ subunit expresses in myotubes and that IGF-1 increases the expression of DHPR ₁ subunit protein. The IGF-1 concentration (20 ng/ml) and the exposure time (3 days in differentiation medium) used in the present work have been found optimal to enhance DHPR _1S expression in rat skeletal muscle primary culture (6). Fig. 1 shows a band at 210 kDa corresponding to the DHPR ₁ subunit in C2C12 myotubes. The size of the band was determined using molecular weight markers as explained under "Experimental Procedures." The size of the DHPR ₁ subunit band is similar to that reported previously (32-34). This band is enhanced in myotubes treated with IGF-1 but not in cells treated with IGF-1 plus the IGF-1R tyrosine kinase inhibitor I-OMe-AG538 (25 µM). It is also apparent that the inhibitor by itself does not modify the expression of the DHPR ₁ subunit (Fig. 1). For these experiments, 500 µg of total proteins extracted from C2C12 cells lysates were used for immunoprecipitation with goat anti-DHPR _1S. The precipitates were analyzed in SDS-PAGE gels and detected by Western blot as described above ("Experimental Procedures"). To explain the increase in DHPR ₁ protein we examined whether IGF-1 enhances DHPR _1S mRNA in C2C12 cells.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   IGF-1 increases DHPR 1 subunit expression. Immunoprecipitation and Western blot analysis for DHPR 1 subunit expression in C2C12 myotubes. Total proteins (500 µg) were extracted from lysates of control myotubes (untreated cells), and myotubes treated with 20 ng/ml IGF-1, I-OMe-AG538 alone, or I-OMe-AG538 plus IGF-1 and immunoprecipitated with goat anti-DHPR 1 subunit antibody. The precipitates were analyzed in SDS-PAGE gel and detected by Western blot. The band at 210 kDa corresponds to the DHPR 1 subunit.

IGF-1 Enhances DHPR _1S Gene Expression-- Repeated RPA analysis revealed that a 140-bp protected fragment corresponding to DHPR _1S mRNA was present in total RNA samples from C2C12 myotubes (Fig. 2). Molecular weight was determined using radiolabeled RNA markers loaded on the same gel as described above. RPA was performed using specific cRNA probes for mouse DHPR _1S and 28 S rRNA (see "Experimental Procedures"). DHPR _1S expression in C2C12 myotubes was recorded in four experiments in which 25 µg of RNA from proliferating and differentiated cells corresponding to the same cell passage were analyzed. These results together with data on mouse skeletal muscle (24) support the concept that differentiation provides the conditions for DHPR _1S expression. Fig. 2 also shows that IGF-1 significantly increased DHPR _1S expression, a phenomenon that can be prevented by I-OMe-AG538. No effects of this agent by itself were recorded by DHPR _1S expression (Fig. 2).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   IGF-1 increases DHPR 1S expression detected by ribonuclease protection assay. Total RNA (25 µg) extracted from control (untreated), and treated with I-OMe-AG538 alone or IGF-1 plus I-OMe-AG538 C2C12 myotubes, were assessed separately by RPA using specific cRNA probes for mouse DHPR 1S (140 bp plus 82 bp corresponding to the vector fragment). Results were normalized to the levels of 28 S rRNA (115 plus 67 bp of the vector). The ratio between cells treated with I-OMe-AG538 or IGF-1 plus I-OMe-AG538 and control was 1.55, 1.53, and 1.46, respectively.

IGF-1 Enhances the Expression of DHPR _1S Promoter Deletion Constructs in Muscle Cells-- To analyze the DHPR _1S 5'-flanking region elements that may play a role in controlling IGF-1-dependent enhancement of DHPR _1S gene transcription, a gene chimera was created by cloning the 5'-flanking sequence and a portion of exon 1 of the DHPR ₁ subunit (1076 to +129) upstream of a luciferase reporter gene. Chimera deletion constructs were made starting at the 5'-end of the chimera and progressing in the 3' direction. Full and deletion constructs were subcloned into pGL3/basic plasmid and transfected into C2C12 cells. The pGL3/basic vector containing the luciferase reporter gene and lacking the DHPR _1S DNA was used as a control. Fig. 3 illustrates the full-length 5'-flanking region (1076/+128)-luciferase reporter gene and 3 deletion constructs. The most distal 5'-end nucleotide from the transcription start site identifies each construct.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   IGF-1 enhances the expression of DHPR 1S promoter deletion constructs expressed in C2C12 cells. The 5'-flanking region was progressively deleted from the 5'-end and fused to the pGL3/basic vector. The deletion constructs are numbered relative to the transcription start site. The relative luciferase activity was normalized to the luminescence signal of the Luc/P-1076 construct (100%). *, indicates the statistically significant difference compared with myotubes treated with IGF-1 for each deletion construct. The results presented are the mean ± S.E.

The expression of Luc/P-1076 has been shown to be dependent on cell differentiation and subtype (24). In our prior study (24) we have shown that the maximum luciferase activity was recorded at day 5 in differentiation medium in transfected C2C12 cells with the Luc/P-1076 construct. In the present work, we selected day 3 for luciferase activity recording because the C2C12 cells exposed to IGF-1 started to detach from the bottom of the chamber thereafter. Fig. 3 shows the relative luciferase activity recorded in the four 5'-flanking promoter-luciferase gene constructs transfected simultaneously into different groups of C2C12 cells. The relative luciferase activity was normalized to the Luc/P-1076 construct luminescence signal. Luciferase activity was much greater for Luc/P-1076 than for any of the other three constructs (p < 0.001) (n = 5). Enhancer and repressor elements in the 1.2 kb of the 5'-flanking region of the DHPR _1S have been discussed previously (24). Fig. 3 also shows that IGF-1 increases significantly the luciferase activity in the cells transfected with the four chimeric constructs and that even the shortest construct (Luc/P-146) tested exhibits IGF-1 potentiation.

Blockade of IGF-1 Effects on DHPR _1S Expression-- Recombinant IGF-1 has been used in the experiments described above, however, C2C12 cells can synthesize and secrete IGF-1 and express IGF-1R (35). Therefore, we examined whether endogenous, in addition to exogenous, recombinant IGF-1 modulate the expression of DHPR _1S. To this end, untreated and IGF-1-treated cells expressing the full promoter-luciferase construct (Luc/P-1076) were examined for the expression of DHPR _1S in the presence or absence of 10-100 µM I-OMe-AG538 (36). I-OMe-AG538 has been selected among several tyrosine kinase inhibitors because of its reported action on IGF-1R kinase (36) and to its significantly less pronounced inhibition of C2C12 cell differentiation. Fig. 4A shows that I-OMe-AG538 decreases luciferase activity at concentrations equal or greater than 50 µM (double asterisks), whereas recombinant IGF-1 significantly enhances this signal in the absence of I-OMe-AG538 (single asterisk). The IGF-1-mediated enhancement of DHPR _1S expression was blocked by the inhibitor in the whole range of concentrations tested (10-100 µM) (n = 5). These experiments indicate that 25 µM, the I-OMe-AG538 concentration used in this work, blocks exogenous IGF-1 effects but does not alter the basal DHPR _1S expression. To investigate whether CREB is the only IGF-1 regulatory element, we tested the effect of IGF-1 on luciferase activity in myotubes transfected with the Luc/P-1076/CREB-deleted construct (Fig. 4B). Luciferase activity was significantly different in control (Luc/P-1076), the CREB-deleted construct, and the CREB-deleted construct plus 25 µM I-OMe-AG538. IGF-1 enhanced luciferase activity only in the full control construct but not in the CREB-deleted construct in the presence or absence of the inhibitor (Fig. 4B). These results support the concept that CREB mediates IGF-1 regulatory effects on DHPR _1S gene expression.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Blockade of IGF-1 effects on CREB-mediated DHPR 1S expression. A, transient expression of the full promoter-luciferase construct (Luc/P-1076) in C2C12 cells treated with a combination of IGF-1 and the inhibitor I-OMe-AG538 (10-100 µM). The firefly luciferase activity was normalized to co-transfected pRL-TK renilla values. *, indicates statistically significant differences compared with myotubes treated with IGF-I plus inhibitor (10-100 µM). **, indicates significant difference with other groups of cells not treated with IGF-1 plus inhibitor (0-25 µM). B, deletion of the CREB binding site in the full-length Luc/P-1076 construct prevents the effects of IGF-1 and I-OMe-AG538 on DHPR 1S expression. Asterisks indicate the statistically significant differences with control cells transfected with Luc/P-1076 constructs treated and untreated with IGF-1.

Role of CREB in DHPR _1S Transcription-- To determine the role of specific transcription factors in DHPR _1S transcription, deletion constructs of the Luc/P-146 construct were produced (Fig. 5A). Luc/P-146 is the shortest construct that exhibits reliable and reproducible luciferase signal and is significantly enhanced by IGF-1. Therefore, this is the construct that was used for mutational analysis. Fig. 5B shows the relative luciferase activity for three different constructs consisting of 4-bp deletions at the center of CREB, GATA-2, and MZF1 binding site sequences, respectively. Deletions in the binding sequence for CREB and GATA-2 led to significant reductions in luciferase activity in untreated cells (p < 0.01) (n = 5), underscoring the importance of these factors in DHPR _1S transcription (24). The Luc/P-146 GATA-Mut construct showed a significant potentiation of the luciferase activity in the group of cells treated with IGF-1 for 3 days; however, this effect was not recorded in cells transfected with the Luc/P-146 CREB Mut. These results support the concept that CREB binding to its consensus sequence in the DHPR _1S promoter region is needed for IGF-1 enhancement of the DHPR _1S gene transcription. Deletion in the consensus sequence for MZF-1 resulted in potentiation of luciferase activity compared with Luc/P-146 (p < 0.05) (n = 5). No explanation for this effect is obvious at the present time. IGF-1 enhanced the luciferase activity in cells transfected with Luc/P-146 MZF-1-Mut. These results do not support a role for MZF-1 in the IGF-1-mediated potentiation of DHPR _1S gene expression. Control experiments using the inhibitor I-OMe-AG538 alone or in combination with IGF-1 give support to the conclusion that IGF-1 modulates DHPR _1S expression by acting on the CREB element of the DHPR _1S promoter region.

View larger version (45K): [in this window] [in a new window]   Fig. 5.   IGF-1 enhances DHPR 1S expression by acting on the CREB element. Site mutations on the Luc/P-146 construct consisting of the 4-nucleotide deletion in the core binding site show that the CREB mutant (A) is the only construct that lacks IGF-1 enhancing activity on DHPR 1S gene expression (B). *, indicates the statistically significant difference with control (no IGF-I treatment) for control Luc/P-146, GATA-del, and MZF-del constructs. The effect of IGF-1 was blocked by the inhibitor I-OMe-AG538.

The low level of activity of Luc/P-146 GATA-Mut could suggest that GATA is important for DHPR _1S expression. To further assess this issue, the effects of sense and antisense for GATA-2, in the presence and absence of IGF-1, were examined in cells transfected with the Luc/P-1076 construct (Fig. 6). IGF-1 enhances the luciferase activity in myotubes pretreated with either sense or antisense for GATA-2. These results confirm that GATA-2 does not mediate the effects of IGF-1 on DHPR _1S expression. The significant difference between cells treated with sense or antisense oligonucleotides supports a role for GATA-2 in basal (not mediated by IGF-1) DHPR _1S expression (24).

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Effect of sense or antisense oligonucleotides for GATA-2 on DHPR 1S expression. The effect of IGF-1 on Luc/P-1076 luciferase activity was measured in myotubes pretreated with sense or antisense against GATA-2. *, indicates the statistically significant differences between IGF-1 treated and nontreated constructs expressed in myotubes incubated in GATA-2 sense oligonucleotide (p < 0.05). **, compares the IGF-1-treated and nontreated cells exposed to GATA-2 antisense (p < 0.01); ***, indicates the statistically significant differences between IGF-1-treated and nontreated cells incubated in GATA-2 sense or antisense oligonucleotides (p < 0.05).

Nuclear Proteins Involved in IGF-1-mediated Enhancement of DHPR _1S Gene Transcription-- To determine the key elements involved in IGF-1-mediated enhancement of the DHPR _1S gene transcription, we performed gel shift assays as an alternative approach to the deletion constructs described above. This procedure allows us to identify the nuclear proteins involved in IGF-1 effects on the DHPR _1S gene. Nuclear proteins from IGF-1-treated myotubes were extracted as described (23). To further support that CREB is binding to the promoter, a double stranded oligonucleotide for the putative CREB site in the promoter was used as the probe for the assay (D8). As additional tests of binding specificity, the effects of competition with 50-fold excess CREB, unlabeled consensus CREB oligonucleotide, or mutated oligonucleotide (D8mut) have been studied. Fig. 7A shows the DNA-protein complexes resolved on 10% native polyacrylamide gel electrophoresis and visualized in x-ray film. The a and b bands disappear in the presence of 50-fold excess of unlabeled CREB or 50-fold excess of D8 oligonucleotide, but not in the presence of 50-fold excess of mutant D8 (D8mut). We repeated the experiment four times with consistent results. The band corresponding to the free probe is missing in Fig. 7 because the gel was run for 90 min to better separate the a and b bands. These experiments provide further evidence for the role of CREB in IGF-1-mediated enhancement of the DHPR _1S gene transcription. As a further control Fig. 7B shows a gel mobility assay for nuclear extracts (5 µg) from IGF-1-treated and untreated (control) myotubes.

View larger version (76K): [in this window] [in a new window]   Fig. 7.   Identification of protein-DNA complex stimulated by IGF-I and localized on the 122/95 sequence of the DHPR 1S promoter. A, gel shift competition analysis for nuclear extracts (5 µg) from IGF-1-treated C2C12 myotubes (IGF-1/NE). The 32P-labeled D8 oligonucleotide was used as the probe with no competitor (lane 1), with a 50-fold molar excess of unlabeled CREB consensus oligonucleotide as competitor (lane 2), with a 50-fold molar excess of the unlabeled D8 oligonucleotide as competitor (lane 3), or with a 50-fold molar excess of the unlabeled oligonucleotide D8 mut as competitor (lane 4). The protein-DNA complexes are indicated as a and b. The sequences for both probe and competitor oligonucleotides are indicated under "Experimental Procedures." B, gel mobility assay for nuclear extracts (5 µg) from IGF-1-treated and untreated (control) myotubes.

Effects of Antisense Oligonucleotide for CREB on Charge Movement-- As a functional expression of the DHPR ₁ subunit we recorded charge movement in differentiated myotubes after 36-48 h incubation in 1 µM sense or antisense oligonucleotides for CREB plus IGF-1 and the results were compared with recordings in control cells not incubated in sense or antisense. Although the DHPR ₁ subunit accounts for only 70% of the total nonlinear capacity of the membrane (27), we preferred the recording of charge movement to calcium current because of the direct relationship between the integral of charge movement and the levels of channel expression in the sarcolemma (37). Also, the amplitude of the L-type calcium current varies in response to channel regulation and percentage of silent channels (2, 38). As the charge movement reported here is similar to that recorded previously with the patch clamp (39) but higher than that recorded with other techniques (20, 40, 41), we studied both linear capacitive transients for the voltage steps from 80 to 90 mV, and the unsubtracted current traces for the test and control steps. No current in the subpulses was detected in either control or test fibers (n = 4 fibers per group). Differences in the recording technique and time after current blockade could account for the variability in the reported maximum charge movement. IGF-1 did not exert any effects per se on muscle fiber capacitance (control or untreated myotubes: 1309 ± 114; IGF-1-treated myotubes: 1297 ± 125; p > 0.05; n = 20).

Fig. 8A shows the Q_on  voltage relationship for the cells treated with 20 ng/ml IGF-1 alone (filled circles), sense (triangles), or antisense (squares) for CREB plus IGF-1 and control (nontreated) cells (open circles). The effects of sense or antisense oligonucleotides against CREB on C2C12 cells in the absence of IGF-1 have been communicated previously (24). In that publication, we reported that antisense oligonucleotides significantly decreased while sense did not modify maximum charge movement.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Effects of antisense oligonucleotide for CREB on charge movement. Charge movement recorded in differentiated myotubes incubated in 1 µM sense or antisense oligonucleotides for CREB plus IGF-1. Results are compared with records in control cells not incubated in sense or antisense oligonucleotides. A, Qon-membrane voltage relationship for cells treated with 20 ng/ml IGF-1 alone (filled circles), sense (triangles), or antisense (squares) for CREB plus IGF-1 and control (open circles) C2C12 cells. Data points were fitted to a Boltzmann equation (see text, Equation 1). Charge movement recordings were in the 30 to 30 mV range for control (B), IGF-1 plus CREB-antisense (C), IGF-1 (D), and IGF-1 plus CREB sense-treated cells. The best fitting parameters for Qmax, VQ1/2, and K recorded in the four experimental groups are included in Table I. Dotted lines indicate the baseline.

For the analysis of the voltage dependence of the charge, data points were fitted to a Boltzmann equation in the form,

(Eq. 1)

where Q_max is the maximum charge, V_m is the membrane potential, V_Q1/2 is the charge movement half-activation potential, and K is the steepness of the curve. Fig. 8, B-E, illustrates charge movement in the 30 to 30 mV range (every 20 mV) corresponding to the steepest part of the current-voltage curve. It is apparent that IGF-1 significantly enhances charge movement. The antisense nucleotide for CREB significantly decreases the maximum charge movement at voltages more positive than 20 mV without affecting the voltage distribution of the charge, which indicates that the number but not the properties of the DHPR ₁ subunits are modified. No significant differences were found between cells exposed to sense oligonucleotide for CREB and control untreated cells. The values recorded in cells treated with CREB sense in the presence of IGF-1 were significantly different from the cells treated with IGF-1 alone in the absence of CREB oligonucleotides. The best fitting parameters for Q_max, V_Q1/2, and K recorded in the four experimental groups are included in Table I.

View this table: [in this window] [in a new window]   Table I Best fitting parameters describing the voltage dependence of charge movement in C2C12 muscle cells Experimental data were fit to the Boltzmann equation described in the text. Qmax; maximum charge movement; VQ1/2, charge movement half activation potential; K, the steepness of the curve. S, sense; AS, antisense oligonucleotides. IGF-1, 20 ng/ml. CREB-S and AS, 1 µM. Asterisks indicate the statistically significant difference compared with control cells (p <0.05). n, number of cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present study, we identified CREB as the transcription factor involved in IGF-1 regulation of the DHPR _1S promoter-luciferase fusion plasmids. Gel shift competition assays and experiments using the IGF-1R inhibitor I-OMe-AG538 demonstrated that IGF-1 enhanced the levels of phosphorylated CREB. Patch clamp recording showed that antisense oligonucleotide against CREB decreases charge movement and prevents the effect of IGF-1 on DHPR _1S expression in C2C12 myotubes.

IGF-1 Increases DHPR _1S Expression in Muscle Cells-- A series of experiments included in the present study demonstrate that IGF-1 increases the expression of the DHPR ₁ subunit protein as a result of increasing gene transcription in C2C12 cells. These results are consistent with previous studies performed in mouse skeletal muscle and rat muscle primary culture that demonstrate that IGF-1 increases DHPR _1S nuclear transcription (6, 31). We investigated whether endogenous or recombinant IGF-1 has an effect on DHPR _1S gene expression. To this end, we measured the effects of the IGF-1R tyrosine kinase inhibitor I-OMe-AG538 (36) on luciferase activity in transfected myotubes with various DHPR _1S promoter-luciferase fusion constructs. This recently synthesized compound inhibits IGF-1R autophosphorylation and the activation of the downstream targets protein kinase B and Erk2 (36). IC₅₀ concentration (25 µM) (Fig. 4) (36) was used for these experiments. Using this concentration of I-OMe-AG538, no effects on luciferase activity or cell differentiation were observed. This is in contrast with marked effects of the protein kinase inhibitor genistein on cell differentiation (data not shown). A more specific effect of I-OMe-AG538 on the IGF-1R could explain the differential effects on cell differentiation. These experiments support a role for IGF-1 on DHPR _1S expression. Whether muscle differentiation and modulation of DHPR _1S expression share common signaling pathways cannot be addressed at the present time.

CREB Mediates IGF-1-dependent Enhancement of DHPR _1S Gene Transcription-- We have recently reported the cloning of the mouse L-type/DHPR _1S subunit gene 5'-flanking sequence and the specific sequences necessary for basal transcription and control of the DHPR _1S expression. Deletion analysis of the 5'-flanking region in the DHPR-luciferase fusion gene indicates that cis-acting regulatory elements in the proximal 146 bp appear to be essential for skeletal muscle cell-specific expression of the DHPR _1S subunit gene. Transfection of the deletion construct Luc/P-146 in C2C12 cells resulted in significant luciferase activity. CREB and GATA-2 consensus sequences in the 146 bp upstream of the transcription start site are critical for DHPR _1S expression (24). In the present study we investigated the role of CREB, GATA, and MZF-1 as potential mediators of IGF-1-dependent enhancement of DHPR _1S transcription. The study of the consensus sequence for these transcription factors is based on their expression in the first 146 bp upstream of the transcription start site. We have found that mutation in the consensus sequence for CREB and GATA significantly decreases luciferase activity. These results are consistent with a recent report from our laboratory (24). Significant potentiation of the DHPR _1S expression was induced by IGF-1 on the cells transfected with the GATA mutant but not with CREB. Ablation of the myeloid zinc finger consensus MZF-1 binding sequences did not significantly modify luciferase activity (24). These results support the concept that CREB mediates IGF-1 potentiation of DHPR _1S transcription. Several consensus sequences have been described in the DHPR _1S promoter region spanning from nucleotides 146 to 1076, SOX-5 being one of them (24). We have reported previously that SOX-5 regulates DHPR _1S transcription. The role of SOX-5 in DHPR _1S transcription was not analyzed in the present work because Luc/146, a construct lacking a consensus binding sequence for SOX-5, exhibits potentiation of the DHPR _1S transcription evoked by IGF-1. Ongoing studies are exploring the role of specific signaling pathways linking IGF-1R activation and CREB binding to the DHPR _1S promoter region. Phosphorylation at a single Ser¹³³ residue greatly enhances the activity of CREB bound to the response element CRE. Therefore, phosphorylated CREB is the active form that regulates gene transcription. Whether CREB is phosphorylated by protein kinase A, C, or any other calcium-dependent protein kinase in response to IGF-1R activation is not currently known.

CREB-mediated IGF-1 Potentiation of Charge Movement-- Charge movements are currents arising on movement of charge molecules dwelling in the membrane (42). The integral of the recordings are directly related to the number of moving charged molecules (37). The maximum control charge movement reported here is similar to that reported by us previously (24) but 1.35- and 2.3-fold higher than that reported by other groups (40, 43), using microelectrode techniques. Although several voltage-gated channels contribute to charge movement, these recordings represent mainly the activity of the DHPR ₁ subunit (27). We have performed high affinity radioligand binding assays in muscle cells treated with IGF-1 to determine whether the effect on charge movement results from an increase in DHPR ₁ subunits or on the remaining 30% of the charge not attributable to the L-type Ca²⁺ channel (18). In that publication (18), we reported a significant increase in binding sites in the cells treated with the same concentration of IGF-1 used in the present study.

In the present work we have recorded a significant effect of antisense oligonucleotides for CREB on charge movements as an indication of down-regulation of the number of DHPR ₁ subunits expressed in the sarcolemma. We have also reported that IGF-1 does not enhance charge movement in the presence of CREB-antisense. These results, together with the data on DHPR ₁ subunit expression and DHPR _1S mRNA, support the concept that IGF-1 enhances the charge movement arising from DHPR ₁ subunits. Whether antisense oligonucleotides for CREB result in decreased expression of other skeletal muscle voltage-gated ion channels is not known. This possibility cannot be ruled out at the present time.

	FOOTNOTES

^* This work was supported by NIA National Institutes of Health Grants AG18755, AG13934, and AG15820, and a grant from the Muscular Dystrophy Association of America (to O. D.).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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF343753.

To whom correspondence should be addressed: Dept. of Physiology and Pharmacology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Tel.: 336-716-9802; Fax: 336-716-7359; E-mail: odelbono@wfubmc.edu.

Published, JBC Papers in Press, October 28, 2002, DOI 10.1074/jbc.M210526200

	ABBREVIATIONS

The abbreviations used are: IGF-1, insulin-like growth factor-1; CREB, cyclic AMP response element-binding protein; DHPR, dihydropyridine receptor; Erk, extracellular signal-regulated kinase; K, steepness of the current-voltage relationship; MZF, myeloid zinc finger binding sequence; Q_max, maximum charge movement; SOX, Sry-type HMG box; V_m, membrane potential, V_Q1/2, charge movement half-activation potential; RPA, RNase protection assays.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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