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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Developmental regulation of the mouse IGF-I exon 1 promoter region by calcineurin activation of NFAT in skeletal muscle

Division of Molecular Cardiovascular Biology, Cincinnati Children's Medical Center, Cincinnati, Ohio

Submitted 29 September 2006 ; accepted in final form 11 January 2007

	ABSTRACT

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Skeletal muscle development and growth are regulated through multiple signaling pathways that include insulin-like growth factor I (IGF-I) and calcineurin activation of nuclear factor of activated T cell (NFAT) transcription factors. The developmental regulation and molecular mechanisms that control IGF-I gene expression in murine embryos and in differentiating C2C12 skeletal myocytes were examined. IGF-I is expressed in developing skeletal muscle, and its embryonic expression is significantly reduced in embryos lacking both NFATc3 and NFATc4. During development, the IGF-I exon 1 promoter is active in multiple organ systems, including skeletal muscle, whereas the alternative exon 2 promoter is expressed predominantly in the liver. The IGF-I exon 1 promoter flanking sequence includes two highly conserved regions that contain NFAT consensus binding sequences. One of these conserved regions contains a calcineurin/NFAT-responsive regulatory region that is preferentially activated by NFATc3 in C2C12 skeletal muscle cells and NIH3T3 fibroblasts. This NFAT-responsive region contains three clustered NFAT consensus binding sequences, and mutagenesis experiments demonstrated the requirement for two of these in calcineurin or NFATc3 responsiveness. Chromatin immunoprecipitation analyses demonstrated that endogenous IGF-I genomic sequences containing these conserved NFAT binding sequences interact preferentially with NFATc3 in C2C12 cells. Together, these experiments demonstrated that a NFAT-rich regulatory element in the IGF-I exon 1 promoter flanking region is responsive to calcineurin signaling and NFAT activation in skeletal muscle cells. The identification of a calcineurin/NFAT-responsive element in the IGF-I gene represents a potential mechanism of intersection of these signaling pathways in the control of muscle development and homeostasis.

insulin-like growth factor I; nuclear factor of activated T cells c3; skeletal muscle; gene regulation

The IGF-I gene is highly conserved among mice, rats, and humans and contains six exons that are subject to alternative splicing and differential promoter usage (40, 46). Transcripts initiated by promoter sequences adjacent to exon 1 are the major isoforms in skeletal muscle and most other tissues, whereas the exon 2-initiated transcripts are predominantly expressed in the liver and represent the majority of circulating IGF-I (34, 47, 52). However, the developmental regulation of IGF-I gene expression from these alternative promoters in skeletal muscle and other organ systems subject to IGF-I growth control is not well characterized. The sequence of exon 1 is highly conserved among mammalian species and contains multiple initiation sites and regulatory sequences in the untranslated region that contribute to gene expression from the adjacent promoter (28, 51). Among these exon 1 promoter regulatory elements are sequences responsive to muscle regulatory factors (MRFs), cAMP, and prostaglandins that function in skeletal myocytes (26, 50). Additional IGF-I exon 1 promoter proximal regulatory sequences 5' of the transcriptional start site also function in myocytes, but the specific sequences or DNA binding proteins involved have not been identified (17, 25). More distal sequences in the IGF-I gene locus required for normal expression related to development and growth are beginning to be uncovered, but these have not been thoroughly investigated (9).

In addition to IGF-I, calcineurin signaling and nuclear factor of activated T cell (NFAT) activation are important for skeletal muscle differentiation, fiber type specialization, and hypertrophy (42). In vertebrates, there are four NFATc family members with distinct functions and target specificity that are expressed in a variety of cell types (39). In skeletal muscle, calcineurin signaling and NFAT activation are required for myogenic differentiation and slow muscle fiber type development (10, 12, 15). In cultured skeletal myoblasts, NFATc3 is the first NFAT to be translocated to the nucleus during differentiation, and targeted loss of NFATc3 in mice leads to decreased primary myogenesis and reduced skeletal muscle mass (1, 19). The regulatory relationships between calcineurin and IGF-I signaling mechanisms in skeletal muscle differentiation and hypertrophy have not been completely established. In skeletal muscle cell cultures, IGF-I induces myocyte hypertrophy through calcineurin signaling and NFAT activation (32, 45). However, induction of calcineurin signaling can augment IGF-I promoter activity in transfected C2C12 myocytes (25). In the present study, regulatory sequences containing multiple NFAT consensus binding sequences were identified flanking the IGF-I exon 1 promoter and respond to calcineurin activation of NFATc3 in skeletal muscle cells.

	MATERIALS AND METHODS

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In situ hybridization. The mouse IGF-I sequence (979 bp, NM_184052 [GenBank] ) containing shared exons 3–6 common to transcripts initiated from either exon 1 or exon 2 promoters was amplified from the embryonic day (E)12.5 head by RT-PCR using primers 5'-GGCGGCTGCTTGTCCAACTTTTCT-3' and 5'-TCCTTCCCTTCCTCCCCCATCGTC-3'. The IGF-1 exon 1 sequence (+1 to +385) was amplified from E14.5 heart RNA using primers 5'-TGTCACGGTGCCCAAAAA-3' and 5'-TTCAAGAAGTCACAGAGGCAGA-3'. The IGF-I exon 2 sequence (991 bp) was amplified using primers 5'- CCTTTTGATCACTGGCCCTA-3' and 5'- GGGTCGTTTACACAGCAGGT-3'. The amplified fragments were inserted into pGEM T-vectors (Promega) and verified by sequencing. The mouse IGF-I plasmid template was linearized with NotI, and the digoxigenin-labeled antisense RNA probe was synthesized with T7 polymerase. Exon 1 and exon 2 plasmid templates were linearized with NcoI, and the antisense RNA probe was synthesized using SP6 RNA polymerase. Mouse embryos were isolated at E10.5 or E14.5 for in situ hybridizations of whole embryos (44) or paraffin-embedded histological sections (22) as previously described. Nfatc3^+/–Nfatc4^–/– and Nfatc3^–/–Nfatc4^–/– embryos were generated and genotyped as described by Bushdid et al. (7).

RT-PCR. RNA was isolated from heart, brain, or liver tissue of E10.5, E14.5, or E18.5 mouse embryos, and 3 µg of RNA were included in RT reactions as previously described (21). Oligonucleotide primer sequences were designed to amplify transcripts originating from either the exon 1 or exon 2 promoter of the mouse IGF-I gene as described above. Amplification reactions were performed with 30 cycles of 94°C for 1 min, 55°C for 1.5 min, and 72°C for 3 min, and amplification of the ribosomal protein L7 was performed in parallel as a loading control (7). Amplified DNA fragments from each set of primers were confirmed by sequence analysis.

Isolation and mutagenesis of mouse IGF-I promoter sequences. The mouse IGF-I promoter sequence [–1745 to +175 relative to +1, as reported by Hall et al. (17)] was amplified from FVBN strain genomic DNA using primers 5'-GTGGAAGCCTTGGGTTACT-3' and 5'-TTTAGCAAGCAGAAGAGGGATTTA-3'. The amplified sequence was ligated into a pGEM T-vector, and the sequence was verified. Sequences within –1567 to +153 cut with HindIII were inserted into the HindIII site of the pGL3-basic plasmid (Promega). –1192IGFI-luc was generated using NcoI, and –514IGFI-luc was generated using BglII in the IGF-I promoter sequence and religating. Mutations were introduced into conserved region (CR)1 NFAT consensus sequences by site-directed mutagenesis of the –1192IGFI-luc plasmid using the QuikChange site-directed mutagenesis kit (Stratagene) and the following primers (upper strand represented with mutated nucleotides underlined): M1 5'-GGCAAGTCTGGCTCAAGAGTATCTCCCCTGGGAAAG-3', M2 5'-CATTTCCATCTCCCC TGTTCAAGCACACCTGG-3', and M3 5'-GGAGAGATATCCGTTTCAAGCATGCAGCGTC-3'. All mutations were verified by sequencing.

Transient transfections and reporter assays. NIH3T3 cells and C2C12 myoblasts were cultured and transfected as previously described (21, 36). C2C12 myoblasts maintained in 15% FBS were transfected at subconfluency, and lysates were prepared from nearly confluent cultures. Cells were transfected with 500 ng luciferase reporter construct, 100 ng expression vector, and 10 ng pRL-TK reference plasmid (Promega) using FuGENE 6 transfection reagent (Roche). Expression vectors for NFATc3, NFATc1, activated calcineurin (CnA), GATA4, and MEF2A have been described previously (2, 21, 29). Control groups included corresponding empty vector expression plasmids or the pGL3 promoter lacking the IGF-I genomic sequence. Reporter gene activity in transfected cell lysates was measured using the Dual Luciferase Assay system (Promega) and normalized to Renilla luciferase activity.

Chromatin immunoprecipitation assays. Chromatin immunoprecipitation (ChIP) assays were performed using the ChIP assay kit (Upstate) according to the manufacturer's instructions. Formaldehyde-treated nuclear lysates were generated from subconfluent C2C12 myoblasts maintained in 15% FBS. DNA protein complexes containing NFATc3 were precipitated using the M-75 antibody (sc-8321, Santa Cruz Biotechnology), and NFATc1-containing complexes were precipitated with the 7A6 antibody (sc-7294, Santa Cruz Biotechnology). Immunoprecipitations with rabbit normal IgG (Santa Cruz Biotechnology) were performed in parallel as a specificity control. IGF-I CR1 sequences were amplified by PCR using primers 5'-CTATCCATGGGGCAGCGTAAAGA-3' and 5'-ATGTCATTCAAATCCCTCAACTG-3'. Positive control precipitations of RNA polymerase II complexes were amplified with primers directed against proximal promoter sequences of mouse -actin (41).

	RESULTS

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Embryonic expression of mouse IGF-I is dependent on NFATc3. The expression of IGF-I during the initial stages of myogenesis was examined by whole mount in situ hybridization of E10.5–E11.5 embryos (Fig. 1). Total IGF-I expression was assessed using an in situ probe encompassing exons 3–6 present in both exon 1- and exon 2-initiated transcripts. In E11.5 embryos, IGF-I is expressed in differentiating somites and in the somitic-derived musculature (Fig. 1A). IGF-I expression also was apparent in the limb buds, branchial arches, and developing face, but was not predominant in cardiac muscle at these stages. The dependence of IGF-I expression on NFATc3 and NFATc4 was examined in embryos with combinatorial disruption of these genes (7). Embryos lacking both NFATc3 and NFATc4 do not survive beyond E10.5 due to defects in heart formation. In E10.5 Nfatc3^+/–Nfatc4^–/– embryos, IGF-I is expressed in somites and branchial arches, but IGF-I expression was not detectable in Nfatc3^–/–Nfatc4^–/– littermates (Fig. 1, B and C). Myogenic differentiation, as indicated by sarco(endo)plasmic reticulum Ca²⁺-ATPase 2 gene and myosin heavy chain gene expression, occurred in Nfatc3^–/–Nfatc4^–/– embryos (data not shown), but IGF-1 expression was dependent on an intact Nfatc3 allele. Together, these experiments indicate that IGF-I is expressed in differentiating somitic muscle and branchial arches and that its embryonic expression is dependent on NFATc3 and NFATc4.

View larger version (101K): [in this window] [in a new window]   Fig. 1. The mouse IGF-I exon 1 promoter is active during skeletal myogenesis and IGF-I expression is absent in nuclear factor of activated T cell (Nfat)c3–/–Nfatc4–/– embryos. A–C: whole mount in situ hybridization was performed with a mouse IGF-I antisense probe corresponding to common exons 3–6. A: IGF-1 expression was apparent in somites and skeletal musculature (arrows) and in branchial arches (*) at embryonic day (E)11.5. B and C: IGF-1 expression was apparent in somites and in the face of Nfatc3+/–Nfatc4–/– embryos at E10.5 but was absent in Nfatc3–/–Nfatc4–/– littermates. D–F: IGF-I exon 1- or exon 2-specific probes were hybridized to paraffin-embedded histological sections of E14.5 embryos. D: exon 1 transcripts were expressed in the forelimb musculature (m) but were less apparent in the humerus (h) and radius (r) primordia. E: relatively decreased expression of exon 2 transcripts was observed in serial sections. F: expression of the exon 1 promoter was apparent in intercostal muscles (im) with lower levels in the sternum (s).

Regulation of IGF-1 gene expression initiated from exon 1 or exon 2 promoters in other organ systems was examined by RT-PCR of tissue from dissected mouse embryos (Fig. 2). RNA was isolated from E10.5, E14.5, or E18.5 heart, brain, or liver tissues and subjected to RT-PCR with primers specific for exon 1- or exon 2-initiated IGF-I transcripts. IGF-I expression from the exon 1 promoter was widespread in the developing embryo and was detected in all tissues tested. In contrast, transcripts initiated from the exon 2 promoter were expressed predominantly only in a subset of developing tissues, including the brain at E10.5 and the liver at E14.5 and E18.5. These data are consistent with those of a previous study (40) demonstrating that exon 1 promoter-initiated transcripts encode the major isoform expressed in most tissues and that the exon 2 promoter is active predominantly in the liver, where circulating IGF-I is produced. However, the expression of exon 2-initiated transcripts in the E10.5 brain may be indicative of additional IGF-I-regulatory mechanisms active in the embryonic nervous system.

View larger version (32K): [in this window] [in a new window]   Fig. 2. RT-PCR analysis of IGF-I exon 1 and exon 2 promoter usage during mouse embryogenesis. RNA isolated from the embryonic heart, brain, or liver at the indicated stages was subjected to RT-PCR with primers specific to IGF-1 exon 1 or exon 2 sequences as well as L7 loading control sequences. Amplified DNA sequences were subjected to agarose gel electrophoresis and visualized using ethidium bromide staining.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Conserved NFAT consensus binding sites are present in IGF-I exon 1 5' flanking sequences. A: alternative exon 1 and exon 2 promoter usage and splicing are indicated, as reported for the rat IGF-I gene (52). Regions of conservation in the exon 1 flanking sequence (CR1 and CR2) were identified based on rVISTA and Clustal analyses. B: nucleotide sequences of mouse CR1 (–1071 to –1022) and CR2 (–414 to –400) align with comparable regions of the rat and human IGF-I genes and contain conserved NFAT and GATA consensus binding sequences.

View larger version (20K): [in this window] [in a new window]   Fig. 4. IGF-I reporter constructs are activated by calcineurin and NFATc3 in transfected C2C12 myocytes and NIH3T3 fibroblasts. The indicated IGF-I promoter reporter constructs or pGL3 promoter plasmids were cotransfected with activated calcineurin (CnA), NFATc3 (A), or NFATc1 (B) expression plasmids [wild type (wt) or constitutively active (ca)] in C2C12 or NIH3T3 cells. Relative expression was calculated as the fold change relative to empty vector controls for each reporter plasmid represented in the histogram for pGL3, –1192IGFI-luc, and –514IGFI-luc. Statistical significance of observed differences was calculated by Student's t-test relative to pGL3 levels for each group (*P < 0.05).

The cell type specificity of IGF-I reporter gene induction by CnA and NFATc3 was examined in NIH3T3 cells (Fig. 4A). In NIH3T3 fibroblasts, specific activation of –1192IGFI-luc was observed with CnA or NFATc3, although the levels of induction were lower than in C2C12 myoblasts (7- vs. 18-fold). In contrast to C2C12 cells, increased induction of –1192IGFI-luc was observed with CnA and NFATc3 together, which is likely indicative of lower levels endogenous NFATc3 expression in NIH3T3 fibroblasts. The differential induction of –1192IGFI-luc relative to –514IGFI-luc by CnA alone was not observed in NIH3T3 cells. However, cotransfection of NFATc3 with CnA induced –1192IGFI-luc expression to levels comparable with induction in C2C12 cells (18- vs. 22-fold). In contrast, the induction of –514IGFI-luc by CnA and NFATc3 was significantly less than that of –1192IGFI-luc (9- vs. 18-fold). However, limited levels of –514IGFI-luc induction by CnA and NFATc3 together were observed in NIH3T3 fibroblasts that were not apparent in C2C12 myocytes. Together, these experiments demonstrate specific CnA/NFATc3 responsiveness of –1192IGFI-luc, but not –514IGFI-luc, in NIH3T3 cells. In addition, the requirement for cotransfected NFATc3 for full induction of –1192IGFI-luc is consistent with lower levels of endogenous NFATc3 expression in NIH3T3 fibroblasts relative to C2C12 myocytes.

The specificity of NFAT family members in activating IGF-I gene expression was examined in cotransfections with NFATc1 expression vectors (Fig. 4B). Cotransfection of either wild-type NFATc1 or constitutively active NFATc1 resulted in limited activation (5- to 6-fold) of –1192IGFI-luc and no specific activation of –514IGFI-luc in C2C12 cells. Interestingly, cotransfection of wild-type NFATc1 with CnA resulted in decreased induction of –1192IGFI-luc relative to cotransfection with CnA alone. The inhibition of CnA induction of –1192IGFI-luc by cotransfected NFATc1 in C2C12 cells may be indicative of competition for endogenous NFATc3 that is more effective in activating the –1192IGFI reporter. In support of the relative inability of NFATc1 to activate –1192IGFI sequences, cotransfection of CnA and NFATc1 in NIH3T3 cells resulted in significantly lower induction of the –1192IGFI reporter than cotransfection with NFATc3 (11- vs. 22-fold). In a previous study (21), these same expression constructs were used to identify a NFATc1-responsive element in the Down syndrome critical region 1 promoter, indicating that differential NFATc family member target gene regulation occurs in these assays. Together, these studies indicate that –1192IGFI-luc is preferentially activated by NFATc3 versus NFATc1 and that C2C12 myoblasts appear to have endogenous NFATc3 activity that can be competed by transfected NFATc1 in the activation of IGF-I exon 1 promoter region regulatory elements.

The ability of GATA4 and MEF2A to activate IGF-I exon 1 flanking sequences was examined. Conserved GATA consensus binding sequences are present in close proximity to CR1 and also are adjacent to the NFAT consensus site in CR2 (Fig. 3). A highly conserved MEF2 site is present near CR1 within the –1192 IGF-I flanking sequence, but there are no conserved MEF2 consensus sites in the –514 IGF-I flanking sequence. Transactivation of IGF-I exon 1 flanking sequences by GATA4 or MEF2A was examined in cotransfections with IGF-I reporter constructs in NIH3T3 cells (Fig. 5). In these experiments, there was extremely weak or no transactivation of either –1192IGFI-luc or –514IGFI-luc after cotransfection with MEF2A or GATA4 expression vectors. The 2- to 3-fold levels of transactivation that were observed for IGF-I reporter genes are much lower than the 10- to 20-fold activation observed for confirmed GATA4 or MEF2A targets in fibroblast cell lines (2, 29). Synergistic activation of IGF-I-luciferase reporter genes by GATA4 or MEF2A with NFATc3 was also not observed. Interestingly, the weak activation of IGF-I sequences by MEF2A was only observed for the –1192IGFI-luc reporter gene but not the –514IGFI-luc reporter gene, which does not contain conserved MEF2A consensus recognition sites. In contrast, weak activation of both –1192IGFI-luc and –514IGFI-luc constructs was observed for GATA4, consistent with the presence of conserved GATA consensus sites in both regions. However, the levels of transactivation with GATA4 or MEF2A were very low and likely do not represent conventional activation mechanisms of IGF-I-regulatory sequences by GATA4 of MEF2A individually or with NFATc3.

View larger version (14K): [in this window] [in a new window]   Fig. 5. IGF-I reporter constructs are relatively insensitive to transactivation by GATA4 or MEF2A. Indicated IGF-I promoter reporter constructs or pGL3 promoter plasmids were cotransfected with GATA4, MEF2A, or NFATc3 expression plasmids in NIH3T3 cells. Relative expression and statistical significance were calculated as described in Fig. 4.

View larger version (21K): [in this window] [in a new window]   Fig. 6. NFAT consensus sequences are required for induction by calcineurin and NFATc3. The indicated nucleotides in NFAT consensus sequences of IGF-I CR1 were mutated for each site (sites 1–3) individually within the context of –1192IGFI-luc. The resulting constructs (Mu1, Mu2, and Mu3) were cotransfected with CnA or NFATc3 expression plasmids in C2C12 cells. Statistical significance of observed differences was calculated relative to pGL3 levels for each group (*P < 0.05). Note that the expression of Mu3 transfected with the NFATc3 expression vector results in a statistically significant decrease in expression relative to nonspecific levels of the pGL3 promoter control plasmid.

View larger version (51K): [in this window] [in a new window]   Fig. 7. NFATc3 interacts with endogenous IGF-I gene CR1-containing sequences in C2C12 myocytes. Chromatin immunoprecipitation assays were performed with antisera directed against NFATc3 or NFATc1. Input or immunoprecipitated DNA was amplified with primers specific to IGF-I CR1 or the -actin promoter as a negative control. Immunoprecipitations with rabbit normal IgG were included as an additional specificity control.

	DISCUSSION

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The IGF-I gene is regulated through alternative promoter usage and splicing in different tissues and in response to hormonal or mechanical stimuli (16). During primary differentiation of C2C12 cells, IGF-I expression from the exon 1 promoter is induced (26). Previous studies (25, 26) have demonstrated that IGF-I reporter gene expression in skeletal muscle cells is regulated in part by proximal promoter sequences within exon 1, including an E box. However, these sequences are not sufficient for the induction of IGF-I expression during muscle differentiation. Additional conserved regulatory sequences within the untranslated region of exon 1 include cAMP- and prostaglandin E-responsive elements located at +193 to +215 (49). However, the presence of these known IGF-I exon 1 regulatory elements does not alter baseline reporter gene activity or calcineurin responsiveness in transfected C2C12 cells (data not shown). It is likely that the regulation of IGF-I gene expression in skeletal muscle involves multiple regulatory elements responsive to distinct conditions such as developmental stage, electrical stimulation, loading, and exercise (16). The identification of calcineurin/NFAT-responsive sequences within the exon 1 promoter 5' flanking region may be indicative of IGF-I gene regulatory mechanisms active during muscle development and adaptation.

The calcineurin-responsive region of the IGF-I exon 1 flanking region contains a cluster of three NFAT sites, two of which are required for full induction by calcineurin signaling and NFATc3 activation in C2C12 muscle cells. The observation that mutation of either site 2 or site 3 resulted in loss of calcineurin/NFATc3 responsiveness suggests that NFATc3 proteins must interact with both sites to induce gene expression. NFATs are often in DNA binding protein complexes with other transcription factors such as activated protein-1, GATA, or MEF2 (29, 39, 54). Although conserved GATA and MEF2 sites are present in the IGF-I CR1 sequence, relatively little transactivation of IGF-I promoter sequences was observed when cotransfected with GATA4 or MEF2A expression vectors in NIH3T3 cells. There is emerging evidence that NFATs can also act through clustered NFAT binding sites. An extreme example is the presence of eight conserved NFAT consensus binding sites in the calcineurin-responsive regulatory region of the modulatory calcineurin-interacting protein/Down syndrome critical region 1 gene (21, 55). However, the necessity of individual NFAT sites within the context of this element has not been determined. Mutagenesis of the IGF-I NFAT consensus sequences in CR1 provides evidence that NFAT proteins bound to adjacent DNA sites are necessary for transcriptional regulatory function in the context of IGF-I exon 1 promoter region. Further studies are necessary to examine NFAT protein-protein interactions necessary and sufficient for downstream target gene regulation.

In this study, we demonstrated that the IGF-I gene is expressed in E10.5 Nfatc3^+/–Nfatc4^–/– embryos but is not expressed in littermates lacking both NFATc3 and NFATc4. In addition, IGF-I exon 1 promoter regulatory sequences are preferentially activated by NFATc3 relative to NFATc1 in C2C12 myocytes. During embryogenesis, NFATc3 is required for skeletal myogenesis, whereas NFATc1-null mice have apparently normal skeletal muscle but exhibit embryonic lethal defects in cardiac valve formation (11, 19, 38). NFATc4 mutant mice are apparently normal and have no reported defects in skeletal muscle development or homeostasis (53). In human primary myoblasts, NFATc3 is the predominant nuclear NFAT, whereas NFATc1 is expressed at lower levels and remains cytoplasmic (1). In C2C12 myocytes, expression of constitutively active calcineurin leads to nuclear localization of NFATc3 but not other NFATc family members, which may explain the observation that NFATc3 is more effective than NFATc1 in activation of IGF-I reporter gene expression in these cells (12). NFATc1 and NFATc3 also exhibit differential nucleocytoplasmic shuttling in adult skeletal muscle and distinct DNA binding activity to NFAT-responsive elements (18, 48). The preferential induction and interaction of NFATc3 relative to NFATc1 with IGF-I exon 1 promoter region regulatory sequences further support the preferential regulation of muscle gene expression by specific NFATc family members. These observations add to the accumulating evidence that NFATc3, rather than NFATc1, is the predominant NFAT protein active during differentiation and maturation of skeletal myocytes during embryogenesis.

Both IGF-I signaling and CnA/NFAT activation have been implicated in skeletal muscle development and homeostasis. Each of these pathways has been implicated in skeletal muscle differentiation in cell culture, and targeted mutagenesis of either IGF-I or NFATc3 in mice results in compromised skeletal myogenesis (12, 19, 23, 56). The regulatory relationships between IGF-I and NFATs appear to be complex. In this study, we demonstrated that embryonic expression of IGF-I is responsive to NFATc3 and that the IGF-I exon 1 promoter region contains a conserved NFAT-responsive regulatory region. Previous studies (32, 45) have demonstrated that IGF-I can induce skeletal muscle maturation and activate NFATc1 in cultured myocytes. However, IGF-I treatment does not induce increased activity of a NFAT reporter gene during skeletal muscle hypertrophy in transgenic mice (35). Ectopic expression of localized IGF-I in skeletal muscle of transgenic mice is sufficient to induce a hypertrophic response, and there is extensive evidence for the regulation of skeletal muscle adaptation and growth by this pathway (13, 16, 31). In contrast, muscle-specific expression of CnA does not result in skeletal muscle hypertrophy but does promote slow fiber formation (33). Therefore, the interplay between IGF-I expression and calcineurin signaling is likely to be differentially regulated and modulated during distinct stages of skeletal muscle differentiation, maturation, and homeostasis.

	GRANTS

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This work was supported by National Institutes of Health (NIH) Grant P01-HL-069779. H. J. Evans-Anderson was supported by NIH Grant T32-ES-07051.

	ACKNOWLEDGMENTS

 
We thank Jeff Molkentin and Brian Black for providing expression plasmids and Nadia Rosenthal for PCR primer sequence information and helpful discussions. We acknowledge Paul Bushdid, who performed the initial analysis of the IGF-I genomic sequence.

	FOOTNOTES

 

Address for reprint requests and other correspondence: K. E. Yutzey, Div. of Molecular Cardiovascular Biology, Cincinnati Children's Medical Center, ML 7020, 3333 Burnet Ave., Cincinnati, OH 45229 (e-mail: katherine.yutzey{at}cchmc.org)

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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