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

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 immuno-precipitation 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.