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

2007 ◽  
Vol 292 (5) ◽  
pp. C1887-C1894 ◽  
Author(s):  
Christina M. Alfieri ◽  
Heather J. Evans-Anderson ◽  
Katherine E. Yutzey

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.

2005 ◽  
Vol 568 (1) ◽  
pp. 229-242 ◽  
Author(s):  
Malcolm Grohmann ◽  
Emily Foulstone ◽  
Gavin Welsh ◽  
Jeff Holly ◽  
Julian Shield ◽  
...  

2013 ◽  
Vol 305 (2) ◽  
pp. E183-E193 ◽  
Author(s):  
Hannah Crossland ◽  
Abid A. Kazi ◽  
Charles H. Lang ◽  
James A. Timmons ◽  
Philippe Pierre ◽  
...  

Focal adhesion kinase (FAK) is an attachment complex protein associated with the regulation of muscle mass through as-of-yet unclear mechanisms. We tested whether FAK is functionally important for muscle hypertrophy, with the hypothesis that FAK knockdown (FAK-KD) would impede cell growth associated with a trophic stimulus. C2C12 skeletal muscle cells harboring FAK-targeted (FAK-KD) or scrambled (SCR) shRNA were created using lentiviral transfection techniques. Both FAK-KD and SCR myotubes were incubated for 24 h with IGF-I (10 ng/ml), and additional SCR cells (±IGF-1) were incubated with a FAK kinase inhibitor before assay of cell growth. Muscle protein synthesis (MPS) and putative FAK signaling mechanisms (immunoblotting and coimmunoprecipitation) were assessed. IGF-I-induced increases in myotube width (+41 ± 7% vs. non-IGF-I-treated) and total protein (+44 ± 6%) were, after 24 h, attenuated in FAK-KD cells, whereas MPS was suppressed in FAK-KD vs. SCR after 4 h. These blunted responses were associated with attenuated IGF-I-induced FAK Tyr397 phosphorylation and markedly suppressed phosphorylation of tuberous sclerosis complex 2 (TSC2) and critical downstream mTOR signaling (ribosomal S6 kinase, eIF4F assembly) in FAK shRNA cells (all P < 0.05 vs. IGF-I-treated SCR cells). However, binding of FAK to TSC2 or its phosphatase Shp-2 was not affected by IGF-I or cell phenotype. Finally, FAK-KD-mediated suppression of cell growth was recapitulated by direct inhibition of FAK kinase activity in SCR cells. We conclude that FAK is required for IGF-I-induced muscle hypertrophy, signaling through a TSC2/mTOR/S6K1-dependent pathway via means requiring the kinase activity of FAK but not altered FAK-TSC2 or FAK-Shp-2 binding.


2006 ◽  
Vol 26 (6-8) ◽  
pp. 409-417 ◽  
Author(s):  
Koichi Ojima ◽  
Yasuko Ono ◽  
Shoji Hata ◽  
Suguru Koyama ◽  
Naoko Doi ◽  
...  

2011 ◽  
Vol 317 (3) ◽  
pp. 356-366 ◽  
Author(s):  
Ai Shima ◽  
Jennifer Pham ◽  
Erica Blanco ◽  
Elisabeth R. Barton ◽  
H. Lee Sweeney ◽  
...  

1991 ◽  
Vol 11 (1) ◽  
pp. 267-280 ◽  
Author(s):  
H Lin ◽  
K E Yutzey ◽  
S F Konieczny

The quail fast skeletal troponin I (TnI) gene is a member of the contractile protein gene set and is expressed exclusively in differentiated skeletal muscle cells. TnI gene transcription is controlled by an internal regulatory element (IRE), located within the first intron, that functions as a muscle-specific enhancer. Recent studies have shown that the TnI IRE may interact directly with the muscle regulatory factors MyoD, myogenin, and Myf-5 to produce a muscle-specific expression pattern, since these factors trans-activate cotransfected TnI gene constructs in C3H10T1/2 fibroblasts. In this study, we have examined the protein-IRE interactions that are responsible for transcriptionally activating the TnI gene during skeletal muscle development. We demonstrate that the helix-loop-helix muscle regulatory factors MyoD, myogenin, Myf-5, and MRF4, when complexed with the immunoglobulin enhancer-binding protein E12, interact with identical nucleotides within a muscle regulatory factor-binding site (MRF site) located in the TnI IRE. The nuclear proteins that bind to the MRF site are restricted to skeletal muscle cells, since protein extracts from HeLa, L, and C3H10T1/2 fibroblasts do not contain similar binding activities. Importantly, the TnI MRF site alone is not sufficient to elicit the full enhancer activity associated with the IRE. Instead, two additional regions (site I and site II) are required. The proteins that interact with site I and site II are expressed in both muscle and nonmuscle cell types and by themselves are ineffective in activating TnI gene expression. However, when the MRF site is positioned upstream or downstream of site I and site II, full enhancer activity is restored. We conclude that helix-loop-helix muscle regulatory factors must interact with ubiquitously expressed proteins to generate the active TnI transcription complex that is present in differentiated muscle fibers.


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