Submitted on September 29, 2008
Revised on June 2, 2009
Accepted on June 22, 2009
FoxO1 induces apoptosis in skeletal myotubes in a DNA binding-dependent manner
Thomas J. McLoughlin1*, Sierra M Smith1, Alissa D. DeLong1, Hengbing Wang2, Terry G. Unterman3, and Karyn A. Esser4
1 The University of Toledo
2 The University of Illinois at Chicago
3 UIC School of Medicine
4 University of Kentucky
* To whom correspondence should be addressed. E-mail: thomas.mcloughlin{at}utoledo.edu.
Recent studies indicate that FoxO transcription factors play an important role in promoting muscle atrophy. To study mechanisms mediating effects of FoxO proteins in muscle wasting, FoxO1-estrogen receptor fusion proteins that are activated by treatment with 4-hydroxytamoxifen (4 OH-T) were stably transfected in C2C12 skeletal myoblasts using the pBABE retroviral system and grown into multinucleated skeletal myotubes. Activation of FoxO1 resulted in significant muscle atrophy, which was accompanied by DNA fragmentation evidenced by TUNEL labeling. Cells expressing a DNA binding-deficient form of FoxO1 also exhibited significant atrophy upon FoxO1 activation but without hallmark signs of apoptosis. FoxO1 activation resulted in a significant increase in MAFbx/Atrogin-1, Murf-1 and Bim gene expression, with no significant increase in BNip3 gene expression. Although the ability of FoxO1 to induce Murf-1 gene expression appeared to be independent of DNA binding, the expression of MAFbx/Atrogin-1 and Bim was significantly blunted in cells expressing DNA binding-deficient FoxO1. BNip3 gene expression was significantly elevated in DNA-binding deficient mutant cells. These findings indicate that FoxO1 promotes skeletal muscle atrophy through induction of proteolytic and apoptotic machinery via DNA binding-dependent and -independent mechanisms.
