FoxO1a-Cyclic GMP-Dependent Kinase I Interactions Orchestrate Myoblast Fusion

Abstract

The regulatory circuits that orchestrate mammalian myoblast cell fusion during myogenesis are poorly understood. The transcriptional activity of FoxO1a directly regulates this process, yet the molecular mechanisms governing FoxO1a activity during muscle cell differentiation remain unknown. Here we show an autoregulatory loop in which FoxO1a directly activates transcription of the cyclic GMP-dependent protein kinase I (cGKI) gene and where the ensuing cGKI activity phosphorylates FoxO1a and abolishes its DNA binding activity. These findings establish the FoxO1a-to-cGKI pathway as a novel feedback loop that allows the precise tuning of myoblast fusion. Interestingly, this pathway appears to operate independently of muscle cell differentiation programs directed by myogenic transcription factors.Fusion of cell membranes is required for the proper function and morphogenesis of several mammalian cell types, including macrophages, trophoblasts, spermatozoa and oocytes, and myoblasts that form skeletal muscle. While the mechanisms regulating intracellular membrane fusion events have been studied in some detail (4, 17), very little is known regarding the mechanisms governing mammalian cell-to-cell fusion events.Complex programs orchestrate myogenesis, which involve coordinated myogenic cell migration, recognition, adhesion, alignment, and finally membrane fusion. The FoxO family of transcription factors (FoxO1a, FoxO3, and FoxO4a) regulates diverse cellular responses, including apoptosis, cell cycle arrest, differentiation, DNA repair, and oxidative stress (1,3,7,34). While the ability of FoxO's to regulate divergent processes such as apoptosis and differentiation seems somewhat paradoxical, some of the cellular alterations associated with skeletal muscle cell differentiation do share a high degree of similarity with many phenotypic changes usually associated with apoptosis. Indeed, cytoskeleton remodeling and actin fiber disassembly are features shared by these two processes (10,14,16,29,32), and myosin light chain kinase, a conserved regulator of muscle cell contraction, is also essential for the blebbing of cell membranes observed during apoptosis (23). Furthermore, matrix metalloproteinases are required for both apoptosis and muscle cell fusion (28, 36), and activation of caspase 3, a key effector apoptotic protease, is also required for skeletal muscle cell differentiation (11).FoxO1a is essential for normal development, as evidenced by the fact that knockout mice lacking FoxO1a die at embryonic day 9.5 (E9.5) to E10.5 due to defects in vasculogenesis, whereas targeted deletions of the close FoxO1a homologs FoxO3 and FoxO4a lead to limited, if any, developmental phenotypes (8, 18). Interestingly, endothelial cells lacking FoxO1a fail to reorganize into vessel-like structures when stimulated with vascular endothelial growth factor (12), suggesting that FoxO1a directs cytoskeleton remodeling and cell-cell interactions during the formation of the vasculature. In addition, FoxO1a is both necessary and sufficient to regu...

Rho/Rho-associated Kinase Signal Regulates Myogenic Differentiation via Myocardin-related Transcription Factor-A/Smad-dependent Transcription of the Id3 Gene

“…The interaction of FKHR with cyclic GMP-dependent protein kinase I causes myoblast fusion by influencing the fusion rate (22). Despite these findings, conflicting results have been reported; normal myogenic differentiation is impaired by the forced expression of constitutively active or wild-type FKHR in skeletal muscle cells (57,58).…”

“…In various other cell types, FKHR that is phosphorylated by Akt, but not by ROCK, is also exported from the nucleus, leading to the inhibition of FKHR-mediated transcription (23). However, the target partners of FKHR and its function in myogenic differentiation remain unknown, except for a Foxo1a-cyclic GMP-dependent kinase I interaction (22).…”

“…One of the forkhead family transcription factors, FKHR (also named Foxo1), is required for myocyte fusion (15,21,22). Because ROCK directly phosphorylates FKHR in myocytes, resulting in the export of FKHR from the nucleus, the inactivation of RhoA/ROCK signaling is prerequisite for the nuclear localization of FKHR and myocyte fusion (15).…”

Structure/function relationships underlying regulation of FOXO transcription factors

“…The cGK1-induced phosphorylation seems to be specific for FOXO1 because the cluster of serine residues preceding the helix H1 is unique to FOXO1, and the residue Ser184 is absent in FOXO4 sequence (Figure 3c). Phosphorylation of FOXO1 by cGK1 efficiently inhibits its DNA-binding activity, in agreement with the fact that both these regions make polar and van der Waals interactions with DNA backbone, and the introduction of negatively charged phosphate groups would be expected to disrupt these contacts (Bois et al, 2005;Tsai et al, 2006Tsai et al, , 2007.…”

“…Recently, it has been shown that the cyclic GMPdependent kinase-1 (cGK1) regulates FOXO1 activity during muscle cell fusion (Bois et al, 2005). FOXO1 directly activates cGK1 transcription and cGK1 then in turn harnesses FOXO1 activity by phosphorylating cluster of serine residue located upstream of the first helix H1 (Ser152-155 of FOXO1) and Ser184 in the N-terminal part of helix H2 (Figures 3c and 7).…”