Abstract
BackgroundMice lacking MyoD exhibit delayed skeletal muscle regeneration and markedly enhanced numbers of satellite cells. Myoblasts isolated from MyoD-/- myoblasts proliferate more rapidly than wild type myoblasts, display a dramatic delay in differentiation, and continue to incorporate BrdU after serum withdrawal.MethodsPrimary myoblasts isolated from wild type and MyoD-/- mutant mice were examined by microarray analysis and further characterized by cell and molecular experiments in cell culture.ResultsWe found that NF-κB, a key regulator of cell-cycle withdrawal and differentiation, aberrantly maintains nuclear localization and transcriptional activity in MyoD-/- myoblasts. As a result, expression of cyclin D is maintained during serum withdrawal, inhibiting expression of muscle-specific genes and progression through the differentiation program. Sustained nuclear localization of cyclin E, and a concomitant increase in cdk2 activity maintains S-phase entry in MyoD-/- myoblasts even in the absence of mitogens. Importantly, this deficit was rescued by forced expression of IκBαSR, a non-degradable mutant of IκBα, indicating that inhibition of NF-κB is sufficient to induce terminal myogenic differentiation in the absence of MyoD.ConclusionMyoD-induced cytoplasmic relocalization of NF-κB is an essential step in linking cell-cycle withdrawal to the terminal differentiation of skeletal myoblasts. These results provide important insight into the unique functions of MyoD in regulating the switch from progenitor proliferation to terminal differentiation.
Highlights
Mice lacking MyoD exhibit delayed skeletal muscle regeneration and markedly enhanced numbers of satellite cells
Given that NF-κB is normally relocalized to the cytoplasm during myogenic differentiation, we sought to determine if NF-κB remains in the nucleus during induction of differentiation in myoblasts lacking MyoD
(See figure on previous page.) Figure 6 Nuclear localization of NF-κB inhibits terminal differentiation. (A) MyoD-/- myoblasts were infected with empty capsid or virus expressing IκBα-SR
Summary
Mice lacking MyoD exhibit delayed skeletal muscle regeneration and markedly enhanced numbers of satellite cells. Myoblasts isolated from MyoD-/- myoblasts proliferate more rapidly than wild type myoblasts, display a dramatic delay in differentiation, and continue to incorporate BrdU after serum withdrawal. Cell survival and differentiation is regulated by NF-κB, a family of ubiquitously expressed transcription factors comprising RelA/p65, c-Rel, RelB, p50 (processed form of p105), and p52 (processed form of p100) [1]. All family members contain a DNA-binding domain, a proteinprotein dimerization domain, and a nuclear localization sequence (NLS). Sub-cellular localization of NF-κB is regulated by ‘inhibitor of κB’ proteins: IκBα, IκBβ, and IκB [3]. The released NF-κB enters the nucleus, binds DNA, and regulates gene transcription. This process is normally activated by molecules such as cytokines, growth factors, and bacterial products [4]
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