Dual-specificity phosphatase 4 is upregulated during skeletal muscle atrophy and modulates extracellular signal-regulated kinase activity.

Skeletal muscle atrophy results from disparate physiological conditions, including denervation, corticosteroid treatment, and aging. The purpose of this study was to describe and characterize the function of dual-specificity phosphatase 4 (Dusp4) in skeletal muscle after it was found to be induced in response to neurogenic atrophy. Quantitative PCR and Western blot analysis revealed that Dusp4 is expressed during myoblast proliferation but rapidly disappears as muscle cells differentiate. The Dusp4 regulatory region was cloned and found to contain a conserved E-box element that negatively regulates Dusp4 reporter gene activity in response to myogenic regulatory factor expression. In addition, the proximal 3'-untranslated region of Dusp4 acts in an inhibitory manner to repress reporter gene activity as muscle cells progress through the differentiation process. To determine potential function, Dusp4 was fused with green fluorescent protein, expressed in C2C12 cells, and found to localize to the nucleus of proliferating myoblasts. Furthermore, Dusp4 overexpression delayed C2C12 muscle cell differentiation and resulted in repression of a MAP kinase signaling pathway reporter gene. Ectopic expression of a Dusp4 dominant negative mutant blocked muscle cell differentiation and attenuated MAP kinase signaling by preferentially targeting the ERK1/2 branch, but not the p38 branch, of the MAP kinase signaling cascade in skeletal muscle cells. The findings presented in this study provide the first description of Dusp4 in skeletal muscle and suggest that Dusp4 may play an important role in the regulation of muscle cell differentiation by regulating MAP kinase signaling.

Isolation, expression analysis and characterization of NEFA-interacting nuclear protein 30 and RING finger and SPRY domain containing 1 in skeletal muscle.

Muscle atrophy results from a range of physiological conditions, including immobilization, spinal cord damage, inflammation and aging. In this study we describe two genes, NEFA-interacting nuclear protein 30 (Nip30) and RING Finger and SPRY domain containing 1 (Rspry1), which have not previously been characterized or shown to be expressed in skeletal muscle. Furthermore, Nip30 and Rspry1 were transcriptionally induced in response to neurogenic muscle wasting in mice and were also found to be expressed endogenously at the RNA and protein level in C2C12 mouse muscle cells. Interestingly, during analysis of Nip30 and Rspry1 it was observed that these genes share a 230 base pair common regulatory region that contains several putative transcription regulatory elements. In order to assess the transcriptional activity of the Nip30 and Rspry1 regulatory regions, a fragment of the promoter of each gene was cloned, fused to a reporter gene, and transfected into cells. The Nip30 and Rspry1 reporters were both found to have significant transcriptional activity in cultured cells. Furthermore, the Nip30-Rspry1 common regulatory region contains a conserved E-box enhancer, which is an element bound by myogenic regulatory factors that function in the regulation of muscle-specific gene expression. Therefore, in order to determine if the predicted E-box was functional, Nip30 and Rspry1 reporters were transfected into cells ectopically expressing the myogenic regulatory factor, MyoD1, resulting in significant induction of both reporter genes. In addition, mutation of the conserved E-box element eliminated MyoD1 activation of the Nip30 and Rspry1 reporters. Finally, GFP-tagged Nip30 was found to localize to the nucleus, while GFP-tagged Rspry1 was found to localize to the cytoplasm of muscle cells.