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Fam83d is Induced During Neurogenic Skeletal Muscle Atrophy and Modulates MAP Kinase and AKT Signaling
Author(s) -
Waddell David,
Cooper Lisa,
Kavanagh Jack
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.06737
Subject(s) - myogenesis , denervation , biology , myocyte , muscle atrophy , reporter gene , skeletal muscle , microbiology and biotechnology , myod , dysferlin , ectopic expression , microarray analysis techniques , atrophy , gene expression , gene , endocrinology , genetics
Skeletal muscle atrophy is a potentially debilitating condition resulting in muscle weakness and a decrease in muscle mass in response to a wide range of physiological stimuli including aging, cancer, denervation, and corticosteroid exposure. To better characterize the molecular genetic events of neurogenic atrophy, a previous study isolated mouse gastrocnemius muscle from mice at 3 and 14 days following sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that Fam83d is expressed in skeletal muscle and is significantly induced in response to denervation. Quantitative PCR (qPCR) and Western blot analysis revealed that Fam83d is more highly expressed in proliferating myoblasts compared to differentiated myotubes. Ectopic expression of myc‐tagged Fam83d revealed that the protein is rapidly turned over in a 26S proteasome‐dependent manner as myoblasts begin to differentiate. Characterization of the transcriptional regulation of Fam83d was analyzed by fusing the proximal promoter region to a reporter gene. The proximal promoter region contains putative E‐box consensus elements that facilitate binding of myogenic regulatory factors (MRFs) and transfection of the reporter construct in combination with MyoD inhibited Fam83d reporter gene activity. To assess subcellular localization, Fam83d was fused with green fluorescent protein (GFP) and the fusion protein was expressed in C 2 C 12 mouse myoblasts cells, visualized by confocal microscopy, and observed to localize exclusively to the cytoplasm. In addition, Fam83d was ectopically expressed in cultured muscle cells and markers of muscle cell differentiation, MAP Kinase signaling, and AKT/mTOR signaling were analyzed. Fam83d overexpression resulted in significantly repressed levels of myosin heavy chain and myogenin expression, while levels of phosphorylated ERK, phosphorylated AKT and total AKT were dramatically repressed. Interestingly, disruption of a putative phospholipase D‐like (PLD‐like) domain significantly attenuated the Fam83d‐mediated repression of both MAPK and AKT/mTOR signaling. Interestingly, it was also observed that Fam83d physically interacts with and destabilizes CKIα in a PLD‐like domain‐dependent manner. The discovery that Fam83d is expressed in skeletal muscle combined with the observation that Fam83d, a potential modulator of MAP Kinase and AKT signaling, is induced in response to neurogenic atrophy helps further our understanding of the molecular and cellular events of skeletal muscle wasting. Support or Funding Information The work described in this study was supported by UNF Transformational Learning Opportunity grants, Academic Affairs Faculty Enhancement grants, a Dean’s Leadership Council Fellowship Award, a UNF Foundation Board Grant, and research start‐up funds from the University of North Florida.