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PKC? as a novel promoter of skeletal muscle differentiation and regeneration.

ABSTRACT: Satellite cells are muscle resident stem cells and are responsible for muscle regeneration. In this study we investigate the involvement of PKC? during muscle stem cell differentiation in vitro and in vivo. Here, we describe the identification of a previously unrecognized role for the PKC?-HMGA1 signaling axis in myoblast differentiation and regeneration processes.PKC? expression was modulated in the C2C12 cell line and primary murine satellite cells in vitro, as well as in an in vivo model of muscle regeneration. Immunohistochemistry and immunofluorescence, RT-PCR and shRNA silencing techniques were used to determine the role of PKC? and HMGA1 in myogenic differentiation.PKC? expression increases and subsequently re-localizes to the nucleus during skeletal muscle cell differentiation. In the nucleus, PKC? blocks Hmga1 expression to promote Myogenin and Mrf4 accumulation and myoblast formation. Following in vivo muscle injury, PKC? accumulates in regenerating, centrally-nucleated myofibers. Pharmacological inhibition of PKC? impairs the expression of two crucial markers of muscle differentiation, namely MyoD and Myogenin, during injury induced muscle regeneration.This work identifies the PKC?-HMGA1 signaling axis as a positive regulator of skeletal muscle differentiation.

SUBMITTER: Di Marcantonio D

PROVIDER: S-EPMC5130411 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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PKCε as a novel promoter of skeletal muscle differentiation and regeneration.

Di Marcantonio D D Galli D D Carubbi C C Gobbi G G Queirolo V V Martini S S Merighi S S Vaccarezza M M Maffulli N N Sykes S M SM Vitale M M Mirandola P P

Experimental cell research 20150930 1

<h4>Introduction</h4>Satellite cells are muscle resident stem cells and are responsible for muscle regeneration. In this study we investigate the involvement of PKCε during muscle stem cell differentiation in vitro and in vivo. Here, we describe the identification of a previously unrecognized role for the PKCε-HMGA1 signaling axis in myoblast differentiation and regeneration processes.<h4>Methods</h4>PKCε expression was modulated in the C2C12 cell line and primary murine satellite cells in vitro ...[more]

PMID: 26431586

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Project description:BackgroundCREG1 (cellular repressor of E1A-stimulated genes 1) is a protein involved in cellular differentiation and homeostasis regulation. However, its role in skeletal muscle satellite cells differentiation and muscle regeneration is poorly understood. This study aimed to investigate the role of CREG1 in myogenesis and muscle regeneration.MethodsRNA sequencing data (GSE8479) was analysed from the Gene Expression Omnibus database (GEO, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi). We generated Creg1 knockdown and skeletal muscle satellite cells specific Creg1 overexpression mice mediated by adeno-associated virus serotype 9 (AAV9), skeletal muscle mature myofibre Creg1 knockout mice (myoblast/Creg1MKO), and control mice Creg1flox/flox (Creg1fl/fl) as in vivo models. The mice were injected into tibialis anterior (TA) muscle with 100 μL of 10 μM cardiotoxin to establish a muscle regeneration model. Creg1fl/fl and Creg1MKO mice were treated with AAV-sh-C-Cbl (2 × 1010 genomic copies/mouse) to silence C-Cbl in the TA muscle. 293T and C2C12 cells were transfected with plasmids using lipofectamine RNAi MAX in vitro. Mass spectrometry analyses and RNA sequencing transcriptomic assay were performed.ResultsWe analysed the transcriptional profiles of the skeletal muscle biopsies from healthy older (N = 25) and younger (N = 26) adult men and women in GSE8479 database, and the results showed that Creg1 was associated with human sarcopenia. We found that Creg1 knockdown mice regenerated less newly formed fibres in response to cardiotoxin injection (~30% reduction, P < 0.01); however, muscle satellite cells specific Creg1 overexpression mice regenerated more newly formed fibres (~20% increase, P < 0.05). AMPKa1 is known as a key mediator in the muscle regeneration process. Our results revealed that CREG1 deficiency inhibited AMPKa1 signalling through C-CBL E3-ubiquitin ligase-mediated AMPKa1 degradation (P < 0.01). C-CBL-mediated AMPKa1 ubiquitination was attributed to the K48-linked polyubiquitination of AMPKa1 at K396 and that the modification played an important role in the regulation of AMPKa1 protein stability. We also found that Creg1MKO mice regenerated less newly formed fibres compared with Creg1fl/fl mice (~30% reduction, P < 0.01). RNA-seq analysis showed that CREG1 deletion in impaired muscles led to the upregulation of inflammation and DKK3 expression. The TA muscles of Creg1MKO mice were injected with AAV-vector or AAV-shC-Cbl, silencing C-CBL (P < 0.01) in the skeletal muscles of Creg1MKO mice significantly improved muscle regeneration induced by CTX injury (P < 0.01).ConclusionsOur findings suggest that CREG1 may be a potential therapeutic target for skeletal muscle regeneration.

Project description:BackgroundCell-specific expression of the gene that encodes brain-derived neurotrophic factor (BDNF) is required for the normal development of peripheral sensory neurons and efficient synaptic transmission in the mature central and peripheral nervous system. The control of BDNF gene expression involves multiple tissue and cell-specific promoters that are differentially regulated. The molecular mechanisms that are responsible for tissue and cell-specific expression of these promoters are still incompletely understood.ResultsThe cloning and analysis of three additional zebrafish (Danio rerio) BDNF gene exons and two associated promoters, is reported. Among them are two exons that generate a novel tripartite mature transcript. The exons were located on the transcription unit, whose overall organization was determined by cloning, Southern blot hybridization and sequence analysis, and compared with the pufferfish (Fugu rubripes) and mammalian BDNF loci, revealing a conserved but more compact organization. Structural and functional analysis of the exons, their adjacent promoters and 5' flanks, showed that they are expressed cell-specifically. The promoter associated with the 5' exon of the tripartite transcript is GC-rich, TATA-less and the 5' flank adjacent to it contains multiple Sp1, Mef2, and AP1 elements. A fusion gene containing the promoter and 1.5 KB of 5' flank is directed exclusively to skeletal muscle of transiently transfected embryos. The second promoter, whose associated 5' exon contains a 25-nucleotide segment of identity with a mammalian BDNF gene exon, was transiently expressed in yolk of the early embryo. RT-PCR analysis of total RNA from whole juvenile fish and adult female skeletal muscle revealed tissue-specific expression of the 5' exons but the novel exon could not be detected even after two rounds of nested PCR.ConclusionThe zebrafish BDNF gene is as complex as the mammalian gene yet much more compact. Its exons are expressed in an independently regulated and cell-specific fashion. An initial structural and functional analysis has shown that the regions controlling zebrafish BDNF gene expression have been cloned and identified. They can now be subjected to detailed molecular and genetic analyses to identify the cellular mechanisms by which the transcription factors that act on these regions control BDNF gene expression.

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PKC? as a novel promoter of skeletal muscle differentiation and regeneration.

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PKCε as a novel promoter of skeletal muscle differentiation and regeneration.

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