2018 Fiscal Year Research-status Report
Vitamin D - vitamin D receptor axis affects muscle differentiation and improves muscle regeneration
| Project/Area Number |
18K16236
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| Research Institution | National Center of Neurology and Psychiatry |
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Principal Investigator |
MARIA TSOUMPRA 国立研究開発法人国立精神・神経医療研究センター, 神経研究所 遺伝子疾患治療研究部, 科研費研究員 (80756198)
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| Project Period (FY) |
2018-04-01 – 2020-03-31
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| Keywords | vitamin D3 / muscle / dystrobrevin alpha / myogenic differentiation |
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| Outline of Annual Research Achievements |
The active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 [VD3] exerts its tissue-specific actions through binding to its vitamin D receptor (VDR). I elucidated the regulatory role of VD3/VDR axis on Dystrobrevin alpha (Dtna), a member of dystrophin associated protein complex (DAPC). I demonstrated that Dtna expression was dose-dependently upregulated upon short or long term VD3 administration during early or late stages of myogenic differentiation in C2C12 cells. Dtna was also upregulated after long term administration and/or VDR/RXR transfection in mdx52 cells. Silencing of MyoD1, an E-box binding myogenic transcription factor, or presence of a MAPK inhibitor in the media did not altere the VD3-mediated Dtna induction but silencing of VDR abolished this effect. Dtna and VDR protein levels were elevated in VD3-treated C2C12 myotubes. I cloned fragments of murine muscle-specific Dtna promoter region, transfected them in C2C12 myoblasts and measured relative induction via dual luciferase assay. The induction of each construct upon 48 hr VD3 administration in reduced serum conditions was dependent on the number of the included putative vitamin D response elements (VDREs) revealed via in silico analysis. I then used the most active construct to generate point mutations targeting individual VDRE regions and identified the mutation that greatly abolished the observed VD3-stimulated induction. This binding region was confirmed by ChIP assay. The above data indicate that the positive regulation of Dtna by VD3 during murine myogenic differentiation is VDR-mediated.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The Dtna upregulation via Vitamin D is a novel concept and all assays, especially the promoter assays, needed optimization. I have characterized the response of various Dystrobrevin constructs (Dtna1, Dtna3, Dtna2B, brain specific, synaptic specific) to VD3 administration via qPCR, western blot and promoter assays in three cell lines, so this was a very laborious task to undertake. I am now preparing to submit my first manuscript describing the dystrobrevin regulation by VD3/VDR axis in myogenic (C2C12, mdx52) and neuronal (Neuro2A) cells.
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| Strategy for Future Research Activity |
The Dtna upregulation by VD3 administration may have beneficial effects in dystrophic murine phenotype where DAPC complex is destabilized- a theory that I am currently testing by administering VD3 in our mdx52 as well as the mdx52/SR1 double knockout dystrophic mouse model. I aim to characterize the rapid response of Dtna to VD3 in younger (week 4-6) mdx52 mice, time when VDR is still expressed and detectable via IP administration. My latest research has shown that deletion of Dtna in C2C12 cells interferes with myogenin response during myogenic differentiation. Furthermore Dtna knockdown negatively influenced dystrophin protein expression, whereas presence of VD3 in the medium partially rescued this effect. I am now cloning the muscle specific Dtna (Dtna3) as well as the Dtna1 (muscle and brain expressed) genes and will overexpress them in C2C12 cells in order to confirm the Dtna/dystrophin expression. If this response is confirmed, I aim to fully elucidate the dystrophin/dystrobrevin interaction. I also wish to test whether Dtna overexpression may have a positive influence on exon skipping treatment for DMD and have the tools to do so, as my current lab is leading the exon skipping research in Japan. At the same time I aim to characterize the VD3 regulatory effect on biglycan and utrophin during myogenic differentiation. The availability of VDR in muscle seems to strongly regulate biglycan and utrophin expression and promoter assays will shed a light to this complicated mechanism.
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[Journal Article] Activation of unliganded FGF receptor by extracellular phosphate potentiates proteolytic protection of FGF23 by its O-glycosylation2019
Author(s)
Takashi Y, Kosako H, Sawatsubashi S, Kinoshita Y, Ito N, Tsoumpra MK, Nangaku M, Abe M, Matsuhisa M, Kato S, Matsumoto T, Fukumoto S.
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Journal Title
Proc Natl Acad Sci U S A.
Volume: 116
Pages: 11418-11427
DOI
Peer Reviewed / Open Access / Int'l Joint Research
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