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A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation
Published on: March 15, 2018
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Leptin inhibits AMPKα2 down-regulation induced decrease in the osteocytic MLO-Y4 cell proliferation and the
1Department of Pediatric Orthopedics, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine Shanghai, P. R. China.
International Journal of Clinical and Experimental Pathology
|January 23, 2020
Summary
AMP-activated protein kinase alpha 2 (AMPKα2) knockdown inhibited osteocytic cell proliferation and osteogenic marker expression. Leptin treatment, however, enhanced these processes, suggesting AMPKα2
Area of Science:
- Biochemistry
- Cell Biology
- Bone Biology
Background:
- AMP-activated protein kinase (AMPK) regulates energy homeostasis.
- AMPKα1 is involved in bone formation, but AMPKα2's role in osteogenesis is unclear.
Purpose of the Study:
- To investigate the role of AMPKα2 in osteocytic MLO-Y4 cell proliferation and osteogenic marker expression.
- To determine the effect of leptin on these processes in the presence or absence of AMPKα2.
Main Methods:
- AMPKα2 was silenced in MLO-Y4 cells using pLKO.1-AMPKα2-shRNA.
- Cell proliferation was assessed using CCK-8 assays.
- Osteogenic marker expression (OPG, OCN, OPN, ALP, BMP6) was analyzed via Real-time PCR and Western blot.
- Global transcriptomic changes were evaluated using RNA-seq.
Main Results:
- AMPKα2 knockdown significantly reduced MLO-Y4 cell proliferation and expression of osteogenic markers (OPG, OCN, OPN, ALP, BMP6) and p-Smad5/Smad5.
- Leptin treatment increased cell proliferation and osteogenic marker expression, irrespective of AMPKα2 levels.
- RNA-seq revealed distinct transcriptional changes upon AMPKα2 silencing and leptin treatment.
Conclusions:
- AMPKα2 plays a crucial role in promoting osteocytic proliferation and osteogenesis in vitro.
- Leptin can counteract the inhibitory effects of AMPKα2 knockdown on osteogenesis.
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