LAUSR

Background Alcohol is regarded as a leading risk factor of osteopenia. Our previous works indicated Akt/GSK-3β/β-catenin pathway plays crucial role in the ethanol-induced anti-osteogenic effect in bone mesenchymal stem cells (BMSCs). It was acknowledged that PI3K/Akt is negatively regulated by the phosphatase and tensin homologue (PTEN) phosphatase. PTEN expression was reported to be upregualted in ethanol-administrated animals. Objectives In this study, we explored the molecular mechanisms underlying alcohol-induced osteopenia and investigated the role of PTEN and Akt/GSK-3β/β-catenin axis in this pathological process. Methods In vitro, Western blotting, separation of nucleus and cytosolic extracts, confocal scanning, RT-PCR were used to investigate the inhibition of ethanol on Akt/GSK3β/β-catenin signalling pathway via upregulation of PTEN in hBMSCs. In vivo, micro-computerised tomography, hematoxylin and eosin (H and E) staining, Van Gieson staining, Masson’s trichrome and fluorochrome labelling were employed to reveal that PTEN inhibition provided protective effects against ethanol on bone tissue. Results We found that ethanol increased PTEN expression both in BMSCs and in bone tissue of ethanol-administrated rats. PTEN upregulation impaired the recruitment of Akt to the plasma membrane, and suppressed Akt phosphorylation at Ser473, there by inhibiting the Akt/GSK3β/β-catenin signalling pathway in BMSCs and inhibited the expression of osteogenic genes COL1 and OCN both in vitro and in vivo. To counteract the inhibitory effect of ethanol, two selective PTEN inhibitors were introduced. The result of micro-computerised tomography, hematoxylin and eosin (H and E) staining, Van Gieson staining, Masson’s trichrome and fluorochrome labelling indicated PTEN inhibition provided protective effects against ethanol on bone tissue. Interestingly, our data revealed that the mRNA of PETN, paralleled with PTENP1, was increased in a time-dependent manner upon ethanol stimulation, which resulted in increasing PTEN protein level. In addition, ethanol increased PTEN expression while decreased p-PTEN expression in a time-dependent manner, which indicated the generation of more functional PTEN. Conclusions Taken together, dual regulations of PTEN by ethanol via transcriptional and post-transcriptional process impaired the downstream signalling of Akt/GSK3β/β-catenin and osteogenic differentiation of hBMSC. Therefore, we propose that PTEN inhibition treatment for Akt/GSK3β/β-catenin activation could be tested in the clinic as a potential therapeutic approach to preventing the development of alcohol-induced osteopenia. References [1] Song MS, Salmena L Pandolfi PP. The functions and regulation of the PTEN tumour suppressor. Nature reviews. Molecular cell biology2012;13:283–296. doi:10.1038/nrm3330 [2] Xian L, et al. Matrix IGF-1 maintains bone mass by activation of mTOR in mesenchymal stem cells. Nature medicine2012;18:1095–1101. doi:10.1038/nm.2793 [3] Vanhaesebroeck B, Stephens L, Hawkins P. PI3K signalling: the path to discovery and understanding. Nature reviews. Molecular cell biology2012;13:195–203. doi:10.1038/nrm3290 Acknowledgements The current study was supported by grants from the National Natural Science Foundation of China (no. 81272003 and no. 81301572) and the SMC-Chen Xing Plan for Splendid Young Investigators of Shanghai Jiao Tong University. Disclosure of Interest None declared