ECTS Abstracts (2015) 1 P183

Extracellular ATP and PTH Work in Concert to Inhibit the Sclerostin Expression by Osteosarcoma Cell Lines

Mohd Azuraidi Osman1,2, Katarzyna Goljanek-Whysall1, Peter J Wilson1, Nick P Rhodes1 & James A Gallagher1

1Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK, 2Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.

Sclerostin is expressed by SOST in osteoblast/octeocyte lineage cells. Sclerostin is anti-anabolic for bone formation through inhibition of the WNT signalling pathway. The aim of this study was to determine how SOST expression is regulated by ATP and PTH at different stages of osteoblastic development. Three human osteosarcoma cell lines representing different stages of osteoblast differentiation were used (MG-63 the least differentiated, TE-85 intermediate and SaOS-2 the most mature). Cells were grown in DMEM/10% FCS in 6 well plates until 90% confluent. Cells were serum deprived for 48 hours before treated with 10 μM ATP, 10 ng/ml PTH and 10 μM ATP +10 ng/ml PTH, respectively and incubated at 37 °C for 17 hours. mRNA was extracted and reverse transcribed to cDNA. Expression of sclerostin was monitored by qRT-PCR. Results were analysed using SPSS. In untreated cells, SaOS-2 cells had the highest expression of sclerostin (p<0.05) compared with MG63 and TE85. Expression of sclerostin was intermediate in TE85 cells but significantly higher than MG63 (p<0.05). Interestingly, sclerostin expression was barely detectable in MG-63 but abundantly expressed in SaOS-2 cells. Sclerostin transcripts were present in TE85 cells but at least 8 fold less than in SaOS-2 cells. In both TE85 and SaOS-2 cells, ATP, PTH and ATP+PTH treatment decreased the expression of sclerostin. The results reinforce previous reports on the relative differentiation status of these commonly used osteoblastic cell models. The robust expression of sclerostin in SaOS-2 suggests that this cell type represents a mature stage of the osteoblast / osteocyte lineage. The findings of this study shows that inhibition of sclerostin by ATP and PTH occurs at all stages of bone development. As ATP can be released by osteoblasts through mechanical stimulation, better understanding of the regulation of sclerostin should contribute to the development of new therapeutic approaches for preventing bone loss.

Disclosure: The authors declared no competing interests. This work was supported by the Ministry of Education Malaysia and Universiti Putra Malaysia.

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