ECTS Abstracts (2015) 1 P162

Homocysteine Modulates Mineralisation of Osteoblastic Cells

Norbert Hassler1, Silvia Spitzer1, Florian Haider1, Roman Thaler2,1, Eleftherios Paschalis1, Klaus Klaushofer1 & Franz Varga1

1Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 1st Medical Department, Hanusch Hospital, Vienna, Austria; 2Departments of Orthopedic Surgery and Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, USA.

Hyperhomocysteinemia is associated with several pathologies such as bone fragility, cardiovascular disease, diabetes, and atherosclerosis. We have recently demonstrated that homocysteine (Hcys) alters collagen cross-linking, perturbs triple-helix formation and regulates expression of genes found in osteoblastic cells, possibly via the inflammation related gene SAA3. Concerning cardiovascular disease, it was demonstrated that Hcys is related to aortic mineralisation in patients with ischaemic heart disease. This finding poses the question whether Hcys influences the deposition of mineral in bone cell cultures as well. For our experiments, we used the pre-osteoblastic cell line MC3T3-E1, which in long-term culture differentiated into mature, mineral depositing osteoblasts. As a second system we cultured MLO-A5 cells. These cells are late osteoblasts, which also deposit mineral, however, already after 10 days. MC3T3-E1 and MLO-A5 cells were cultured up to 5 and up to 3 weeks, respectively. The cultures were treated either with Hcys or ß-glycerophosphate (ßGP) or in combination and mineralisation was determined by Alizarin-red staining. Gene expression was addressed by genome-wide expression analysis (GeneChip, Affymetrix) and expressions of interesting genes were confirmed by RT-qPCR. Long-term cultures of MC3T3-E1 cells revealed that Hcys in combination with ßGP strongly increased the deposition of mineral after 4 and 5 weeks of culture. In MLO-A5 cultures, however, the sole treatment with ßGP stimulated the deposition of mineral, and Hcys had no additional effect. Genome-wide expression analysis and RT-qPCR of Hcys treated cells demonstrated an increase of Phospho1 (phosphatase, orphan 1) and Alpl (alkaline phosphatase), both genes, which are involved in the mineralisation process. Our data suggests that Hcys by up-regulating expression of phosphatases increases the concentration of inorganic phosphate, which accelerates mineralisation of osteoblastic MC3T3-E1 cell cultures. These results also suggest that Hcys can modulate physiological as well as pathological mineralisation processes.

Disclosure: The authors declared no competing interests.

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