Artikel
Modulation of Transient Receptor Potential Channels TRPC3 and TRPC6 influences activity of osteoclasts and quality of bone
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Autoren
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Birgit Mentrup
- Institute of Musculoskeletal Medicine, University Hospital Münster, Münster, Germany
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Melanie Timmen
- Institute of Musculoskeletal Medicine, University Hospital Münster, Münster, Germany
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Sebastian Klein
- Department of Translational Genomics, Medical Faculty, University of Cologne, Cologne, Germany
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Daniel Kronenberg
- Institute of Musculoskeletal Medicine, University Hospital Münster, Münster, Germany
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Richard Stange
- Institute of Musculoskeletal Medicine, University Hospital Münster, Münster, Germany
| Veröffentlicht: | 22. Oktober 2019 |
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Gliederung
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Objectives: The differentiation of bone-resorbing osteoclasts depends on the level of intracellular calcium, which stimulates calcineurin to dephosphorylate and thereby facilitate the translocation of NFAT, the designated master transcription factor of osteoclastogenesis, to the nucleus. A range of membrane channels, including the Transient Receptor Potential Channels TRPC3 and TRPC6, control the entry of calcium into the cells. Analyzing the bone phenotype of TRPC6-deficient mice reveals a so far unknown impact of these channels on bone quality.
Methods: Lumbar vertebrae of TRPC6 knock out mice (n=7) and wild type littermates (n=10) were analyzed by microCT. Primary bone marrow mononuclear cells (PBMMCs) of these mice were isolated und differentiated to osteoclasts. RNA was isolated with RNeasy Kit (Qiagen), transcribed into cDNA (Applied Biosystems) and expression was analyzed by qPCR. Resorptive activity of differentiated PBMMCs was determined on Osteo Assay Surface Plates (Corning). TRAP was stained in PFA-fixed cells (Sigma). To confirm the data obtained from primary cells, RAW264.7 cells (ECACC-91062702) were manipulated by CRISPR/Cas9 technology to knock out TRPC6. Cells were infected with lentivirus after subcloning a sgRNA addressing exon 2 into the vector lentiCRISPRv2 (Addgene # 52961).
Intracellular calcium levels were measured photometrically after stimulation with 3 µM Fura-2AM (Thermofisher Scientific). Luciferase activity was determined after transient transfection of pGL3-NFAT (Addgene # 17870). Activity of TRPC3 was inhibited by incubating the cells with 10 µM Pyr3 (Sigma).
Results and conclusion: TRPC6 knock out mice have an impaired bone structure with significant decreases of bone volume per tissue volume, trabecular thickness and trabecular numbers when compared to wild type littermates. This is reflected on the cellular level, where more and bigger osteoclasts were observed on the surface of trabecular bone. In vitro we detected enhanced osteoclastic differentiation of TRPC6-deficient PBMMCs and increased resorptive activity. This could be confirmed by analyzing TRPC6-deficient RAW264.7 cells. So far it was not known, that osteoclasts express TRPC6. Beside TRPC6 we could also detect expression of TRPC3, even significantly upregulated in TRPC6-deficient cells. The increased intracellular calcium level and the elevated luciferase activity after transfection observed in TRPC6-deficient osteoclasts led to the idea to inhibit the upregulated TRPC3 in these cells to counteract the negative effect of TRPC6 deficiency on osteoclasts. Pyr3, a specific inhibitor of TRPC3, reduced not only the intracellular calcium level and luciferase activity, it also decreased the osteoclastic differentiation of TRPC6-deficient cells and their resorptive activity.
This indicates an interesting new approach to modify osteoclast activity and thereby address bone quality.
Experiments were approved by LANUV (84-02.05.50.15.005).
This research was supported by Elsbeth Bonhoff Foundation.
