Article
Absence of the sensory neuropeptide Substance P alters osteoblast and osteoclast metabolism
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Published: | October 23, 2017 |
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Objectives: Phenotyping of sensory nerve fibers innervating bone, bone marrow and periosteum has demonstrated the presence of signaling molecules including the sensory neuropeptide substance P (SP) indicating that the sensory nervous system may be a part of the endo- and paracrine control system of bone metabolism. In previous studies, we observed impaired biomechanical bone properties and inferior bone matrix quality in mice lacking the sensory neurotransmitter SP. Recently, we found changes in cell survival and activity of osteoblasts and osteoclasts isolated from wildtype (WT) mice when stimulated with SP. In this study we investigated and compared cell metabolism of osteoblasts and osteoclasts isolated from SP-deficient mice and WT controls.
Methods: Isolation of bone marrow macrophages (BMM) and isolation/osteogenic differentiation (for 14/21 days) of osteoblasts from female Tac1-/- (no SP) and WT control (C57Bl/6J) mice according to established protocols. Cell migration of osteoblast-like cells out of femoral bone chips was analyzed by cristal violet staining, Proliferation by BrdU incorporation, viability using WST-1 reagent, caspase 3/7-activity for determination of apoptosis rate and protein expression of SP receptor (NK1R) by western blot. Alkaline phosphatase (ALP) activity reflects osteoblast bone formation activity and cathepsin K activity was measured to determine osteoclast resorption activity. SP production was measured by ELISA.
Results and Conclusion: We found reduced numbers of BMM from Tac1-/- mice after initial seeding compared to WT but no changes in viability. Osteoblast-like cells from Tac1-/- mice tend to migrate out faster from bone chips compared to WT. Viability was reduced in Tac1-/- osteoblast-like cells at the beginning of osteogenic differentiation compared to WT. Osteoblasts and osteoclasts from WT mice endogenously synthesize and secrete SP. NK1R protein expression profile in osteoclasts and osteoblasts was similar in Tac1-/- and WT mice. Cathepsin K activity was reduced in osteoclasts from Tac1-/- mice and apoptosis rate in osteoclasts from Tac1-/- mice was lower compared to WT. Survival rate was higher after 14 days in osteoblasts of Tac1-/- mice compared to WT. Proliferation was not affected. ALP activity of Tac1-/- osteoblasts was higher after 7 days and reduced after 21 days of osteogenic culture.
Reduced BMM number and altered osteoblast cell migration and viability may contribute to inferior bone properties of Tac1-/- mice compared to WT. Osteoclast resorption activity via cathepsin K activity is reduced in Tac1-/- mice, however may be compensated by increased survival rate of osteoclasts. Increased ALP activity during early osteogenic differentiation may compensate increased apoptosis of Tac1-/- osteoblasts. SP seems to have anabolic and catabolic effects on osteoclast and osteoblast cell metabolism depending on osteogenic differentiation time point and cell type.