Related Experiment Videos
Protein kinase C inhibitor H-7 blocks effects of tumor necrosis factor on bone cells
1Research Laboratory of Natural and Synthetic Drugs, School of Pharmacy, Second Military Medical University, Shanghai, China.
Aim:
To study the effects of tumor necrosis factor (TNF) on the neonatal mouse osteoblast-enriched calvarial cells and effects of protein kinase C (PK C) inhibitor, 1-(5-isoquinolinesulfonyl)-2 -methylpiperazine (H-7) on the TNF actions.
Methods:
[3H]TdR uptake by the osteoblasts was used to measure cell proliferation. Cellular alkaline phosphatase (AIP) and tartrate resistant acid phosphatase (trAcP) activities were determined spectrophotometrically.
Results:
TNF (1-100 kU . L-1) inhibited both proliferation and expression of AIP activity, but stimulated trAcP activity. These TNF-induced actions were blocked by simultaneous addition of H-7 (5-20 mumol . L-1).
Conclusion:
TNF has potent effects on the osteoblasts, and the blockade of TNF actions by H-7 suggests that TNF exert its effects through PK C.
Insights
Tumor necrosis factor (TNF) affects neonatal mouse osteoblasts, inhibiting proliferation and alkaline phosphatase (ALP) while stimulating tartrate-resistant acid phosphatase (TRAP). A protein kinase C (PKC) inhibitor blocked these TNF effects.
Area of Science:
- Cell Biology
- Biochemistry
- Immunology
Background:
- Tumor necrosis factor (TNF) is a key inflammatory cytokine with known roles in bone metabolism.
- Osteoblasts are crucial cells responsible for bone formation.
- The signaling pathways through which TNF influences osteoblasts are not fully elucidated.
Purpose of the Study:
- To investigate the impact of TNF on neonatal mouse osteoblast-enriched calvarial cells.
- To determine the role of protein kinase C (PKC) in mediating TNF's effects on these cells.
- To examine the influence of the PKC inhibitor H-7 on TNF-induced cellular responses.
Main Methods:
- Cell proliferation was assessed using [3H]thymidine ([3H]TdR) uptake.
- Cellular alkaline phosphatase (ALP) activity was measured spectrophotometrically.
- Tartrate-resistant acid phosphatase (TRAP) activity was determined spectrophotometrically.
Main Results:
- TNF significantly inhibited osteoblast proliferation and ALP activity in a dose-dependent manner.
- TNF stimulated TRAP activity in the osteoblast cultures.
- The PKC inhibitor H-7 effectively blocked all TNF-induced effects on proliferation, ALP, and TRAP activity.
Conclusions:
- TNF exerts potent effects on neonatal mouse osteoblasts, influencing both proliferation and differentiation markers.
- The findings strongly suggest that TNF mediates its actions on osteoblasts via the protein kinase C (PKC) signaling pathway.
- Inhibition of PKC by H-7 demonstrates a critical role for this kinase in TNF's osteoblast modulation.