BMC musculoskeletal disorders · 2025
Priming of mouse pre-osteoblasts with Interleukin-3 attenuates TNF-α-induced-inhibition of osteoblast differentiation.
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Piprode Vikrant, Behera Shubhanath, Karhade Juilee, Mhaske Suhas T, Wani Mohan R
Аннотация
BACKGROUND: In inflammatory bone loss disorders, such as rheumatoid arthritis and post-menopausal osteoporosis, TNF-α is an important effector cytokine that promotes osteoclastogenesis and inhibits osteoblastogenesis. Currently, clinical interventions primarily include anti-osteoclastic agents to prevent bone loss, however, anabolic agents are scarce and there's an utmost need. Previously, IL- 3 has been shown to enhance osteogenic differentiation of human bone marrow-derived mesenchymal stem cells in vitro. However, its role remains unclear in inflammatory pathologies, where TNF-α negatively impacts osteoblast differentiation. This study aims to investigate IL- 3's impact on osteogenic differentiation process under the negative influence of TNF-α.
METHODS: Calvarial pre-osteoblasts were isolated from BALB/c mice pups and in vitro osteoblast differentiation was performed as per established protocols. TNF-α was used as a negative regulator of osteoblast differentiation and the effect of IL- 3 on this differentiation was studied by assessing matrix mineralization using Alizarin red S staining. Osteoblast differentiation markers, including, alkaline phosphatase and osteocalcin were evaluated using qPCR. ALP activity was measured using the pNPP colorimetric assay. Furthermore, Western blotting was employed to deduce the mechanism(s) of IL- 3 action on TNF-α-induced-inhibition of osteoblast differentiation.
RESULTS: Alizarin red S staining revealed that IL- 3 priming protects pre-osteoblasts from TNF-α-induced-inhibition of osteoblast differentiation and that this protective effect is irreversible. Mechanistically, IL- 3 pretreatment suppresses TNF- α-induced activation of NF-κB/SMURF1 axis thus preventing β-CATENIN degradation, which drives osteogenic gene expression.
CONCLUSIONS: Priming mouse pre-osteoblasts with IL- 3 prevents TNF-α-induced of osteoblast differentiation in vitro, and, thus, could be a novel candidate in the treatment of inflammatory bone loss disorders.
METHODS: Calvarial pre-osteoblasts were isolated from BALB/c mice pups and in vitro osteoblast differentiation was performed as per established protocols. TNF-α was used as a negative regulator of osteoblast differentiation and the effect of IL- 3 on this differentiation was studied by assessing matrix mineralization using Alizarin red S staining. Osteoblast differentiation markers, including, alkaline phosphatase and osteocalcin were evaluated using qPCR. ALP activity was measured using the pNPP colorimetric assay. Furthermore, Western blotting was employed to deduce the mechanism(s) of IL- 3 action on TNF-α-induced-inhibition of osteoblast differentiation.
RESULTS: Alizarin red S staining revealed that IL- 3 priming protects pre-osteoblasts from TNF-α-induced-inhibition of osteoblast differentiation and that this protective effect is irreversible. Mechanistically, IL- 3 pretreatment suppresses TNF- α-induced activation of NF-κB/SMURF1 axis thus preventing β-CATENIN degradation, which drives osteogenic gene expression.
CONCLUSIONS: Priming mouse pre-osteoblasts with IL- 3 prevents TNF-α-induced of osteoblast differentiation in vitro, and, thus, could be a novel candidate in the treatment of inflammatory bone loss disorders.