Dose-specific effects of tumor necrosis factor alpha on osteogenic differentiation of mesenchymal stem cells - PubMed (original) (raw)
Dose-specific effects of tumor necrosis factor alpha on osteogenic differentiation of mesenchymal stem cells
H Huang et al. Cell Prolif. 2011 Oct.
Abstract
Objectives: To investigate tumor necrosis factor alpha (TNF-α)-induced changes in osteogenic differentiation from mesenchymal stem cells (MSCs).
Materials and methods: Blockade of nuclear factor-κB (NF-κB) was achieved in ST2 murine MSCs via overexpression of the NF-κB inhibitor, IκBα. Osteogenic differentiation was induced in IκBα-overexpressing ST2 cells and normal ST2 cells when these cells were treated with TNF-α at various concentrations. Expression levels of bone marker genes were determined using real time RT-PCR and ALP activity assay. In vitro mineralization was performed to determine long-term exposure to TNF-α on mineral nodule formation. MTT assay was used to determine the changes in cell proliferation/survival.
Results: Levels of Runx2, Osx, OC and ALP were up-regulated in cell cultures treated with TNF-α at lower concentrations, while down-regulated in cell cultures treated with TNF-α at higher concentrations. Blockade of NF-κB signaling reversed the inhibitory effect observed in cell cultures treated with TNF-α at higher concentrations, but showed no effect on cell cultures treated with TNF-α at lower concentrations. In contrast, long-term treatment of TNF-α at all concentrations induced inhibitory effects on in vitro mineral nodule formation. MTT assay showed that TNF-α inhibits proliferation/survival of mesenchymal stem cells when the NF-κB signaling pathway is blocked.
Conclusions: The binding of TNF-α to its receptors results in the activation of multiple signaling pathways, which actively interact with each other to regulate the differentiation, proliferation, survival and apoptosis of MSCs.
© 2011 Blackwell Publishing Ltd.
Figures
Figure 1
The NF‐κB signaling pathway was successfully inhibited via the overexpress of NF‐κB antagonist IκBα. Western blot analysis was performed to determine the cytosolic protein level of IκBα and the nuclear protein level of the NF‐κB subunit, p65, in the ST2 cells stably infected with pBABE‐puro or pBABE‐puro‐IκBα‐mut. (A) Changes in the cytosolic protein level of IκBα. Data were represented as mean ± SEM. *, P < 0.05, ST2‐pBABE‐puro‐IκBα‐mut cells versus ST2‐pBABE‐puro cells. (B) Changes in the nuclear protein level of p65. a, P <0.05, versus ST2‐pBABE‐puro cells treated without TNF‐α; b, P <0.05, versus ST2‐pBABE‐puro‐IκBα‐mut cells TNF‐α.
Figure 2
Effects of short‐term TNF‐α treatment on osteogenic gene expression in MSCs. ST2 cells stably infected with pBABE‐puro (a) or pBABE‐puro‐IκBα‐mut (b) were induced to differentiate towards osteoblasts with the osteogenic medium, which was supplemented with murine TNF‐α at concentrations of 0, 0.01, 0.1, 1, 10 or 100 ng/ml. Forty‐eight hours after the treatment, real time RT‐PCR was performed to determine mRNA levels of Runx2, Osx, OC and ALP in these cells. Data were represented as mean ± SEM. *, P < 0.05, versus cells treated with 0 ng/ml TNF‐α.
Figure 3
Effects of short‐term TNF‐α treatment on ALP activity in MSCs diffentiating towards osteogenic lineage cells. ST2 cells stably infected with pBABE‐puro (a) or pBABE‐puro‐IκBα‐mut (b) were induced to differentiate towards osteoblasts with the osteogenic medium, which was supplemented with murine TNF‐α at concentrations of 0, 0.01, 0.1, 1, 10 or 100 ng/ml. Forty‐eight hours after the treatment, alkaline phosphatase (ALP) activity was determined using an established colorimetric assay. Data were represented as mean ± SEM. *, P < 0.05, versus cells treated with 0 ng/ml TNF‐α.
Figure 4
Long‐term treatment of TNF‐α mainly induced inhibitory effects on in vitro mineral nodule formation. ST2 cells stably infected with pBABE‐puro or pBABE‐puro‐IκBα‐mut were induced to differentiate towards osteoblasts with the osteogenic medium for 4 weeks. During the 4‐week osteogenic induction, the cells were also incubated with murine TNF‐α at concentrations of 0, 0.1, 1 or 100 ng/ml. Four weeks after the osteogenic induction, in vitro mineralization was monitored using Alizarin red staining. (a) Demonstrative photos of osteogenesis in ST2 cells stably infected with pBABE‐puro (upper panel) or pBABE‐puro‐IκBα‐mut (lower panel). (b) Quantification of positively stained area by Alizarin red in ST2 cells stably infected with pBABE‐puro. Data were represented as mean ± SEM. *, P < 0.05, versus cells treated with 0 ng/ml TNF‐α. (c) Quantification of positively stained area by Alizarin red in ST2 cells stably infected with pBABE‐puro‐IκBα‐mut. Data were represented as mean ± SEM. *, P < 0.05, versus cells treated with 0 ng/ml TNF‐α.
Figure 5
TNF‐α inhibits proliferation/survival of MSCs with the bloackade of NF‐κB signaling pathway. ST2 cells stably infected with pBABE‐puro (a) or pBABE‐puro‐IκBα‐mut (b) were seeded on 96‐well plates at a cell density of 2 × 103 cells/well and treated with murine TNF‐α at concentrations of 0, 0.01, 0.1, 1, 10 or 100 ng/ml. At 24, 48, and 72 h after the TNF‐α treatment, the proliferation/survival of the cells was evaluated using the methylthiazolyldiphenyl‐tetrazolium bromide (MTT) test. Data were represented as mean ± SEM.
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