Tumor necrosis factor alpha promotes the proliferation of human nucleus pulposus cells via nuclear factor-κB, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase

Abstract

Although tumor necrosis factor alpha (TNF-α) is known to play a critical role in intervertebral disc (IVD) degeneration, the effect of TNF-α on nucleus pulposus (NP) cells has not yet been elucidated. The aim of this study was to explore the effect of TNF-α on proliferation of human NP cells. NP cells were treated with different concentrations of TNF-α. Cell proliferation was determined by cell counting kit-8 (CCK-8) analysis and Ki67 immunofluorescence staining, and expression of cyclin B1 was studied by quantitative real-time RT-PCR. Cell cycle was measured by flow cytometry and cell apoptosis was analyzed using an Annexin V-fluorescein isothiocyanate (FITC) & propidium iodide (PI) apoptosis detection kit. To identify the mechanism by which TNF-α induced proliferation of NP cells, selective inhibitors of major signaling pathways were used and Western blotting was carried out. Treatment with TNF-α increased cell viability (as determined by CCK-8 analysis) and expression of cyclin B1 and the number of Ki67-positive and S-phase NP cells, indicating enhancement of proliferation. Consistent with this, NP cell apoptosis was suppressed by TNF-α treatment. Moreover, inhibition of NF-κB, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) blocked TNF-α-stimulated proliferation of NP cells. In conclusion, the current findings suggest that the effect of TNF-α on IVD degeneration involves promotion of the proliferation of human NP cells via the NF-κB, JNK, and p38 MAPK pathways.

Keywords: Tumor necrosis factor alpha; intervertebral disc degeneration; mitogen-activated protein kinase; nuclear factor-κB; nucleus pulposus cell; proliferation.

Figure 1

Effect of TNF-α on proliferation of human nucleus pulposus cells. The proliferation of NP cells was measured by CCK-8 analysis (a) and Ki67 immunofluorescence staining (b, c). TNF-α (20, 40, 80, 120 ng/mL) increased NP cell proliferation. n = 27. Bar = 100 µm. (A color version of this figure is available in the online journal.)

Figure 2

Effect of TNF-α on cyclin B1 expression in NP cells. Expression of cyclin B1 was studied using quantitative real-time RT-PCR. Treatment with TNF-α (100 ng/mL) for two days resulted in a significant increase in cyclin B1 mRNA levels. n = 3

Figure 3

Effect of TNF-α on cell cycle distribution of NP cells. Treatment with TNF-α (10 or 100 ng/mL) for 24 h increases the S phase population. n = 3

Figure 4

Effect of TNF-α on apoptosis in NP cells. Cell apoptosis was demonstrated by flow cytometry using annexin V-FITC/PI. Treatment with TNF-α (10 or 100 ng/mL) for 24 h reduced apoptosis in NP cells. n = 3

Figure 5

Effect of signaling pathway inhibitors on TNF-α induction of NP cell proliferation. CCK-8 analysis showed that treatment with inhibitors of NF-κB (pyrrolidinedithiocarbamate ammonium [PDTC]; 100 µmol/L), p38 (SB203580 [SB]; 10 µmol/L), and JNK (SP60025 [SB]; 10 µmol/L) blocked TNF-α-induced proliferation of NP cells, with the exception of Erk1/2 (U0126; 10 µmol/L) inhibition, which had no effect on the TNF-α-induced proliferation of NP cells. n = 4

Figure 6

Effect of TNF-α on levels of activated NF-κB, JNK, and p38 in NP cells. Western analysis indicated that treatment of NP cells with TNF-α for less than 15 min induced phosphorylation of p65, JNK, and p38, with no appreciable effect on the overall cellular levels of p65, JNK, and p38