Mechanisms of tumor necrosis factor-alpha-induced interleukin-6 synthesis in glioma cells

Abstract

Background: Interleukin (IL)-6 plays a pivotal role in a variety of CNS functions such as the induction and modulation of reactive astrogliosis, pathological inflammatory responses and neuroprotection. Tumor necrosis factor (TNF)-alpha induces IL-6 release from rat C6 glioma cells through the inhibitory kappa B (IkappaB)-nuclear factor kappa B (NFkappaB) pathway, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). The present study investigated the mechanism of TNF-alpha-induced IL-6 release in more detail than has previously been reported.

Methods: Cultured C6 cells were stimulated by TNF-alpha. IL-6 release from the cells was measured by an enzyme-linked immunosorbent assay, and the phosphorylation of IkappaB, NFkappaB, the MAP kinase superfamily, and signal transducer and activator of transcription (STAT)3 was analyzed by Western blotting. Levels of IL-6 mRNA in cells were evaluated by real-time reverse transcription-polymerase chain reaction.

Results: TNF-alpha significantly induced phosphorylation of NFkappaB at Ser 536 and Ser 468, but not at Ser 529 or Ser 276. Wedelolactone, an inhibitor of IkappaB kinase, suppressed both TNF-alpha-induced IkappaB phosphorylation and NFkappaB phosphorylation at Ser 536 and Ser 468. TNF-alpha-stimulated increases in IL-6 levels were suppressed by wedelolactone. TNF-alpha induced phosphorylation of STAT3. The Janus family of tyrosine kinase (JAK) inhibitor I, an inhibitor of JAK 1, 2 and 3, attenuated TNF-alpha-induced phosphorylation of STAT3 and significantly reduced TNF-alpha-stimulated IL-6 release. Apocynin, an inhibitor of NADPH oxidase that suppresses intracellular reactive oxygen species, significantly suppressed TNF-alpha-induced IL-6 release and mRNA expression. However, apocynin failed to affect the phosphorylation of IkappaB, NFkappaB, p38 MAP kinase, SAPK/JNK or STAT3.

Conclusion: These results strongly suggest that TNF-alpha induces IL-6 synthesis through the JAK/STAT3 pathway in addition to p38 MAP kinase and SAPK/JNK in C6 glioma cells, and that phosphorylation of NFkappaB at Ser 536 and Ser 468, and NADPH oxidase are involved in TNF-alpha-stimulated IL-6 synthesis.

Figure 1

Effects of TNF-α on IκB phosphorylation and degradation and NFκB phosphorylation. Cultured cells were stimulated with 10 ng/ml TNF-α for the indicated period. Cell extracts were analyzed by Western blotting using antibodies against phospho-specific IκB, IκB, phospho-specific NFκB, NFκB or GAPDH. Similar results were obtained with two additional and different cell preparations.

Figure 2

Effects of wedelolactone on TNF-α-induced phosphorylation and degradation of IκB. Cultured cells were pretreated with various concentrations of wedelolactone or vehicle for 60 min, and then stimulated with 10 ng/ml TNF-α or vehicle for 10 min. Cell extracts were analyzed by Western blotting using antibodies against phospho-specific IκB, IκB or GAPDH. Similar results were obtained with two additional and different cell preparations.

Figure 3

Effects of wedelolactone on TNF-α-induced phosphorylation of NFκB. Cultured cells were pretreated with various concentrations of wedelolactone or vehicle for 60 min, and then stimulated with 10 ng/ml TNF-α or vehicle for 10 min. Cell extracts were analyzed by Western blotting using antibodies against phospho-specific NFκB or NFκB. Similar results were obtained with two additional and different cell preparations.

Figure 4

Effects of wedelolactone on TNF-α-induced IL-6 release. Cultured cells were pretreated with various concentrations of wedelolactone for 60 min, and then stimulated with 10 ng/ml TNF-α (closed circle) or vehicle (open circle) for 36 h. Each value represents the mean ± SD of triplicate independent determinations of a representative experiment carried out three times. Similar results were obtained with two additional and different cell preparations. *P < 0.05 in comparison to the value of TNF-α alone.

Figure 5

Effects of TNF-α on STAT3 phosphorylation. Cultured cells were stimulated with 10 ng/ml TNF-α for the indicated period. Cell extracts were analyzed by Western blotting using antibodies against phospho-specific STAT3 or STAT3. Similar results were obtained with two additional and different cell preparations.

Figure 6

Effect of JAK inhibitor I on TNF-α-induced IL-6 release. (A) Cultured cells were pretreated with 10 nM JAK inhibitor I for 60 min, and then stimulated with 10 ng/ml TNF-α or vehicle for 36 h. Each value represents the mean ± SD of triplicate independent determinations of a representative experiment carried out three times. *P < 0.05 in comparison to the value of TNF-α alone. (B) Cultured cells were pretreated with various concentrations JAK inhibitor I or vehicle for 60 min, and then stimulated with 10 ng/ml TNF-α or vehicle for 150 min. Cell extracts were analyzed by Western blotting using antibodies against phospho-specific STAT3 or STAT3. Similar results were obtained with two additional and different cell preparations.

Figure 7

Effect of apocynin on TNF-α-induced IL-6 release. Cultured cells were pretreated with various concentrations apocynin for 60 min, and then stimulated with 10 ng/ml TNF-α (closed circle) or vehicle (open circle) for 36 h. Each value represents the mean ± SD of triplicate independent determinations of a representative experiment carried out three times. Similar results were obtained with two additional and different cell preparations. *P < 0.05 in comparison to the value of TNF-α alone.

Figure 8

Effect of apocynin on TNF-α-induced IL-6 mRNA expression. Cultured cells were pretreated with 0.1 mM apocynin or vehicle for 60 min, and then stimulated with 10 ng/ml TNF-α for 6 h. Total RNA was isolated and transcribed into cDNA. The expressions of IL-6 mRNA and GAPDH mRNA were quantified by real-time RT-PCR. IL-6 mRNA levels were normalized with those of GAPDH mRNA. Each value represents the mean ± SD of triplicate independent determinations of a representative experiment carried out three times. Similar results were obtained with two additional and different cell preparations. *P < 0.05 in comparison to the value of unstimulated cells. **P < 0.05 in comparison to the value of TNF-α alone.

Figure 9

Effect of apocynin on TNF-α-induced the phosphorylation of IκB, NFκB, MAP kinases and STAT3. Cultured cells were pretreated with 0.1 mM apocynin for 60 min, and then stimulated with 10 ng/ml TNF-α for 10 min (A) or 150 min (B). Cell extracts were analyzed by Western blotting using antibodies against phospho-specific IκB, phospho-specific NFκB, phospho-specific p38 MAP kinase, phospho-specific SAPK/JNK, GAPDH, phospho-specific STAT3 or STAT3. Similar results were obtained with two additional and different cell preparations.