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. 2015 Sep;36(3):661-8.
doi: 10.3892/ijmm.2015.2280. Epub 2015 Jul 10.

Autophagy attenuates the catabolic effect during inflammatory conditions in nucleus pulposus cells, as sustained by NF-κB and JNK inhibition

Kang Xu  1 , Weijian Chen  2 , Xiaofei Wang  3 , Yan Peng  3 , Anjing Liang  3 , Dongsheng Huang  3 , Chunhai Li  3 , Wei Ye  3
Affiliations

Autophagy attenuates the catabolic effect during inflammatory conditions in nucleus pulposus cells, as sustained by NF-κB and JNK inhibition

Kang Xu et al. Int J Mol Med. 2015 Sep.

Abstract

Proteoglycan degradation contributing to the pathogenesis of intervertebral disc (IVD) degeneration is induced by inflammatory cytokines, such as tumor necrosis factor‑α (TNF‑α) and interleukin‑1β (IL‑1β). Cell autophagy exists in degenerative diseases, including osteoarthritis and intervertebral disc degeneration. However, the autophagy induced by TNF‑α and IL‑1β and the corresponding molecular mechanism appear to be cell‑type dependent. The effect and mechanism of autophagy regulated by TNF‑α and IL‑1β in IVDs remains unclear. Additionally, the impact of autophagy on the catabolic effect in inflammatory conditions also remains elusive. In the present study, autophagy activator and inhibitor were used to demonstrate the impact of autophagy on the catabolic effect induced by TNF‑α. A critical role of autophagy was identified in rat nucleus pulposus (NP) cells: Inhibition of autophagy suppresses, while activation of autophagy enhances, the catabolic effect of cytokines. Subsequently, the autophagy‑related gene expression in rat NP cells following TNF‑α and IL‑1β treatment was observed using immunofluorescence, quantitative polymerase chain reaction and western blot analysis; however, no association was present. In addition, nuclear factor κB (NF‑κB), c‑Jun N‑terminal kinase (JNK), extracellular signal‑regulated kinases and p38 mitogen‑activated protein kinase inhibitors and TNF‑α were used to determine the molecular mechanism of autophagy during the inflammatory conditions, and only the NF‑κB and JNK inhibitor were found to enhance the autophagy of rat NP cells. Finally, IKKβ knockdown was used to further confirm the effect of the NF‑κB signal on human NP cells autophagy, and the data showed that IKKβ knockdown upregulated the autophagy of NP cells during inflammatory conditions.

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Figures

Figure 1
Figure 1
Autophagy is an important regulator of the catabolic effects induced by interleukin-1β (IL-1β) in nucleus pulposus (NP) cells. mRNA expression of (A) matrix metalloproteinase 3 (MMP3) and (B) MMP9 in NP cells was induced by IL-1β and significantly repressed by additional rapamycin (Rapa). (C) MMP2 mRNA expression was not regulated by IL-1β, however, it was suppressed by additional Rapa. (D) A disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS4) and (E) COX2 mRNA expression in NP cells induced by IL-1β repressed by additional Rapa. Data represent mean + standard error of three independent experiments. *P<0.05; ns, not significant.
Figure 2
Figure 2
Autophagy represses the catabolic effects induced by tumor necrosis factor-α (TNF-α) in nucleus pulposus (NP) cells at the mRNA level. mRNA expression of (A) matrix metalloproteinase 3 (MMP3) and (B) MMP9 in NP cells was induced by TNF-α and markedly repressed by additional rapamycin (Rapa). (C) MMP2 mRNA expression was downregulated by Rapa but was not regulated by TNF-α. (D) A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS4) and (E) COX2 mRNA expression in NP cells upregulated by TNF-α and subsequently suppressed by additional Rapa. Data represent mean + standard error of three independent experiments. *P<0.05; ns, not significant.
Figure 3
Figure 3
Autophagy regulates the catabolic effects induced by tumor necrosis factor-α (TNF-α) in nucleus pulposus (NP) cells at the protein level. (A) Western blot analysis showed cell protein COX2, condition medium protein and matrix metalloproteinase 3 (MMP3) expression in NP cells treated with autophagy inhibitor and activator. Densitometry showed protein expression of medium (B) MMP3 protein and (C) COX2 induced by TNF-α was significantly promoted by 3-methyladenine (3-MA) and markedly repressed by rapamycin (Rapa) in NP cells. Data represent mean + standard error of three independent experiments. *P<0.05. CM, condition medium; Ctr, control.
Figure 4
Figure 4
Autophagy of nucleus pulposus (NP) cells is refractory to tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) stimulation. (A) Rat nucleus pulposus (NP) cells cultured with serum-free medium showed robust expression of LC3-II in cytoplasm (magnification, ×20). Rat NP cells were treated with TNF-α or IL-1β, and (B) Beclin-1 and (C) LC3-II mRNA showed no change in response to cytokine treatment. (D) Western blot analysis and corresponding (E and F) densitometric analyses of NP cells treated with TNF-α and IL-1β. No significant change was observed in (E) Beclin-1 protein and (F) LC3 protein turnover. Data represent mean + standard error of three independent experiments. *P<0.05. PI, propidium iodide; ns, not significant; Ctr, control.
Figure 5
Figure 5
Autophagy of nucleus pulposus (NP) cells is activated by the inhibition of the nuclear factor κB (NF-κB) and c-Jun N-terminal kinase (JNK) signaling pathway in inflammatory conditions. (A) Rat NP cells were treated with tumor necrosis factor-α (TNF-α) and inhibitors; LC3-II mRNA significantly increased in response to SM7368, PD98059, SP600125 or SB203580 in inflammatory conditions. (B) Western blot analysis and (C) corresponding densitometric analyses of NP cells treated with TNF-α and inhibitors showed that only SM7368 and SP600125 increased the protein LC3-II expression. (D) Acridine orange staining (AO) showed more autophagic vacuoles in the cells treated with SM7368 compared with the control. NP cells were transfected with NF-κB promoter construct and treated with cytokines. (E) TNF-α and (F) interleukin-1β (IL-1β) increased the activity of NF-κB promoter construct. Data represent mean + standard error of three independent experiments. SM, SM7368; PD, PD98059; SP, SP600125; SB, SB203580; Ctr, control; NRE, NF-κB promoter construct. *P<0.05.
Figure 6
Figure 6
Stable silencing of IKKβ increases the autophagy-related protein expression in human nucleus pulposus (NP) cells. (A) Western blot analysis showed that the Beclin-1, LC3-II and IKKβ protein expression in NP cells was transduced with LV-shCtr or LV-shIKKβ in the tumor necrosis factor-α (TNF-α) condition. (B) Densitometric analyses of NP cells transduced with LV-shCtr or LV-shIKKβ; IKKβ expression levels were suppressed by LV-shIKKβ in the TNF-α condition. Compared to cells transduced with LV-shCtr, NP cells transduced with LV-shIKKβ demonstrated a higher expression of (C) LC3 protein turnover and an increase of (D) Beclin-1 protein in TNF-α condition. Data represent mean + standard error of three independent experiments. *P<0.05. Ctr, control.
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