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. 2019 Mar;52(2):e12542.
doi: 10.1111/cpr.12542. Epub 2018 Nov 14.

Nuclear factor-kappa B-dependent X-box binding protein 1 signalling promotes the proliferation of nucleus pulposus cells under tumour necrosis factor alpha stimulation

Lu Chen  1 , Zhi-Yang Xie  1 , Lei Liu  1 , Lei Zhu  1 , Feng Wang  1 , Pan Fan  1 , Arjun Sinkemani  1 , Cong Zhang  1 , Xin Hong  1 , Xiao-Tao Wu  1
Affiliations

Nuclear factor-kappa B-dependent X-box binding protein 1 signalling promotes the proliferation of nucleus pulposus cells under tumour necrosis factor alpha stimulation

Lu Chen et al. Cell Prolif. 2019 Mar.

Abstract

Objectives: Tumour necrosis factor alpha (TNF-α) expressed by nucleus pulposus cells (NPCs) plays a critical role in intervertebral disc (IVD) degeneration. A key unfolded protein response (UPR) component, X-box binding protein 1 (XBP1) and nuclear factor-kappa B (NF-κB) are essential for cell survival and proliferation. The aim of our study was to elucidate the roles of XBP1 and NF-κB in IVD degeneration (IDD).

Materials and methods: Rat NPCs were cultured with TNF-α in the presence or absence of XBP1 and NF-κB-p65 small interfering RNA. The associated genes and proteins were evaluated through quantitative real-time PCR, Western blot analyses and immunofluorescence staining to monitor UPR and NF-κB signalling and identify the regulatory mechanism of p65 by XBP1. Cell counting kit-8 assay, cell cycle analysis and related gene and protein expression were performed to examine the proliferation of NPCs.

Results: The acute exposure of TNF-α accelerated the proliferation of rat NPCs by activating the UPR/XBP1 pathway. XBP1 signalling favoured the phosphorylation and nuclear translocation of p65 subunit of NF-κB. The activation of NF-κB in the later phase also enhanced NPC proliferation.

Conclusions: Unfolded protein response reinforces the survival and proliferation of NPCs under TNF-α stimulation by activating the XBP1 pathway, and NF-κB serves as a vital mediator in these events. The XBP1 signalling of UPR can be a novel therapeutic target in IDD.

Keywords: NF-κB; TNF-α; nucleus pulposus; proliferation; unfolded protein response.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1
Tumour necrosis factor alpha (TNF‐α) enhanced the proliferation of rat nucleus pulposus cells (NPCs). A, Cell proliferation was evaluated through CCK‐8 analysis at different time points upon exposure to gradient concentrations of TNF‐α (5, 10, 20 and 40 ng/mL). B, NPC proliferation under TNF‐α (0, 5, 10 and 20 ng/mL) stimulation for 24 h was measured through Ki67 immunofluorescence staining (original magnification, ×200). C, Ki67‐positive cells were quantified upon exposure to different TNF‐α concentrations at 24 h. D,E, The proportions of cells in each cycle were measured through flow cytometry with different concentrations of TNF‐α stimulation at 24 h. F,G, After TNF‐α treatment (0, 5, 10 and 20 ng/mL) for 24 h, the mRNA expression levels of cyclin D1 and cyclin B1 were detected through qRT‐PCR and normalized to that of β‐actin. H,I, Cyclin D1 and cyclin B1 expression levels in NPCs subjected to different treatments were measured through Western blot analysis and normalized to that of vinculin. J,K, ACAN and COL2 expression levels in NPCs subjected to different treatments were measured through Western blot analysis and normalized to that of vinculin. The results were presented as mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001
Figure 2
Figure 2
Activation of unfolded protein response (UPR) signalling after tumour necrosis factor alpha (TNF‐α) treatment in rat nucleus pulposus cells (NPCs) and X‐box binding protein 1 (XBP1) siRNA inhibition of the TNF‐α‐induced activation of the XBP1 pathway. A, After treatment with 10 ng/mL TNF‐α, the activation of UPR signalling was assessed in terms of the related protein expression at different time intervals (0, 6, 12 and 24 h). B, UPR markers in NPCs were analysed through Western blot, and the protein expression levels of BiP were normalized to that of vinculin. The protein expression levels of p‐eIF2α and XBP1s were normalized to those of total eIF2α (t‐eIF2α) and XBP1u. NPCs were treated with control siRNA (siCtrl) or XBP1 siRNA (siXBP1) for 24 h and subsequently exposed to 10 ng/mL TNF‐α for another 24 h. C, The mRNA expression of XBP1 in NPCs was quantified through qRT‐PCR. The mRNA expression levels were normalized to that of β‐actin. D,E, XBP1s was evaluated through Western blot, and the protein expression levels were normalized to those of XBP1u. The results were presented as mean ± SD (n = 3). *P < 0.05; **P < 0.01
Figure 3
Figure 3
Role of inositol‐requiring enzyme 1/X‐box binding protein 1 (XBP1) pathway in rat NPC proliferation induced by tumour necrosis factor alpha (TNF‐α). Nucleus pulposus cells (NPCs) were treated with siCtrl or siXBP1. At 24 h post‐transfection, the cells were treated with 10 ng/mL TNF‐α for additional 24 h. A, Cell proliferation was evaluated through CCK‐8 analysis at different time points. B,C, The proliferating antigen Ki67 was subjected to immunofluorescence staining (original magnification, ×200), and Ki67‐positive cells were quantified. D,E Cell cycle distribution was measured through flow cytometry. F,G, The mRNA expression levels of cyclin D1 and cyclin B1 were detected through qRT‐PCR and normalized to that of β‐actin. H,I, Cyclin D1 and cyclin B1 expression levels were measured through Western blot analysis in NPCs subjected to different treatments and normalized to the expression level of vinculin. The results were presented as mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001
Figure 4
Figure 4
Kinetics of nuclear factor‐kappa B activation by tumour necrosis factor alpha (TNF‐α) and tunicamycin (TM). Nucleus pulposus cells (NPCs) were treated with either 10 ng/mL TNF‐α or 100 ng/mL TM at various time points. A,B, p65 protein phosphorylation with TNF‐α treatment was analysed through Western blot, and proteins were extracted to detect the expression of p‐p65. C,D, p65 protein phosphorylation with TM treatment was examined through Western blot. The protein expression levels of p‐p65 were normalized to that of p65. E, The nuclear translocation of p‐p65 under the TNF‐α stimulus was subjected to immunofluorescence studies (original magnification, ×400). F, p‐p65‐positive cells in the nucleus were quantified. The results were presented as mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001
Figure 5
Figure 5
X‐box binding protein 1 (XBP1) regulates nuclear factor‐kappa B signalling by regulating p65 transcription and p‐p65 expression. The cells were exposed to 10 ng/mL tumour necrosis factor alpha with or without pretreatment with siCtrl and siXBP1 for 24 h. A, In immunofluorescence double staining, the cells were stained initially with XBP1 antibody, Alexa Fluor 488 (green), p‐p65 antibody and Alexa Fluor 647 (red), and the nucleus was counterstained with DAPI (blue) and examined through fluorescence microscopy (original magnification, ×400). B, XBP1‐ and p‐p65‐positive cells were quantified. C, Total RNA was extracted, and the mRNA expression of p65 was measured through qRT‐PCR. D,E, Proteins were extracted to detect the expression of p‐p65 and p65 through Western blot analysis, and the protein expression levels of p‐p65 were normalized to that of p65. The results were presented as mean ± SD (n = 3). *P < 0.05; **P < 0.01
Figure 6
Figure 6
Role of nuclear factor‐kappa B (NF‐κB) signalling in rat NPC proliferation induced by tumour necrosis factor alpha (TNF‐α). The cells were exposed to 10 ng/mL TNF‐α with or without p65 siRNA (siP65) pretreatment for 24 h. A,B, NF‐κB‐p65 interference was verified through Western blot analysis. Proteins were extracted to detect the expression of p‐p65 and p65. The expression levels of p‐p65 were normalized to that of p65. C, Cell proliferation was evaluated through CCK‐8 analysis at different time points. D,E, Immunofluorescence staining of Ki67 (original magnification, ×200) and quantification of Ki67‐positive cells. F,G, The proportions of cells in each phase were examined through flow cytometry. H,I, Cyclin D1 and cyclin B1 expression levels in NPCs subjected to different treatments were measured through Western blot analysis and normalized to that of vinculin. The results were presented as mean ± SD (n = 3). *P < 0.05; **P < 0.01
Figure 7
Figure 7
Schematic diagram proposed by the present research. Tumour necrosis factor alpha‐α‐mediated IRE1/XBP1 pathway induces NPC proliferation via NF‐κB signalling. ATF4, activating transcription factor 4; ATF6, activating transcription factor 6; eIF2α, eukaryotic translation initiation factor 2α; ER, endoplasmic reticulum; ERAD, ER‐associated degradation; IRE1, inositol‐requiring enzyme 1; PERK, protein kinase RNA‐like ER kinase; XBP1, X‐box binding protein 1
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