Collagen-derived N-acetylated proline-glycine-proline upregulates the expression of pro-inflammatory cytokines and extracellular matrix proteases in nucleus pulposus cells via the NF-κB and MAPK signaling pathways

Feng,Chencheng; He,Jinyue; Zhang,Yang; Lan,Minghong; Yang,Minghui; Liu,Huan; Huang,Bo; Pan,Yong; Zhou,Yue

doi:10.3892/ijmm.2017.3005

Collagen-derived N-acetylated proline-glycine-proline upregulates the expression of pro-inflammatory cytokines and extracellular matrix proteases in nucleus pulposus cells via the NF-κB and MAPK signaling pathways

Authors:
- Chencheng Feng
- Jinyue He
- Yang Zhang
- Minghong Lan
- Minghui Yang
- Huan Liu
- Bo Huang
- Yong Pan
- Yue Zhou
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Affiliations: Department of Orthopaedics, Xinqiao Hospital, The Third Military Medical University, Chongqing 400037, P.R. China
Published online on: May 29, 2017 https://doi.org/10.3892/ijmm.2017.3005
Pages: 164-174
Copyright: © Feng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

N-acetylated proline-glycine-proline (N-Ac-PGP) is a chemokine involved in inflammatory diseases and is found to accumulate in degenerative discs. N-Ac-PGP has been demonstrated to have a pro-inflammatory effect on human cartilage endplate stem cells. However, the effect of N-Ac-PGP on human intervertebral disc cells, especially nucleus pulposus (NP) cells, remains unknown. The purpose of this study was to investigate the effect of N-Ac-PGP on the expression of pro-inflammatory factors and extracellular matrix (ECM) proteases in NP cells and the molecular mechanism underlying this effect. Therefore, Milliplex assays were used to detect the levels of various inflammatory cytokines in conditioned culture medium of NP cells treated with N-Ac-PGP, including interleukin-1β (IL-1β), IL-6, IL-17, tumor necrosis factor-α (TNF-α) and C-C motif ligand 2 (CCL2). RT-qPCR was also used to determine the expression of pro-inflammatory cytokines and ECM proteases in the NP cells treated with N-Ac-PGP. Moreover, the role of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways in mediating the effect of N-Ac-PGP on the phenotype of NP cells was investigated using specific signaling inhibitors. Milliplex assays showed that NP cells treated with N-Ac-PGP (10 and 100 µg/ml) secreted higher levels of IL-1β, IL-6, IL-17, TNF-α and CCL2 compared with the control. RT-qPCR assays showed that NP cells treated with N-Ac-PGP (100 µg/ml) had markedly upregulated expression of matrix metalloproteinase 3 (MMP3), MMP13, a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS4), ADAMTS5, IL-6, CCL-2, CCL-5 and C-X-C motif chemokine ligand 10 (CXCL10). Moreover, N-Ac-PGP was shown to activate the MAPK and NF-κB signaling pathways in NP cells. MAPK and NF-κB signaling inhibitors suppressed the upregulation of proteases and pro-inflammatory cytokines in NP cells treated with N-Ac-PGP. In conclusion, N-Ac-PGP induces the expression of pro-inflammatory cytokines and matrix catabolic enzymes in NP cells via the NF-κB and MAPK signaling pathways. N-Ac-PGP is a novel therapeutic target for intervertebral disc degeneration.

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1	Roberts S, Evans H, Trivedi J and Menage J: Histology and pathology of the human intervertebral disc. J Bone Joint Surg Am. 88(Suppl 2): 10–14. 2006.PubMed/NCBI
2	Battié MC, Videman T, Kaprio J, Gibbons LE, Gill K, Manninen H, Saarela J and Peltonen L: The twin spine study: contributions to a changing view of disc degeneration. Spine J. 9:47–59. 2009. View Article : Google Scholar
3	Xing QJ, Liang QQ, Bian Q, Ding DF, Cui XJ, Shi Q and Wang YJ: Leg amputation accelerates senescence of rat lumbar intervertebral discs. Spine. 35:E1253–E1261. 2010. View Article : Google Scholar : PubMed/NCBI
4	Wang D, Nasto LA, Roughley P, Leme AS, Houghton AM, Usas A, Sowa G, Lee J, Niedernhofer L, Shapiro S, et al: Spine degeneration in a murine model of chronic human tobacco smokers. Osteoarthritis Cartilage. 20:896–905. 2012. View Article : Google Scholar : PubMed/NCBI
5	Stirling A, Worthington T, Rafiq M, Lambert PA and Elliott TS: Association between sciatica and Propionibacterium acnes. Lancet. 357:2024–2025. 2001. View Article : Google Scholar : PubMed/NCBI
6	Park EY and Park JB: Dose- and time-dependent effect of high glucose concentration on viability of notochordal cells and expression of matrix degrading and fibrotic enzymes. Int Orthop. 37:1179–1186. 2013. View Article : Google Scholar : PubMed/NCBI
7	Adams MA and Roughley PJ: What is intervertebral disc degeneration, and what causes it? Spine. 31:2151–2161. 2006. View Article : Google Scholar : PubMed/NCBI
8	Binch AL, Cole AA, Breakwell LM, Michael AL, Chiverton N, Creemers LB, Cross AK and Le Maitre CL: Nerves are more abundant than blood vessels in the degenerate human intervertebral disc. Arthritis Res Ther. 17:3702015. View Article : Google Scholar : PubMed/NCBI
9	Vo V, Hartman RA, Yurube T, Jacobs LJ, Sowa GA and Kang JD: Expression and regulation of metalloproteinases and their inhibitors in intervertebral disc aging and degeneration. Spine J. 13:331–341. 2013. View Article : Google Scholar : PubMed/NCBI
10	Risbud MV and Shapiro IM: Role of cytokines in intervertebral disc degeneration: pain and disc content. Nat Rev Rheumatol. 10:44–56. 2014. View Article : Google Scholar
11	Huang YC, Leung VY, Lu WW and Luk KD: The effects of microenvironment in mesenchymal stem cell-based regeneration of intervertebral disc. Spine J. 13:352–362. 2013. View Article : Google Scholar : PubMed/NCBI
12	Gaggar A, Jackson PL, Noerager BD, O'Reilly PJ, McQuaid DB, Rowe SM, Clancy JP and Blalock JE: A novel proteolytic cascade generates an extracellular matrix-derived chemoattractant in chronic neutrophilic inflammation. J Immunol. 180:5662–5669. 2008. View Article : Google Scholar : PubMed/NCBI
13	O'Reilly P, Jackson PL, Noerager B, Parker S, Dransfield M, Gaggar A and Blalock JE: N-alpha-PGP and PGP, potential biomarkers and therapeutic targets for COPD. Respir Res. 10:382009. View Article : Google Scholar : PubMed/NCBI
14	Koelink PJ, Overbeek SA, Braber S, Morgan ME, Henricks PA, Abdul Roda M, Verspaget HW, Wolfkamp SC, te Velde AA, Jones CW, et al: Collagen degradation and neutrophilic infiltration: a vicious circle in inflammatory bowel disease. Gut. 63:578–587. 2014. View Article : Google Scholar :
15	Feng C, Zhang Y, Yang M, Huang B and Zhou Y: Collagen-derived N-acetylated proline-glycine-proline in intervertebral discs modulates CXCR1/2 expression and activation in cartilage endplate stem cells to induce migration and differentiation toward a pro-inflammatory phenotype. Stem Cells. 33:3558–3568. 2015. View Article : Google Scholar : PubMed/NCBI
16	Pfirrmann CW, Metzdorf A, Zanetti M, Hodler J and Boos N: Magnetic resonance classification of lumbar intervertebral disc degeneration. Spine. 26:1873–1878. 2001. View Article : Google Scholar : PubMed/NCBI
17	Lv F, Leung VY, Huang S, Huang Y, Sun Y and Cheung KM: In search of nucleus pulposus-specific molecular markers. Rheumatology (Oxford). 53. pp. 600–610. 2014, View Article : Google Scholar
18	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar
19	Hiyama A, Sakai D, Risbud MV, Tanaka M, Arai F, Abe K and Mochida J: Enhancement of intervertebral disc cell senescence by WNT/β-catenin signaling-induced matrix metalloproteinase expression. Arthritis Rheum. 62:3036–3047. 2010. View Article : Google Scholar : PubMed/NCBI
20	Wuertz K, Vo N, Kletsas D and Boos N: Inflammatory and catabolic signalling in intervertebral discs: the roles of NF-κB and MAP kinases. Eur Cell Mater. 23:103–119. 2012.
21	Feng C, Liu H, Yang Y, Huang B and Zhou Y: Growth and differentiation factor-5 contributes to the structural and functional maintenance of the intervertebral disc. Cell Physiol Biochem. 35:1–16. 2015. View Article : Google Scholar
22	Kepler CK, Ponnappan RK, Tannoury CA, Risbud MV and Anderson DG: The molecular basis of intervertebral disc degeneration. Spine J. 13:318–330. 2013. View Article : Google Scholar : PubMed/NCBI
23	Ding F, Shao ZW and Xiong LM: Cell death in intervertebral disc degeneration. Apoptosis. 18:777–785. 2013. View Article : Google Scholar : PubMed/NCBI
24	Huang YC, Urban JP and Luk KD: Intervertebral disc regeneration: do nutrients lead the way? Nat Rev Rheumatol. 10:561–566. 2014. View Article : Google Scholar : PubMed/NCBI
25	Weathington NM, van Houwelingen AH, Noerager BD, Jackson PL, Kraneveld AD, Galin FS, Folkerts G, Nijkamp FP and Blalock JE: A novel peptide CXCR ligand derived from extracellular matrix degradation during airway inflammation. Nat Med. 12:317–323. 2006. View Article : Google Scholar : PubMed/NCBI
26	Marcu KB, Otero M, Olivotto E, Borzi RM and Goldring MB: NF-kappaB signaling: multiple angles to target OA. Curr Drug Targets. 11:599–613. 2010. View Article : Google Scholar : PubMed/NCBI
27	Kim HJ, Chang EJ, Kim HM, Lee SB, Kim HD, Su Kim G and Kim HH: Antioxidant alpha-lipoic acid inhibits osteoclast differentiation by reducing nuclear factor-kappaB DNA binding and prevents in vivo bone resorption induced by receptor activator of nuclear factor-kappaB ligand and tumor necrosis factor-alpha. Free Radic Biol Med. 40:1483–1493. 2006. View Article : Google Scholar : PubMed/NCBI
28	Acharyya S, Villalta SA, Bakkar N, Bupha-Intr T, Janssen PM, Carathers M, Li ZW, Beg AA, Ghosh S, Sahenk Z, et al: Interplay of IKK/NF-kappaB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy. J Clin Invest. 117:889–901. 2007. View Article : Google Scholar : PubMed/NCBI
29	Dai S, Hirayama T, Abbas S and Abu-Amer Y: The IkappaB kinase (IKK) inhibitor, NEMO-binding domain peptide, blocks osteoclastogenesis and bone erosion in inflammatory arthritis. J Biol Chem. 279:37219–37222. 2004. View Article : Google Scholar : PubMed/NCBI
30	Huang P, Han J and Hui L: MAPK signaling in inflammation-associated cancer development. Protein Cell. 1:218–226. 2010. View Article : Google Scholar
31	Zarubin T and Han J: Activation and signaling of the p38 MAP kinase pathway. Cell Res. 15:11–18. 2005. View Article : Google Scholar : PubMed/NCBI
32	Nerlich AG, Bachmeier BE, Schleicher E, Rohrbach H, Paesold G and Boos N: Immunomorphological analysis of RAGE receptor expression and NF-kappaB activation in tissue samples from normal and degenerated intervertebral discs of various ages. Ann NY Acad Sci. 1096:239–248. 2007. View Article : Google Scholar : PubMed/NCBI
33	Dimozi A, Mavrogonatou E, Sklirou A and Kletsas D: Oxidative stress inhibits the proliferation, induces premature senescence and promotes a catabolic phenotype in human nucleus pulposus intervertebral disc cells. Eur Cell Mater. 30:89–102. 2015. View Article : Google Scholar : PubMed/NCBI
34	Séguin CA, Bojarski M, Pilliar RM, Roughley PJ and Kandel RA: Differential regulation of matrix degrading enzymes in a TNFalpha-induced model of nucleus pulposus tissue degeneration. Matrix Biol. 25:409–418. 2006. View Article : Google Scholar : PubMed/NCBI
35	Wako M, Ohba T, Ando T, Arai Y, Koyama K, Hamada Y, Nakao A and Haro H: Mechanism of signal transduction in tumor necrosis factor-like weak inducer of apoptosis-induced matrix degradation by MMP-3 upregulation in disc tissues. Spine. 33:2489–2494. 2008. View Article : Google Scholar : PubMed/NCBI
36	Kyriakis JM and Avruch J: Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev. 81:807–869. 2001.PubMed/NCBI
37	Kim JS, Ellman MB, An HS, Yan D, van Wijnen AJ, Murphy G, Hoskin DW and Im HJ: Lactoferricin mediates anabolic and anti-catabolic effects in the intervertebral disc. J Cell Physiol. 227:1512–1520. 2012. View Article : Google Scholar
38	Kim JH, Studer RK, Vo V, Sowa GA and Kang JD: p38 MAPK inhibition selectively mitigates inflammatory mediators and VEGF production in AF cells co-cultured with activated macrophage-like THP-1 cells. Osteoarthritis and cartilage/OARS. Osteoarthritis Res Soc. 17:1662–1669. 2009. View Article : Google Scholar
39	Séguin CA, Pilliar RM, Madri JA and Kandel RA: TNF-alpha induces MMP2 gelatinase activity and MT1-MMP expression in an in vitro model of nucleus pulposus tissue degeneration. Spine. 33:356–365. 2008. View Article : Google Scholar : PubMed/NCBI