Current treatments to alleviate chronic low back pain are principally surgical with very variable outcomes. For example, a 10–40% failure rate was observed in lumbar surgeries with or without spinal fusion, and similar results were obtained in patients whom underwent a discectomy of herniated disc with recurrence after two years of surgery [21–24]. Innovative strategies for IVD with regenerative medicine and/or pharmacological inhibitors of disc degeneration are urgently wanted.
The present study investigated the contribution of ERK signaling in in vitro inflammation-induced human NPC by TNFα and whether ERK inhibition using selective mitogen-activated protein kinase kinase enzyme (MEK) inhibitor can reverse the inflammatory phenotype of these cells. We found that TNFα stimulation in NPC induced a pro-inflammatory microenvironment of human IVD cells, in particular NPC, characterized by increased expression of important catabolic events and pro-inflammatory mediators. Herein, the expression of matrixproteinases including MMP3 and MMP13 was upregulated in TNFα-stimulated NPC. MMP are believed to be the major proteolytic enzymes responsible for ECM degradation in the IVD leading to disc degeneration [25]. The expression of numerous metalloproteinases at the transcript and protein levels has been studied in several human IVD as well in experimental animal models revealing the catabolic changes and their mediation in the progression of IVD degeneration [26–28].
This study further supports that the TNFα cytokine is a potent mediator of inflammatory response in IVD and in particular in NPC leading to ECM degradation through increased catabolism and therefore to disc degeneration. Inflammatory processes exacerbated by TNFα and/or IL1β are believed to trigger disc degeneration and in later stages low back pain. For instance, surgical samples obtained from patients with history of low back pain revealed higher levels of TNFα-positive cells than autopsy from healthy controls [11]. In addition, these intradiscal pro-inflammatory cytokines are implicated in the onset and progression of IVD degeneration and discogenic pain, and are produced by native IVD cells including NPC and AFC as well to infiltrating macrophages [5, 9–10]. Taken together, TNFα is an essential initiator of a pro-inflammatory environment in IVD tissue and cells which leads to the tissue ECM degradation and disorganization, and therefore to disc degeneration and painful spine. The importance of TNFα cytokine in discogenic pain led to multiple clinical trials using TNFα inhibitors, which resulted in mixed results [29–31] and highlighting, therefore, for further research studies [12]. In particular, monoclonal antibodies against TNFα have shown promise for mitigating disc degeneration and relieving low back pain. Anti- TNFα treatment significantly decreases the concentration and activity of MMP1 and MMP3 in ex vivo IVD tissues isolated from patients with herniated discs [32]. Despite obvious benefit of TNFα mAbs, some patients do not respond to them and/or many will develop recurrent disease despite continuing dosing which hampers the clinical use of these antibodies. [33]. In addition, Infliximab which is a TNFα blocker and a chimeric IgG1 antibody did not appear to interfere with spontaneous resorption of disc herniation over a prolonged period based on MRI diagnosis in a randomized controlled study [34]. Therefore, further research to elucidate the mechanism by which inflammatory cascade is initiated through TNFα is required for targeted pharmacological treatment of IVD degeneration.
Within this study, we aimed to block the TNFα downstream signaling pathway by targeting the ERK from the MAPK family. First, we reproduced an in vitro inflammatory environment in NPC and subsequently the ERK pathway was blocked through U0126. The current study sought to elucidate the role of ERK1/2 signaling pathway in TNFα-mediated catabolic effect in NPC.
We used the resazurin red assay to examine the possibility of any cytotoxic effect of the ERK inhibitor on NPC. It was found that U0126 was cytotoxic in NPC at large concentrations translated by a dose-dependent reduction of cells’ viability with increasing U0126 molarity. This finding further supports the critical role of MAPK, in particular ERK1/2 pathway, in the regulation of mammalian cell proliferation as previously documented [35].
Blocking the ERK pathway in inflammation-induced human NPC resulted in a down-regulation of MMP3 and MMP13 to similar levels observed in control NPC, at least for the mRNA levels. It was previously demonstrated that CCAAT/enhancer binding protein beta (C/EBPβ) in the TNFα promoter region was suppressed in the presence of an ERK inhibitor PD98059 and the p38-MAPK inhibitor SB202190, but not the JNK inhibitor SP600125 in rat NPC [36]. In addition, the C/EBPβ and MMP13 expression was co-localized in chondrocytes in inflammatory arthritic patients [37]and that pro-inflammatory cytokines such as IL1β and TNFα binds to MMP3 and MMP13 promoter regions and stimulates their expression [37–38]. Similarly, treatment of rat NPC with ERK1/2 inhibitors (PD98059 and U0126) abolished the antagonistic effect of TGF-β1 on TNFα mediated MMP3 catabolic response [39], which further supports our finding on the implication of ERK pathway in inflammatory human NPC. Taken together, TNFα induces an inflammatory cascade in mammalian cells, in particular NPC, by up-regulation and modulation of MMP family members such as MMP3/MMP13 through ERK1/2 pathway, and inhibition of ERK signaling can reverse this catabolic effect. ERK1/2 is, therefore, considered a downstream signaling pathway of TNFα and this MAPK might be a target for the increased MMP enzymatic activity. Although, the enzymatic activities mediated by the gelatinases MMP2/9 were visible on gel zymography in NPC cell layer lysates, we could not detect significant variations of MMP2/9 following treatment of NPC with TNFα. This could be explained by an inappropriate model for the detection of MMP2/9, the intracellular gelatinases MMPs are normally secreted in the extracellular compartment for a variety of cell lineages [40–42]. Therefore, it is more relevant to assess the incorporated gelatinases in culture supernatant rather than within the cells lysates [43] as also detected previously for IVD cells [44].
The activation of an inflammatory microenvironment through stimulation of NPC with TNFα resulted in an increased ADAMTS5 expression as compared to controls. Blocking the ERK1/2 pathway with U0126, however, did not alter the expression of ADAMTS5. This observation might be explained by a differentiational regulation of aggrecanase-mediated proteoglycan degradation including ADAMTS4-5 which is mediated through NF-𝑘B activation and not ERK1/2 in bovine NPC [45]. Similarly, it was suggested that ADAMTS4 expression and promoter activity increased in NPC following TNFα and IL-1β treatments [46] and treatment of the cells with NF-𝑘B inhibitor abolished this inductive effect of the cytokines on ADAMTS4 mRNA and protein expression. This further supports that modulation of ADAMTS5 in NPC is mediated through NF-𝑘B which might explain our observation on the role of ERK1/2 in ADAMTS5 expression.
The results of this study revealed a trend towards an increase in transcripts levels of the NP-specific markers including COL2 and ACAN, but also a significant increase of KRT19 in inflammatory NPC that were treated with U0126. In addition, increased expression of anabolic genes like IGF1 was observed suggesting a restoration of the NPC phenotype following the inhibition of the ERK pathway in inflammatory cells. Wei et al. [17] observed an imbalance between anabolic and catabolic events in rat AFC activated with IL1, and ERK inhibition significantly blocked the catabolic and inflammatory effects of IL1 in AFC.
Within this study, we provided evidence that TNFα-mediated inflammation in human NPC is triggered through the ERK1/2 pathway which in terms increased pro-inflammatory mediators like MMPs and decreased anabolic genes characteristic of a degenerated IVD cells. The ERK1/2 pathway was modulated through TNFα as observed by Western Blot assay and simultaneous treatment of NPC with TNFα and U0126 abolished this effect. We also showed that U0126 is a specific inhibitor of ERK1/2 pathway.
There are some limitations of the current study. First, due to the difficulty and availability in obtaining healthy and non-degenerated human disc tissues samples, the results were limited to gene expression analysis of some anabolic and catabolic genes, and we did not investigate their protein levels more in details. Second, we did not address the contribution of other MAPK like p38-MAPK and JNK in inflammation, although some pilot data were generated (data not shown). In addition, we used in vitro monolayer cultures of NPC, which might differ from 3D cultures like cells-seeded scaffolds or ex vivo IVD samples and the combined effects of inflammatory mediators with biomechanical stimuli was not addressed which is the aim for further investigations.