Inhibition of the extracellular signal‐regulated kinase pathway reduces the inflammatory component in nucleus pulposus cells

Abstract Intervertebral disc (IVD) degeneration is a spinal disorder that triggers an inflammatory response and subsequent development of spinal pseudoarthrosis. The aim of the present study is to elucidate the role of the extracellular signal‐regulated kinase (ERK) pathway in inflammation‐induced IVD cells. Inflammatory human nucleus pulposus (NP) cells (NPCs) were induced using tumor necrosis factor‐α and the ERK pathway was blocked using a selective molecule‐based inhibitor U0126. Gene expression of catabolic and anabolic markers, proinflammatory, and NPCs markers was investigated. The enzymatic activity of matrix metalloproteinases (MMP)2/MMP9 was determined by gelatin zymography and nitrite production was assessed by Griess reaction. The NPC metabolic activity and viability were assessed using resazurin sodium‐salt and live/dead assays, and subsequently, the specificity of U0126 on ERK1/2 signaling was determined. The catabolic enzyme MMP3 (p = 0.0001) and proinflammatory cytokine interleukin 6 (p = 0.036) were downregulated by U0126 in NPCs under inflammatory conditions. A significant increase of the cytokeratin 19 (p = 0.0031) was observed, suggesting a partial and possible recovery of the NP phenotype. U0126 does not seem to have an effect on prostaglandin production, aggrecanases, or other anabolic genes. We confirmed that U0126 selectively blocks the ERK phosphorylation and only affects the cell metabolic activity without the reduction of viable cells. Inhibition of ERK signaling downregulates important metalloproteinases and proinflammatory cytokines, and upregulates some NP markers, suggesting its potential to treat IVD degeneration.


| INTRODUCTION
Low back pain affects millions of people in industrialized societies every year, making it a leading cause of disability with significant economic and social burdens. [1][2][3] Chronic low back pain is strongly associated with intervertebral disc (IVD) degeneration which results in increasing pain during the execution of daily spinal movements. The IVD consists of an inner nucleus pulposus (NP) surrounded by the annulus fibrosus (AF) tissue, and hyaline articular cartilage that is located at the endplates between the IVD and two adjacent vertebral bodies. The NP is a gelatinouslike and avascular connective tissue containing a highly organized extracellular matrix (ECM) rich in proteoglycans and collagens with few numbers of cells. The nucleus pulposus cells (NPCs) actively regulate homeostasis in the ECM by several growth factors and cytokines through autocrine and paracrine signals.
The early stage of disc degeneration is often asymptomatic 4 and chronic inflammation tends to develop within the spine. This produces high stresses in the disc tissue, which begins with the synthesis of catabolic enzymes, 5  Noninvasive interventions including medications, steroid injection, and physical therapy are recommended most often but have limited long-term efficacy 6,7 since they do not maintain or restore the native tissue structure in degenerative discs. Current surgical treatments aim to eliminate discogenic pain by replacing the injured tissue with a functional biological substitute or prosthesis. However, surgery for disc degeneration also yielded mixed clinical outcomes and often results in incomplete interbody fusion. 8 Interleukin 1β (IL1β) and tumor necrosis factor-α (TNF-α) are proinflammatory cytokines known to be key mediators in the development and progression of disc degeneration and low back pain. 5 Many studies have shown that TNF-α is highly expressed in degenerative IVD tissues 9,10 and surgical samples obtained from patients with a history of low back pain revealed higher levels of TNF-αpositive cells than autopsies from healthy controls. 11 TNF-α causes an upregulation of ECM-degrading enzymes and decreases the expression of matrix-specific matrix proteins. 12 Thus, identifying and targeting signaling pathways responsible for the TNF-α-mediated inflammation could be a promising therapeutic option for IVD tissue regeneration.
Extracellular signal-regulated kinase (ERK), a downstream signal of the mitogen-activated protein kinase (MAPK) signaling cascade, is an important inflammatory pathway that plays a critical role in the production of inflammatory cytokines and the activation of procatabolic responses induced by TNF-α in chondrocyte-lineage cells. 13 Inhibition of MAPK signaling attenuates the decrease of collagen type II (COL2A1) and aggrecan (ACAN) without inducing apoptosis in primary rats and immortalized chondrocytes. 14 In agreement with this, inhibition of MAPK/ERK activity enhances chondrogenesis of mesenchymes, 15 and TNF-α-induced nuclear factor-κB (NF-kB) DNA binding in chondrocytes is reduced by inhibition of MAPK signaling. 14 Recent studies suggest ERK is a catabolic pathway in the degeneration of IVD. In AF cells, ERK was shown to mediate IL1-induced upregulation of cyclooxygenase (COX), 16

| Induction of proinflammatory environment and ERK inhibition
NPCs were seeded at a density of 5 × 10 4 cells/well in 24-well plates in the proliferation medium and left overnight for cell adherence.
Induction of a proinflammatory microenvironment in NPCs was performed by addition of 10 ng/ml of human recombinant TNF-α (Peprotech) to the wells as previously described. 19

| NPC metabolic activity
The NPCs were seeded at a density of 2 × 10 3 cells/well in 96-well plates in the proliferation medium and left overnight for cell adherence. To address whether blocking the ERK pathway or a combination of TNF-α and ERK inhibition may reduce cell metabolism, NPCs were stimulated with 10 ng/ml TNF-α and increasing concentration of a U0126 ranging from 0.1 to 10 µM after a pretreatment of 1 h. The cultures were collected after 3 days and cell metabolic activity was determined with a resazurin red solution (Sigma-Aldrich) as previously described. 22 Briefly, the cultures were incubated with 50 µM resazurin red solution in a humidified atmosphere (5% CO 2 , 37°C) for 2 h. The absorbance was measured at 580 nm on a microplate reader (SpectraMax M5; Bucher Biotec).

| NPC viability assay
The NPCs were seeded at a density of 2 × 10 4 cells/well in 24-well plates and stimulated with 10 ng/ml TNF-α and increasing con-

| Analysis of gene expression
Total RNA was extracted as previously described 23

| Nitric oxide assay
The levels of nitric oxide (NO) production in the NPCs culture medium were determined as the concentration of its stable nitrite oxidation product using the Griess Reaction Reagent Kit (Promega) according to the manufacturer's instruction.

| Western blot analysis
Primary human NPCs (2.5 × 10 4 cells/well) were seeded into six-well culture plates and grown for 1 week in the proliferation medium without additional factors to allow for cell adherence. The medium was changed and the cells were treated with either TNF-α, a combination of TNF-α and U0126 (0.1, 0.5, 1, and 5 μM), or left as controls in dimethyl sulfoxide.   Gene  Forward sequence  Reverse sequence   18S  CGA TGC GGC GGC GTT ATT C  TCT GTC AAT CCT GTC CGT GTC C   ACAN  TCT GGA GTA GAG GAC ATC  AGG AAG TTC ACT GAC ATC   COL2  AGC AGC AAG AGC AAG GAG AA  GTA GGA AGG TCA TCT GGA   KRT19  TCT TGC TGC TGA TGA CTT  CCT CTT CGT GGT TCT TCT   IGF1  The TNF-α-stimulated NPCs were morphologically different, spindleshaped with a fibroblast-like appearance and less dense compared to untreated cells observed microscopically.

| Gene expression
The effect of TNF-α stimulation followed by ERK inhibition was in-   NPCs. MMPs are believed to be the major proteolytic enzymes responsible for ECM degradation in the IVD leading to disc degeneration. 30 The expression of numerous metalloproteinases at the transcript and protein levels has been studied in several human IVD and experimental animal models, revealing catabolic changes and their mediation in the progression of IVD degeneration. [31][32][33] This study further supports that the TNF-α cytokine is a potent mediator of inflammatory response in the IVD, particularly in NPCs, and leads to disc degeneration through increased catabolism due to ECM degradation. Inflammatory processes exacerbated by TNF-α and/or

| Gelatinases expression and nitric oxide production
IL1β are believed to trigger disc degeneration and, in later stages, low back pain. For instance, surgical samples obtained from patients with a history of low back pain revealed higher levels of TNF-α-positive cells than autopsy from healthy controls. 11 In addition, these intradiscal proinflammatory cytokines are implicated in the onset and progression of IVD degeneration and discogenic pain, and are produced by native IVD cells including NPCs and AFCs as well by infiltrating macrophages. 5,9,10 Taken together, TNF-α is an essential initiator of the proinflammatory environment in IVD tissue and cells that leads to the tissue ECM degradation and disorganization, and, therefore, to disc degeneration and back pain. The importance of TNF-α in discogenic pain led to multiple clinical trials using TNF-α inhibitors which resulted in mixed results, [34][35][36] highlighting the need for further research studies. 12 In particular, monoclonal antibodies (mAbs) against TNF-α have shown promise for mitigating disc degeneration and relieving low back pain. Anti-TNF-α treatment significantly decreases the concentration and activity of MMP1 and MMP3 in ex vivo IVD tissues isolated from patients with herniated discs. 37 Despite the obvious benefit of TNF-α mAbs, some patients do not respond to them and many will develop recurrent disease despite continuing dosing, which hampers the clinical use of these antibodies. 38 In addition, Infliximab, a TNF-α blocker and a chimeric immunoglobulin G1 antibody, did not appear to interfere with F I G U R E 4 Western blot of phosphorylated ERK1/2 (p-ERK) and their respective total ERK1/2 in NPCs after TNF-α stimulation. (A) The NPCs were stimulated with TNF-α (10 ng/ml) and the cells lysates were collected at different time points (minutes until 2 h) with control being time 0 min before stimulation. (B) p-ERK and total ERK were determined using Western blot, the signal intensities from a representative experiment were quantified, and the p-ERK were normalized to total ERK. ERK, extracellular signal-regulated kinase; NPC, nucleus pulposus cell; TNF-α, tumor necrosis factor-α spontaneous resorption of disc herniation over a prolonged period based on magnetic resonance imaging diagnosis in a randomized controlled study. 39 Therefore, further research to elucidate the mechanism by which inflammatory cascade is initiated through TNF-α is required for targeted pharmacological treatment of IVD degeneration. observed an imbalance between anabolic and catabolic events in rat AFCs activated with IL1, and ERK inhibition significantly blocked the catabolic and inflammatory effects of IL1 in AFCs.
Within this study, we provided evidence that the ERK1/2 pathway triggers TNF-α-mediated inflammation in human NPCs and leads to increased proinflammatory mediators, such as MMPs, and some decreased anabolic genes characteristic of degenerated IVD cells. A Western blot assay conveyed that the ERK1/2 pathway was modulated through TNFα, while simultaneous treatment of NPCs with TNF-α and U0126 abolished this effect. This reveals that U0126 is a specific inhibitor of the ERK1/2 pathway.
Unfortunately, there are some limitations of the current study.
First, due to the difficulty and availability in obtaining healthy and nondegenerated 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 in more detail. Second, we did not address the contribution of other MAPKs, such as p38-MAPK and JNK, in inflammation, although some pilot data was generated. In addition, we used in vitro monolayer cultures of NPCs, which might differ from three-dimensional cultures such as cells-seeded scaffolds or ex vivo IVD samples. Furthermore, the combined effects of inflammatory mediators and biomechanical stimuli were not addressed in this study; however, future investigations aim to explore this.
In conclusion, the current study provides evidence that the ERK pathway is implicated in NPCs inflammatory processes and ERK1/2 inhibition could provide some protection against the adverse effects of TNF-α in the IVD.

ACKNOWLEDGMENTS
This study was supported by the Swiss National Science Foundation (Project # 310030_153411) and from the Marie Skłodowska Curie International Training Network "disc4all" (https://cordis.europa.eu/ project/id/955735, accessed April 28, 2021). The authors would like to thank all the patients enrolled in this study. Open access funding provided by Universitat Bern.