EGFR is a pivotal regulator of thrombin-mediated inflammation in primary human nucleus pulposus culture

We found that the coagulation and cytokine pathways were important mechanisms involve in the degeneration of intervertebral discs (IVD) using a microarray approach to analyze gene expression in different grades of specimens. Furthermore, using a cytokine/chemokine array, a significant increase in CXCL8 expression was observed in human nucleus pulposus (NP) cells after thrombin treatment. The enhancement of CXCL8 expression by thrombin was activated by the PAR1 receptor. Importantly, analysis of degenerated human NP tissue samples showed that EGFR expression positively correlated with the grade of tissue degeneration. In NP cells, thrombin caused an increase in phosphorylation of the EGFR at the Tyr1068, and treatment with the pharmacological EGFR inhibitor, AG1473 effectively blocked thrombin-enhanced CXCL8 production. Surprisingly, inhibition of STAT3 for 24 h decreased expression of EGFR. Treatment with thrombin also increased Akt and GSK3α/β activation; this activation was also blocked by EGFR inhibitor. Although c-Src, ERK, and FAK were activated by thrombin, only c-Src and ERK were involved in the STAT3/CXCL8 induction. Our findings indicate that stimulation of an inflammatory response in NP cells by thrombin is part of a specific pathophysiology that modulates the EGFR activation through activation of Src/ERK/STAT3 signaling.

thrombin dynamically regulates cell growth, development, and response to injury in the central and peripheral nervous systems 17 . Low concentrations of thrombin induce tolerance against various insults while high concentrations of thrombin induce apoptotic cell death 18,19 . In various types of nervous system injuries and diseases, secretion of thrombin is associated with cell degeneration, which involves numerous cytokines and chemokines 17, 20 . A clinical study has shown that levels of prothrombin, the thrombin precursor, increase with age 21 . It has also been reported that spinal cord parenchymal accumulation of thrombin results in blood-spinal cord barrier breakdown in amyotrophic lateral sclerosis patients 22 . Spinal motor neurons express the thrombin receptor PAR-1, and activation of this receptor induces neuronal cell degeneration and death 23,24 . Furthermore, activation of the PAR-1 receptor in the lamina I and II regions of the spinal cord modulates neuropathic pain 25 .
Chemokines constitute of a group of pro-inflammatory cytokines with potent chemotaxis activity that can target specific inflammatory cells 26 and recruit them from the blood into tissues 27 , and in this way drive the chronic inflammatory process. Several cytokines and chemokines are involved in the development of inflammation and are responsible for the pathology of IVD degeneration 28 . CXCL8 has been considered to participate in the pathomechanism of nerve root inflammation in lumbar disc herniations and to be associated with development of radicular pain by back extension 29 . A recent study has reported that patients with lumbar radicular pain due to disc herniation have increased serum levels of CXCL8 30 . Substance P, a neurotransmitter that acts as an inflammatory mediator, stimulates CXCL8 upregulation in NP cells 31 . Importantly, the concentration of CXCL8 has been found to be significantly higher in patients with spinal cord injury than in healthy controls, and was negatively correlated with the duration of spinal cord injury 32 . The increased level of CXCL8 within the nucleus pulposus has been reported to be related to neovascularization in patients with discogenic pain 33 , and a CXCL8 inhibitor has been suggested as a promising therapeutic agent for disc herniation that causes chronic radicular neuropathic pain 34 . Importantly, the protein expression of CXCL8 by NP cells is significantly increased concordant with the increasing severity of degenerative tissue changes in human NP 35 . Gene expression analysis has also indicated that CXCL8 expression was greater in IVDs with immune cell infiltration compared to that in nondegenerative IVD 35 . Although the roles of chemokines in immune cell chemotaxis have been described in detail, the regulation of CXCL8 during disc degeneration is still not clear.
In this study, immunohistochemistry revealed that higher levels of the EGFR were found in NP cells and were associated with higher clinical pathological stages of IVD in degenerative patients. Our results also show that EGFR-activated Akt/GSK3 signaling promotes CXCL8 expression. Stimulation by thrombin upregulates EGFR-dependent CXCL8 expression through the Src/ERK/STAT3 signaling pathways in NP cells, which may contribute to immune cell recruitment and disc degeneration.

Materials and Methods
All methods were performed in accordance with the relevant guidelines and regulations.
Human tissue collection and grading. Human IVD samples (Table 1) were obtained during spinal surgery with approval of the Taichung Tzu-Chi hospital ethics committee (REC102-14 and REC103-55). IVD degenerative specimens were obtained from patients who underwent surgical discectomy for cervical, lumbar, or sacral
discs (Table 2). These IVD degenerative specimens were obtained as surgical waste from patients who underwent surgical discectomy for lumbar discs. All informed consents were obtained from patients themselves, or their family members, prior to tissue collection. Assessment of the disease state was performed using Pfirrmann grading 36 . This scheme uses a T2-weighted magnetic resonance imaging with image analysis by three independent observers. The Pfirrmann grade covers the spectrum from a healthy disc (Grade I) to discs with advanced degeneration (Grade V, the most advanced stage of degeneration). The patients' age, gender, spinal level, NP tissue grade, and surgical diagnosis of contained or non-contained disc, are listed in Tables 1 and 2. Additionally, we assessed the disc degenerative condition using another scoring system based on pathologic results 37 . The Boos score analyzes the degenerative condition based on chondrocyte proliferation, mucus degeneration, cell death, tear and cleft formation, and granular changes and is scored from 0 to 22. We also compared the Pfirrmann grade from the MRI analysis with the pathological Boos score and they were found to be closely correlated (Fig. 1B).
Isolation and cell culture of NP and AF from tissue specimens. Human NP and AF cells were isolated used a non-enzymatic method that were modified from previous studies 38,39 , which did not cause damage to cell surface receptors and preserved its phenotypes [39][40][41] . Human disc tissue (minimally degenerative) was obtained from patients undergone cervical spinal surgeries as per an approved protocol at Taichung Tzu Chi Hospital, Buddhist Tzuchi Medical Foundation. Tissues were anonymized, with only the patient age, gender, and grade recorded. Disc tissues were rinsed with PBS and grossly separated into AF and nucleus NP. Any other non-disc materials such as endplate bone or cartilage in the surgical sample were discarded prior to tissue incubation. Separated AF and NP tissues were further washed three times with a washing medium [DMEM basal medium with 100 μg/mL kanamycin (Sigma, St. Louis, USA) and 165 μg/mL gentamycin (Gibco, Grand Island, NY, USA), and 1.25 μg/mL fungizone (Gibco)] and cut into small pieces (the average size of the tissue explant was 1-2 mm 3 for AF, and 3-5 mm 3 for NP). NP and AF cells were maintained in DMEM/F12 supplemented with antibiotics and 10% FBS. The culture medium was changed every two days. Once cells had migrated out of the tissue and had been expanded for three to four weeks (to about 80% of confluence), the tissue explants were transferred to a new flask for another round of incubation, with the remaining cells in the original flask being ready for harvesting. In general, incubated tissues could be transferred up to ten times or until cell migration ceased.
Patients sample microarray. We selected sixteen patients (the clinical-pathological data are summarized in Table 3) with IVD degenerative specimens and performed a cDNA microarray. RNA labeling and hybridization were performed using a kit from Welgene Biotech Co., Ltd (Welgene Biotech Co., Ltd., Taipei, Taiwan) according to the manufacturer's instructions. Briefly, 0.2 μg of total RNA was amplified using a Low Input Quick-Amp Labeling kit (Agilent Technologies) and labeled with Cy3 (CyDye, Agilent Technologies) during the in vitro transcription process. Cy3-labeled cRNA (0.6 μg) was fragmented to an average size of about 50-100 nucleotides by incubation with fragmentation buffer at 60 °C for 30 min. The fragmented labeled cRNA was then pooled and hybridized to an Agilent SurePrint G3 Human V2 GE 8 × 60 K Microarray (Agilent Technologies, USA) at 65 °C for 17 h. After washing and then drying using a nitrogen gun, microarrays were scanned with an Agilent microarray scanner (Agilent Technologies) at 535 nm to detect the Cy3 signal. Scanned images were analyzed using the Feature extraction 10.5.1.1 software (Agilent Technologies). Image analysis and normalization software was used to quantify the signal and background intensity for each feature.
Non-degenerative human NP cells. Healthy human NP cells from the human intervertebral disc were purchased from Sciencell Research Laboratories (Cat# 4800, Carlsbad, CA, USA). Cells were cultured in Nucleus Pulposus Cell Medium with supplements (NPCM, Sciencell, Cat.#4801) on poly-L-lysine coated tissue culture dishes. The media was changed every two days and cells were passaged when they were over 90% confluent. Cells were cultured in medium supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 °C, and incubated in a humidified atmosphere consisting of 5% CO 2 and 95% air.  Immunohistochemical Staining. The protocol for immunohistochemistry was slightly modified from our previous report 42 . Briefly, tissue sections measuring 4 μm were dewaxed and rehydrated, and endogenous peroxidases were quenched and, following heat-mediated antigen retrieval, blocked in normal goat serum. Sections were incubated overnight at 4 °C with primary antibody against EGFR. Pre-immune rabbit IgG was used as a negative control. Binding was detected using a biotinylated secondary antibody and an ABC standard kit. Visualization was performed using 0.05% diaminobenzene. After slices were stained with hematoxylin staining for counter staining, slices were mounted, and visualized with Zeiss microscope. Slides were evaluated independently by a pathologist who was blinded to the patients' outcomes. Cytokine/chemokine array. Supernatants collected from NP cells were then removed and clarified with a 20,000 g spin (10 s at 4 °C), and applied to each of two separate cytokine array membranes. Membranes were processed following the manufacturer's instructions. Chemi-luminescence was detected using LAS-3000 film (Fuji; Tokyo, Japan). Manual background subtraction was performed prior to quantitation, and "fold change" was calculated as the difference between the average values for both cytokine spots on thrombin-treated versus untreated arrays.
Western blot analysis. The western blotting procedure has been previously described 43 . Briefly, cells were lysed with immunoprecipitation assay buffer on ice. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk and then probed with appropriate primary antibody. The blots were visualized using enhanced chemiluminescence and LAS-3000 film. The blots were subsequently stripped by incubation in a stripping buffer and re-probed for β-actin as a loading control. Quantitative data were obtained using a densitometer and ImageJ software.
ELISA Assays. Production of CXCL8 in the culture supernatant was measured using a commercial kit (R&D Systems) according to the manufacturer's instructions. The absorbance was determined using a microplate reader. Statistical Analysis. Statistical analysis was performed using the GraphPad Prism 6 (GraphPad Software

RNA extraction and
Inc., San Diego, CA, USA). The values are presented as mean ± SEM. Statistical differences between the experimental group and the control groups were assessed using Student's t-test. Statistical comparisons between more  than two groups were performed using a one-way analysis of variance with a Bonferroni post-hoc test. The difference was considered significant if the p-value was <0.05.

Results
Patient characteristics and microarray. The demographics of the patients recruited into this study are presented in Tables 1 and 2. Pfirrmann grades I/II was used as the control/normal group, and grades III to V were used as the degenerative group. Degenerative grading was determined by a radiologist who independently evaluated the MRI according to the classification scale 36 . Disc pathology (contained and non-contained) were also analyzed by MRI. Contained disc herniation included disc protrusion, while non-contained disc herniation included disc extrusion or sequestration. In total, eighty-seven disc samples with Pfirrmann grade I/II (n = 10), grade III (n = 40), grade IV (n = 28) and grade V (n = 9) were collected. Typical MRI images of patients with Pfirrmann grades II-V are shown in Fig. 1A. The patient characteristics, including age (range 25-85 years), gender, surgical level, and disc pathology are listed in Table 2. The mean ages in years for each Pfirrmann grade were as follows: II, 48.7 ± 10.4; III, 52.1 ± 12.6; IV, 60.7 ± 10.7; V, 69.2 ± 11.7. There was a significant difference between age distribution and Pfirrmann grade (p < 0.001), but no significant difference in gender or contained vs. non-contained disc groups among the different Pfirrmann grades (p = 0.96 and 0.97). Furthermore, there was a high correlation (p < 0.001) between the Boos scores and the Pfirrmann grade (Fig. 1B). The NP cell numbers significantly decreased with the increasing IVD degenerative process (p = 0.001, Fig. 1C); specifically, the number of NP cartilage-like cells were 14.4 ± 4.99 (Pfirrmann grade II), 11.35 ± 4.8 (Pfirrmann grade III), 9.14 ± 4.47 (Pfirrmann grade IV), and 6.33 ± 3.16 (Pfirrmann grade V). To elucidate the possible mechanism underlying IVD degeneration and the decrease in NP cell number, we used a microarray approach to analyze gene expression of cell cultures from the different grades of NP and AF tissues. Since a decline of cellularity with clinical grade and the IVD exhibit a very limited capability for cell proliferation for self-repair 44 , we used a non-enzymatic method for isolating IVD cells, which preserves the phenotype of nucleus pulposus cells 39 , and approached for a microarray analysis. As shown in Fig. 1D, genes altered in NP and AF cells from each grade revealed a core of 172 genes that were changed 2-fold comparing AF with NP. Use of a KEGG pathway analysis of biological function with a strict p value cutoff of p < 0.01 resulted in the identification of eleven pathways (Fig. 1E). Because of IVD degeneration-caused back pain has been linked to ageing, excessive manual labour and genetic factors 45 , several previous clinical reports were focused on discovering the mechanisms of IVD cell death, cell cycle, DNA replication or DNA repair. However, there are other underlying events, such as injury of NP results in recruitment of immune cells to the disc which promotes low back pain 46 . From this perspective, in the present study, we would like to explore novel biomarkers for chemoattraction of immune cells. Therefore, we elected to exclude pathways involved in the modification of cell cycle, DNA function, reproduction, and cancer signaling, but instead we focused on the coagulation and cytokine pathways. We hypothesized that coagulation factors modulate the inflammatory mediators in the degenerative microenvironment of IVD.

Thrombin effectively modulates cytokine and chemokine expression in NP cells. In order to
validate the microarray observations, we performed a cytokine/chemokine array to examine the levels of expression of cytokine/chemokines after the addition of thrombin, the most important coagulation factor, to NP cells. As shown in Fig. 2A, thrombin increased the levels of a variety of inflammatory mediators expressed in NP cells culture supernatants, including CXCL1, IL-6, CXCL8, IL-27 and CCL2, whereas thrombin slightly decreased CD154, IL-16, IL-23 and IL-13 (right-hand table, Fig. 2A). The microarray data of CXCL1, IL-6, CXCL8, IL-27, CCL2 and CXCL8 in the different grades of NP tissue were shown in Table 4. Important to this study, the levels of CXCL8 were increased to the largest extent by 24 h thrombin stimulation compared to the vehicle control group.
To further validate that thrombin enhances cytokines/chemokines and pro-inflammatory mediators expression, we treated NP cells with recombinant thrombin and analyzed the mRNA expression of inflammation related molecules. Treatment with thrombin significantly increased the expression of IL-6 and CXCL8, but not the levels of other pro-inflammatory mediators including ANXA2, CCL2, ICAM1 and tPA (Fig. 2B). Moreover, treatment with thrombin upregulated IL-6 and CXCL8 mRNA expression in a time-and dose-dependent manner (Fig. 2C). Treatment with thrombin also increased CXCL8 secretion as measured by ELISA assay (Fig. 2D). To further confirm this up-regulation of CXCL8 by thrombin receptor in NP cells, we used a thrombin inhibitor PPACK, which has been previously demonstrated to be efficacious in limiting thrombin-induced platelet activation 47,48 . As shown in Fig. 2E, we found that PPACK completely inhibited thrombin-induced CXCL8 production. To further determine which PAR receptor was required for thrombin-induced CXCL8 expression, NP cells were transfected with siRNAs against PAR1, PAR2, PAR3, or PAR4 receptors and stimulated with thrombin. As shown in Fig. 3A, the thrombin-mediated induction of CXCL8 expression was abolished by the PAR1 receptor siRNA but not by PAR2, PAR3, or PAR4 siRNAs. In addition, these siRNAs specifically knockdowned the target genes more than 70%, but not crossed to other subtypes (Fig. 3B). Moreover, thrombin did not affect PAR1 expression itself in NP cells (Fig. 3C). Taken together, these findings demonstrate that the induction of CXCL8 by thrombin depends on PAR1 receptor in NP cells.

Thrombin-stimulated CXCL8 expression is mediated through activation of EGF receptor.
In the microarray assay, inspection of the common genes altered in grade II to IV IVD degenerative specimens revealed five EGF receptor ligand genes that were increased two-fold in NP cells (EGF, EPGN, EREG, AREG and TGFA) but only two genes in AF cells (EPGN and HBEGF) (Fig. 4A). Next, we examined the expression of the EGF receptor in human degenerative IVD specimens using immuno-histochemical analysis. As shown in Fig. 4B, higher expression of the EGF receptor was found to be associated with a higher clinical pathological stage in the IVD degenerative specimens. To establish that the EGF receptor mediates thrombin-induced CXCL8 expression, AG1478, a specific antagonist of the EGF receptor, was used. The thrombin-induced increase in CXCL8 expression was reduced by treatment with AG1478, the EGF receptor antagonist, in a concentration-dependent manner (Fig. 4C). Furthermore, the levels of the phosphorylated-EGF receptor (Tyr 1068 ) were significantly increased in response to thrombin treatment (Fig. 4D). Phosphorylation of the EGFR achieved a maximum at 30-60 min and was sustained up to 120 min after thrombin stimulation. These results suggest that the stimulatory effects of thrombin on CXCL8 were mediated through the EGF receptor in NP cells.
We further investigated the signaling pathway involved in the EGFR-mediated stimulation of CXCL8 expression by thrombin. Surprisingly, administration with a STAT3 inhibitor for 24 h reduced EGF receptor expression (Fig. 4E). As shown in Fig. 3F, stimulation of NP cells with thrombin also resulted in the time-dependent phosphorylation of STAT3 without any effect on STAT3 protein levels. The phosphorylation of STAT3 reached a maximum 30-60 min after thrombin stimulation. In addition, the phosphorylation of both JAK1 and JAK2, which lie upstream of STAT3, were also increased in response to thrombin stimulation (Fig. 4F). Thrombin-enhanced CXCL8 expression was significantly reduced by treatment with AG490, a selective JAK2 inhibitor (Fig. 4G).
We then explored other signaling pathways that could also be involved in the up-regulation of CXCL8 after thrombin stimulation. As shown in Fig. 5A, treatment of NP cells with thrombin resulted in the time-dependent phosphorylation of ERK, which was initiated at 5 min, achieved a maximum at 30-60 min, and was sustained for up to 120 min after thrombin stimulation. In addition, thrombin also mildly induced FAK and Src phosphorylation in NP cells (Fig. 5A). As shown in Fig. 5B, stimulation with thrombin also increased STAT3 phosphorylation and this enhanced STAT3 activation was antagonized by inhibitors of Src (PP2) and ERK (U0126) but not of FAK (PF573228) (Fig. 5C). The same inhibitors were used to further confirm the signaling pathways that mediate thrombin-induced CXCL8 expression; the thrombin-induced increase in CXCL8 expression was markedly antagonized by treatment with PP2 and U0126 but not PF573228 (left-hand panel, Fig. 5D). In parallel, a siRNA against FAK did not affect thrombin-enhanced CXCL8 expression (right-hand panel, Fig. 5D). Taken together, these results suggest that thrombin stimulates ERK, c-Src, and STAT3 activation, and activation of the EGFR pathway is required for the up-regulation of CXCL8 in NP cells.

Involvement of Akt and GSK3α/β signaling pathways in EGF receptor-mediated CXCL8
up-regulation. We then examined whether Akt and GSK3α/β, which are downstream of the EGF receptor, are also important in leading to increased CXCL8 expression in response to thrombin. As shown in Fig. 6A, treatment of NP cells with thrombin resulted in the time-dependent phosphorylation of Akt and GSK3α/β. Dual phosphorylation of GSK3α/β at Ser21 and Ser9 reached a maximum at 30 min and was sustained up to 120 min after thrombin stimulation. Moreover, treatment with AG1478 attenuated the thrombin-induced phosphorylation of Akt and GSK3α/β (Fig. 6B). To further confirm that these signaling pathways mediated the thrombin-induced CXCL8 expression, inhibitors of Akt (LY294002) and GSK3 (SB216763) were used. The thrombin-induced increase in CXCL8 expression was markedly antagonized by treatment with LY294002 and SB216763 (Fig. 6C). Additionally, thrombin-enhanced ERK phosphorylation was reduced by treatment with ERK (U0126) and Src (PP2) inhibitors but not FAK, Akt, and GSK3 inhibitors (Fig. 6D). Taken together, these data provide evidence that the downstream molecules Akt and GSK3α/β are involved in thrombin-induced EGFR activation and CXCL8 production in NP cells.

Discussion
There is no curative strategy for degenerative disc disease (DDD) and the molecular mechanisms controlling IVD degeneration and accelerated cell death are not well understood. Given this lack of knowledge, the fact that   there are no known biomarkers is hardly surprising. A potential therapeutic target that may offer the opportunity for medical intervention to the degenerating disc is therefore a widely sought goal. A previous microarray analysis of patients with IVD degeneration indicated that EGF expression in NP cells might play a key role in inflammation-associated IVD degeneration 49,50 . In addition, a previous report also showed that EGF is expressed and produced by NP cells, both in vivo and in 3D culture, and this expression was significantly increased after inflammatory stimulation 51 . A recent study has also reported that periodic mechanical stress stimulates the phosphorylation of the EGFR in NP cells 52 . Importantly, the levels of EGF and CXCL8 correlated with pain and clinical parameters, including assessment of tear stability and tear production in patients with severe dry-eye disease. There are many transmembrane growth factor precursors that have been described as ligands for the EGFR, including EGF, heparin-binding EGF-like growth factor (HBEGF), amphiregulin (AREG), transforming growth factor-α (TGFA), betacellulin (BTC), epiregulin (EREG) and epigen (EPGN) 53 . In the microarray assay performed here, these EGFR binding ligands were up-regulated to a greater degree in NP cells than in AF cells (Fig. 3A). In addition, an analysis of patient IVD specimens showed that EGFR expression was positively correlated with the grade of tissue degeneration (Fig. 3B). In addition, thrombin enhanced EGFR phosphorylation, and treatment with an EGFR inhibitor effectively reduced thrombin-induced CXCL8 expression (Fig. 3C). Our results also support previous reports that thrombin stimulates pathological and biological functions through EGFR activation 54,55 . Our results provide a clue that EGFR might be a potential biological target in IVD diagnosis and for development of a therapeutic drug. EGFR is a receptor tyrosine kinase (RTK) which can be transactivated by G protein-coupled receptors (GPCRs) through an EGF ligand-independent manner, which suggested to be regulated through intracellular signaling pathways 56 . Daub et al., 1996 firstly provided the evidence that transactivation of EGFR was following the addition of the GPCR agonists such as thrombin 57 , which activation was blocked by the EGFR kinase inhibitor AG1478 57 . This regulatory effect could be classified into two major mechanisms, including through the triple membrane passing signal (TMPS) pathway and through the EGF-like ligands-independent pathway 56,58,59 . The EGFR transactivation through TMPS pathway controlled by the activation of membrane-bound matrix metalloproteases has been reported in many cell types following activation by various agonists like thrombin 60 . On the other hand, the ligand-independent transactivation of EGFR is phosphorylated EGFR in its cytosolic domain by the activation of intracellular protein tyrosine kinases like Src family proteins 58,59 . It has also been reported that Src activation plays a permissive role for PAR1-mediated transactivation of EGFR in colon cancer cell growth 61 . Previous report also found that FAK plays an essential role for the transactivation of EGFR 62 . Present study supported previous reports that phosphorylation of EGFR is a critical for thrombin-induced CXCL8 overexpression, and the EGFR kinase inhibitor could attenuate the enhancement effect. Our results also found that Src and FAK activation are downstream of thrombin stimulation. However, the key kinases directly modulate the transactivation of EGFR in NP cells need further investigation.
IVD degeneration is associated with inflammation, which can become chronic 63 . Beyond the potential application to new therapeutics, the novel hypotheses and mechanistic studies portrayed here have the potential to describe how the pathophysiologic state in IVD is modulated. Thrombin is generated following joint inflammation in osteoarthritis patients 64 and following the abnormal proliferation of synovial cells in rheumatoid arthritis 65 . Importantly, the use of a thrombin antagonist has been proposed as a novel therapeutic strategy for the suppression of inflammation and degeneration in nervous system injury [66][67][68] . In this regards, a means of attenuating the untoward effects of thrombin is a goal of current research efforts. Patients with radicular pain caused by lumbar spinal stenosis, who received epidural injections of the anti-IL-6R monoclonal antibody tocilizumab, showed improvement in lower back and leg pain. Moreover, epidural administration of the TNF inhibitor etanercept to the spinal nerve provided pain relief in patients experiencing radicular pain arising as a result of lumbar spinal stenosis 69 . In a randomized controlled study, the anti-TNF antibody adalimumab 70 and infliximab 71 showed  a marked improvement in leg pain and a reduction in the number of patients with IVD-herniation-induced sciatica. Furthermore, the combination of anti-IL-8/CXCL8 antibodies enhances the effect of an anti-TNFα antibody on mechanical hyperalgesia in both the autologous IVD autograft model and the spinal nerve ligation model 72 .
Higher levels of the inflammatory mediator C-reactive protein (CRP) have been found in patients with sciatic neuropathy versus healthy controls 73 . Previous studies have suggested that NP cells may release many mediators that contribute to the pain experience. Inflammatory agents that might be released from NP cells could affect the dorsal root ganglion to enhance the primary afferent excitation of nociception 74 . In addition, NP cells applied onto the dorsal nerve roots enhance the spinal C-fiber response and macrophage activation 75 . Importantly, the release of CXCL8 from the NP plays a critical role in IVD degeneration and immune cell infiltration 35 . Clinically, over-expression of the chemokine CCL3 has been shown to be positively correlated with the grade of tissue degeneration by recruiting macrophages 76 . Clinical observations have also shown that serum levels of CXCL8 are associated with long-lasting pain in patients with disc herniation 77 and increased levels of CXCL8 in the cerebrospinal fluid are also correlated with disc herniation 78 and worsening of fibromyalgia-related pain severity 79 . Moreover, degenerated and herniated human intervertebral discs may secrete high levels of CXCL8 80 . It has also been reported that intra-plantar CXCL8 injection induces a persistent hypersensitivity 81 , which can be blocked using a CXCL8 receptor antagonist 34 .
A previous study has reported that ERK1/2 phosphorylation in NP cells has been observed in degenerated human intervertebral disc 82 . Moreover, thrombin also enhances CXCL8 expression through ERK activation 83 . It has also been demonstrated that transcriptional activation of the phosphoinositide 3 (PI3)-kinase/Akt pathway is involved in lumbar disc degeneration 84 and thrombin activates the PI3 kinase/Akt signaling in neural retina 85 . A recent report, using bioinformatics, also indicated that ERK, PI3 kinase/Akt, and EGFR (ErbB) signaling pathways are potentially involved in intervertebral disc degeneration 50 . Beyond these, Src and FAK have been reported as being key early signaling molecules that mediate neurite growth in response to growth factor stimulation 86 and thrombin has also been reported to induce c-Src activation 87 .
In conclusion, using standard molecular biological tools we have identified CXCL8 as a critical cytokine/ chemokine important to IVD cells. Using a microarray of human tissues and in vitro stimulation of healthy NP cells, we confirmed a role for thrombin and the EGF receptor signaling pathways in the onset of IVD. This study suggests that targeting the EGFR pathway may be of crucial importance in the preventive treatment of disc Figure 6. The Akt and GSK3α/β signaling pathways are involved in thrombin-enhanced EGFR activation in NP cells. (A) NP cells were treated with thrombin for indicated periods, cell lysates were separated by SDS-PAGE and immunoblotted with anti-phosphorylated-Akt and anti-phosphorylated-GSK3α/β. (B) NP cells were treated with AG1478 (3 μM) for 30 min followed by treatment with thrombin for 30 min, and the levels of phosphorylated-Akt, Akt, phosphorylated GSK3α/β, and GSK3α/β were determined by western blotting. Similar results were obtained from four independent experiments. (C) NP cells were treated with LY294002 (10 μM), or SB216763 (12.5 μM) for 30 min followed by treatment with thrombin (10 U/mL) for 24 h. The production of CXCL8 was determined by ELISA. Each bar represents the mean ± S.E.M. from at least three independent experiments performed in duplicate. *p < 0.05 compared with the control group. # p < 0.05 compared with the thrombin-treated group. (D) NP cells were treated with U0126 (1 μM), PF573228 (1 μM), PP2 (1 μM), LY294002 (10 μM), or SB216763 (12.5 μM) for 30 min followed by treatment with thrombin for 60 min, and phosphorylated-ERK and ERK2 levels were determined by western blot. Similar results were obtained from three independent experiments. degeneration that leads to chronic low back pain. The results of this study may also contribute to the development of post-operation treatments and/or a diagnosis for the IVD degenerative disease.