Roles of p38 MAPK signalling in intervertebral disc degeneration

Abstract Intervertebral disc degeneration (IVDD) is a common degenerative disease mediated by multiple factors. Because of its complex aetiology and pathology, no specific molecular mechanisms have yet been identified and no definitive treatments are currently available for IVDD. p38 mitogen‐activated protein kinase (MAPK) signalling, part of the serine and threonine (Ser/Thr) protein kinases family, is associated with the progression of IVDD, by mediating the inflammatory response, increasing extracellular matrix (ECM) degradation, promoting cell apoptosis and senescence and suppressing cell proliferation and autophagy. Meanwhile, the inhibition of p38 MAPK signalling has a significant effect on IVDD treatment. In this review, we first summarize the regulation of p38 MAPK signalling and then highlight the changes in the expression of p38 MAPK signalling and their impact on pathological process of IVDD. Moreover, we discuss the current applications and future prospects of p38 MAPK as a therapeutic target for IVDD treatment.


| INTRODUCTION
Low back pain (LBP) is a widespread health problem that is a leading contributor to disability as well as the global burden of disease. 1 Intervertebral disc (IVD) degeneration (IVDD) is a common degenerative musculoskeletal disease that is regarded as the main cause of LBP.
Normal IVD consists of avascular tissue and is primarily composed of an outer annulus fibrosus (AF), an internal nucleus pulposus (NP) and cranially and caudally cartilaginous endplates (CEPs). 2,3 Due to ageing, mechanical overloading, nutritional deficiency, oxidative stress and other factors, the above-mentioned structures can exhibit pathological changes and loss of biological function, including reduction in proteoglycan and water content, the decline in IVD height, endplate sclerosis and osteophyte formation and decreased ability to withstand compression load. [3][4][5] IVDD progression may lead to spinal degenerative diseases, including disc herniation, spinal canal stenosis, spondylolisthesis and degenerative scoliosis, which can cause pain and disability. 6 Although IVDD has been studied to a certain extent in recent years, the underlying mechanisms remain unclear, and effective treatment approaches are not available. Therefore, the need to explore the underlying mechanisms and develop novel therapeutic approaches for the early recovery of IVDD is a matter of urgency.
Mitogen-activated protein kinases (MAPKs) are serine and threonine (Ser/Thr) protein kinases that convert extracellular stimuli into a broad range of cellular responses. MAPKs are highly conserved regulatory mechanisms in mammals and are divided into three main groups, namely extracellular signal-regulated kinases (ERKs), c-Jun Nterminal kinases (JNKs) and p38 isoforms (α, β, γ and δ). 6,7 As a member of the MAPK family, p38 MAPK signalling is involved in multiple diseases, such as inflammatory, 8 cardiovascular 9 and neurodegenerative diseases, 10 as well as cancer. 11 Meanwhile, a growing body of evidence indicates that p38 MAPK signalling is a suitable therapeutic target and a potential treatment strategy for several diseases. [12][13][14] For the past few years, p38 MAPK signalling has received considerable attention in the field of IVD research. An increasing number of studies have shown that dysregulation of p38 MAPK signalling is associated with IVDD progression, including IVD cell death, inflammatory response, imbalance of the extracellular matrix (ECM) homeostasis and other cell phenotypes. [15][16][17] Simultaneously, targeting p38 MAPK signalling has shown promising therapeutic potential in alleviating IVDD. In the following sections, we will focus on describing the regulation of p38 MAPK signalling, changes in its expression and its role in the process of IVDD. Moreover, we will discuss the approaches for regulating p38 MAPK signalling as a treatment strategy for IVDD.

| p38 MAPK SIGNALLING
p38 MAPK is a class of evolutionarily conserved MAPKs that transduce extracellular signals to regulate multiple cellular processes. The p38 MAPK family has four isoforms, consisting of p38α (MAPK14), p38β (MAPK11), p38γ (MAPK12) and p38δ (MAPK13), which are encoded by distinct genes. 18,19 Among the four isoforms, sequence identity is conserved within the kinase domains, and there is sequence homology. For example, p38α shares 75% sequence identity with p38β and 60% with p38γ and p38δ. In addition, p38γ and p38δ have approximately 70% similarity. 20,21 Despite their high sequence homology, these isoforms have notable differences in tissue expression, sensitivity to chemical inhibitors and cellular functions. For example, p38α and p38β are ubiquitously expressed in most of cells and tissues, whereas p38γ and p38δ have more restricted expression patterns. 22 Significantly, the four isoforms have different sensitivity to chemical inhibitors, such as SB203580. 21 In addition, because genetic ablation of p38α (MAPK14) results in embryonic lethality in mice, p38α has been proven to be the only p38 kinase essential for mouse embryo development. 23 However, ablation of other isoforms, or disruption of single p38δ and p38γ, does not result in major abnormalities in live mice. 24,25 Overall, current evidence suggests that p38α and p38β act together in heart development, sex determination, mitotic entry inhibition and induction of T-cell immunity. [26][27][28][29] p38γ and p38δ can cooperate in tissue regeneration and immune responses in certain cell lines. 30 Interestingly, some research has shown that a lack of p38α can promote the activation of p38γ or p38δ. 31,32 Taken together, based on the complex relationship between the isoforms, the biological functions of this intricate signalling pathway require further exploration.

| REGULATION OF p38 MAPK SIGNALLING
In mammals, p38 MAPK can be activated by many extracellular stimuli such as oxidative and osmotic stress, ultraviolet radiation, hypoxia, ischaemia, interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-α). 19 Activated p38 MAPK regulates embryonic development, immune responses, cell cycle, endocytosis, metabolism and cytoskeleton dynamics by phosphorylating downstream substrates. 33 Generally, the activation of p38 MAPK is associated with the MAPK kinase kinase (MAP3K)-MAP kinase kinase (MKK) pathway ( Figure 1). First, extracellular stimuli, such as oxidative stress and cytokines can activate MAP3Ks via phosphorylation or by promoting their interaction with the Ras homologous (Rho) protein, cell division control protein 45 (CDC45) and Rac small GTPases. Next, the activation of MAP3Ks can phosphorylate MAP2Ks, including MKK3, MKK4 and MKK6. Notably, among active MAP3Ks, only apoptosis signal-regulating kinase 1 (ASK1) can activate MKK4 that specifically promotes the activation of p38α. 34 Moreover, the phosphorylation of MKK3 activates p38α and p38β together, and the phosphorylation of MKK6 is responsible for the activation of p38γ and p38δ. 35,36 In addition to the above regulatory pattern, activation of p38α can also depend on the autophosphorylation of p38α. This activation mechanism is independent of MAP2K. Mechanistically, TAK1-binding protein 1 (TAB1) can directly interact with p38α and promote autophosphorylation on Thr 180 and Tyr 182, resulting in full activation of p38α. 37 Furthermore, p38α and p38β can also be activated by the T cell antigen receptor (TCR) signalling pathway in addition to the MAP2K-dependent mechanism. 38 Overall, phosphorylated p38 MAPK is located in the cytoplasm and nucleus, which, in turn, can activate a series of substrates, such as transcription factors, protein kinases and cytosolic and nuclear proteins.
Contrarily, the activated p38 MAPK can be dephosphorylated and thereby deactivated by protein phosphatases such as dualspecificity phosphatases, serine/threonine phosphatases and tyrosine phosphatases. 39 A series of post-translational modifications can also mediate the deactivation of p38 MAPK, including ubiquitination, acetylation and methylation. 40 Moreover, one study showed that the phosphorylation of p38α can negatively regulate the expression of the p38γ protein via the ubiquitin-proteasome pathways. 32 This result represents a novel mechanism by which p38 MAPK isoforms can be regulated by each other. Taken together, the regulation of p38 MAPK is associated with multiple regulatory mechanisms. Therefore, identifying specific mechanisms that are responsible for this regulation in certain cellular processes is a great challenge.

| EXPRESSION CHANGES OF p38 MAPK IN IVDD
Current evidence indicates that the expression level of p38 MAPK changes with the degree of IVDD and shows a significant difference in NP versus AF tissue. Yang et al. 41 first found that the expression level of p38 MAPK in the NP tissue was higher than that in the AF tissue. Moreover, the expression levels of the four p38 MAPK isoforms also showed a considerable difference. The expression levels of p38α and p38β were remarkably higher in the IVDD tissue compared with that in the normal NP tissue, while the expression level of p38δ was lower in the IVDD tissue compared with that in the normal NP tissue and could only be detected in half of the IVDD tissue; further, the expression level of p38γ was lower than that of p38δ. In vivo, Dai et al. 42 showed that the expression level of the activated p38 MAPK was significantly higher in the rat IVDD groups compared with the control group ( p < 0.01). Cheng et al. 43 found a significant increase in the expression level of activated p38 in degenerated rat NP tissues. In addition, a series of reports confirmed that the expression level of activated p38 was higher in degenerative NP cells than that in normal NP cells. From the above results, we can conclude that p38 MAPK may play a more important role in the NP tissue rather than in the AF tissue. In contrast, though the expression of p38α and p38β is higher than that of p38δ and p38γ, there is still no specific evidence as to which whether p38α or p38β is mainly responsible for the progress of IVDD. Therefore, it is necessary to further explore the individual functions of p38α and p38β in IVDD progression.

| ACTIVATING p38 MAPK SIGNALLING CONTRIBUTES TO IVDD
As mentioned previously, p38 MAPK activation is involved in IVDD progression. Meanwhile, increasing evidence has suggested that activated p38 MAPK is associated with the pathophysiological characteristics of IVDD, including the inflammatory response, ECM degradation, IVD cell apoptosis, proliferation and senescence and level of autophagy ( Figure 2). Below, we address the mechanisms and functions of p38 MAPK signalling in IVDD.

| Mediating inflammatory response
An inflammatory response is a crucial factor in the progression of IVDD.
Degenerated IVD tissue contains inflammatory-like cells that can spontaneously produce chemokines, including monocyte chemoattractant protein (MCP)-1 and C-C motif chemokine ligand 4 (CCL4). These chemokines can recruit macrophage infiltration that can release inflammatory cytokines to promote IVDD progression. 44,45 Moreover, several reports suggest that the activation of p38 MAPK signalling is associated with mediating the inflammatory response in IVDD. For example, Yang et al. 41 indicated that the knockdown of p38α can completely suppress macrophage M1 polarization, and the downregulation of p38β or p38δ can partially inhibit macrophage M1 polarization. In addition, it is well known that macrophage M1 polarization has pro-inflammatory functions. 46 A recent study found that the expression of CCL4 was elevated in degenerated NP tissue and associated with macrophage infiltration.
Mechanically, the expression level of CCL4 was regulated by p38 MAPK and NF-κB, and using the inhibitors of p38 MAPK and NF-κB can suppress the upregulation of CCL4 expression. 47 Moreover, inhibiting the activation of p38 MAPK signalling suppresses the production of inflammatory cytokines. Park et al. 48  signalling inhibitor, SB202190, can decrease the production of IL-6, IL-8 and TNF-α and inhibit IVDD progression. Genistein, a type of protein tyrosine kinase inhibitor, has been proven to suppress the expression of IL-1β and TNF-α by inhibiting the p38 MAPK signalling. 49 In addition, Studer et al. 16 found that p38 MAPK signalling was involved in nitric oxide (NO), prostaglandin E2 (PGE2) and IL-6 production. Inhibition of p38 MAPK was found to decrease PGE2 and IL-6 accumulation. Overall, there is increasing evidence that activated p38 MAPK signalling can promote the inflammatory response. Therefore, inhibition of p38 MAPK may be a promising strategy for alleviating IVDD progression.

| Increasing ECM degradation
In healthy IVD, the anabolism and catabolism of ECM are in balance. and ADAMTSs (ADAMTS-4 and ADAMTS-5) but decreased the expression of tissue inhibitors of metalloproteinases (TIMPs) in the IVD tissue of diabetic rats. 43 In addition, a recent study showed that the umbilical cord mesenchymal stem cell-conditioned medium (MSC-CM) can alleviate NP mesenchymal stem cells' (NPMSCs') degeneration via high glucose levels. Specifically, they found that the use of MSC-CM inhibited the activation of p38 MAPK signalling to promote the levels of collagen II and aggrecan. 51 Taken together, the above results suggest that inhibiting the activation of p38 MAPK signalling can promote the restoration of ECM homeostasis in IVDD.

| Promoting apoptosis and senescence
IVD cell apoptosis is widely considered to play a crucial role in the process of IVDD. Excessive IVD cell apoptosis can accelerate the decrease in cell density and catabolism of the ECM. Therefore, a decreased rate of cell apoptosis could improve the progression of F I G U R E 2 Functions of activated p38 MAPK signalling in the progression of intervertebral disc degeneration (IVDD). The activation of p38 MAPK signalling is associated with the progression of IVDD, including mediating the inflammatory response, increasing extracellular matrix (ECM) degradation, promoting cell apoptosis and senescence and suppressing cell proliferation and autophagy.
IVDD. In the past few years, growing evidence has demonstrated that the activation of p38 MAPK signalling is involved in IVD cell apoptosis. For example, IL-1β and TNF-α can induce NP cell apoptosis by promoting the expression of caveolin-1 to activate Wnt/β-catenin signalling. However, the p38 MAPK inhibitor can reverse IL-1β or TNFα-mediated apoptosis by inhibiting the activity of caveolin-1/ β-catenin cox. 52 Xu et al. 53

| Suppressing proliferation
The dysregulation of IVD cell proliferation is also considered an indicator of IVDD. It has been reported that p38 MAPK signalling can regulate IVD cell proliferation. For example, Wu et al. 60 revealed that IL-1β could activate p38 MAPK signalling to decrease the expression of histone deacetylase 4 (HDAC4), which resulted in an aggravation of the inflammatory response and cell cycle arrest. However, the use of the p38 MAPK inhibitor, SD0006, can suppress inflammation and promote cell proliferation by restoring the expression of HDAC4 and inhibiting p38 MAPK signalling. In another report, IL-2 was also found to inhibit cell proliferation by promoting the activation of p38 MAPK signalling. 61 Moreover, the study showed that oxidative stress can lead to a G1 cell cycle delay and decrease cell proliferation by activating p38 MAPK signalling. 62 Taken together, the activation of p38 MAPK signalling has a negative effect on IVD cell proliferation. However, the mechanism by which IVD cell proliferation regulates is still unclear, and further exploration is necessary.

| Inhibiting autophagy
Autophagy is a conserved intracellular degradation process that maintains metabolism and homeostasis. In recent years, autophagy has been proven to play a crucial role in the progress and treatment of IVDD. 63

| TARGETING p38 MAPK SIGNALLING FOR THE TREATMENT OF IVDD
Over the past few years, developing p38 MAPK inhibitors has been a potential strategy for the treatment of multiple diseases, such as rheumatoid arthritis, chronic obstructive pulmonary disease and cancer.
Certainly, the use of p38 MAPK inhibitors results in an obvious improvement in the prognosis and survival rates for some diseases. In recent years, targeting p38 MAPK by the application of p38 MAPK inhibitors has been shown to alleviate the progression of IVDD in vitro and in vivo ( Table 1).

| p38 MAPK inhibitors
At present, p38 MAPK inhibitors are only used in disease models for the IVDD treatment because of their limitations in safety and method of use. For example, SB202190, as a p38 MAPK inhibitor, has been found to inhibit the inflammatory response and ECM degradation to degenerate NP cells. 16,48 SB203580 is another p38 MAPK inhibitor that was found to decrease the rate of cell apoptosis and inhibit the production of inflammatory cytokines, which, in turn, alleviated the progression of IVDD in a rat IVDD model. 47,66,72,73 A recent study showed that another p38 MAPK inhibitor, SD0006, could also inhibit the inflammatory response and promote NP cell proliferation by regulating the p38 MAPK/HDAC4 pathway. 60 Moreover, genistein, as a protein tyrosine kinase inhibitor, has been identified to inhibit the inflammatory response and delay the process of IVDD by inhibiting p38 MAPK. 49 In addition to specific p38 MAPK inhibitors, there are T A B L E 1 Applications of p38 MAPK inhibitors in intervertebral disc degeneration (IVDD) treatment.