Identification and characterization of the transient receptor potential (TRP) channels in the intervertebral disc

Abstract

Low back pain (LBP) has a high prevalence and is a major public health problem globally. Intervertebral disc degeneration (IVD), which is the central cause of LBP, is a natural process with an early onset. Due to the rapidly aging population in the western society, it is predicted that occurrences of LBP as well as its related costs will further increase, causing a high economic burden on the individual and the society. Biomechanical, biological and genetic factors have an impact on the development of painful IVD degeneration (also called degenerative disc disease). However, treatment options are limited. Therefore, there is a clear need for advancing our understanding of IVD degeneration and designing novel treatment strategies. Transient receptor potential (TRP) channels constitute a family of Ca2+ permeable cell membrane receptors and have recently emerged as potential contributors to tissue homeostasis, inflammation and pain. TRP channels were investigated as biomarkers and potential therapeutic targets in many other tissues, but so far, their role remains largely unknown in the IVD. This thesis attempts to address this knowledge gap. The objective of this thesis was to identify and characterize TRP channels and proinflammatory factors in the IVD, with the goal to evaluate their therapeutic potential. In the first study, the occurrences of inflammatory processes and the expression of TRPC6 and TRPV4 in human degenerated tissue from the lumbar and cervical IVDs were measured and compared. Aside from genes with known implication in degenerative disc disease (DDD) or disc herniation (DH), four previously not recorded genes from the interferon and TRP families (IFNα1, IFNα8, IFNβ1, TRPC6) could be detected in the human degenerated IVD tissue. A correlation between gene expression and age (IL-15) and IVD degeneration grade (IFNα1, IL-6, IL-15, TRPC6), but not Modic grade, was identified. Significant differences were found between cervical and lumbar IVDs (IL-15), nucleus and annulus (IL-6, TNF-α, TRPC6), single-level and multi-level surgery (IL-6, IL-8) as well as DDD and DH (IL-8), while sex had no effect. Multiple gene-gene pair correlations, either between different cytokines (e.g. IL-6 and IL-8) or between cytokines and TRP channels (e.g. TRPC6 and IL-15/IL-8/IL-6), exist in the disc. Presented findings support the importance of IL-6 and IL-8 during IVD degeneration, but additionally demonstrate a potential pro-inflammatory role of IL-15 and interferon type I as well as a mechanistic role of TRPC6 in the disease. This study concluded that despite existing tissue- or patient-specific differences, novel anti-inflammatory or TRP-modulatory strategies for the treatment of disc pathologies may be applicable independently of the spinal region. In the second study, the gene and protein expression pattern of currently known TRP channels in non-degenerated and degenerated human IVD tissue was analyzed. Patient- and tissue- specific characteristics, such as age, IVD degeneration grade, pain intensity and chronicity and others were included in the analysis. For the first time we could show that 26 out of 28 currently known TRP channels are expressed in the IVD on the gene level, thereby revealing novel therapeutic candidates from the TRPC, TRPM and TRPML subfamilies. TRPC6, TRPM2 and TRPML1 displayed enhanced gene and protein expression in degenerated IVDs as compared with non-degenerated IVDs. Furthermore, the gene expression of TRPC6 and TRPML1 was influenced by the IVD degeneration grade. Pain intensity and/or chronicity affected the gene and/or protein expression of TRPC6, TRPM2 and TRML1, therefore suggesting their therapeutic potential. Interestingly, decreased gene expression of TRPM2 was observed in patients treated with steroids. This is the first study to present the link between TRP channels and pain in the IVD. Thus, these results represent the current benchmark for the TRP channel expression pattern in the IVD. Our findings support the importance of TRP channels in IVD homeostasis and pathology, and their possible application as pharmacological targets for the treatment of IVD degeneration and LBP. The objective of the third study was to investigate the effect of hypo-osmotic stress, which is one of the main hallmarks of IVD degeneration, on the gene expression pattern and calcium signaling of TRPV4 and TRPM7 channels in bovine IVD cells. As the first in the field, we presented a complete screening of currently known TRP channel in bovine IVD cells. In line with the results obtained in human IVD tissue, TRPC1, TRPM7 and TRPV4 were overall the most highly expressed TRP channels in bovine IVD cells. We determined that TRPV4 gene expression was downregulated in hypo-osmotic condition, whereas its Ca2+ flux increased. RNA sequencing identified over 3000 up- or down-regulated targets, from which we selected aggrecan, ADAMTS9 and IL-6 and investigated whether their altered gene expression is mediated through either the TRPV4 or TRPM7 channel, using specific activators and inhibitors (GSK1016790A/ GSK2193874 for TRPV4 and Naltriben/ NS8593 for TRPM7). Application of TRPV4 selective activator (GSK1016790A) induced the expression of IL-6 under iso-osmotic condition, proportionally to hypo-osmotic stimulation alone, indicating that this effect might be TRPV4-mediated. However, the application of TRPV4 selective blocker (GSK2193874) failed to inhibit the increase of IL-6 under hypo-osmotic condition. A treatment with TRPM7-activator did not cause significant changes in the gene expression of tested targets. We concluded that although TRPV4 and TRPM7 are likely involved in osmosensing in the IVD, neither of them mediates hypo-osmotically- induced gene expression changes of aggrecan, ADAMTS9 and IL-6. This thesis has made several significant contributions to the field of spine research. Through a detailed identification and characterization of TRP channels in the IVD, it revealed their important role in IVD degeneration. It demonstrated that the expression levels of TRP channels and pro-inflammatory factors may depend on the specific characteristics of the IVD tissue (e.g. lumbar versus cervical, degeneration grade) or patients (e.g. pain intensity/chronicity). Importantly, results of these experiments highlighted novel therapeutic targets from the TRPC, TRPM and TRPML family, which are not only linked to low back pain intensity or chronicity, but also to the level of inflammation. These findings may contribute to the design of new biomarker-based tests and help develop novel pharmacological treatments for painful IVD degeneration and LBP. Moreover, RNA sequencing data presented in this thesis can serve spine scientist as a source for further research. In conclusion, IVD tissue- and/or patient-dependent expression pattern of TRP channels presented over the course of this study suggests that TRP channels have a potential to become novel therapeutic targets in painful IVD degeneration. Their pharmacological modulation may prove beneficial in treating pain and inflammation, could contribute to the regulation of the IVD tissue homeostasis and might ultimately change clinical practice related to discogenic back pain in the future.