Abstract
Painful intervertebral disc (IVD) degeneration is an age-related process characterized by reduced tissue osmolarity, increased catabolism of the extracellular matrix, and elevated levels of pro-inflammatory molecules. With the aging population and constantly rising treatment costs, it is of utmost importance to identify potential therapeutic targets and new pharmacological treatment strategies for low back pain. Transient receptor potential (TRP) channels are a family of Ca2+ permeable cell membrane receptors, which can be activated by multitude of stimuli and have recently emerged as contributors to joint disease, but were not investigated closer in the IVD. Based on the gene array screening, TRPC1, TRPM7, and TRPV4 were overall the most highly expressed TRP channels in bovine IVD cells. We demonstrated that TRPV4 gene expression was down-regulated in hypo-osmotic condition, whereas its Ca2+ flux increased. No significant differences in Ca2+ flux and gene expression were observed for TRPM7 between hypo- and iso-osmotic groups. Upon hypo-osmotic stimulation, we overall identified via RNA sequencing over 3,000 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). GSK1016790A induced the expression of IL-6 under iso-osmotic condition, alike to hypo-osmotic stimulation alone, indicating that this effect might be TRPV4-mediated. However, using the TRPV4 blocker GSK2193874 failed to prevent 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. In conclusion, while 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.
Highlights
The intervertebral disc (IVD) is a mechanically loaded structure composed of two main tissues – the centrally located and highly hydrated nucleus pulposus (NP), which is surrounded by circular lamellar rings known as annulus fibrosus (AF) (Cassinelli et al, 2001)
The mRNA expression of 14 Transient receptor potential (TRP) channels (TRPC1-C7, TRPM3, TRPM7, TRPM8, TRPV2-V2, and TRPV6) was tested in bovine NP and AF cells obtained directly from tissue digest or passaged cells collected at P2
We could demonstrate that TRPC1 was consistently well expressed in bovine NP and AF cells, and its expression was unaffected by passaging, which is consistent with earlier results on human non-degenerated NP and AF tissue (Sadowska et al, 2019) as well as on human articular chondrocytes (Gavenis et al, 2009)
Summary
The IVD is a mechanically loaded structure composed of two main tissues – the centrally located and highly hydrated nucleus pulposus (NP), which is surrounded by circular lamellar rings known as annulus fibrosus (AF) (Cassinelli et al, 2001). The NP contains a small population of cells embedded in a loose matrix of collagen (COL) type II and proteoglycans (PGs), which enable the NP’s high water content (60–99%) and subsequent osmotic pressure (Cassinelli et al, 2001; Roughley et al, 2002). Biochemical and structural changes to the IVD affect its mechanical function and can promote inflammation. Inflammation in the IVD can be characterized by an increased expression of pro-inflammatory molecules such as interleukin (IL)-6, IL-1b, and tumor necrosis factor alpha (TNF-a), and contributes to the development of degenerative disc disease (DDD) and low back pain (LBP) (Molinos et al, 2015). There is a clear need to develop new treatment strategies, and to identify specific therapeutic targets, which mediate and/or promote homeostatic or inflammatory processes in the IVD
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