Abstract
Intervertebral disc (IVD) degeneration is associated with local inflammation and increased expression of neurotrophins. Acidic microenvironment is believed to cause the progression of IVD degeneration. However, there is a paucity of information regarding the relationship between acidic microenvironment and the inflammation and expression of neurotrophins in IVD. G-protein-coupled receptor 4 (GPR4) is a pH-sensing receptor, which can activate the inflammation and increase the expression levels of nerve growth factor in acidic microenvironment. In this study, culture media with pH 7.2 (representing the normal IVD-like acidic condition) and pH 6.5 (degenerated IVD-like acidic condition) were prepared. The gene and protein expression levels of GPR4 in SD rat nucleus pulposus cells were determined under the acidic conditions. And cyclic AMP (cAMP), the second messenger of GPR4, was assayed. Furthermore, the expression levels of receptor activator of nuclear factor κ B (RANK), RANKL ligand (RANKL), osteoprotegerin (OPG), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) were also determined. To clarify the involvement of GPR4 in the upregulation of the expression of RANK/RANKL/OPG system and neurotrophins, gene knockdown and forced expression of GPR4 and inhibiting its downstream cAMP accumulation and Ca2+ mobilization were performed. The alternation of the expression levels of matrix metalloproteinase-3 (MMP-3), MMP-13, and aggrecanase-2 (ADAMTS-5) were evaluated by RT-PCR and western blot. The results showed that GPR4 was expressed in rat nucleus pulposus cells, and the expression was upregulated under the degenerated IVD-like acidic microenvironment. cAMP accumulation levels were increased under the degenerated IVD-like acidic culture conditions. The expression levels of RANK, RANKL, OPG, NGF, and BNDF were significantly upregulated under the degenerated IVD-like acidic microenvironment. GPR4 knockdown and reduction of cAMP by the inhibitor SQ22536 abolished the upregulation of the expression of RANK, RANKL, OPG, NGF, and BNDF under the degenerated IVD-like acidic microenvironment. On the opposite, acidosis-induced cAMP accumulation and upregulation of RANK, RANKL, OPG, NGF, and BNDF were further promoted by GPR4 overexpression. The expression levels of MMP-3, MMP-13, and ADAMTS-5 were upregulated under the degenerated IVD-like acidic condition, which can be promoted or attenuated by GPR4 overexpression or knockdown, respectively. We concluded that GPR4-mediated cAMP accumulation was involved in the increased expression of RANK/RANKL/OPG system and neurotrophins by nucleus pulposus cells under the degenerated IVD-like acidic microenvironment.
Highlights
Low back pain is one of the most common and costly musculoskeletal problems worldwide, which increases a severe economic burden to the patients and the society [1, 2]
We found that Gprotein-coupled receptor 4 (GPR4) knockdown inhibited the cyclic AMP (cAMP) accumulation
We found that GPR4 was expressed by the nucleus pulposus cells and the expression level was increased in the degenerated Intervertebral disc (IVD)-like acidic condition when compared to the normal IVD-like acidic condition
Summary
Low back pain is one of the most common and costly musculoskeletal problems worldwide, which increases a severe economic burden to the patients and the society [1, 2]. The specific molecular mechanism of IVD degeneration remains elusive, local inflammation [3, 6] and elevated expression of neurotrophins [5, 7] have been identified as important players in the progression of disc degeneration. Tremendous amount of studies evidenced the abnormal expression of proinflammatory cytokines in the disc contribute to upregulate the expression of matrix-degrading enzymes [8, 9]. The upregulation of RANK/RANKL/OPG can result in the biosynthesis of extracellular matrix-degrading enzymes, including matrix metalloprotease-3 (MMP-3) and MMP-13 in degenerated IVD [11]. The increased expression of neurotrophins in severely degenerate discs has been implicated in chronic low back pain associated with the progression of disc degeneration [15, 16]
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