Abstract
AMD3100 is a small-molecule inhibitor of the C-X-C motif chemokine ligand 12/C-X-C chemokine receptor type 4 (CXCL12/CXCR4) axis, while its role in aggrecan metabolism is unclear. We hypothesized that the AMD3100 modulates the transforming growth factor-β1 (TGF-β1)-induced expression of tissue inhibitor of metalloproteinase-3 (TIMP-3) in chondrocytes. We evaluated expression of CXCL12/CXCR4 and TIMP-3 in the knee joints of rats with and without osteoarthritis (OA) by immunohistochemistry, immunofluorescence, Western blotting, and enzyme-linked immunosorbent assay (ELISA). The rats were divided into sham control, destabilization of the medial meniscus/AMD3100-treated (DMM/AMD3100-treated), and DMM/phosphate-buffered saline (PBS)-treated groups. After 6 weeks, the rats were euthanized and subjected to histological and immunohistochemical analyses. Also, interleukin (IL)-1-pretreated primary chondrocytes were cultured in the presence of empty control (−, −), CXCL12a (+,−), CXCL12a + small interfering RNA (siRNA) CXCR4 (+,+), or CXCL12a + siNC (+NC), and the expression levels of target markers were evaluated by Western blotting and real-time reverse transcription PCR (RT-PCR). The CXCL12/CXCR4 levels were higher, and the expression of TIMP-3 was lower, in the OA rats compared to the healthy control rats. The rats in the DMM/AMD3100-treated group revealed a markedly decreased immunological response and mild pathology. Treatment with CXCL12a increased expression of aggrecan and disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5) and suppressed that of TIMP-3 in IL-1-pretreated primary chondrocytes. TGF-β1 increased expression of TIMP-3, and this increase was reversed by CXCL12a via the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Moreover, these effects were inhibited by the CXCR4 antagonist AMD3100 and the PI3K inhibitor LY303511. In conclusion, inhibition of the CXCL12a/CXCR4 signaling axis maintained TIMP-3 expression via the PI3K/Akt pathway. Our findings provide insight into the mechanism by which AMD3100 prevents OA.
Highlights
Osteoarthritis (OA) is a common chronic disease and a leading cause of chronic pain and physical disability, afflicting over 15% of the elderly population (Yelin et al, 2019)
Expression of Tissue inhibitor of metalloproteinase-3 (TIMP-3) Was Low and That of the CXCL12/CXCR4 Axis Was High in Rats With OA
Immunofluorescence staining showed that 92.2% of chondrocytes and 62.7% of synoviocytes in the OA rats were positive for CXCR4, compared to 11.2 and 5.2%, respectively, in the healthy control rats (Figures 1A, B)
Summary
Osteoarthritis (OA) is a common chronic disease and a leading cause of chronic pain and physical disability, afflicting over 15% of the elderly population (Yelin et al, 2019). Progressive deterioration and loss of articular cartilage is crucial in the pathogenesis of OA (Onuora, 2015; Bortoluzzi et al, 2018). Type II collagen and aggrecan are important structural components of the cartilage extracellular matrix (ECM) and are the primary determinants of the mechanical properties of cartilage. Type II collagen has a three-helical network structure and provides the structural strength and elasticity of articular cartilage. Multiple aggrecan monomers bind to hyaluronan and to link proteins to fill the interstices of the collagen network, which plays a role in lubrication and resistance to compression. Because aggrecan prevents degradation of collagen fibrils, its loss is an important event in early stage OA (Wilusz et al, 2014; Carballo et al, 2017). Matrix metalloproteinases (MMPs), the collagenase matrix metalloproteinase 13 (MMP-13), are involved in degradation of type II collagen (Wang et al, 2013a; Onitsuka et al, 2018), and disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-5) is responsible for degradation of aggrecan (Malfait et al, 2010; Verma and Dalal, 2011; Larkin et al, 2015)
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