Abstract
Background: Cartilage regeneration requires a balance of anabolic and catabolic processes. Aim: To examine the susceptibility of fibromodulin (FMOD) and lumican (LUM) to degradation by MMP-13, ADAMTS-4 and ADAMTS-5, the three major degradative proteinases in articular cartilage, in cartilage development and in osteoarthritis (OA). Methods: Immunolocalization of FMOD and LUM in fetal foot and adult knee cartilages using an FMOD matrix metalloprotease (MMP)-13 neoepitope antibody (TsYG11) and C-terminal anti-FMOD (PR184) and anti-LUM (PR353) antibodies. The in vitro digestion of knee cartilage with MMP-13, A Disintegrin and Metalloprotease with Thrompospondin motifs (ADAMTS)-4 and ADAMTS-5, to assess whether FMOD and LUM fragments observed in Western blots of total knee replacement specimens could be generated. Normal ovine articular cartilage explants were cultured with interleukin (IL)-1 and Oncostatin-M (OSM) ± PGE3162689, a broad spectrum MMP inhibitor, to assess FMOD, LUM and collagen degradation. Results and Discussion: FMOD and LUM were immunolocalized in metatarsal and phalangeal fetal rudiment cartilages and growth plates. Antibody TsYG11 localized MMP-13-cleaved FMOD in the hypertrophic chondrocytes of the metatarsal growth plates. FMOD was more prominently localized in the superficial cartilage of normal and fibrillated zones in OA cartilage. TsYG11-positive FMOD was located deep in the cartilage samples. Ab TsYG11 identified FMOD fragmentation in Western blots of normal and fibrillated cartilage extracts and total knee replacement cartilage. The C-terminal anti-FMOD, Ab PR-184, failed to identify FMOD fragmentation due to C-terminal processing. The C-terminal LUM, Ab PR-353, identified three LUM fragments in OA cartilages. In vitro digestion of human knee cartilage with MMP-13, ADAMTS-4 and ADAMTS-5 generated FMOD fragments of 54, 45 and 32 kDa similar to in blots of OA cartilage; LUM was less susceptible to fragmentation. Ab PR-353 detected N-terminally processed LUM fragments of 39, 38 and 22 kDa in 65–80-year-old OA knee replacement cartilage. FMOD and LUM were differentially processed in MMP-13, ADAMTS-4 and ADAMTS-5 digestions. FMOD was susceptible to degradation by MMP-13, ADAMTS-4 and to a lesser extent by ADAMTS-5; however, LUM was not. MMP-13-cleaved FMOD in metatarsal and phalangeal fetal rudiment and growth plate cartilages suggested roles in skeletogenesis and OA pathogenesis. Explant cultures of ovine cartilage stimulated with IL-1/OSM ± PGE3162689 displayed GAG loss on day 5 due to ADAMTS activity. However, by day 12, the activation of proMMPs occurred as well as the degradation of FMOD and collagen. These changes were inhibited by PGE3162689, partly explaining the FMOD fragments seen in OA and the potential therapeutic utility of PGE3162689.
Highlights
The prevalence of knee osteoarthritis (OA) has doubled since the mid-twentieth century despite concerted efforts of the research and pharmaceutical industries over the last five decades to develop a therapeutic solution [1]
We initially examined the immunolocalization of FMOD and matrix metalloprotease (MMP)-13-cleaved FMOD in human developmental phalangeal and metatarsal cartilage rudiments and metatarsal growth plates (Figure 1)
While FMOD fragmentation has been reported in RA and OA articular cartilage [28,70] and with ageing [71], we have recently found minimal fragmentation compared with other small leucine-rich proteoglycan (SLRP) in OA knee menisci and knee and hip articular cartilage when a C-terminal antibody was used for FMOD detection [72]
Summary
The prevalence of knee osteoarthritis (OA) has doubled since the mid-twentieth century despite concerted efforts of the research and pharmaceutical industries over the last five decades to develop a therapeutic solution [1]. The in vitro digestion of knee cartilage with MMP-13, A Disintegrin and Metalloprotease with Thrompospondin motifs (ADAMTS)-4 and ADAMTS-5, to assess whether FMOD and LUM fragments observed in Western blots of total knee replacement specimens could be generated. Normal ovine articular cartilage explants were cultured with interleukin (IL)-1 and Oncostatin-M (OSM) ± PGE3162689, a broad spectrum MMP inhibitor, to assess FMOD, LUM and collagen degradation. Ab TsYG11 identified FMOD fragmentation in Western blots of normal and fibrillated cartilage extracts and total knee replacement cartilage. In vitro digestion of human knee cartilage with MMP-13, ADAMTS-4 and ADAMTS-5 generated FMOD fragments of 54, 45 and 32 kDa similar to in blots of OA cartilage; LUM was less susceptible to fragmentation. Ab PR-353 detected N-terminally processed LUM fragments of 39, 38 and 22 kDa in 65–80-year-old OA knee replacement cartilage. These changes were inhibited by PGE3162689, partly explaining the FMOD fragments seen in OA and the potential therapeutic utility of PGE3162689
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