Abstract
ObjectiveThe aggrecanase ADAMTS‐5 and the collagenase matrix metalloproteinase 13 (MMP‐13) are constitutively secreted by chondrocytes in normal cartilage, but rapidly endocytosed via the cell surface endocytic receptor low‐density lipoprotein receptor–related protein 1 (LRP‐1) and subsequently degraded. This endocytic system is impaired in osteoarthritic (OA) cartilage due to increased ectodomain shedding of LRP‐1. The aim of this study was to identify the LRP‐1 sheddase(s) in human cartilage and to test whether inhibition of LRP‐1 shedding prevents cartilage degradation in OA.MethodsCell‐associated LRP‐1 and soluble LRP‐1 (sLRP‐1) released from human cartilage explants and chondrocytes were measured by Western blot analysis. LRP‐1 sheddases were identified by proteinase inhibitor profiling and gene silencing with small interfering RNAs. Specific monoclonal antibodies were used to selectively inhibit the sheddases. Degradation of aggrecan and collagen in human OA cartilage was measured by Western blot analysis using an antibody against an aggrecan neoepitope and a hydroxyproline assay, respectively.ResultsShedding of LRP‐1 was increased in OA cartilage compared with normal tissue. Shed sLRP‐1 bound to ADAMTS‐5 and MMP‐13 and prevented their endocytosis without interfering with their proteolytic activities. Two membrane‐bound metalloproteinases, ADAM‐17 and MMP‐14, were identified as the LRP‐1 sheddases in cartilage. Inhibition of their activities restored the endocytic capacity of chondrocytes and reduced degradation of aggrecan and collagen in OA cartilage.ConclusionShedding of LRP‐1 is a key link to OA progression. Local inhibition of LRP‐1 sheddase activities of ADAM‐17 and MMP‐14 is a unique way to reverse matrix degradation in OA cartilage and could be effective as a therapeutic approach.
Highlights
Shed soluble form of LRP-1 (sLRP-1) bound to ADAMTS-5 and matrix metalloproteinase 13 (MMP-13) and prevented their endocytosis without interfering with their proteolytic activities
Shedding of lipoprotein receptor–related protein 1 (LRP-1) is a key link to OA progression
The main cause of the disease is degradation of articular cartilage due to elevated activities of matrix metalloproteinases (MMPs) and ADAMTS. While both ADAMTS-4 and ADAMTS-5 have been considered to participate in aggrecan degradation in human OA [2,3], recent studies by Larkin et al with neutralizing monoclonal antibodies have shown that ADAMTS-5 is more effective than ADAMTS-4 in aggrecan degradation in human OA cartilage and nonhuman primates in vivo [4]
Summary
Cell-associated LRP-1 and soluble LRP1 (sLRP-1) released from human cartilage explants and chondrocytes were measured by Western blot analysis. Degradation of aggrecan and collagen in human OA cartilage was measured by Western blot analysis using an antibody against an aggrecan neoepitope and a hydroxyproline assay, respectively. Address correspondence to Kazuhiro Yamamoto, PhD, University of Oxford, Kennedy Institute of Rheumatology, Roosevelt Drive, Oxford OX3 7FY, UK. Human cartilage tissue preparation and isolation of chondrocytes. Tissue specimens were obtained from 9 patients (6 males, ages 9–57 years, mean age 35.5 years; 3 females, ages 13–19 years, mean age 15.7 years). Human OA articular cartilage was obtained from patients following total knee replacement surgery. Tissues were obtained from 16 patients (8 males, ages 51–86 years, mean age 75.1 years; 8 females, ages 50–82 years, mean age 68.1 years). Primary chondrocytes were used in the experiments to compare normal and OA chondrocytes, and passaged cells were used in the experiments to identify the LRP-1 sheddase
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