Neutrophil gelatinase–associated lipocalin is expressed in osteoarthritis and forms a complex with matrix metalloproteinase 9

Abstract

Objective. Expression of matrix metalloproteinase 9 (MMP-9) is up-regulated in osteoarthritis (OA) and usually presents as multiple bands when synovial fluid (SF) from OA patients is analyzed by zymography. Among these bands is an ϳ125-130-kd band for high molecular weight (HMW) gelatinase, which has not been characterized. This study was undertaken to characterize the HMW MMP activity in OA SF.Methods. MMP activity in OA SF was determined by gelatin zymography. Recombinant MMPs were used to identify MMP activity on the zymogram. Western immunoblotting, immunoprecipitation, and immunodepletion analyses were performed using antibodies specific for human MMP-9 and human neutrophil gelatinase-associated lipocalin (NGAL). Human cartilage matrix degradation was determined by dimethylmethylene blue assay.Results. Zymographic analysis showed that the HMW gelatinase in OA SF comigrated with a purified NGAL-MMP-9 complex. Results of Western immunoblotting showed that the HMW gelatinase was also recognized by antibodies specific for human NGAL or human MMP-9. These same antibodies also immunoprecipitated the HMW gelatinase activity from OA SF. The NGAL-MMP-9 complex was reconstituted in vitro in gelatinase buffer. In the presence of NGAL, MMP-9 activity was stabilized; in the absence of NGAL, rapid loss of MMP-9 activity occurred. MMP-9-mediated release of cartilage matrix proteoglycans was significantly higher in the presence of NGAL (P < 0.05).Conclusion. Our findings demonstrate that the HMW gelatinase activity in OA SF represents a complex of NGAL and MMP-9. The ability of NGAL to protect MMP-9 activity is relevant to cartilage matrix degradation in OA and may represent an important mechanism by which NGAL may contribute to the loss of cartilage matrix proteins in OA.The developed world's aging population has experienced a dramatic increase in the incidence of joint dysfunction and osteoarthritis (OA), leading to a compromised quality of life. Aging, biomechanical stress, and oxidative stress, in addition to genetic factors and trauma, all appear to be associated with degenerative and progressive cartilage and bone changes in OA, particularly in the weight-bearing joints. Although OA is not considered an inflammatory disease by traditional standards, elevated levels of proinflammatory cytokines, such as interleukin-1␤ (IL-1␤) and tumor necrosis factor ␣, have been observed in OA synovial fluid (SF), supporting the notion that there is an inflammatory component associated with the pathogenesis of OA (for review, see refs. 1-3).The only cell type present in articular cartilage is the chondrocyte, which is embedded within an extracellular matrix (ECM) primarily composed of type II collagen and aggrecan. Chondrocytes arise from a mesenchymal progenitor cell and are responsible for the biosynthesis of ECM proteins and their transport into the space occupied by the ECM. Chondrocytes play an important role in cartilage homeostasis by maintaining the proper balance between anabolic pathways, which Supported in part by the NIH...

Nerve growth factor increases MMP9 activity in annulus fibrosus cells by upregulating lipocalin 2 expression

“…Among the genes identified as being upregulated in the microarray study, Lcn2 was selected for further investigation in view of previous published work that indicates potential roles for this molecule in protecting MMP9 activity on cartilage matrix breakdown [16]. Real-time PCR confirmed increased expression of Lcn2 following NGF stimulation (Fig.…”

“…In basal cultures, gelatin zymography showed that culture media from AF cultures were rich in 62-, 72-, 82-, 92-, and 125-kDa MMP activity. MMP activity at 92-and 82-kDa and 72-and 62-kDa has previously been determined to represent latent and active forms of MMP9 and MMP2, respectively, whereas 125-kDa activity has been demonstrated to be due to an Lcn2-MMP9 complex [16]. When NGF is added to the cultures, the 125-kDa activity was increased significantly and inhibited by addition of the NGF inhibitor Ro 082750 (Fig.…”

“…Lcn2 has been shown to form a complex with MMP9 to stabilize and protect MMP9 activity [16] and hence has the potential to enhance catabolic activity through this mechanism. To establish whether this was potentially the case in our model system, we undertook a series of studies using gelatin zymography (Fig.…”

“…NGF influences expression of several catabolic genes in animal and human IVD cells including ch3l1, lipocalin 2 (lcn2), mmp3 and mmp13 according to our previous microarray-based study [15]. Since Lcn2 protein has been reported as forming a complex with MMP9 to protect it from degradation and thereby enhancing its catabolic function in osteoarthritic cartilage [16], the current study was undertaken to test the hypothesis that NGF regulates expression of lipocalin 2 in AF cells resulting in an increase in MMP9 activity which would potentially contribute to IVD degeneration.…”

Proteomic characterization of mouse cartilage degradation in vitro

“…Our data showing that NGAL is secreted in response to IL-1 stimulation, while being absent from normal cartilage (27), indicates that NGAL is a potential biomarker for arthritic disease. In addition, NGAL binds and inhibits the autocatalysis of MMP-9, an interaction cited as a potential therapeutic target in vascular injury, cancer, and arthritis (28)(29)(30). Another strongly IL-1␣-induced protein was CH3L1 (YKL-40), a known target of inflammatory cytokines in human chondrocyte cultures (31).…”