Osteoarthritis afflicts millions of individuals across the world resulting in impaired quality of life and increased health costs. To understand this disease, physicians have been studying risk factors, such as genetic predisposition, aging, obesity, and joint malalignment; however have been unable to conclusively determine the direct etiology. Current treatment options are short-term or ineffective and fail to address pathophysiological and biochemical mechanisms involved with cartilage degeneration and the induction of pain in arthritic joints. OA pain involves a complex integration of sensory, affective, and cognitive processes that integrate a variety of abnormal cellular mechanisms at both peripheral and central (spinal and supraspinal) levels of the nervous system Through studies examined by investigators, the role of growth factors and cytokines has increasingly become more relevant in examining their effects on articular cartilage homeostasis and the development of osteoarthritis and osteoarthritis-associated pain. Catabolic factors involved in both cartilage degradation in vitro and nociceptive stimulation include IL-1, IL-6, TNF-α, PGE2, FGF-2 and PKCδ, and pharmacologic inhibitors to these mediators, as well as compounds such as RSV and LfcinB, may potentially be used as biological treatments in the future. This review explores several biochemical mediators involved in OA and pain, and provides a framework for the understanding of potential biologic therapies in the treatment of degenerative joint disease in the future.

The Runx2 (CBFA1/AML3/PEBP2␣A) transcription factor promotes skeletal cell differentiation, but it also has a novel cell growth regulatory activity in osteoblasts. We addressed here whether Runx2 activity is functionally linked to cell cycle-related mechanisms that control normal osteoblast proliferation and differentiation. We found that the levels of

Excessive release of basic fibroblast growth factor (bFGF) during loading and/or injury of the cartilage matrix may contribute to the onset or progression of osteoarthritis. This pathological role may be related to the ability of bFGF to decrease proteoglycan synthesis and to antagonize the activity of anabolic growth factors in cartilage such as insulin-like growth factor-1 and bone morphogenetic protein 7 (BMP7 or OP-1). Matrix metalloproteinase-13 (MMP-13), a catabolic cartilage-degrading enzyme, is dramatically up-regulated by inflammatory cytokines or by fibronectin fragments in articular chondrocytes. In this study, we investigated MMP-13 production by bFGF using human articular chondrocytes. Endogenous concentration of bFGF in synovial fluids collected from arthritis patients and asymptomatic subjects showed a good linear correlation with the endogenous levels of MMP-13. bFGF stimulation of MMP-13 was mediated at the transcriptional level and, at least in part, by stimulation of interleukin-1 production. Also, our findings suggest that bFGF stimulation of MMP-13 required the activation of multiple MAPKs (ERK, p38, and JNK) by bFGF, and more importantly, bFGF activation of protein kinase C (PKC) ␦ played a key role in the MMP-13 stimulation. Indeed, PKC␦ is the only isoform associated with MMP-13 stimulation among the PKC isoforms tested. PKC␦ controls the bFGF response by regulating multiple MAPK pathways. Our results suggest that PKC␦ activation is a principal rate-limiting event in the bFGF-dependent stimulation of MMP-13 in human adult articular chondrocytes. We propose that deregulation of cross-talk between MAPK and PKC␦ signaling may contribute to the etiology of osteoarthritis in human patients. Osteoarthritis (OA)2 involves the progressive destruction of the cartilage extracellular matrix (ECM) by a pathological imbalance in the normal metabolic functions of articular chondrocytes. Under normal conditions, chondrocytes maintain a dynamic equilibrium between synthesis and degradation of ECM components. Although the causative events in the etiology of OA remain to be clearly defined, OA is characterized by a disruption of matrix equilibrium leading to progressive loss of cartilage tissue and clonal expansion of cells in the depleted regions. In the early stages of OA, cells respond with a transient induction of matrix synthesis (e.g. increases in the expression and/or protein secretion of insulin-like growth factor-1 (IGF-1) and bone morphogenetic protein 7 (BMP7 or OP-1)). This de novo ECM synthesis cannot overcome the concurrent catabolic processes (1, 2) that involve the excess production of matrixdegrading enzymes, including matrix metalloproteinases (MMPs), aggrecanases, and other proteinases by chondrocytes. The resulting degradation of cartilage ECM may exacerbate the imbalance by enhancing the local activity of systemic regulatory factors, including growth factors and cytokines.Matrix metalloproteinase-13 (MMP-13 or collagenase-3) is the most potent enzyme that degrades type II collagen (the...

Objective While transforming growth factor β (TGFβ) signaling plays a critical role in chondrocyte metabolism, the TGFβ signaling pathways and target genes involved in cartilage homeostasis and the development of osteoarthritis (OA) remain unclear. Using an in vitro cell culture method and an in vivo mouse genetic approach, we undertook this study to investigate TGFβ signaling in chondrocytes and to determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFβ signaling. Methods TGFβ receptor type II (TGFβRII)–conditional knockout (KO) (TGFβRIICol2ER) mice were generated by breeding TGFβRIIflox/flox mice with Col2-CreER–transgenic mice. Histologic, histomorphometric, and gene expression analyses were performed. In vitro TGFβ signaling studies were performed using chondro-genic rat chondrosarcoma cells. To determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFβ signaling, TGFβRII/matrix metalloproteinase 13 (MMP-13)– and TGFβRII/ADAMTS-5–double-KO mice were generated and analyzed. Results Inhibition of TGFβ signaling (deletion of the Tgfbr2 gene in chondrocytes) resulted in up-regulation of Runx2, Mmp13, and Adamts5 expression in articular cartilage tissue and progressive OA development in TGFβRIICol2ER mice. Deletion of the Mmp13 or Adamts5 gene significantly ameliorated the OA-like phenotype induced by the loss of TGFβ signaling. Treatment of TGFβRIICol2ER mice with an MMP-13 inhibitor also slowed OA progression. Conclusion Mmp13 and Adamts5 are critical downstream target genes involved in the TGFβ signaling pathway during the development of OA.

Matrix metalloproteinase-13 (collagenase-3), a member of the family of matrix metalloproteinases (MMPs), plays a major pathological role in the cartilage destruction of arthritis. A dramatic up-regulation of MMP-13 by inflammatory cytokines such as interleukin (IL)-1␤ or by fibronectin fragments has been observed in chondrocytes. In this study, we investigated the inhibitory effects of insulin-like growth factor-1 (IGF-1) and osteogenic protein-1 (OP-1) on the expression of MMP-13, which was induced by fibronectin fragment or IL-1␤ in human immortalized or human primary chondrocytes. IGF-1 and OP-1 each significantly reduced the basal level as well as fibronectin fragment-or IL-1␤-stimulated transcription of the MMP-13 gene in a dosedependent fashion with the corresponding decreases in the protein level of MMP-13. The most prominent suppressive effect was observed by the combination of IGF-1 and OP-1, which decreased the basal promoter activity by 60% and almost completely abrogated the fibronectin fragmentstimulated MMP-13 promoter activity. OP-1 was found to enhance mRNA levels of IGF-1 and the IGF-1 receptor, the latter of which appeared to be responsible for the combined effect of IGF-1 and OP-1. The suppressive effect of IGF-1 and OP-1 on MMP-13 expression was due in part to downregulation of the expression of pro-inflammatory cytokines and the activity of their intermediate molecules, including NF-B and AP-1 factors. We propose that IGF-1 and OP-1 could be key physiological regulators of MMP-13 gene expression and that the combination of IGF-1 and OP-1 may be useful in controlling the excess catabolic activity in arthritis.Cartilage homeostasis is a well synchronized balance between anabolic and catabolic processes. During the development of osteoarthritis, this balance is disrupted resulting in progressive degradation of the articular cartilage (1). Studies have demonstrated that members of the matrix metalloproteinase (MMP) 1 family are the major pathophysiological mediators of the cartilage destruction process in osteoarthritis (2). Recently, in vitro, clinical, and transgenic studies have provided evidence that chondrocyte MMP-13 (collagenase-3) is a leading candidate enzyme mediating the degradation of type II collagen in osteoarthritis (2-4). A dramatic up-regulation of MMP-13 gene expression in response to inflammatory cytokines, such as interleukin-1␤ (IL-1␤) (5), or by fibronectin fragment (Fn-f) (6) has been observed in chondrocytes. However, there is limited knowledge of the cellular mechanisms that regulate MMP-13 gene expression in chondrocytes. Accumulating evidence demonstrates that the bone morphogenetic proteins (BMPs), a subfamily of the transforming growth factor-␤ superfamily, are inhibitors of MMP-13 expression in human fetal chondrocytes (7) or rat osteoblast-enriched cells (8, 9). Upon ligand binding, the specific serine/threonine kinase activity of BMP receptors (type I or II) transduce signals (10) by allowing the association of Smad1 and Smad4 in the cytoplasm followed by the tran...

Objective. The receptor for advanced glycation end products (RAGE) binds multiple ligands, including S100 proteins, high mobility group box chromosomal protein 1 (HMGB-1), and AGEs, all of which are present in articular cartilage. Stimulation of RAGE signaling can lead to MAP kinase activation and increased NF-B activity. The objective of the present study was to determine if chondrocytes express functional RAGE.Methods. The presence of chondrocyte RAGE was analyzed by immunohistochemistry using normal and osteoarthritic (OA) cartilage from young and old monkeys and humans, immunoblotting of chondrocyte lysates and human cartilage extracts, and reverse transcription-polymerase chain reaction (RT-PCR) analysis of RNA from chondrocytes treated with interleukin-1 (IL-1) and fibronectin fragments. RAGE signaling was evaluated by stimulating chondrocytes with S100B and HMGB-1 and analyzing for activation of the ERK MAP kinase and NF-B. The ability of S100B and HMGB-1 to stimulate matrix metalloproteinase 13 (MMP-13) production was also assessed. A pull-down assay using biotin-labeled S100B was used to demonstrate binding to RAGE.Results. RAGE was detected in sections of monkey knee cartilage and human knee and ankle cartilage. Increased immunostaining for RAGE was noted in cartilage from older adult monkeys and humans and was further increased in OA tissue. RAGE was also detected by immunoblotting and by RT-PCR, where IL-1␤ and fibronectin fragments were found to stimulate RAGE expression. Stimulation of chondrocytes with S100B or HMGB-1 increased phosphorylation of the ERK MAP kinase and the p65 subunit of NF-B and increased the production of MMP-13. This signaling was inhibited in cells pretreated with soluble RAGE, and S100B was shown to bind to chondrocyte RAGE.Conclusion. Articular chondrocytes express functional RAGE. The increase in RAGE noted in OA cartilage and the ability of RAGE ligands to stimulate chondrocyte MAP kinase and NF-B activity and to stimulate MMP-13 production suggests that chondrocyte RAGE signaling could play a role in OA.

Fibronectin fragments (FN-f) that bind to the α5β1 integrin stimulate chondrocyte-mediated cartilage destruction and could play an important role in the progression of arthritis. The objective of this study was to identify potential cytokine mediators of cartilage inflammation and destruction induced by FN-f and to investigate the mechanism of their stimulation. Human articular chondrocytes, isolated from normal ankle cartilage obtained from tissue donors, were treated with a 110-kDa FN-f in serum-free culture, and expression of various cytokine genes was analyzed by cDNA microarray and by a cytokine protein array. Compared with untreated control cultures, stimulation by FN-f resulted in a >2-fold increase in IL-6, IL-8, MCP-1, and growth-related oncogene β (GRO-β). Constitutive and FN-f-inducible expression of GRO-α and GRO-γ were also noted by RT-PCR and confirmed by immunoblotting. Previous reports of IL-1β expression induced by FN-f were also confirmed, while TNF expression was found to be very low. Inhibitor studies revealed that FN-f-induced stimulation of chondrocyte chemokine expression was dependent on NF-κB activity, but independent of IL-1 autocrine signaling. The ability of FN-f to stimulate chondrocyte expression of multiple proinflammatory cytokines and chemokines suggests that damage to the cartilage matrix is capable of inducing a proinflammatory state responsible for further progressive matrix destruction, which also includes the chemoattraction of inflammatory cells. Targeting the signaling pathways activated by FN-f may be an effective means of inhibiting production of multiple mediators of cartilage destruction.

Objective. To verify the biologic links between progressive cellular and structural alterations within knee joint components and development of symptomatic chronic pain that are characteristic of osteoarthritis (OA), and to investigate the molecular basis of alterations in nociceptive pathways caused by OA-induced pain.Methods. An animal model of knee joint OA pain was generated by intraarticular injection of monoiodoacetate (MIA) in Sprague-Dawley rats, and symptomatic pain behavior tests were performed. Relationships between development of OA with accompanying pain responses and gradual alterations in cellular and structural knee joint components (i.e., cartilage, synovium, meniscus, subchondral bone) were examined by histologic and immunohistologic analysis, microscopic examination, and microfocal computed tomography. Progressive changes in the dynamic interrelationships between peripheral knee joint tissue and central components of nociceptive pathways caused by OA-induced pain were examined by investigating cytokine production and expression in sensory neurons of the dorsal root ganglion and spinal cord.Results. We observed that structural changes in components of the peripheral knee joint correlate with alterations in the central compartments (dorsal root ganglia and the spinal cord) and symptomatic pain assessed by behavioral hyperalgesia. Our comparative gene expression studies revealed that the pain pathways in MIA-induced knee OA may overlap, at least in part, with neuropathic pain mechanisms. Similar results were also observed upon destabilization of the knee joint in the anterior cruciate ligament transection and destabilization of the medial meniscus models of OA.Conclusion. Our results indicate that MIAinduced joint degeneration in rats generates an animal model that is suitable for mechanistic and pharmacologic studies on nociceptive pain pathways caused by OA, and provide key in vivo evidence that OA pain is caused by central sensitization through communication between peripheral OA nociceptors and the central sensory system. Furthermore, our data suggest a mechanistic overlap between OA-induced pain and neuropathic pain.