Identification of new alternative pathways and therapeutic targets in tnf-α-mediated inflammation and osteolysis induced by ultra-high molecular weight polyethylene

s / Osteoarthritis and Cartilage 20 (2012) S54–S296 S134 matrix metalloproteinase (MMP)-13 in cartilage explant culture. Although the intracellular signaling that leads to cartilage destruction is mediated by a cluster of catabolic pathways including mitogen-activated protein kinases (MAPKs), the effect of CB12-II on MAPK pathways remains unclear. Hyaluronan (HA) of high molecular weight is widely used in the treatment of osteoarthritis (OA) by intra-articular injection. An increasing body of evidence indicates that HA suppresses catabolic actions by proinflammatory cytokine like interleukin-1. However, little is known of HA effect on actions of CB12-II through interaction with HA receptor such as intercellular adhesion molecule-1 (ICAM-1). Purpose: This study was aimed to examine activation of p38 MAPK in associationwith MMP-13 production by CB12-II and its inhibition by HA in chondrocytes. Methods: Cartilage explants harvested from OA knee joints or isolated chondrocytes in monolayer were incubated with CB12-II or its scramble peptide with or without pretreatment with 2700 kDa HA. In another set of experiments, following preincubation with anti-ICAM-1 antibody or nonspecific IgG, cartilage explants were incubated with or without HA, followed by coincubation with CB12-II or the scramble peptide. Enzyme-linked immunosorbent assay for phosphorylated p38 and MMP13 was performed using total cell lysates and culture supernatants, respectively. Results: When cartilage explants or chondrocytes in monolayer were incubated with CB12-II, the peptide activated p38 in association with enhanced MMP-13 production. Inhibition studies with the specific inhibitor (SB203580) indicated the requirement of p38 for CB12-II-induced MMP-13 production. Pretreatment with HA resulted in significant suppression of CB12-II-stimulated MMP-13 production in cartilage as well as in chondrocyte monolayer cultures. HA suppressed p38 activation by CB12-II, leading to a decrease in MMP-13 production. Anti-ICAM-1 antibody reversed HA effect on CB12-II action. Conclusions: The present study clearly demonstrated that HA suppressed CB12-II-activated p38 via ICAM-1 in OA articular chondrocytes. HA could down-regulate the catabolic action of type II collagen fragments in osteoarthritic joints through the mechanism demonstrated in this study. 258 IDENTIFICATION OF NEW ALTERNATIVE PATHWAYS AND THERAPEUTIC TARGETS IN TNF-a-MEDIATED INFLAMMATION AND OSTEOLYSIS INDUCED BY ULTRA-HIGH MOLECULAR WEIGHT POLYETHYLENE Z. He , D.J. Leong , R.J. Majeska , J.A. Hardin , N.J. Cobelli , H.B. Sun . Albert Einstein Coll. of Med., Bronx, NY; 2 The City Coll. of New York, New York, NY, USA Purpose: Osteolysis is the most common cause of failure for joint replacement surgeries. Wear debris of prosthetic implants provoke inflammatory responses, leading to osteoclast differentiation, osteolysis and ultimately, implant loosening. Debris from implants, including ultrahigh molecular weight polyethylene (UHMWPE), one of the most common materials used in arthroplasty, induce a variety of inflammatory responses, including increased secretion of pro-inflammatory cytokine TNF-a (Tumor necrosis factor alpha). A large body of literature has established the critical role of TNF-a in UHMWPE-induced inflammation and osteolysis, predominantly mediated via the RANKL/NF-kB pathway. However, extensive efforts to prevent and treat prosthetic wear-induced osteolysis by interfering with the TNF-a-associated RANKL/NF-kB pathway has seen limited success. This suggests the existence of alternative pathways for TNF-a-mediated inflammation and osteolysis. Accordingly, in the current studies, we explored alternative molecular pathways by which TNF-a might mediate inflammation and osteolysis induced by UHMWPE. Methods: Osteoclast differentiation induction, UHMWPE treatment and TNF-a inhibition: Bone marrow was harvested from TNF-a knockout and wild-type C57/BL6 mice (Jackson Laboratories) according to our IACUCapproved protocol and cultured. Marrow cells were induced for osteoclast differentiation by M-CSF and RANKL. After 2 days of differentiation induction, the cells were treated with UHMWPE particles (53-75mm, Sigma) alone or with TNF-a inhibitor Infliximab (Centocor Biotech). For some experiments, cultures were treated with NF-kB inhibitor for 72 hrs (Santa Cruz). Cellular and molecular assays: The cells were harvested for qPCR and Western blot to measure targeted gene expression, or subject to TRAP staining (kit from Clontech) to determine osteoclast differentiation. Cells without UHMWPE treatment were used as controls. Results: Exposure to UHMWPE promotes differentiation of wild-type marrow cells to osteoclasts (increased number of TRAP-positive osteoclastlike cells), increases expression of TNF-a, TNF-a receptor 1 (TNF-aR1), and multiple TNF targets including Snail1 and survivin in these cells, over control (non-UHMWPE-treated) cultures. UHMWPE exposure fails to enhance osteoclast differentiation, or increase expression of TNF receptor or its downstream targets in TNF-a knockout cells, confirming the critical mediating role of TNF-a in UHMWPE-induced inflammation and osteolysis. Interestingly, TNF inhibitor-a Infliximab suppresses the osteoclast differentiation and the upregulated expression of factors induced by UHMWPE treatment in wild type cell culture; however, the UHMWPE upregulated expression of Snail1 and survivin appeared not affected by NF-kB inhibition. Conclusions: Our results, in addition to substantiating the critical role of TNF-a in UHMWPE-induced inflammation and osteolysis, identified several downstream targets of TNF-a that are activated in response to UHMWPE but are beyond the NF-kB pathway typically implicated in osteoclastogenesis. These TNF-a targets have been shown to serve important and relevant functions. Snail1 is critical for inflammatoryinduced cell migration, and survivin is an apoptosis inhibitor. Our results, although preliminary, provide evidence of alternative pathway/factors in addition to NF-kB through which TNF-a mediates UHMWPE-induced inflammation and osteolysis. Our results are supported by recent reports of TNF-a’s involvement in regulation of some signaling molecules, whose expressions are not directly regulated by NF-kB. Further elucidation of these alternative pathways will faciliate our understanding of the osteolytic process induced by UHMWPE. The identification of new TNF-a targets may also promote prevention and treatment of osteolysis and the consequent implant loosening induced by UHMWPE.