Studies of signaling pathways in cellular responses to titanium particles: Implications of cyclooxygenase-2/prostaglandin E2/RANK ligand induction in fibroblasts during wear debris-induced osteolysis

Bone loss adjacent to the prosthetic implant is a major cause of joint arthroplasty failure. In prosthetic loosening, wear debris-induced osteolysis occurs by resorption that is induced by wear debris particles generated from the artificial joint articulation. The cellular and molecular responses to microscopic wear debris particles are unclear and little is known about the role of fibroblastic cells in this process. In vitro experiments examined the signaling pathways through which fibroblasts mediate a cellular response to wear particles. RANKL (Receptor Activator of Nuclear Factor kappa B Ligand), a critical activator of osteoclastogenesis, was stimulated in fibroblasts exposed to titanium particles. This effect occurs through a COX-2 (cyclooxygenase-2)/PGE2 (Prostaglandin E2) dependent pathway. The PGE2 receptor EP4 is involved in this pathway downstream of PGE2. Fibroblast cultures activated with either titanium particles or PGE2 enhance osteoclast formation, confirming the functional importance of RANKL induction. Subsequent experiments showed that titanium particles induce COX-2 gene expression by activating NFkappaB (Nuclear Factor kappa B) signaling pathway. Ti particles resulted in the degradation of inhibitor of NFkappaB (IkappaBalpha), permitting active NFKkappaB to translocate into the nucleus where it induces the COX-2 transcription. Surprisingly, IkappaBalpha degradation is not associated with the classical IKK (IkappaB kinase) dependent IkappaBalpha phosphorylation and is independent of the 26S proteasome degradation. Instead, a proteasome independent mechanism was responsible for NFkappaB activaton. Through multiple methods we showed that Ti particles stimulated reactive oxygen species and activated the cysteine protease calpain which targets the C-terminal region of IkappaB. Altogether, our studies demonstrate signaling pathways by which titanium particles induce oxidative stress, stimulate calpain-mediated NFkappaB activation, and activate target gene expression, including COX-2. The COX-2 metabolite, PGE2, induces RANKL expression through EN receptor, and enhances osteoclastogenesis. Finally, a mouse genetic model, FSP1-Cre (Fibroblast specific protein-1), was characterized as a model in which specific genes in fibroblasts can be deleted and offers a method to delete the EP4 receptor specifically in fibroblasts using Cre-Lox technology. Thus, these findings define the synovial fibroblast as an important mediator of osteolysis and present novel critical cellular and molecular targets for therapeutic strategies of prosthetic loosening.