The effects of three glucocorticoid-induced molecules MIF, GILZ and AnxA1 on osteoclasts and arthritic bone erosion

Abstract

Inflammatory diseases, such like RA, are commonly associated with systemic and/or local bone loss. Excessive bone resorption is contributed to by immune cells, directly and indirectly. Glucocorticoids (GC) remain major anti-inflammatory treatments for RA. GC decrease bone formation, and enhance osteoclast activity, resulting in an unbalanced bone remodelling that favours of bone resorption. About 50% of RA patients receiving GCs treatment for over 6 months develop glucocorticoids-induced osteoporosis (GIOP). However, the precise mechanisms of how GC affects osteoclasts are not fully understood. Due to the side effects of GC, alternative treatments that can inhibit unwanted inflammation without the adverse effects of GC are needed. GC utilise many pathways of actions, including inducing the expression of anti-inflammatory molecules, such as glucocorticoid-induced leucine zipper (GILZ) and Annexin A1 (AnxA1), and even a pro-inflammatory molecule, macrophage migration inhibitory factor (MIF). The effects of MIF, GILZ and AnxA1 have been reported in inflammatory arthritis studies. However, their role in osteoclastogenesis has not been defined. Studies presented in this thesis examined the effects of MIF, GILZ and AnxA1 on osteoclastogenesis. Understanding the effects of these GC-induced molecules on osteoclasts is important for the potential of translating these molecules into therapies. In Chapter 3, the effects of MIF on osteoclastogenesis were examined. MIF deficiency or neutralization significantly reduced RANKL-induced osteoclastogenesis, and NF-κB, NFAT and ERK MAPK activity. Administration of rMIF did not alter osteoclastogenesis in WT or RAW 264.7 cells, but restored RANKL-induced osteoclast formation in MIF deficient cells. The role of CD74, a cell surface binding protein for MIF, in osteoclastogenesis was examined in Chapter 4. Reduced RANKL-induced osteoclastogenesis and ERK phosphorylation were observed in CD74-/- cells. However, rMIF did not restore osteoclastogenesis in CD74-/- cells. Moreover, CD74-/- BMM were still able to internalize rMIF, suggesting CD74 may mediate the effects with mechanisms that are partially independent of MIF. The anti-inflammatory effect of GILZ has been recently reported in a model of RA. In Chapter 5, the effects of GILZ on osteoclastogenesis were examined. GILZ deficiency did not alter trabecular bone mass in vivo, or RANKL-induced osteoclastogenesis in vitro. However, overexpressing GILZ with a GILZ-rAAV significantly reduced RANKL-induced osteoclastogenesis, accompanied by decreased RANKL-induced NF- κB and NFAT activity. In Chapter 6, the effect of AnxA1 on osteoclastogenesis was examined. Using AnxA1-/- bone marrow cells, a comparable level of osteoclastogenesis was observed between WT and AnxA1-/- cultures. However, an AnxA1 receptor ligand, compound 43, significantly inhibited RANKL-induced osteoclast formation. This was accompanied by decreased osteoclast-relevant gene expression. RANKL-induced NFAT, but not NF- κB activity, was also inhibited by compound 43. In summary, studies presented in thesis demonstrated an opposing effect at MIF compared to GILZ and AnxA1 in osteoclastogenesis. Drug with previously reported anti-inflammatory effects of blocking MIF or inducing GILZ or AnxA1 in inflammatory arthritis, the anti-resorptive effects of such treatments suggest them as possible therapies for RA.