Although anti-tumor necrosis factor (TNF)-alpha treatments efficiently block inflammation in ankylosing spondylitis (AS), they are inefficient to prevent excessive bone formation. In AS, ossification seems more prone to develop in sites where inflammation has resolved following anti-TNF therapy, suggesting that TNF-alpha indirectly stimulates ossification. In this context, our objectives were to determine and compare the involvement of Wnt proteins, which are potent growth factors of bone formation, in the effects of TNF-alpha on osteoblast function. In human mesenchymal stem cells (MSCs), TNF-alpha significantly increased the levels of Wnt10b and Wnt5a. Associated with this effect, TNF-alpha stimulated tissue-non specific alkaline phosphatase (TNAP) and mineralization. This effect was mimicked by activation of the canonical beta-catenin pathway with either anti-Dkk1 antibodies, lithium chloride (LiCl) or SB216763. TNF-alpha reduced, and activation of beta-catenin had little effect on expression of osteocalcin, a late marker of osteoblast differentiation. Surprisingly, TNF-alpha failed to stabilize beta-catenin and Dkk1 did not inhibit TNF-alpha effects. In fact, Dkk1 expression was also enhanced in response to TNF-alpha, perhaps explaining why canonical signaling by Wnt10b was not activated by TNF-alpha. However, we found that Wnt5a also stimulated TNAP in MSCs cultured in osteogenic conditions, and increased the levels of inflammatory markers such as COX-2. Interestingly, treatment with anti-Wnt5a antibodies reduced endogenous TNAP expression and activity. Collectively, these data suggest that increased levels of Dkk1 may blunt the autocrine effects of Wnt10b, but not that of Wnt5a, acting through non-canonical signaling. Thus, Wnt5a may be potentially involved in the effects of inflammation on bone formation. (C) 2012 Elsevier Inc. All rights reserved.