Cell-specific effects of TNF-α and IL-1β on alkaline phosphatase: implication for syndesmophyte formation and vascular calcification

Tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta stimulate tissue non-specific alkaline phosphatase (TNAP) activity and mineralization in cultures of vascular smooth muscle cells (VSMCs). They are, therefore, considered as stimulators of vascular calcification in the context of atherosclerosis and diabetes type 2. In contrast, although ankylosing spondylitis (AS) leads to the formation of syndesmophytes, which are ectopic ossifications from entheses (where ligaments, tendons and capsules are attached to bone), anti-TNF-alpha therapies fail to block bone formation in this disease. In this context, our aims were to compare the effects of TNF-alpha and IL-1 beta on TNAP activity and mineralization in entheseal cells and VSMCs. Organotypic cultures of mouse ankle entheses were treated or not with TNF-alpha and IL-1 beta for 5 days. Micro-computed tomography was performed to determine trabecular bone parameters, and histology to assess TNAP activity and mineralization. Human mesenchymal stem cells cultured in pellets in chondrogenic conditions and human VSMCs were also used to determine the effects of cytokines on TNAP activity and expression, measured by quantitative PCR. In organotypic cultures, TNF-alpha and IL-1 beta significantly reduced the tibia BV/TV ratio. They also inhibited TNAP activity in entheseal chondrocytes in situ, and in mouse and human chondrocytes in vitro. In contrast, TNF-alpha stimulated TNAP expression and activity in human VSMCs. These differences were likely due to cell-specific effects of peroxisome proliferator-activated receptor gamma (PPAR gamma), which is inhibited by TNF-alpha. Indeed, in human chondrocytes and VSMCs, the PPAR gamma inhibitor GW-9662 displayed the same opposite effects as TNF-alpha on TNAP expression. In conclusion, whereas TNF-alpha and IL-1 beta stimulate TNAP activity in VSMCs, they inhibit it in entheseal cells in situ and on chondrocytes in vitro. The identification of PPAR gamma as a likely mediator of cytokine effects deserves consideration for future research on the mechanisms of ectopic ossification. Laboratory Investigation (2011) 91, 1434-1442; doi:10.1038/labinvest.2011.83; published online 9 May 2011