Oxygen Tension Regulates the Expression of ANK (Progressive Ankylosis) in an HIF-1-Dependent Manner in Growth Plate Chondrocytes

Abstract

The proximal promoter region of ANK, a gene that codes for a protein that regulates the transport of inorganic pyrophosphate, contains two hypoxia responsive elements (HREs); therefore, we studied the expression and function of ANK at different oxygen tensions. ATDC5 and N1511 clonal chondrocytic cells were cultured in either hypoxia (2% O(2)) or normoxia (21% O(2)). Transcript and protein levels of ANK were depressed in hypoxic conditions, as were levels of extracellular pyrophosphate (ePPi). To determine whether HIF-1 was involved in the oxemic response, Hif-1alpha knockdown cells were exposed to varying oxygen conditions and ANK expression was assessed. Knockdown of Hif-1alpha resulted in low levels of expression of ANK in hypoxia and normoxia. Chromatin immunoprecipitation (ChIP) assays explored the binding of Hif-1alpha to ANK HREs and showed that Hif-1alpha is able to bind to the HREs of ANK more avidly in normoxia than in hypoxia. Furthermore, functional studies of Hif-1alpha activity using luciferase reporter assays of wildtype and mutagenized HREs showed that only HRE-1 binds Hif-1alpha in normoxia. Expression of ANK in growth plate and articular cartilage was low in hypoxic regions of the tissues, and higher levels of ANK expression were observed in the synovium and meniscus in regions that have a normally higher oxygen tension. The data suggest that ANK expression and function in vitro and in vivo are repressed in hypoxic environments and that the effect is regulated by HIF-1.