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

• Published in: Journal of bone and mineral research Vol. 24; no. 11; pp. 1869 - 1878
• Main Authors: Zaka, Raihana, Dion, Arnold S., Kusnierz, Anna, Bohensky, Jolene, Srinivas, Vickram, Freeman, Theresa, Williams, Charlene J.
• Format: Journal Article
• Language: English
• Published: Washington, DC John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR) 01.11.2009; Wiley; Amer Soc Bone & Mineral Res
• Subjects: Animals; ANK; Base Sequence; Binding Sites; Biological and medical sciences; cartilage biology; Cell Hypoxia - drug effects; Cell Hypoxia - genetics; Chondrocytes - drug effects; Chondrocytes - metabolism; Chondrocytes - pathology; Chromatin Immunoprecipitation; Consensus Sequence; Fundamental and applied biological sciences. Psychology; gene expression; Gene Expression Regulation - drug effects; Gene Knockdown Techniques; Growth Plate - drug effects; Growth Plate - metabolism; Growth Plate - pathology; HIF‐1; Humans; hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit - metabolism; Immunohistochemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mice; Models, Biological; Molecular Sequence Data; Mutagenesis - drug effects; Original; Oxygen - pharmacology; Phosphate Transport Proteins - genetics; Phosphate Transport Proteins - metabolism; Protein Binding - drug effects; Response Elements - genetics; Reverse Transcriptase Polymerase Chain Reaction; Skeleton and joints; Vascular Endothelial Growth Factor A - genetics; Vascular Endothelial Growth Factor A - metabolism; Vertebrates: osteoarticular system, musculoskeletal system; Osteoarticular system; HIF-1; Vertebrata; Oxygen; Ankylosis; Epiphyseal cartilage; Mammalia; Chondrocyte; Hypoxia; cartilage biology; Gene expression; ANK
• ISSN: 0884-0431; 1523-4681
• DOI: 10.1359/jbmr.090512

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% O2) or normoxia (21% O2). 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‐1α knockdown cells were exposed to varying oxygen conditions and ANK expression was assessed. Knockdown of Hif‐1α resulted in low levels of expression of ANK in hypoxia and normoxia. Chromatin immunoprecipitation (ChIP) assays explored the binding of Hif‐1α to ANK HREs and showed that Hif‐1α is able to bind to the HREs of ANK more avidly in normoxia than in hypoxia. Furthermore, functional studies of Hif‐1α activity using luciferase reporter assays of wildtype and mutagenized HREs showed that only HRE‐1 binds Hif‐1α 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.