\u3ci\u3ePTHR1/SOX9\u3c/i\u3e and \u3ci\u3eIDH1/IDH2\u3c/i\u3e Relative Expression in Primary Chondrocyte and Chondrosarcoma Cells Under the Synergistic Influence of Inducible Hypoxia and Extracellular Acidosis

Abstract

Cartilage cells (Chondrocytes) grow in rather unique environmental conditions in the human body. Cartilage is avascular tissue and lacks innervation. Its main source of nutrients is derived from the synovial fluid and/or perichondrium. Consequently, these cells must survive and thrive under hypoxic and acidic stressors. Published data suggests that there are a multitude of genes affected from either one of these two stressors or both. However, these factors are frequently overlooked in cartilage research, and results are reported in either normoxia/pH=7.0 conditions, or they only account for one of the conditions. The scope of this study is to examine how these stressors affect gene expression in primary chondrocytes and chondrosarcomas. In this study, one primary chondrocyte cell line (CON5) and two chondrosarcoma grade II cell lines, JJ012-IDH1 mutant and SW1353- IDH2 mutant, were grown in four experimental conditions: hypoxia (5% O2), acidosis (pH=5.5), hypoxia and acidosis, and normoxia/(pH=7). Four genes of interest were analyzed via RT-qPCR relative to the ACTB housekeeping gene: parathyroid hormone receptor-1 (PTHR1), SRY-box transcription factor 9 (SOX9), and isocitrate dehydrogenase 1 and 2 (IDH1/IDH2). PTHR1 and SOX9 keep chondrocytes in a proliferative state and delay their hypertrophy. On the other hand, IDH1 and IDH2 are metabolic enzymes that convert isocitrate to α-ketoglutarate (α-KG). Their mutant counterparts further convert α-KG into a competitive oncometabolite D-2-Hydroxygluterate (D-2-HG). Our colorimetric assay data suggest that D-2-HG concentration levels are 10.5-fold and 6-fold more elevated in JJ012/SW1353 respectively than in the IDH wild type CON5. Our gene expression data indicates that both inducible hypoxia and extracellular acidosis alter gene expression not only separately but also when combined. This study further highlights the importance of these stressors in cartilage biology research.