HIF-1 alpha IN ARTICULAR CARTILAGE IS UP-REGULATED BY TREADMILL RUNNING IN VIVO

Purpose: Articular cartilage is a specialized avascular tissue composed of a small number of cells and an extensive extracellular matrix. Due to its avascular structure, articular cartilage is physiologically hypoxic tissue. The transcription factor hypoxia-inducible factor-1 alpha (HIF-1α) represents one of the important elements in maintaining proper cellular functions under hypoxic condition. HIF-1α is also of great importance by promoting the synthesis of relevant extracellular matrix components in chondrocytes. On the other hand, heat shock protein (HSP) 70 is a member of a family of highly conserved proteins which are synthesized in cells after stress loading, and protects cells from various types of stress including hypoxia or mechanical stress. In our previous study, HSP70 induced by up-regulation of HIF-1α expression has chondroprotective effects under hypoxic condition in vitro. The purpose of this study was to investigate the relationship between mechanical stress and HIF-1α expression on rat articular cartilage. Methods: We used treadmill apparatus as mechanical loading. Twelve-week-old male Wistar rats ran on a treadmill at 12 m/min as moderate treadmill exercise or 20 m/min as intense for 45-min at a single time (n = 16 in each group). Rats in control group were simply kept in their cage (n = 16). All were immediately sacrificed after running, and articular cartilage in right knee was removed from patellar, distal femur, and proximal tibia. The cartilage was homogenized, and total RNA was extracted. We analyzed gene expressions of sox9 as transcription factors, aggrecan and col2a1 as anabolic factors with quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Left knees of rats in control group and the 20 m/min group were removed and stained with immunostaining for HIF-1α. Results: Gene expression of sox9 was significantly higher in the 20 m/min group than the others. Gene expression of aggrecan was tended to increase in a speed dependent manner. There was no significant difference among all groups in gene expression of Col2a1. On immunological staining, HIF-1α in rat knee articular cartilage was more strongly stained in the 20 m/min running group than the control group. The staining of HIF-1α was no difference between medial and lateral sides of the knees. Furthermore, the staining of HIF-1α was particularly enhanced from the superficial to the middle zone of the knee articular cartilage. Conclusions: We have reported that the expression of HSP70 is elevated in chondrocytes with heat stimulation and mechanical stress in vitro, and that the expression of HSP70 had an important role for proteoglycan(PG) and Col2a1 synthesis with heat stimulation in rabbit articular cartilage. In rabbits, we also clarified that HSP70 induced by up-regulation of HIF-1α expression has chondroprotective effects under hypoxic condition in vitro. HIFs which sense extracellular oxygen tension and regulate gene expression play important roles as transcription factors in hypoxic environments. HIF-1α is a transcription factor essential for chondrocyte differentiation and maturation, and for maintenance of chondrocyte phenotypes. On the other hand, the oxygen environment in the joint may change with exercise, because it is known that oxygen partial pressure in articular cartilage is decreased by mechanical stress. In present study, the 20 m/min running increased the expression of sox9. This result show that cartilage metabolism in the joint may be activated from the early stage with excessive exercise. In immunostaining, the expression of HIF-1α is enhanced in rat knee articular cartilage by treadmill running in vivo. These may be reflected to inducing hypoxic environment in joints due to treadmill running. In conclusion, moderate speed running may protect articular cartilage by inducing hypoxic environment in rats.