Identifizierung neuer Coregulatoren von SOX9 und RUNX2 in chondrogenen Progenitorzellen in der Osteoarthrose

Abstract

Osteoarthritis is a degenerative disease of the joints. It is characterized by a successive destruction of the articular cartilage and surrounding structures. Osteoarthritis is the most common musculoskeletal joint disease of humans and affects in particular the elderly. According to estimates, osteoarthritis will be the fourth most common cause of disability by the year 2020. In addition to patients suffering, osteoarthritis causes substantial costs to the health system and contributes to significant economic damage. As an avascular tissue, the natural regeneration of hyaline cartilage is limited due to the low cell density and the low metabolism of the cells. It lacks a perichondrium which could stabilize and supply chondrocytes or chondroblasts by its source of undifferentiated mesenchymal cells. The fibrocartilaginous repair tissue in diseased articular cartilage harbors a population of progenitor cells with chondrogenic properties. The origin of these cells is poorly understood. It is assumed that these cells migrate through blood vessels from the subchondral bone into the damaged areas by vascularization and breaks in the tidemark. These chondrogenic progenitor cells (CPCs) could prove to be accessible to drug therapies to stimulate their potential for regeneration attempts. The aim of this work was to identify co-activators of the chondrogenic transcription factor SOX9 or co-repressors of the osteogenic transcription factor RUNX2. For this approach, CPCs were used, which are stimulated in the production of chondrogenic factors by siRNA-mediated knockdown of RUNX2. The reverse experiment was also carried out. Osteogenic factors, which could serve as a target of inhibitory therapies, should be stimulated by the knockdown of SOX9. At first, the antagonistic relationship between SOX9 and RUNX2 were shown in CPCs. The knockdown of RUNX2 led to an upregulation of SOX9 and thus to an increase in the chondrogenic potential of the cells. Conversely, the knockdown of SOX9 led to an up-regulation of RUNX2 and thus to a reduction in the chondrogenic potential of CPCs. By mass spectral analysis, various proteins associated with signal transduction and transcription could be isolated from immunoprecipitation and pull-down-isolates of SOX9 and RUNX2. In this work, some of the potential co-activators of SOX9 were overexpressed and potential co-repressors of SOX9 were downregulated by siRNA in CPCs. Overexpression of DDX5, HSPA8, RAB5C and YWHAE led to an increase in the gene expression of SOX9. RUNX2 was also upregulated by the overexpression of HSPA8. In contrast, the overexpression of YWHAE downregulated RUNX2 which could have enhanced the chondrogenic potential of CPCs. The upregulation of SOX9 was also achieved by the knockdown of LEMD2 and TMPO. In this case, the expression of ACAN was upregulated while COL1A1 was downregulated. This is an indication of the increased chondrogenic potential of CPCs. In addition, the protein-protein interaction between SOX9 and DDX5 was for the first time examined by co-immunoprecipitation. SOX9 was able to be co-precipitated. However, the reverse experiment was unsuccessful and thus, the interaction could not be validated. Further investigations, e. g. by using another method such as yeast-two-hybrid are needed to confirm the actual protein-protein interaction. These results show that the identification of possible cofactors by affnity chromatographic purification of SOX9 and RUNX2 can be achieved by mass spectrometric analysis of the isolated protein complexes. The extent to which these proteins act, whether directly on SOX9 or as part of an overall transcription machinery needs to be investigated in further studies to answer the question if they are suitable targets for therapeutic approaches. Furthermore, the question needs to be clarified to what extent the identified proteins affect the expression of downstream chondrogenic factors. This work may be relevant to the development of new agents and strategies in osteoarthritis relevant therapies.