To decipher the molecular mechanisms down-regulatingPITX1expression in primary osteoarthritis (OA).Methods
The functional activity of differentPITX1promoter regions was assessed by luciferase reporter assay. Tandem mass spectrometry coupled to protein sequencing was performed using nuclear extracts prepared from OA chondrocytes, in order to identify proteins bound to DNA regulatory elements. Expression analyses of selected candidate proteins were performed by real-time reverse transcription–polymerase chain reaction (RT-PCR) and immunohistochemistry methods, using cartilage sections and articular chondrocytes from non-OA control subjects and patients with OA. Gain-of-function and loss-of-function experiments were performed in normal and OA chondrocytes, respectively, to study their effects onPITX1regulation. The results were validated by real-time RT-PCR and immunohistochemistry in STR/Ort mice, a well-known animal model of OA.Results
PITX1promoter analyses led to the identification of prohibitin 1 (PHB1) bound to a distal E2F1 transcription factor site. Aberrant accumulation of PHB1 was detected in the nuclei of OA articular chondrocytes, and overexpression of PHB1 in control cells was sufficient to inhibit endogenousPITX1expression at the messenger RNA and protein levels. Conversely, knockdown of PHB1 in OA articular chondrocytes resulted in up-regulation ofPITX1.Studies of early molecular changes in STR/Ort mice revealed a similar nuclear accumulation of PHB1, which correlated withPitx1repression.Conclusion
Collectively, these data define an unrecognized role for PHB1 in repressingPITX1expression in OA chondrocytes.