RESUMO
OBJECTIVE: Osteoarthritis (OA) is characterized by articular cartilage erosion, pathological subchondral bone changes, and signs of synovial inflammation and pain. We previously identified p[63-82], a bone morphogenetic protein 7 (BMP7)-derived bioactive peptide that attenuates structural cartilage degeneration in the rat medial meniscal tear-model for posttraumatic OA. This study aimed to evaluate the cartilage erosion-attenuating activity of p[63-82] in a different preclinical model for OA (anterior cruciate ligament transection-partial medial meniscectomy [anterior cruciate ligament transection (ACLT)-pMMx]). The disease-modifying action of the p[63-82] was followed-up in this model for 5 and 10 weeks. DESIGN: Skeletally mature male Lewis rats underwent ACLT-pMMx surgery. Rats received weekly intra-articular injections with either saline or 500 ng p[63-82]. Five and 10 weeks postsurgery, rats were sacrificed, and subchondral bone characteristics were determined using microcomputed tomography (µCT). Histopathological evaluation of cartilage degradation and Osteoarthritis Research Society International (OARSI)-scoring was performed following Safranin-O/Fast Green staining. Pain-related behavior was measured by incapacitance testing and footprint analysis. RESULTS: Histopathological evaluation at 5 and 10 weeks postsurgery showed reduced cartilage degeneration and a significantly reduced OARSI score, whereas no significant changes in subchondral bone characteristics were found in the p[63-82]-treated rats compared to the saline-treated rats. ACLT-pMMx-induced imbalance of static weightbearing capacity in the p[63-82] group was significantly improved compared to the saline-treated rats at weeks 5 postsurgery. Footprint analysis scores in the p[63-82]-treated rats demonstrated improvement at week 10 postsurgery. CONCLUSIONS: Weekly intra-articular injections of p[63-82] in the rat ACLT-pMMx posttraumatic OA model resulted in reduced degenerative cartilage changes and induced functional improvement in static weightbearing capacity during follow-up.
RESUMO
BMP7 is a morphogen capable of counteracting the OA chondrocyte hypertrophic phenotype via NKX3-2. NKX3-2 represses expression of RUNX2, an important transcription factor for chondrocyte hypertrophy. Since RUNX2 has previously been described as an inhibitor for 47S pre-rRNA transcription, we hypothesized that BMP7 positively influences 47S pre-rRNA transcription through NKX3-2, resulting in increased protein translational capacity. Therefor SW1353 cells and human primary chondrocytes were exposed to BMP7 and rRNA (18S, 5.8S, 28S) expression was determined by RT-qPCR. NKX3-2 knockdown was achieved via transfection of a NKX3-2-specific siRNA duplex. Translational capacity was assessed by the SUNsET assay, and 47S pre-rRNA transcription was determined by transfection of a 47S gene promoter-reporter plasmid. BMP7 treatment increased protein translational capacity. This was associated by increased 18S and 5.8S rRNA and NKX3-2 mRNA expression, as well as increased 47S gene promotor activity. Knockdown of NKX3-2 led to increased expression of RUNX2, accompanied by decreased 47S gene promotor activity and rRNA expression, an effect BMP7 was unable to restore. Our data demonstrate that BMP7 positively influences protein translation capacity of SW1353 cells and chondrocytes. This is likely caused by an NKX3-2-dependent activation of 47S gene promotor activity. This finding connects morphogen-mediated changes in cellular differentiation to an aspect of ribosome biogenesis via key transcription factors central to determining the chondrocyte phenotype.
