BMP-7 enhances ribosomal RNA levels in chondrocytes via BAPX1-NKX3.2

Abstract

Purpose: During osteoarthritis (OA) progression the articular chondrocyte undergoes a phenotypic switch in which the chondrocyte acquires a catabolic and hypertrophy-like state. Bone morphogenetic protein (BMP)-7 (or OP-1) is known for its anti-catabolic and pro-anabolic properties in cartilage repair and in OA chondrocytes. In its anabolic state the chondrocyte’s metabolism and protein synthesis are up-regulated. In order to meet this higher demand of protein synthesis, it is expected that the translational capacity of the chondrocyte is increased after exposure to BMP-7. The cellular availability of maturated ribosomal RNAs (rRNA) is rate-limiting in the assembly of ribosomes and previously it has been shown that BMP-7 treatment resulted in increased expression levels of bagpipe homeobox homolog 1 (BAPX-1/NKX3.2). We therefore hypothesize that BMP-7 enhances the translational capacity of articular chondrocytes via BAPX-1/NKX3.2-dependent synthesis of rRNAs. Methods: OA human articular chondrocytes (HACs) were isolated from OA cartilage from total knee arthroplasty. SW1353 cells and OA HACs were exposed to BMP-7 (1 nM) and expression levels of rRNAs (18S, 5.8S, 28S) rRNA processing snoRNAs (RMRP and U3), a crucial co-factor in rRNA transcription (UBF-1) and BAPX-1/NKX3.2 were determined by RT-qPCR (and immunoblotting for BAPX-1/NKX3.2). BAPX-1/NKX3.2 overexpression and knockdown were achieved via transfection of a FLAG-BAPX-1/NKX3.2 plasmid or a BAPX-1/NKX3.2 siRNA duplex. Human OA cartilage explants from total knee arthroplasty were exposed to BMP-7 (1 nM) and gene expression levels of rRNAs were measured via qPCR. Alterations in rRNA gene transcription were determined by transfecting SW1353 cells and OA HACs with a 47S promoter reporter plasmid (Cypridina luciferase) to determine transcriptional activity of the 47S pre-rRNA gene. Translational capacity of the cells was directly determined via puromycilation assays. Results: BMP-7 treatment resulted in increased 18S and 5.8S rRNA levels, increased UBF-1, RMRP and U3 expression. This correlated with increased BAPX-1/NKX3.2 mRNA and protein expression. Overexpression of BAPX-1/NKX3.2 resulted in increased rRNA expression levels and the reciprocal knockdown of BAPX-1/NKX3.2 resulted in decreased rRNA expression levels. Exposure of OA cartilage explants to BMP-7 confirmed our in vitro data (increase of 18S, 5.8S, UBF-1, RMRP, U3 and BAPX-1 expression levels). Treatment of chondrocytic cells with BMP-7 increased 47S pre-rRNA transcription as well as overall translational capacity. Conclusions: Here we show that BMP-7 induces increased cellular levels of maturated rRNAs, with concomitant induction of factors involved in the transcription and maturation of rRNAs. This process is directly influenced by BAPX-1/NKX3.2 in similar ways as BMP-7. In line with these results, 47S pre-rRNA transcription is increased after exposure to BMP-7, as well as the overall translational capacity. In future research the functionality of increased transcriptional activity of the 47S pre-rRNA gene will be determined by quantification of ribosome levels after BMP-7 exposure. Our data provide important novel insight into the mechanism behind the anabolic properties of BMP-7 and may provide a new molecular cue to target the chondrocyte phenotype in OA.