Transcription of ribosomal rnas and translational capacity are regulated by BMP-7, via BAPX1/NKX3.2 in chondrocytic cells

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 OA chondrocytes, and induces a hypertrophy-suppressive cellular change in OA chondrocytes. It is expected that the protein translational capacity of the chondrocyte is increased after exposure to BMP-7 which might explain the pro-anabolic shift. The cellular availability of maturated ribosomal RNAs (rRNA) is rate-limiting in the assembly of ribosomes and thus the cells protein translational capacity. In addition, we have previously shown that the phenotypic changes by BMP-7 in chondrocytes are, at least partly, induced via bagpipe homeobox homolog 1 (BAPX1/NKX3.2). We therefore hypothesize that BMP-7 enhances the translational capacity of articular chondrocytes via BAPX1/NKX3.2-dependent synthesis of rRNAs. Methods: OA human articular chondrocytes (HACs) were isolated from OA cartilage following total knee arthroplasty (with ethical permission). SW1353 cells and OA HACs were exposed to BMP-7 (1 nM) and expression levels of rRNAs (18S, 5.8S, 28S), crucial co-factors in rRNA transcription (upstream binding factor 1 (UBF-1) and treacle ribosome biogenesis factor 1 (TCOF1)), as well as BAPX1/NKX3.2) were determined by RT-qPCR (and immunoblotting for BAPX1/NKX3.2). BAPX1/NKX3.2 overexpression and knockdown were achieved via transfection of a FLAG-BAPX1/NKX3.2 plasmid or a BAPX1/NKX3.2 siRNA duplex. Overall translational capacity following BMP-7 exposure, BAPX1/NKX3.2 overexpression or knockdown was directly assessed by SUNsET assays. Alterations in rRNA gene transcription were determined by transfecting SW1353 cells and OA HACs with a 47S promoter reporter plasmid (pNL1.2[NlucP]) to determine transcriptional activity of the 47S pre-rRNA gene. Results: Exposure to BMP-7 or BAPX1/NKX3.2 overexpression lead to increased overall protein translational capacity. BAPX1/NKX3.2 knockdown resulted in reduced translational capacity, which could not be restored by BMP-7 treatment. BMP-7 stimulation lead to increased 18S and 5.8S rRNA, as well as UBF-1 expression levels, which correlated with increased BAPX1/NKX3.2 mRNA and protein expression. Overexpression of BAPX1/NKX3.2 resulted in increased rRNA and UBF-1 expression levels, and reciprocally knockdown of BAPX1/NKX3.2 resulted in decreased rRNA and UBF-1 expression levels. Finally, BMP-7 exposure or overexpression of BAPX1/NKX3.2 resulted in increased 47S pre-rRNA promoter activity and BAPX1/NKX3.2 knockdown resulted in an antagonistic reduction of 47S pre-rRNA gene transcription. Conclusions: BMP-7 induces the overall protein translational capacity. This enhanced anabolic state is accompanied with increased cellular levels of maturated rRNAs and concomitant induction of factors involved in the transcription of rRNAs. In line with these results, 47S pre-rRNA transcription is increased after exposure to BMP-7 or BAPX1/NKX3.2 overexpression. Here we show that the anabolic shift seen by BMP-7 works via a BAPX1/NKX3.2 dependent manner. 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.