Table 1.
siRNA oligo sequences.
Table 2.
DNA oligo sequences for RT-qPCR.
Fig 1.
BMP7 exposure increases translational capacity of chondrocytic SW1353 cells and is associated with increased rRNA levels Translational capacity was determined using the SUNsET assay in SW1353 cells (A) or human primary chondrocytes (n = 3 inidividual donors) (B), which were exposed for 24 hours to BMP7 (1nM) or control conditions. Puromycilation data were normalized to DNA content and calculated relative to the control condition (A: n = 6 samples per condition, B: n = 3 samples per donor). In similar samples from A and B, expression levels of 18S rRNA, 5.8S rRNA, and 28S rRNA were determined using RT-qPCR analysis in SW1353 cells (C) or human primary chondrocytes (D). Data were normalized to cyclophilin expression and set relative to the control condition (C: n = 3 samples per condition, D: n = 3 samples per donor). E. Translational capacity was determined in SW1353 cells, which were exposed for 24 hours to Actinomycin D (10 ng/ml) and BMP7 (1 nM) or control conditions. Puromycilation data were normalized to DNA content and calculated relative to the control condition (n = 5 samples per condition). F. In similar samples as E, protein content was determined using a BCA assay. Statistical significance was determined using two-tailed unpaired Student’s t-tests for A, C, E, F and two-tailed paired Student’s t-test for B and D. Bars show the mean ±SEM. * P<0.05, ** P<0.01, *** P<0.001 versus control conditions. ns = not significant.
Fig 2.
rDNA promotor reporter activity is increased in SW1353 cells exposed to BMP7.
SW1353 cells were transfected with an NL1.2_47S-rDNA promoter plasmid. Subsequently, cells were exposed to BMP7 (1nM) for 24 hours and Nanoluc luciferase levels were measured (A). Data were normalized to DNA content and calculated relative to control conditions (RLU) (n = 6 samples per condition). Expression levels of UBTF and TCOF1 were determined using RT-qPCR analysis in SW1353 cells (B) or human primary chondrocytes (C). Data were normalized to cyclophilin expression and set relative to the control condition (B: n = 3 samples per condition, C: n = 3 samples per donor). Statistical significance was determined using two-tailed unpaired Student’s t-tests for A and B and two-tailed paired Student’s t-test for C. Bars show the mean ±SEM. * P<0.05, ** P<0.01, *** P<0.001 versus control conditions.
Fig 3.
BMP7-induced promotor reporter activity and rRNA levels are NKX3-2 dependent.
SW1353 cells were transfected with either a scrambled (Control RNAi) or NKX3-2 (NKX3-2 RNAi) siRNA duplex (100nM) and after 24 hours expression levels of RUNX2, COL10A1, 18S rRNA, 5.8S rRNA, 28S rRNA, UBTF and TCOF1 were measured by RT-qPCR. Data were normalized to cyclophilin expression and set relative to the control condition (n = 3 samples per condition) (A). In similar samples from A, translational capacity was determined using the SUNsET assay. Puromycilation data were normalized to DNA content and calculated relative to the control condition (n = 6 samples per condition) (B). SW1353 cells or human primary chondrocytes were transfected with either a scrambled (Control RNAi) or NKX3-2 (NKX3-2 RNAi) siRNA duplex (100nM) and exposed to BMP7 (1nM) for 24 hours after which expression levels of NKX3-2 (C,G) and RUNX2 (D, G) were determined using RT-qPCR analysis. Data were normalized to cyclophilin expression and set relative to the SCR control condition (n = 3 samples per condition). E. In similar samples as Fig 3C, SW1353 cells were subsequently transfected with a pNL1.2_47S-rDNA promoter plasmid and exposed to BMP7 (1nM) for 24 hours after which Nanoluc luciferase levels were measured. Data were normalized to DNA content and calculated relative to SCR control conditions (RLU) (n = 6 samples per condition). F/G. In similar samples from C and G, expression levels of 18S rRNA, 5.8S rRNA, 28S rRNA, UBTF and TCOF were determined using RT-qPCR analysis. H. In similar samples from C, translational capacity was determined using the SUNsET assay. Puromycilation data were normalized to DNA content and calculated relative to the control condition (n = 6 samples per condition) Statistical significance was determined using a 1-way ANOVA with a Bonferroni’s Multiple Comparison test (E,H) or two-tailed Student’s t-tests (unpaired: A-D, F; paired: G). Bars show the mean ±SEM. * P<0.05, ** P<0.01, *** P<0.001. ns = not significant.
Fig 4.
NKX3-2 overexpression increases rRNA levels and is associated with increased translational capacity.
NKX3-2 was overexpressed by transient transfection of a codon usage optimized FLAG-NKX3-2 vector into SW1353 cells (A,B) or primary chondrocytes (n = 3 donors) (C). FLAG-empty vector was used as a negative control. Expression of mRNA for the indicated genes was determined by real-time RT-qPCR. Data were normalized to cyclophilin expression and set relative to the control condition (A-B: n = 3 samples per condition, C: n = 3 samples per donor). D. Translational capacity was determined in SW1353 cells with and without overexpression of NKX3-2. Puromycilation data were normalized to DNA content and calculated relative to the control condition (n = 5 samples per condition). Statistical significance was determined using two-tailed Student’s t-tests (unpaired for: A,B,D; paired for: C). Bars show the mean ±SEM. * P<0.05, ** P<0.01, *** P<0.001 versus control conditions.
Fig 5.
Graphical representation of the elucidated mechanism.
The results of this study demonstrate that BMP7 increases protein translation and promotes rRNA transcription in SW1353 cells. rRNA transcription in SW1353 cells has been demonstrated to be mediated by a BMP7-induced inhibition of RUNX2 in a NKX3-2-dependent manner.