Figure 4. GDF5^W414R shows impaired Bmpr1a signaling in a SBE-Luciferase reporter gene assay.

NIH/3T3 cells were transfected with the BMP type I receptors, Bmpr1a or Bmpr1b, as well as with wild type GDF5 and the GDF5 variants GDF5^W414R, GDF5^R399C and GDF5^E491K. As reporter, the SMAD binding element (SBE) was used and firely luciferase was normalized against TK-Renilla luciferase. A: No Bmp type I receptor was co-expressed which resulted in a weak SBE reporter activation for wild type GDF5 and GDF5^E491K, whereas in case of GDF5^W414R and GDF5^R399C signaling activity was absent. B: Bmpr1a co-expression increased the signaling activity of wild type GDF5 and GDF5^E491K; however, GDF5^W414R and GDF5^R399C were not able to induce reporter gene expression. C: Co-expression of Bmpr1b further increased the signaling activity of wild type GDF5 and GDF5^E491K compared to co-expression with Bmpr1a. In case of GDF5^W414R and GDF5^R399C, Bmpr1b co-expression rescued their signaling activity. The means of triplicate measurements are shown, error bars indicate standard deviation and a represent experiment is shown. Statistical analysis was performed using a two-tailed Student's t test (n.s.: not significant; *p≤0.05; **p≤0.01). Significances are related to the respective wild type GDF5 value.