Inhibition of rhBMP-2-induced ALP activity by intracellular delivery of SMURF1 in murine calvarial preosteoblast cells.

Intracellular protein delivery is a novel tool for functional analysis of protein inside a cell. Several protein delivery reagents with diverse mechanisms have been developed and are commercially available. In this study, we focused on the inhibitory effect of intracellular delivery of SMAD ubiquitination regulation factor 1 (SMURF1) on the recombinant human bone morphogenetic protein-2 (rhBMP-2) signaling pathway. First, three commercially available reagents for intracellular delivery (BioPORTER(®), PULSin(®), and Xfect(TM)) were tested in a murine preosteoblast cell line, MC3T3-E1.4. The biocompatibility of these reagents was examined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and the cellular uptake and delivery efficiency were determined with FITC-antibody and β-galactosidase (β-gal), respectively. BioPORTER(®) provided the best results and was, therefore, chosen for the second aspect of our study: intracellular SMURF1 delivery. SMURF1/BioPORTER(®) complexes were applied to cells prior to rhBMP-2 application. The outcome data suggest intracellular SMURF1 delivery in MC3T3-E1.4 cells significantly inhibited alkaline phosphatase upregulation. This outcome may be useful to off-targets effects of rhBMP-2.