Canonical Wnt And Mechanotransduction Crosstalk In Mineralized Collagen-induced Osteogenesis
Qi Zhou, DDS, PhD1, Kelly X. Huang, HSD1, Xiaoyan Ren, MD, PhD1, Dillon Dejam, BS1, Meiwand Bedar, MD, MSc1, Michelle K. Oberoi, BA, BS1, Natalie J. Dahan, BA1, Brendan A.C. Harley, SB, ScD2, Justine C. Lee, MD, PhD1.
1Division of Plastic and Reconstructive Surgery, University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, CA, USA, 2Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
PURPOSEThe capability of the extracellular matrix to instruct osteoprogenitor differentiation has generated significant interest in developing and characterizing biomimetic materials for bone regeneration. We previously described a novel nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffold that induced osteogenic differentiation of human mesenchymal stem cells (hMSCs) and calvarial bone healing without the addition of exogenous growth factors and progenitor cell priming. We found that MC-GAG stiffness activated the mechanotransduction mediators Yes-associated protein (YAP) and transcription activator with PDZ-binding motif (TAZ) and the canonical Wnt pathway (β-catenin dependent) to stimulate osteogenesis and mineralization. However, the interaction of these pathways remains unknown. The current study aims to elucidate the molecular mechanisms of mechanotransduction and canonical Wnt crosstalk and their contribution to MC-GAG-induced osteogenesis. METHODSBone marrow-derived hMSCs were transfected with combinations of small interfering RNAs (siRNAs) specific for YAP, TAZ, and β-catenin and a scrambled control and seeded on MC-GAG. Osteogenic differentiation was assessed through quantitative RT-PCR and western blots at 7 days of culture. Mineralization was evaluated using micro-computed tomographic analyses at 8 weeks and compared using analysis of variance with post hoc comparisons under the Tukey criterion.
RESULTSGene and protein expression of YAP, TAZ, and β-catenin were diminished only in their respective siRNA conditions. Inhibition of β-catenin led to an increase in gene expression of osteogenic markers RUNX2, COL1A1, and BSPII, as well as BMP receptor ligands BMP2 and BMP4. The combination of YAP/TAZ inhibition downregulated gene expression of COL1A1, whereas the addition of β-catenin inhibition restored COL1A1 expression levels. Compared to YAP/TAZ inhibition alone, YAP/TAZ/β-catenin siRNA combination treatment increased the gene expression of BSPII and BMP4. Meanwhile, there was a reciprocal upregulation of OCN and BMP2 by YAP/TAZ siRNA treatment, but expression levels were reduced upon the additional inhibition of β-catenin. Overall, RUNX2 protein expression and mineralization were not affected by the combined inhibition of the mechanotransduction and canonical Wnt pathways. CONCLUSION The mechanotransduction and the canonical Wnt signaling mediators YAP, TAZ, and β-catenin do not influence each other, and they interact through negative regulatory mechanisms to modulate osteogenic differentiation induced by MC-GAG.
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