Wnt/β-Catenin Signaling Mediates Osteoblast Differentiation Triggered by Peptide-induced α5β1 Integrin Priming in Mesenchymal Skeletal Cells

The alpha 5 beta 1 integrin is a key fibronectin (EN) receptor that binds to RGD-containing peptides to mediate cell adhesion. We previously reported that alpha 5 beta 1 integrin promotes osteogenic differentiation in mesenchymal skeletal cells (MSCs), but the underlying mechanisms are not fully understood. In this study, we determined the signaling mechanisms induced by alpha 5 beta 1 integrin interaction with its high-affinity ligand CRRETAWAC in murine and human MSCs and in vivo. We show that cyclized CRRETAWAC fully displaced MSC adhesion to FN, whereas related peptides lacking the full RRET sequence produced a partial displacement, indicating that RRET acts as an RGD-like sequence that is required to antagonize EN-mediated cell adhesion. However, all peptides increased focal adhesion kinase phosphorylation, OSE2 transcriptional activity, osteoblast gene expression, and matrix mineralization in MSCs, indicating that peptide-induced alpha 5 beta 1 integrin priming can promote osteogenic differentiation independently of the RRET sequence. Biochemical analyses showed that peptide-induced alpha 5 beta 1 integrin priming transiently increased PI3K/Akt phosphorylation and promoted Wnt/beta-catenin transcriptional activity independently of RRET. Consistently, pharmacological inhibition of PI3K activity reduced osteoblast differentiation and abolished Wnt regulatory gene expression induced by alpha 5 beta 1 integrin priming. In vivo, systemic delivery of cyclized GACRETAWACGA linked to (DSS)(6) to allow delivery to bone-forming sites for 6 weeks increased serum osteocalcin levels and improved long bone mass and microarchitecture in SAMP-6 senescent osteopenic mice. The results support a mechanism whereby alpha 5 beta 1 integrin priming by high-affinity ligands integrates Wnt/beta-catenin signaling to promote osteoblast differentiation independently of cell adhesion, which could be used to improve bone mass and microarchitecture in the aging skeleton.