Abstract
The α5β1 integrin is a key fibronectin (FN) receptor that binds to RGD-containing peptides to mediate cell adhesion. We previously reported that α5β1 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 α5β1 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 FN-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 α5β1 integrin priming can promote osteogenic differentiation independently of the RRET sequence. Biochemical analyses showed that peptide-induced α5β1 integrin priming transiently increased PI3K/Akt phosphorylation and promoted Wnt/β-catenin transcriptional activity independently of RRET. Consistently, pharmacological inhibition of PI3K activity reduced osteoblast differentiation and abolished Wnt regulatory gene expression induced by α5β1 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 α5β1 integrin priming by high-affinity ligands integrates Wnt/β-catenin signaling to promote osteoblast differentiation independently of cell adhesion, which could be used to improve bone mass and microarchitecture in the aging skeleton.
Highlights
The mechanisms whereby ␣51 integrin triggers osteogenesis are poorly understood
Cyclized peptides lacking the full RRET sequence partially displaced adhesion on FN in both hMSCs (Fig. 1B) and mMSCs (Fig. 1C) at the same dosage. These results indicate that the RRET sequence acts as an RGD-like sequence that is required for CRRETAWAC to fully antagonize FN-mediated cell adhesion in mesenchymal skeletal cells (MSCs)
To determine the equivalence of RGD and RRET sequences, we investigated the functional roles of the RRET sequence in MSC adhesion and osteogenic differentiation
Summary
Results: CRRETAWAC-mediated ␣51 integrin priming promotes osteoblast differentiation via PI3K/Wnt/-catenin signaling independently of the RGD-like REET sequence. All peptides increased focal adhesion kinase phosphorylation, OSE2 transcriptional activity, osteoblast gene expression, and matrix mineralization in MSCs, indicating that peptide-induced ␣51 integrin priming can promote osteogenic differentiation independently of the RRET sequence. The results support a mechanism whereby ␣51 integrin priming by high-affinity ligands integrates Wnt/-catenin signaling to promote osteoblast differentiation independently of cell adhesion, which could be used to improve bone mass and microarchitecture in the aging skeleton. We report a novel mechanism by which priming of ␣51 integrin in MSCs promotes osteoblast differentiation through activation of PI3K/Akt and Wnt/-catenin signaling, a model that can translate into improved bone microarchitecture in senescent osteopenic mice
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