Priming integrin α5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis

Abstract

Adult human mesenchymal stromal cells (hMSCs) have the potential to differentiate into chondrogenic, adipogenic, or osteogenic lineages, providing a potential source for tissue regeneration. An important issue for efficient bone regeneration is to identify factors that can be targeted to promote the osteogenic potential of hMSCs. Using transcriptome analysis, we found that integrin ␣5 (ITGA5) expression is up-regulated during dexamethasone-induced osteoblast differentiation of hMSCs. Gain-of-function studies showed that ITGA5 promotes the expression of osteoblast phenotypic markers and in vitro osteogenesis of hMSCs. Down-regulation of endogenous ITGA5 using specific shRNAs blunted osteoblast marker gene expression and osteogenic differentiation. Molecular analyses showed that the enhanced osteoblast differentiation induced by ITGA5 was mediated by activation of focal adhesion kinase/ERK1/2-MAPKs and PI3K signaling pathways. Remarkably, activation of endogenous ITGA5 using agonists such as a specific antibody that primes the integrin or a peptide that specifically activates ITGA5 was sufficient to enhance ERK1/2-MAPKs and PI3K signaling and to promote osteoblast differentiation and osteogenic capacity of hMSCs. Importantly, we demonstrated that hMSCs engineered to overexpress ITGA5 exhibited a marked increase in their osteogenic potential in vivo. Taken together, these findings not only reveal that ITGA5 is required for osteoblast differentiation of adult hMSCs but also provide a targeted strategy using ITGA5 agonists to promote the osteogenic capacity of hMSCs. This may be used for tissue regeneration in bone disorders where the recruitment or capacity of hMSCs is compromised. mesenchymal stem cells ͉ bone formation ͉ agonist M esenchymal stromal cells (MSCs) derived from the bone marrow stroma are capable of differentiating into chondroblasts, adipocytes, or osteoblasts (1, 2) under appropriate environmental conditions (3, 4). Adult human MSCs (hMSCs) are considered as a valuable source for bone tissue regeneration in human diseases (5, 6). However, the capacity of autologous hMSCs to differentiate along functional bone-forming osteoblasts remains relatively limited for bone regeneration in vivo (7). An important issue for efficient bone regeneration is therefore to target hMSCs to promote their osteogenic potential for in vivo bone regeneration.The osteogenic differentiation process of MSCs is characterized by the expression of the main osteoblast transcription factor Runx2 and osteoblast markers such as alkaline phosphatase (ALP) and type I collagen (Col1A1) and is typified by ECM mineralization (8-10). The ECM-osteoblast interactions generate important signaling mechanisms that converge to promote early osteoblast-specific gene expression and differentiation (11-13). Cell-matrix interactions involve integrins, a family of transmembrane proteins that induce intracellular signals (14,15). The ␣5␤1 integrin is a cell surface receptor for fibronectin that has been implicated in cell spreading, proliferation, di...

Myocilin Stimulates Osteogenic Differentiation of Mesenchymal Stem Cells through Mitogen-activated Protein Kinase Signaling

“…Activation of p38, but not Erk1/2 or JNK MAPK, and the induction of MSC differentiation into osteoblasts by gold nanoparticles have been reported previously reported (52). At the same time, integrin ␣5 has been shown to induce osteogenic differentiation of MSCs by activating Erk1/2 but not p38 MAPK (53). After addition of myocilin, similar as in other systems, phosphorylated p38 is translocated into the nucleus where it may phosphorylate Dlx5 and Runx2, resulting in increased transcriptional activity of those factors (54,55).…”

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

“…Our finding that pharmacological inhibition of PI3K attenuated the stimulatory effect of the peptides on osteoblast gene expression in mMSCs suggests a role for PI3K/ Akt signaling in the activation of osteogenic differentiation induced by ␣5␤1 integrin priming in MSCs. Thus, in addition to ERK1/2 and IGF2/IGFBP2 signaling (24,32), Wnt signaling activation may contribute in part to osteoblast differentiation induced by ␣5␤1 integrin priming in MSCs.…”

“…Three g of total RNA from each samples were reverse-transcribed using the a high-capacity cDNA reverse transcription kit (Applied Biosystems) in a total volume of 30 l at 37°C for 2 h. Relative mRNA levels were evaluated by quantitative PCR (LightCycler, Roche Applied Science) using a SYBR Green PCR kit (ABgene, Courtaboeuf, France) and specific primers (24,47,49). Signals were normalized to hypoxanthine phosphoribosyltransferase as an internal control.…”

“…We previously reported that MSC osteoblast differentiation mediated by ␣5␤1 integrin in part involves activation of FAK/ERK1/ERK2, PI3K (24), and IGF2/IGFBP2 (32) signaling, suggesting that several pathways may mediate the effect of the integrin. In bone, Wnt signaling is an important pathway that regulates osteoblastogenesis (33,34).…”

“…A peptide ligand with high affinity for ␣5␤1 integrin (CRRETAWAC) was found to act as a direct competitive inhibitor of RGD binding to ␣5␤1 integrin (28 -30). We previously showed that priming ␣5␤1 integrin with CRRETAWAC is able to trigger MSC osteogenic differentiation in vitro and to promote osteogenesis in vivo (24,26). The peptide is known to fit in the binding site of ␣5␤1 integrin and is believed to interact with an overlapping binding site for RGD in the ␣5 integrin subunit propeller domain (29 -31).…”