Role of Aqp1, Sm51 and GATA6 in Differentiation and Migration of Bone Marrow Derived Mesenchymal Stem Cells

Introduction: Bone marrow derived mesenchymal stem cells (BM-MSCs) have high migration and multiple differentiation potentials. Enhanced migration and osteogenic ability of BM-MSCs may be beneficial for promoting bone formation. Our laboratory once compared rat BM-MSCs and rat peripheral blood derived MSCs (PB-MSCs) by microarray and found many differential genes, Aquaporin 1 (Aqp1), small nuclear ribonucleoprotein polypeptide N clone sm51 (Sm51) and GATA-binding factor 6 (GATA6), were differentially expressed in the PB-MSCs. The precise roles of Aqp1, Sm51 and GATA6 in MSCs have not been studied clearly yet. Given that their high differential expression in PB-MSCs, we hypothesize that manipulating expression level of Aqp1, Sm51 and GATA6 will be beneficial for migration and differentiation of MSCs.;Methods: Aqp1, Sm51 and GATA6 overexpressing and knocking down stable MSCs cell lines were established by lentiviral transfection. The migration ability of Aqp1 modified MSCs were assessed through transwell and wound healing assays in vitro and administrated systemically in rats with experimental tibial fracture in vivo. Aqp1 overexpressing rat were then administrated into femora fracture rat intracardiaclly, and the outcome of bone healing were assessed by mechanical testing and micro-CT. The osteogenic potentials of rat Sm51 and GATA6 modified MSCs were assessed using standard osteogenic induction assays in vitro and ectopic bone formation by implanting into nude mouse with HA/TCP scaffolds in vivo.;Results: Knocking down Aqp1 had no effects on osteogenesis, adipogenesis, chondrogenesis and proliferation of MSCs. Overexpression of Aqp1 promoted MSCs migration, while knocking down Aqp1 impaired MSCs migration in vitro. Higher numbers of GFP-MSCs were found at the fracture site in the Aqp1-MSCs treated group compared to the DsRed-MSCs group. The level of beta-catenin and focal adhesion kinase (FAK) increased in the Aqp1-MSCs, and decreased in the Aqp1 knocking down MSCs. Beta-catenin and FAK were co-immunoprecipitated with Aqp1, and the co-localization of FAK and Aqp1 was confirmed by confocal images.;Administration of Aqp1 overexpressing MSCs enhanced the outcome of bone fracture healing in terms of bone stiffness and bone density, while the structure and morphology of callus had no significant difference when compared to DsRed-MSCs group.;Overexpression of Sm51 accelerated osteogenic differentiation of MSCs by enhancing the bone mineralization rate and alkaline phosphatase (ALP) activities. Ectopic bone formation data showed Sm51-MSCs induced more osteoid bone formation than that of DsRed-MSCs control. The expressions of osteogenic markers such as Runx2, Osteocalcin (OCN), Osteopontin (OPN), alkaline phosphatase (ALP), type I collagen increased after overexpressing Sm51 with or without osteogenic induction on mRNA level. In addition, we proved that Sm51 up-regulated expression of Runx2 on protein level, and Sm51 bound to Runx2 RNA precursor directly and specifically, no bounding was seen with other osteogenic genes such as OCN, OPN; adipogenic gene like peroxisome proliferator-activated receptors gamma (PPARgamma) and CCAAT-enhancer-binding proteins (C/EBPs). Finally, knockdown of Runx2 abolished Sm51 effects on osteogenesis of MSCs.;Overexpression of GATA6 enhanced the mineralization of osteogenic differentiation of rat and human BM-MSCs. Depletion of GATA6 in human BM-MSCs led to the reduction of Runx2. In addition, GATA6 was capable of interacting with Runx2 physically demonstrated by coimmunoprecipitation.;Conclusions: The studies demonstrate that Aqp1 enhances MSCs migration ability by affecting expression of beta-catenin and FAK. Aqp1 empowers MSCs move faster to reach the fracture site, which contributes to the better healing outcome. Sm51 promotes osteogenic differentiation of MSCs by modulate the splicing of Runx2, while GATA6 enhances osteogenic differentiation of MSCs by interaction with Runx2.