Role And Mechanism Of Focal Adhesion Kinase In Inducing Osteogenic Differentiation Of Mouse Embryonic Fibroblasts Cells

Background and Objectives:Mesenchymal stem cells?MSCs?are pluripotent stem cells with strong self-proliferation and multi-directional differentiation potential,which can differentiate into bone,cartilage,muscle,fat,etc.Under specific induction conditions,MSCs can direct differentiation to a variety of cells.In the field of regenerative medicine,MSCs are used as ideal seed cells for bone tissue engineering,mainly in the study of osteoporosis,difficult-to-heal fractures and other diseases.Immortalized mouse embryonic fibroblasts?iMEF?has MSCs-related biological properties,has the potential for self-renewal and multi-directional differentiation,and is also able to express allogeneic genes through plasmid transfection or genetic modification.It is a suitable cell model for analyzing gene function,and is used as a substitute stem cell model for interstitial stem cells of bone marrow to study the differentiation of germ cells.Focal adhesion kinase?FAK?is a cytoplasmic non-receptor protein tyrosine kinase that is concentrated near the sticky spot and participates in multiple signal transduction pathways by integrating signals from out-of-cell pressure,nutrition,cell adhesion,etc.Existing studies have shown that FAK can regulate cell migration and angiogenesis,especially in the progression and metastasis of tumor cells.In addition,it has been confirmed that the relevant physical factors involved in the process of bone-forming cell bone differentiation,such as magnetic field,ultrasonic,chemical factors such as nitric oxide,histone H1,and biomechanics,etc,can be integrated by sticky plaque kinase.It is worth mentioning that the research shows that FAK plays a key role in regulating the proliferation of embryonic stem cells and guiding the differentiation of stem cell lineages in the process of high expression in embryonic stem cells and the entire embryonic development process.At present,the research on the bone differentiation of FAK is mostly focused on extracellular signals such as biomechanics,whether it is mediated by FAK and thus affects bone differentiation,how,after the integration of extracellular signals,the way in which the FAK affects embryonic stem differentiation is still lacking in research.This research in order to answer the two scientific questions that whether silent expression of FAK significantly reduces the bone-forming effect of embryonic stem cells,and what mechanisms affect osteogenic differentiation of embryonic stem cells after FAK integration of associated extracellular signals.By directing induced bone differentiation of immortalized mouse embryonic fibroblasts,and applying related treatment,the role and mechanism of FAK in inducing the bone differentiation of immortalized mouse embryonic fibroblasts was explored.Methods:1?Effective morphological and functional identification of iMEF^FAK+/+?iMEF^FAK-/-cells.2?The iMEF^FAK+/+cells and iMEF^FAK-/-cells were induced to differentiate into osteoblasts under the same induction conditions.3?Under the same induction conditions,the iMEF^FAK+/+cells and iMEF^FAK-/-cells treated with PI3K/AKT phosphorylation inhibitor LY294002 or MEK/ERK_1/2phosphorylation inhibitor U0126,were induced to differentiate into osteoblasts.4?Observed the morphological changes of the iMEF^FAK+/+cells and iMEF ^FAK-/-cells at different induction periods under a microscope.5?Detected RUNX2 protein expression levels of the iMEF^FAK+/+cells and iMEF^FAK-/-cells and corresponding P-AKT?P-ERK_1/2 expression levels by Western blot.6?Detected the transcription level of RUNX2 gene of the iMEF^FAK+/+cells and iMEF ^FAK-/-cells by qRT-PCR technology.7?The induced iMEF^FAK+/+cells and iMEF^FAK-/-cells were stained with Cystatin red calcium nodules 21 days after osteogenesis induction.Results:1?The osteogenic effect of the iMEF^FAK-/-cells decreased significantly compared to that of iMEF^FAK+/+cells group under the same induction conditions.2?The P-PI3Kp85?Y458?,P-AKT?Ser473?,P-ERK_1/2?Thr202/Tyr204?levels of iMEF ^FAK-/-cells decreased to varying degrees relative to iMEF ^FAK+/+cells.3?The osteogenic effect of iMEF^FAK+/+and iMEF^FAK-/-cells treated with LY294002decreased significantly compared with the control group.4?The osteogenic effect of iMEF^FAK+/+and iMEF^FAK-/-cells treated with U0126decreased significantly compared with the control group.Conclusion:1?Silent expression of FAK significantly reduces the bone-forming effect of the immortalized mouse embryonic fibroblasts cells.2?Focal adhesion kinase can affect osteogenic differentiation of the immortalized mouse embryonic fibroblasts cells by altering PI3K/AKT,ERK_1/2 phosphorylation levels.