Effects Of Osteopontin On Mesenchymal Stem Cell Motility And Nuclear Mechanical Properties

Mesenchymal stem cells(MSCs) are mesoderm originated adult stem cells that are capable to differentiate into mesoderm or non-mesodern derived cells include but not limited in neuron cells, muscle cells, osteoblasts and adipocytes. MSCs are widely distributed in a range of organic tissues like bone marrow, dental pulp, fat and peripheral blood. In response to chemotactic factors that are secret by tissue injury, MSCs are able to migrate to the damaged sites, where they excert tissue reparing effects via cell proliferation, differentiation and anti-inflammatory effectors secretion. Research on the potential application of MSCs in tissue engineering has been becoming a central issue with great interests for that MSCs are of rich resources, easy cultivation, low immunogenic activity, multi-potent differentiation and no moral issues.Osteopontin(OPN) is a widely distributed matix protein and cytokin that plays significant roles in cell migration, invasion, stem cell niche and stem-like properties of tumor cells maintaining, cancer cell apoptosis avoiding and stem cell homing. Former in vivo studies demonstrates that damaged sites secret OPN to recruit MSCs, following in vitro experiments indicate also that exogenous OPN is able to promote directed migration of MSCs, suggesting the interaction between OPN and MSCs, which might uncover the instinct of stem cell therapy of tissue damage.Cell motility that is essential for physiological processes like embryogenesis, wound healing and cancer metastasis is composed of migration and invasion, stydies of both and the related regulators are helpful to develop new means of stem cell therapy. Cell motility relies on cytoskeleton remodeling, cell-matix adhesion and cell/nuclear mechanical properties, among which, especially, Nuclear stiffness and plastisity are the most prominent factors in 3D migration, determines the velocity and efficiency of cell motility. Cell invasion is a case of migration that is accompanied with proteolysis of matix fibers by Matrix metalloproteinases(MMPs). MMPs expression is controlled by a range of biofactors and in turn influence invasion. Though lots of stydies in migration and invasion of tumor cells are available, little is known about the mechanism of MSCs motility.Given that MSCs has great potential in stem cell therapy and close correlation to OPN, Effects of OPN on MSCs motility are discussed in this article from signal transduction and nuclear mechanics aspects to uncover the underlying mechanism of OPN promoted cell motility and provide theoretical supports for clinical application of OPN and MSCs. Main results are listed as follow.1. Isolation, cultivation and identification of MSCsBone marrow adherent separation method is applied to isolate rat MSCs, the isolated cells are cultivated in L-DMEM medium supplemented with 10% fetal bovin serum. Morphological observation find that individual cell is spindle shaped with high refraction, the cell groups are distributed as swirl shape. Flow cytometry indicates that the cells express CD90 and CD44 but not CD34. The cultivated cells are found to conform general characters of MSCs by the combination of morphological observation and flow cytometry analysis.2. Effects of OPN on MSCs motilityTranswell assay and Matrigel invasion assay are applicated in this article to assess if motility of MSCs is regulated in response to OPN treatment. Results demonstrate that OPN exposure does not affect MSCs proliferation but significantly increased migration and invasion capability of MSCs, suggesting that OPN is able to promote MSCs motility.Western blot demonstrates that OPN exposure significantly increased the phosphoration of FAK and ERK1/2, but FAK inhibitor PF573228 and ERK1/2 inhibitor PD98059 notably suppressed the OPN promoted migratory and invasive behaviors of MSCs. Further gelatin zymograph experiment demonstrates that OPN activates MMP2 while PD98059 and PF573228 treatment significantly reduced the OPN induced MMP2 activation but not MMP9, correlates with the tendency of MSCs invasion. All the results strongly support that OPN promotes MSCs migration and invasion by FAK, ERK1/2, especially, for invasion, MMP2 is also involved and modulated by FAK and ERK1/2.3.Effects of OPN on nuclear mechanical properties of MSCs.To verify if change in nuclear mechanic properties is responsible for OPN induced cell motility, atomic force microscope(AFM) is applied to measure Youngs’ modulus of separated nucleus. Results find that Youngs’ modulus of MSCs nuclear drastically reduced after OPN exposure, but PD98059 and PF573228 treatment inverted the stiffness change to some extent, indicating the involvement of FAK and ERK1/2 in the modulation of nuclear stiffness by OPN.Nuclear skeleton protein LaminA/C is a main determinant of nuclear stiffness. With RT-PCR and Western blot we find that LaminA/C down regulated in both mRNA and protein levels after OPN treatment. Phosphoration inhibiton of FAK and ERK1/2 by respective inhibitor PF573228 and PD98059 significantly supressed the down regulation of LaminA/C by OPN, suggesting that LaminA/C downregulation mediated by activated FAK and ERK1/2 is a potent explanation to Youngs’ modulus change of MSCs nucleus.To sum up, This article proposes that OPN treatment activates FAK, ERK1/2 and MMP2 to modulate cell motility, the probable mechanism underlying is that FAK and ERK1/2 downregulate LaminA/C expression to diminish the nuclear stiffness and promote MSCs motility. Therefore, studies on the regulation of OPN expression would hopefully provide theoretical basis for MSCs related stem cell therapy.