Platelet-derived Growth Factor Receptor Kinase Inhibitor AG-1295Promotes The Osteoblast Differentiation In MC3T3-E1Cells Through Erk Pathway

Platelet-derived growth factor receptors (PDGFRs) are cell surface tyrosinekinase receptors for members of the platelet-derived growth factor (PDGF) family,which are characterized by five immunoglobulin-like domains in theextracellular-ligand-binding domain, a single membrane-spanning motif, and a splitintracellular tyrosine kinase domain[1]. Ligand binding induces the dimerization oftwo receptors and autophosphorylation of specific tyrosine residues in theircytoplasmic domains[2,3]. There are two forms of the PDGFR, alpha and beta eachencoded by a different gene[4]. PDGFR can activate three major signal transductionpathways: the mitogen-activated protein kinase (MAPK)/extracellularsignal-regulated kinase (Erk) pathway, the phosphatidylinositol3-kinase/Aktpathway, and the phospholipase C-γ (PLC-γ) pathway[3,5,6]. MAPK/Erk pathway hasan important role in osteocyte differentiation[7].PDGFR signaling plays an important role in the control of various cellfunctions of skeletal cells.And previous studies reported the controversial effects thatPDGFR signaling could regulate osteoblast mineralization. For example, somerecent experiments suggest PDGF-BB have an positive function in in bone formationand regeneration[8,9]. As well as, Fierro and colleagues[10]have shown that inhibitionof PDGF receptor activity by imatinib mesylate partially suppressed osteogenesis ofMSCs, as assessed by ALP activity measurements and von Kossa staining. On theother hand, several lines of evidence have suggested that PDGF suppressesosteoblast differentiation[11-13]. For example, pharmacological inhibition of PDGFRactivity stimulates osteogenic marker gene expression and mineralized matrixproduction in vitro[14],In accordance with these in vitro data, it was reported thatlong-term inhibition of PDGFR by imatinib mesylate therapy promotes boneformation in chronicmyeloid leukemia (CML) patients[13,14].And, Tokunaga et al.[15]have investigated the effect of PDGFR-β gene knockout in murine MSCs andreported on enhanced osteogenic differentiation, as evidenced by increased ALPactivity and osteogenic marker gene expression. However, a recent study suggestedthat PDGFR inhibition by AG1296has no significantly contribution to osteogenicdifferentiation of Human Mesenchymal Stem Cells[16].Collectively, there exists a lotof controversy concerning the role of PDGFR in osteogenic differentiation.And littleis known about mechanisms responsible for PDGFR affecting osteogenicdifferentiation.In this study, we examined the effection of PDGFR-β inhibition by TyrphostinAG-1295, a potent PDGFR-β blocker[17]on matrix mineralization in MC3T3-E1cells. And we investigated whether PDGF-β-responsive Erk1/2cascades areinvolved in the mineralized regulation. Here, we suggested for the first time thatPDGFR-β signaling could negatively regulate osteoblast mineralization throughErk1/2pathyway.Chapter1: The effect of Tyrphostin AG-1295, a potent PDGFR-β blocker onthe matrix mineralization in MC3T3-E1cells.Objective: we examined the effect of PDGFR-β inhibition by TyrphostinAG-1295, a potent PDGFR-β blocker on matrix mineralization in MC3T3-E1cellsMethods: Perform perturbation experiments with the highly potent and selectivePDGF receptor inhibitor Tyrphostin AG-1295. In the indicate time, we detected thealkaline phosphatase activity，examed the mineralization nodule by alizarin redstaining and osteoblastic markerswere detected by RT-PCR at day3,6,9and14.Results: AG-1295increased the ALP activity at the early stages of osteoblastdifferentiation in MC3T3-E1cells, and enhanced the matrix mineralization at thelate stages of osteoblast differentiation. we also found that the expression of most ofthe osteogenic markers was elevated by AG-1295.Conclusions: AG-1295enhanced osteoblast differentiation of MC3T3-E1cells,which suggests that PDGFR-β pathway has a negative effect on osteoblast differentiation. Chapter2: PDGF-β-Erk1/2pathway negatively regulate matrix mineralization inMC3T3-E1cellsObjective: The aim is to examining whether Erk1/2pathway participates in theregulation of platelet-derived growth factor receptor kinase inhibitor AG-1295promoting the osteoblast differentiation in MC3T3-E1cells.Methods: collect MC3T3-E1cells during osteogenic differentiation2d,10d.Western-blot were peformed to found out the levels of expression andphosphorylation of Erk1/2.Results: phosphorylated Erk1/2protein was detected at10day of the osteogenicinduction, meanwhile no significant variation was observed for the expression levelof total Erk1/2during osteogenic induction. Furthermore, the phosphorylationactivation of Erk1/2was significantly suppressed by20μmol/L Tyrphostin AG-1295in MC3T3-E1cells induced by osteogenic media at day10.Conclusions: Erk1/2pathway participates in the regulation of AG-1295increasing osteoblast differentiation in MC3T3-E1cells.