Effects Of CXCR4Inhibitor AMD3100on Osteogenic Differentiation In MC3T3-E1Cells

Stromal-derived factor1(SDF-1), was also known as CXCL12, which speciallybinds to a G-protein-coupled receptor CXCR4(C-X-C chemokine receptor type4).SDF-1/CXCR4signaling pathway has been found to be important for the process of therecruitment of stem/precursor cells, cellular inflammatory, immune response, bloodhomeostasis, embryonic organ development and postnatal tissue regeneration et al.AMD3100, a synthetic macrocyclic CXCR4antagonist, which can inhibit thebinding of SDF-1to CXCR4and the subsequent signal transductions, has been used as aneffective hematopoietic stem cell mobilization agent in patients with non-Hodgkin’slymphoma (NHL) and multiple myeloma (MM). It has been proposed that AMD3100may also play an important role in the treatment of many other SDF-1/CXCR4-regulatedpathological processes.Recent studies have suggested SDF-1/CXCR4pathway is involved in the boneremodeling and osteoblast differentiation. Kitaori et al found that SDF-1/CXCR4signaling has a critical role in the recruitment of mesenchymal stem cells (MSC) to thefracture site during the skeletal repair in vivo. Zhu et al and Hosogane et al showed thatSDF-1/CXCR4participated in the process of bone morphogenetic protein2(BMP2)-induced osteogenic differentiation of primary MSCs or MSC cells lines in vivo.Moreover, expression of SDF-1and CXCR4has also been detected in stem cells andvarious precursor cells, especially MSCs, but their expression rapidly declines once cellscommit to a differentiation pathway. Kortesidis A et al considered that in vitro, immature preosteogenic cells expressed greater levels of SDF-1and CXCR4when compared withmature cell type, suggesting that SDF-1signaling has a role in the initiation of theirdifferentiation. Therefore, special attention has been given to learn about the efficacy ortoxicity of CXCR4antagonist AMD3100on osteogenesis. Until now, only a few studieshave demonstrated the effect of AMD3100on the osteogenesis of MSC at the early stage.The modulation of AMD3100on the differentiation of preosteoblasts, especially on itsmineralization has not been investigated. In addition, the chemokine SDF-1triggersCXCR4receptor dimerization and activates the JAK/STAT pathway, JAK (Janus kinase)is activated and associated with the CXCR4. This activation enables the recruitment andtyrosine phosphorylation of signal transducer and activator of transcription3(STAT3).Moreover, the regulatory effect of AMD3100might be mediated via intracellular STAT3activation.Objective：The purpose of this study is to explain the detailed mechanism ofSDF-1/CXCR4signaling during the osteogenic differentiation, and to facilitate morein-depth, systematic understanding of the complex network of signaling pathwayinvolved in the osteogenic differentiation. CXCR4antagonist AMD3100was used toinhibit the binding of SDF-1and CXCR4to provide novel insights into the effect ofCXCR4antagonists on the modulation of osteogenesis.Methods: In this study, we utilized the murine pre-osteoblastic cell line MC3T3-E1to observe the effect of CXCR4inhibitor AMD3100on the DEX/AA/β-GP-inducedosteogenic differentiation of MC3T3-E1.(1) Alkaline phosphatase (ALP) activity wasmeasured at3,6,9days of osteogenic induction.(2) To evaluate the calcium depositionin matrix, Alizarin red staining was performed.(3) The mRNA levels of fourosteoblast-specific markers (ALP, Runx2/cbfal, ANK and OCN) affected by CXCR4inhibitor AMD3100were determined by quantitative real time PCR analysis.(4) AndWestern Blot was used to detect the expression of SDF-1/CXCR4pathway downstreamproteins STAT3and phospho-STAT3. Using the above methods, we confirmed the mineralization process of MC3T3-E1, as well as the modification of AMD3100duringthe osteogenesis. In this study, all changes in MC3T3-E1were carefully studied tospeculate the role of SDF-1/CXCR4pathway and its downstream proteins STAT3duringdifferent stages of the osteogenic differentiation.Results:(1) With the osteoblast induction, the ALP activity increased and achievedthe peak at6days, and decreased slightly at9days. When treated with CXCR4antagonist AMD3100, the ALP activity were significantly decreased at3,6,9days.Furthermore, the detection of ALP activity in MC3T3-E1at6days also showed thattreatment with CXCR4antagonist AMD3100decreased the osteogenic media-inducedALP activity in a concentration-dependent manner.(2) Obvious matrix mineralizationcould be observed at the14days of induction, and became extensively at21days.However, cetylpyridinium chloride analysis failed to detect minor increases or decreasesin mineralization by the treatment of AMD3100at50,100μM, respectively.(3)Compared with the osteogenic group, CXCR4inhibitor AMD3100(400μM) suppressedthe expression of both the early osteoblast differentiation markers (ALP, Runx2) and thelate differentiation markers (ANK, OCN) at6and9days.(4) Western-blot resultsshowed that osteogenic media stimulation increased the phosphorylation of STAT3protein at6days compared with the control group, and treatment with CXCR4antagonistAMD3100almost abolished the osteogenic media-induced STAT3phosphorylation.Conclusions: These data suggested that CXCR4inhibitor AMD3100significantlyinhibited the ALP activity during the early stage of osteogenic differentiation inMC3T3-E1, but seemed to have no significant effect on the generation of the matrixmineralization at the terminal stage of osteogenic induction in MC3T3-E1cells. AndCXCR4inhibitor AMD3100suppressed the expression of both the early osteoblastdifferentiation markers (ALP, Runx2) and the late differentiation markers (ANK, OCN).Moreover, our results that blocking of the SDF-1/CXCR4axis inhibited thephosphorylation of STAT3suggested the involvement of STAT3in mediating the SDF-1/CXCR4effect on osteogenic differentiation in MC3T3-E1.