Dexamethasone Regulates EphA5, a Potential Inhibitory Factor with Osteogenic Capability of Human Bone Marrow Stromal Cells

Figure 2

EphA5 is upregulated after repeated passaging and downregulated during osteogenic induction with DEX. (a) Expression of members of the ephrin and Eph receptor families in hBMSCs. P5 BMSCs were differentiated into osteogenic lineages using AA + β-GP + DEX or BMP-2, and quantitative PCR to determine the mRNA expression of ephrin and Eph receptor family members was performed at day 3 of osteogenic induction (). The fold change of gene expression was normalized against the expression level in the nontreated culture. DEX treatment suppressed and BMP treatment increased the mRNA expression of both EphA2 and EphA5, respectively. (b) Cell surface alkaline phosphatase (ALP) expression in hBMSCs at passage 5 was analyzed using flow cytometry (left, ). Positive expression was defined as a level of fluorescence higher than 97% of the fluorescence obtained with the corresponding isotype-matched control antibody. Positive expression rates are displayed as the mean ± SEM (green line, isotype control). DEX-treated cells showed a higher positive rate for ALP expression than untreated cells (). Cell bodies became smaller and showed morphological changes at day 14 of DEX treatment (right, magnification ×40). ((c)–(e)) Effect of cell density on ALP and EphA5 mRNA levels under different osteogenic induction protocols. (c) hBMSCs at P5 were seeded at low cell density, without intercellular contact, or at high cell density, that is, almost at confluency. # shows intracellular contact between P1 and P5 cells. ((d), (e)) Quantitative analyses of (d) ALP and (e) EphA5 mRNA expression. The fold change of gene expression was normalized against the expression in cultures at low density with AA and β-GP treatment (). (f) Quantitative analyses of ALP and EphA5 mRNA expression in BMSCs at passage 5 when treated with DEX. The change in the expression of each gene expression was normalized against the expression in cell cultures prior to DEX addition (). EphA5 expression was transiently reduced after 2 hours of DEX induction and reached a minimum value after 6 h of induction. ALP expression was induced after 24 h of DEX induction, after which EphA5 expression gradually decreased again.

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