Runx2 is essential for osteoblast differentiation and chondrocyte maturation. The expression of Runx2 is the first requisite step for the lineage determination from mesenchymal stem cells to osteoblasts. Although the transcript from Runx2 distal promoter is majorly expressed in osteoblasts, the promoter failed to direct green fluorescent protein (GFP) expression to osteoblasts. To find the regulatory region, we generated GFP reporter mice driven by a bacterial artificial chromosome (BAC) of Runx2 locus, and succeeded in the reproduction of endogenous Runx2 expression. By serially deleting it, we identified a 343-bp enhancer, which directed GFP expression specifically to osteoblasts, about 30 kb upstream of the distal promoter. The sequence of the 343-bp enhancer was highly conserved among mouse, human, dog, horse, opossum, and chicken. Dlx5, Mef2c, Tcf7, Ctnnb1, Sp7, Smad1, and Sox6, which localized on the enhancer region in primary osteoblasts, synergistically upregulated the enhancer activity, whereas Msx2 downregulated the activity in mouse osteoblastic MC3T3-E1 cells. Msx2 was predominantly bound to the enhancer in mouse multipotent mesenchymal C3H10T1/2 cells, whereas Dlx5 was predominantly bound to the enhancer in MC3T3-E1 cells. Dlx5 and Mef2 directly bound to the enhancer, and the binding sites were required for the osteoblast-specific expression in mice, whereas the other factors bound to the enhancer by protein-protein interaction. The enhancer was characterized by the presence of the histone variant H2A.Z, the enrichment of histone H3 mono-and dimethylated at Lys4 and acetylated at Lys18 and Lys27, but the depletion of histone H3 trimethylated at Lys4 in primary osteoblasts. These findings indicated that the enhancer, which had typical histone modifications for enhancers, contains sufficient elements to direct Runx2 expression to osteoblasts, and that Dlx5 and Mef2, which formed an enhanceosome with Tcf7, Ctnnb1, Sp7, Smad1, and Sox6, play an essential role in the osteoblast-specific activation of the enhancer.
Glutathione S-transferase (GST) functions in xenobiotic biotransformation and drug metabolism. Increased expression of GSTpi, an isozyme of GST, has been found in cancer cells resistant to doxorubicin hydrochloride (DOX) or cis-diamminedichloroplatinum (II) (CDDP), and this increase was believed to be correlated with drug resistance of cancer cells. GST is mainly expressed in the cytoplasm; GSTpi in the nucleus has been reported in cancer cells, but the meaning of this result is not known. Here, we studied changes in the amount of nuclear GSTpi after exposure of cancer cells to anticancer drugs, and role of the nuclear GSTpi in drug resistance. We found nuclear GSTpi in cancer cells resistant to DOX, and the amount of nuclear GSTpi was enhanced by treatment of the cancer cells with DOX or CDDP. We also found that a mushroom lectin, an inhibitor of nuclear transport, inhibited the nuclear transfer of GSTpi, suggesting the existence of a specific transport system for the nuclear transfer of GSTpi. Nuclear GSTpi protected DNA against damage by anticancer drugs. These results suggest a possible role of GSTpi in the acquisition of resistance to anticancer drugs by cancer cells.
Runx family proteins, Runx1, Runx2, and Runx3, play important roles in skeletal development. Runx2 is required for osteoblast differentiation and chondrocyte maturation, and haplodeficiency of RUNX2 causes cleidocranial dysplasia, which is characterized by open fontanelles and sutures and hypoplastic clavicles. Cbfb forms a heterodimer with Runx family proteins and enhances their DNAbinding capacity. Cbfb-deficient (Cbfb À/À ) mice die at midgestation because of the lack of fetal liver hematopoiesis. We previously reported that the partial rescue of hematopoiesis in Cbfb À/À mice revealed the requirement of Cbfb in skeletal development. However, the precise functions of Cbfb in skeletal development still remain to be clarified. We deleted Cbfb in mesenchymal cells giving rise to both chondrocyte and osteoblast lineages by mating Cbfb fl/fl mice with Dermo1 Cre knock-in mice. Cbfb fl/fl/Cre mice showed dwarfism, both intramembranous and endochondral ossifications were retarded, and chondrocyte maturation and proliferation and osteoblast differentiation were inhibited. The differentiation of chondrocytes and osteoblasts were severely inhibited in vitro, and the reporter activities of Ihh, Col10a1, and Bglap2 promoter constructs were reduced in Cbfb fl/fl/Cre chondrocytes or osteoblasts. The proteins of Runx1, Runx2, and Runx3 were reduced in the cartilaginous limb skeletons and calvariae of Cbfb fl/fl/Cre embryos compared with the respective protein in the respective tissue of Cbfb fl/fl embryos at E15.5, although the reduction of Runx2 protein in calvariae was much milder than that in cartilaginous limb skeletons. All of the Runx family proteins were severely reduced in Cbfb fl/fl/Cre primary osteoblasts, and Runx2 protein was less stable in Cbfb fl/fl/Cre osteoblasts than Cbfb fl/fl osteoblasts. These findings indicate that Cbfb is required for skeletal development by regulating chondrocyte differentiation and proliferation and osteoblast differentiation; that Cbfb plays an important role in the stabilization of Runx family proteins; and that Runx2 protein stability is less dependent on Cbfb in calvariae than in cartilaginous limb skeletons.
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