Differentiation of human marrow stromal precursor cells: bone morphogenetic protein-2 increases OSF2/CBFA1, enhances osteoblast commitment, and inhibits late adipocyte maturation.

Because regulation of the differentiation to osteoblasts and adipocytes from a common progenitor in bone marrow stroma is poorly understood, we assessed effects of bone morphogenetic protein-2 (BMP-2) on a conditionally immortalized human marrow stromal cell line, hMS(2-6), which is capable of differentiation to either lineage. BMP-2 did not affect hMS(2-6) cell proliferation but enhanced osteoblast differentiation as assessed by a 1.8-fold increase in expression of OSF2/CBFA1 (a gene involved in commitment to the osteoblast pathway), by increased mRNA expression and protein secretion for alkaline phosphatase (ALP), type I procollagen and osteocalcin (OC) (except for OC protein), and by increased mineralized nodule formation. Transient transfection with Osf2/Cbfa1 antisense oligonucleotide substantially reduced BMP-2-stimulated expression of ALP mRNA and protein. The effects of BMP-2 on adipocyte differentiation varied: expression of peroxisome proliferator-activated receptor gamma2 (a gene involved in commitment to the adipocyte pathway) was unchanged, mRNA expression of the early differentiation marker, lipoprotein lipase, was increased, and mRNA and protein levels of the late differentiation marker, leptin, and the formation of cytoplasmic lipid droplets were decreased. Thus, by enhancing osteoblast commitment and by inhibiting late adipocyte maturation, BMP-2 acts to shunt uncommitted marrow stromal precursor cells from the adipocyte to the osteoblast differentiation pathway.     

核心结合因子α1过表达促进脂肪组织来源干细胞向成骨细胞分化

目的 检测核心结合因子α1(Cbfα1)的特异性成骨作用以及脂肪组织来源干细胞的成骨分化潜能.方法 采用Cbfα1重组的腺病毒载体感染脂肪来源干细胞,通过间接免疫荧光的方法检测脂肪来源干细胞中Cbfα1的表达.Cbfα1转染脂肪组织来源干细胞后1、3、7 d利用RT-PCR检测成骨特异性基因和成脂特异性基因的表达变化.同时通过检测Cbfα1转染后7、10 d脂肪来源干细胞碱性磷酸酶活性的变化,观察Cbfα1过表达后诱导脂肪组织来源干细胞向成骨细胞分化.结果 转染后脂肪组织来源干细胞细胞数目增长一倍.在特定诱导条件下,可向成脂细胞、成骨细胞分化.Cbfα1重组的腺病毒载体对脂肪组织来源干细胞的转染效率为82.4%.转染后第1、3、7天脂肪组织来源干细胞中骨钙素、骨桥素、Ⅰ型胶原的表达增强;脂蛋白脂酶的表达逐渐下降.转染Cbfα1后,脂肪组织来源干细胞中碱性磷酸酶活性显著增加.结论 核心结合因子过表达促进脂肪组织来源干细胞向成骨细胞分化、抑制成脂分化.     

Overexpression of core binding factor alpha1 enhancing osteoblastic differentiation in adipose-derived stem cells

OBJECTIVE: To determine whether mouse core binding factor alpha1 (Cbfalpha1) overexpression enhancing osteoblastic differentiation in adipose-derived stem cells (ADSCs). METHODS: ADSCs were harvested from SD rats and transduced with the recombinant adenovirus carrying Cbfalpha1 gene. Untransduced cells and cells transduced with adenovirus carrying the enhanced green fluorescence protein (Ad-EGFP) gene served as controls. Cbfalpha1 expression was assessed by immunofluorescence. Alkaline phosphatase (ALP) activity was assayed in 7 and 10 d. RESULTS: Overexpression of Runx2 inhibited adipogenesis, as demonstrated by suppression of LPL expression. Moreover, ADSCs transduced with Ad-Runx2 underwent rapid and marked osteoblast differentiation as determined by osteoblastic gene expression, alkaline phosphatase activity. CONCLUSIONS: Cbfalpha1 overexpression enhances osteoblastic differentiation in adipose-derived stem cells.     

Overexpression of the ZIP1 zinc transporter induces an osteogenic phenotype in mesenchymal stem cells

Zinc is an essential trace element that is involved in diverse metabolic and signaling pathways. Zinc deficiency is associated with retardation of bone growth. Previous in vitro studies have suggested a direct effect of zinc on both the proliferation and differentiation of osteoblast-like cells. However, the mechanisms for uptake of zinc into osteoblasts have not been examined in detail. Several families of zinc transporters have previously been characterized in mammalian cells; such transporters function in the uptake, intracellular sequestration or efflux of zinc. In the current study, we examined zinc transport in osteoprogenitor cells and have attempted to define a functional role for a zinc transport mechanism in osteogenic differentiation. We identified at least two zinc transporters in both human mesenchymal stem cells (MSCs) and in osteoblastic cells--the ubiquitous zinc transporter, ZIP1, and LIV-1, which was previously characterized as a protein that is expressed in breast cancer cells. The subcellular localization of both these zinc transporters suggested distribution in both the plasma membrane and also diffusely in the cytoplasm. During the differentiation process of pluripotent MSCs into osteoblast-like cells, both zinc uptake and expression of the ZIP1 protein were increased. An adenoviral-mediated overexpression of ZIP1 in MSCs resulted in Alizarin-red-positive mineralization and also increased expression of specific osteoblast-associated markers, such as alkaline phosphatase, and of several osteoblast differentiation genes, including osteopontin, Cbfa1/Runx2, promyelocytic leukemia zinc finger and bone sialoprotein. An siRNA-mediated reduction of ZIP1 protein expression in MSCs caused decreased zinc uptake and inhibition of osteoblastic differentiation under osteogenic culture conditions. Finally, following overexpression of ZIP1 in MSCs, cDNA microarray analysis revealed differential regulation of several genes associated with the proliferation of osteoprogenitor cells and osteoblast differentiation. In conclusion, these studies provide important insights into the role of a plasma membrane zinc transporter in the initiation of an osteogenic lineage from MSCs.     

Reduction in Gsalpha induces osteogenic differentiation in human mesenchymal stem cells

We hypothesized that a decrease in Gsalpha expression occurs with osteogenic differentiation and that when Gsalpha expression was decreased by antisense oligonucleotides or direct inhibition of protein kinase A there was a concomitant increase in Runx2/Cbfa1. We also investigated the mechanism involved in the change in Runx2/Cbfa1 levels and whether the expression of other genes known to be involved in bone formation was altered. There was a decrease in Gsalpha expression with osteogenic differentiation and antisense oligonucleotides, and protein kinase A inhibition led to increased expression and DNA binding of the osteoblast-specific Runx2/Cbfa1. Additionally, with decreased Gsalpha expression or protein kinase A inhibition, Runx2/Cbfa1 protein was serine phosphorylated and ubiquitinated less. Microarray analysis, after the addition of antisense Gsalpha, showed a more than 10-fold increase in collagen Type I Alpha 2 mRNA (a target of Runx2/Cbfa1). These data show that reduced expression of Gsalpha can induce an osteoblast-like phenotype. The results also indicate a potential pathophysiologic role in patients with heterozygous inactivating mutations in GNAS1, the gene for the alpha chain (Gsalpha) of the heterotrimeric G protein, present in three disorders with ectopic intramembranous bone: Albright's hereditary osteodystrophy, progressive osseous heteroplasia, and osteoma cutis.