Vitamin K2 modulates proliferation and function of osteoblastic cells in vitro.

A human osteosarcoma cell line, HOS TE85 cells, and a mouse osteoblastic cell line, MC3T3-E1 cells, were cultured for 3 days in a medium containing various concentrations of menaquinone-4 (vitamin K2). As a result, the proliferation of HOS cells was suppressed by vitamin K2 in a dose dependent manner up to 56% of control by 10(-7)M of vitamin K2 and that of MC3T3-E1 cells was suppressed to 84% of control by 10(-6)M of vitamin K2. Vitamin K2 increased alkaline phosphatase activity in both kinds of cells. Warfarin counteracted the effect of vitamin K2 on osteoblastic cell proliferation. Our results show that vitamin K2 modulates proliferation and function of osteoblastic cells by some mechanisms including gamma-carboxylation system.     

Pregnane X Receptor Knockout Mice Display Aging-Dependent Wearing of Articular Cartilage

Steroid and xenobiotic receptor (SXR) and its murine ortholog, pregnane X receptor (PXR), are nuclear receptors that are expressed at high levels in the liver and the intestine where they function as xenobiotic sensors that induce expression of genes involved in detoxification and drug excretion. Recent evidence showed that SXR and PXR are also expressed in bone tissue where they mediate bone metabolism. Here we report that systemic deletion of PXR results in aging-dependent wearing of articular cartilage of knee joints. Histomorphometrical analysis showed remarkable reduction of width and an enlarged gap between femoral and tibial articular cartilage in PXR knockout mice. We hypothesized that genes induced by SXR in chondrocytes have a protective effect on articular cartilage and identified Fam20a (family with sequence similarity 20a) as an SXR-dependent gene induced by the known SXR ligands, rifampicin and vitamin K2. Lastly, we demonstrated the biological significance of Fam20a expression in chondrocytes by evaluating osteoarthritis-related gene expression of primary articular chondrocytes. Consistent with epidemiological findings, our results indicate that SXR/PXR protects against aging-dependent wearing of articular cartilage and that ligands for SXR/PXR have potential role in preventing osteoarthritis caused by aging.     

Prostaglandin F2 alpha stimulates proliferation of clonal osteoblastic MC3T3-E1 cells by up-regulation of insulin-like growth factor I receptors.

Prostaglandin F2 alpha (PGF2 alpha) stimulates proliferation of clonal osteoblastic MC3T3-E1 cells mainly via the stimulation of phospholipase C. These cells constitutively produced and secreted insulin-like growth factor I (IGF-I). In addition, a neutralizing anti-IGF-I antibody completely abolished DNA synthesis stimulated by PGF2 alpha in MC3T3-E1 cells, suggesting that IGF-I indeed mediates the PGF2 alpha effect. However, PGF2 alpha decreased the expression of IGF-I mRNA and the secretion of immunoreactive IGF-I into the medium, whereas progression activity in the conditioned medium was not affected by PGF2 alpha. Although IGF-I alone did not stimulate DNA synthesis in MC3T3-E1 cells, when PGF2 alpha was added to the cultures, IGF-I stimulated their proliferation. Thus, PGF2 alpha may potentiate the action of IGF-I. At the same time, PGF2 alpha increased the number of high affinity binding sites (molecular mass of 130 kDa) for IGF-I in a dose-dependent manner. The increase in IGF-I-binding site number preceded the elevation of DNA synthesis by approximately 3 h. Furthermore, MC3T3-E1 cells secreted at least three species of IGF-binding proteins (IGFBPs) with molecular masses of 24, 30, and 34 kDa. In the early period of PGF2 alpha exposure, PGF2 alpha attenuated the secretion of all of these IGFBPs, whereas thereafter, it markedly increased their secretion, especially that of the 34-kDa IGFBP, suggesting a modulation of metabolism and action of IGF-I. These effects of PGF2 alpha on IGF-I receptor number and IGFBP secretion may play a role in the synergism between PGF2 alpha and IGF-I that results in the stimulation of DNA synthesis in MC3T3-E1 cells.     

Genomic organization of the mouse OSF-1 gene.

The mouse OSF-1 protein (also known as pleiotrophin, HB-GAM, HBGF-8, or HBNF) gene was isolated from a mouse genomic library and sequenced. OSF-1 is a 15-kD secreted protein specifically expressed in bone and brain, and is believed to play a role in brain development and osteogenesis. The mouse OSF-1 gene consists of at least 5 exons and 4 introns and spans > 32 kb. Computer analysis of approximately 4 kb of 5'-flanking sequence of the OSF-1 gene revealed two candidate promoter regions. One candidate promoter contains a thyroid hormone/retinoic acid-responsive element and the other contains two glucocorticoid-responsive elements. DNA sequence analysis of novel OSF-1 cDNA clones indicates that two promoters can be utilized in MC3T3-E1 osteoblastic cells. The overall organization of the mouse OSF-1 gene is similar and the locations of the three exon-intron junctions within the coding region are identical to the mouse gene encoding the differentiation-related factor midkine (MK). Based on this similarity and on the high degree of nucleotide sequence homology (approximately 55%) of mouse OSF-1 and mouse MK, we conclude that OSF-1 and MK are generated from a common ancestral gene and are members of a family of structurally and probably functionally related proteins.     

Activation of Human Stearoyl-Coenzyme A Desaturase 1 Contributes to the Lipogenic Effect of PXR in HepG2 Cells

The pregnane X receptor (PXR) was previously known as a xenobiotic receptor. Several recent studies suggested that PXR also played an important role in lipid homeostasis but the underlying mechanism remains to be clearly defined. In this study, we found that rifampicin, an agonist of human PXR, induced lipid accumulation in HepG2 cells. Lipid analysis showed the total cholesterol level increased. However, the free cholesterol and triglyceride levels were not changed. Treatment of HepG2 cells with rifampicin induced the expression of the free fatty acid transporter CD36 and ABCG1, as well as several lipogenic enzymes, including stearoyl-CoA desaturase-1 (SCD1), long chain free fatty acid elongase (FAE), and lecithin-cholesterol acyltransferase (LCAT), while the expression of acyl:cholesterol acetyltransferase(ACAT1) was not affected. Moreover, in PXR over-expressing HepG2 cells (HepG2-PXR), the SCD1 expression was significantly higher than in HepG2-Vector cells, even in the absence of rifampicin. Down-regulation of PXR by shRNA abolished the rifampicin-induced SCD1 gene expression in HepG2 cells. Promoter analysis showed that the human SCD1 gene promoter is activated by PXR and a novel DR-7 type PXR response element (PXRE) response element was located at -338 bp of the SCD1 gene promoter. Taken together, these results indicated that PXR activation promoted lipid synthesis in HepG2 cells and SCD1 is a novel PXR target gene.