Elemene, the essential oil of Curcuma wenyujin, inhibits osteogenic differentiation in ankylosing spondylitis.

OBJECTIVE: To explore the effects of Elemene, the essential oil of Curcuma wenyujin, on Bone morphogenetic protein/drosophila mothers against decapentaplegic proteins (BMP/SMADs) signal pathway in ankylosing spondylitis (AS) fibroblasts. METHODS: Hip joint capsules were obtained from AS patients (n=10) receiving total hip replacement. Healthy hip joint capsules from patients with hip fracture (n=10) receiving surgery were included as a control. Primary fibroblast cell lines were established from these tissue samples. Fibroblasts were incubated with Elemene for 48 hours. The protein expression was detected by Western blot. The mRNA expression was detected by real-time fluorescent quantitative polymerase chain reaction (PCR). RESULTS: The results showed that the expression of proteins including SMAD1, pSMAD1, SMAD4 and Runt-related transcription factor 2 (RUNX2), and mRNA of RUNX2, which were over-expressed in AS fibroblasts were decreased in the AS fibroblasts cultured in medium with Elemene. CONCLUSIONS: Ele could have a hand in anti-osteogenic differentiation of AS fibroblasts by inhibiting the BMP/SMADs signal pathway and subsequently blocking expression of ossification marker genes RUNX2 that initiate the osteogenic differentiation.

Runx regulation of sphingolipid metabolism and survival signaling.

The Runx genes (Runx1, 2, and 3) regulate cell fate in development and can operate as either oncogenes or tumor suppressors in cancer. The oncogenic potential of ectopic Runx expression has been shown in transgenic mice that develop lymphoma in potent synergy with overexpressed Myc, and in established fibroblasts that display altered morphology and increased tumorigenicity. Candidate oncogenic functions of overexpressed Runx genes include resistance to apoptosis in response to intrinsic and extrinsic stresses. In a search for gene targets responsible for this aspect of Runx phenotype, we have identified three key enzymes in sphingolipid metabolism (Sgpp1, Ugcg, and St3gal5/Siat9) as direct targets for Runx transcriptional regulation in a manner consistent with survival and apoptosis resistance. Consistent with these changes in gene expression, mass spectrometric analysis showed that ectopic Runx reduces intracellular long-chain ceramides in NIH3T3 fibroblasts and elevated extracellular sphingosine 1 phosphate. Runx expression also opposed the activation of c-Jun-NH(2)-kinase and p38(MAPK), key mediators of ceramide-induced death, and suppressed the onset of apoptosis in response to exogenous tumor necrosis factor alpha. The survival advantage conferred by ectopic Runx could be partially recapitulated by exogenous sphingosine 1 phosphate and was accompanied by reduced phosphorylation of p38(MAPK). These results reveal a novel link between transcription factor oncogenes and lipid signaling pathways involved in cancer cell survival and chemoresistance.

Runx expression is mitogenic and mutually linked to Wnt activity in blastula-stage sea urchin embryos.

BACKGROUND: The Runt homology domain (Runx) defines a metazoan family of sequence-specific transcriptional regulatory proteins that are critical for animal development and causally associated with a variety of mammalian cancers. The sea urchin Runx gene SpRunt-1 is expressed throughout the blastula stage embryo, and is required globally during embryogenesis for cell survival and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: Depletion of SpRunt-1 by morpholino antisense-mediated knockdown causes a blastula stage deficit in cell proliferation, as shown by bromodeoxyuridine (BrdU) incorporation and direct cell counts. Reverse transcription coupled polymerase chain reaction (RT-PCR) studies show that the cell proliferation deficit is presaged by a deficit in the expression of several zygotic wnt genes, including wnt8, a key regulator of endomesoderm development. In addition, SpRunt-1-depleted blastulae underexpress cyclinD, an effector of mitogenic Wnt signaling. Blastula stage cell proliferation is also impeded by knockdown of either wnt8 or cyclinD. Chromatin immunoprecipitation (ChIP) indicates that Runx target sites within 5' sequences flanking cyclinD, wnt6 and wnt8 are directly bound by SpRunt-1 protein at late blastula stage. Furthermore, experiments using a green fluorescent protein (GFP) reporter transgene show that the blastula-stage operation of a cis-regulatory module previously shown to be required for wnt8 expression (Minokawa et al., Dev. Biol. 288: 545-558, 2005) is dependent on its direct sequence-specific interaction with SpRunt-1. Finally, inhibitor studies and immunoblot analysis show that SpRunt-1 protein levels are negatively regulated by glycogen synthase kinase (GSK)-3. CONCLUSIONS/SIGNIFICANCE: These results suggest that Runx expression and Wnt signaling are mutually linked in a feedback circuit that controls cell proliferation during development.

Expression of AML/Runx and ETO/MTG family members during hematopoietic differentiation of embryonic stem cells.

Runx1/AML1 plays important roles in hematopoiesis, including the commitment of cells to hematopoiesis during embryonic development, and in the maintenance of hematopoietic cell populations. It is also one of the most common genes involved in chromosomal translocations related to leukemia. One such translocation is t(8;21), which fuses the Runx1 gene to the MTG8/ETO gene and generates the Runx1-MTG8 (AML1-ETO) fusion gene. Both Runx1 and MTG8 have two additional family members that are much less studied in hematopoiesis. Here we report the expression of every member of the Runx and MTG families as well as the Runx heterodimerization partner CBFbeta during hematopoietic differentiation of murine embryonic stem cells. We observed substantially increased expression of Runx1, Runx2, and MTG16 during hematopoietic differentiation. Furthermore, the increase in Runx2 expression is delayed relative to Runx1 expression, suggesting their possible sequential contribution to hematopoiesis.

[Experimental study on osteoking in promoting gene expression of core binding factor alpha 1 in necrotic femoral head of rabbits].

OBJECTIVE: To study the effects of Osteoking in promoting gene expression of core binding factor alpha 1 (cbfalpha 1) in necrotic femoral head of rabbits. METHODS: Rabbit model of femoral head necrosis (FHN) was induced by intravenous injection of lipopolysaccharide (LPS) 10 microg/kg body weight twice with an interval of 24 h and intramuscular injection with methyl prednisone (MPS) 20 mg/kg body weight 3 times. The dynamic changes of cbfalpha 1 gene expression in the femoral head were observed with immunohistochemistry and real-time quantitative PCR. RESULTS: The protein expression of cbfa 1 gene was negative in both model and treatment groups at the 4th week, it turned to weakly positive in the treatment group at the 8th and 12th week but still negative in the model group. The mRNA expression of cbfalpha 1 in the treatment group was 2.87 times that in the model group at the 12th week. There was no significant difference in cbfalpha 1 expression between the normal rabbits with or without Osteoking treatment. CONCLUSION: Osteoking could promote the endogenous cbfalpha 1 expression in the FHN, the effect is better along with the prolonging of the time applied. But it showed no affection on cbfalpha 1 expression in the normal femoral head of rabbits.

[Effects of OGP(10-14) and its derivative G38I on proliferation and differentiation of rat osteoblasts in vitro].

OBJECTIVE: To investigate the effects of the carboxyl end of osteogenic growth peptide (OGP)-OGP((10-14)) and its derivative G38I on the proliferation and differentiation of osteoblasts (OBs). METHODS: Osteoblasts were isolated from the calvariae of newborn SD rats and cultured to G3. OGP((10-14)) or G38I of the concentrations of 10(-15) to 10(-7) mol/L were added to culture medium for 48 hours respectively. The number of cells was counted and MTT analysis was used to examine the proliferation of the cells. The ultrastructure of cells was investigated by electron microscopy. The osteoblasts of G3 were divided into experimental groups, treated with OGP((10-14)) or G38I of the concentration of 10(-11) mol/L for 48 hours, and control group. The alkaline phosphatase activity in the culture medium was measured. The protein expression level of type-I collagen was evaluated by immunohistochemistry. The core binding factor 1 (Cbfa1) and type-I collagen mRNA level of osteoblasts were determined by RT-PCR. RESULTS: With a biphasic effect on, OGP((10-14)) and G38I stimulated the number enhancement of OBs dose-dependently at low concentration and inhibited it at high concentration. The numbers of OB were the highest (37 x 10(4)/ml +/- 7 x 10(4)/ml and 30 x 10(4)/ml +/- 5 x 10(4)/ml respectively) when treated by OGP((10-14)) or G38I of the concentration of 10(-11)mol/L. The rough endoplasm net was flourishing and the secreting vesicle was abounding in the experimental cells. There was calcium crystal in the control cells. The activity of alkaline phosphatase in the culture medium of the OGP (10(-14)) and G38I groups were higher than that in the control group (4.47 U/g and 3.82 U/g vs 2.21 U/g). The protein expression level of type-I collagen was higher and the mRNA levels of Cbfa1 and type-I collagen were higher in the OGP((10-14)) and G38I groups were increased in the experimental groups in comparison with the control group (P < 0.05, P < 0.01, and P < 0.05). CONCLUSION: They stimulated cell number enhancement dose dependently at low concentration and followed by inhibition at high concentration. Just as the native OGP, OGP((10-14)) and its derivative G38I stimulate the proliferation of osteoblasts, and improve their activity, up-regulate the Cbfa1 and type-I collagen mRNA expression levels and increase the collagen synthesis, thus promoting the differentiation and osteogenic effect of osteoblasts.