Inhibition of osteoclast differentiation and bone resorption by a novel lysophosphatidylcholine derivative, SCOH

Osteoclasts are multinucleated cells formed by multiple steps of cell differentiation from progenitor cells of hematopoietic origin. Intervention in osteoclast differentiation is considered as an effective therapeutic approach to the treatment for bone diseases involving osteoclasts. In this study, we found that the organic compound (S)-1-lyso-2-stearoylamino-2-deoxy-sn-glycero-3-phosphatidylcholine (SCOH) inhibited osteoclast differentiation. The inhibitory effect of SCOH was observed in mouse bone marrow cell cultures supported either by coculturing with osteoblasts or by adding macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL). M-CSF and RANKL activate the ERK, Akt, and NF-kappaB signal transduction pathways, and SCOH suppressed this activation. SCOH also inhibited the bone resorptive activity of differentiated osteoclasts. It attenuated bone resorption, actin ring formation, and survival of mature osteoclasts. Reduced activation of Akt and NF-kappaB and decreased induction of XIAP were observed in mature osteoclasts treated with SCOH. Thus, this novel phosphatidylcholine derivative may be useful for treating bone-resorption diseases.     

RAW264.7细胞在体外分化成破骨细胞的研究

目的:观察小鼠单核巨噬细胞RAW264.7的一般生物学特征及在重组细胞核因子kB受体活化因子配基(RANKL)诱导下形成成熟的有骨吸收能力的破骨细胞的可行性.方法:培养RAW264.7后,RANKL诱导RAW264.7,用抗酒石酸酸性磷酸酶(TRAP)染色法观察阳性多核细胞,电镜下扫描检测电镜骨吸收陷窝.结果:RANKL诱导RAW264.7第3天开始出现少数多核巨细胞,随时间的延长细胞数目逐渐增多,第7天多核巨细胞数达峰值,多核巨细胞TRAP染色阳性,电镜下可见骨片上的吸收陷窝呈圆形或椭圆形,边界轮廓清晰,陷窝底部纤维腐蚀吸收的纹路清晰可见,提示多核巨细胞具有骨吸收功能.结论:RAW264.7是一种较好的破骨前体细胞模型.RANKL诱导RAW264.7形成破骨细胞方法简便易行、可重复性好.     

PF2401-SF, standardized fraction of Salvia miltiorrhiza and its constituents, tanshinone I, tanshinone IIA, and cryptotanshinone, protect primary cultured rat hepatocytes from bile acid-induced apoptosis by inhibiting JNK phosphorylation.

Bile acid-induced hepatocyte apoptosis plays an important role in cholestatic liver disease, and the role of apoptosis may be of therapeutic interest in preventing liver disease. The dried root of Salvia miltiorrhiza Bunge (Labiatae) has been used traditionally to treat liver diseases. We investigated the antiapoptotic effects of a standardized fraction of S. miltiorrhiza (PF2401-SF) and its components, tanshinone I, tanshinone IIA, and cryptotanshinone, in primary cultured rat hepatocytes. PF2401-SF was enriched with tanshinone I (11.5%), tanshinone IIA (41.0%), and cryptotanshinone (19.1%). Glycochenodeoxycholic acid (GCDC)-induced apoptosis, as shown by DNA fragmentation, poly(ADP-ribose) polymerase cleavage, and activation of caspases-8, -9, and -3. PF2401-SF and its components, tanshinone I, tanshinone IIA, and cryptotanshinone showed antiapoptotic activity. Treatment with PF2401-SF or with its components significantly inhibited the generation of intracellular reactive oxygen species. Hydrophobic bile acids activate c-Jun-NH(2)-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPK), and extracellular signal-regulated kinase 1/2, and PF2401-SF inhibited the phosphorylation of JNK and p38. All three components of PF2401-SF inhibited JNK phosphorylation. Addition of inhibitors of MAPK showed that inhibition of JNK decreased apoptosis. These data indicate that PF2401-SF and its components protect hepatocytes from GCDC-induced apoptosis in vitro by inhibiting JNK.     

Inhibition of osteoclast formation by 3-methylcholanthrene, a ligand for arylhydrocarbon receptor: suppression of osteoclast differentiation factor in osteogenic cells

We investigated the effects of 3-methylcholanthrene (3MC), a ligand for arylhydrocarbon receptor (AhR), on osteoclastogenesis. Osteoclast-like cells, in cocultures with mouse spleen cells and clonal osteogenic stromal ST2 cells, are formed from spleen cells by a combination of the receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) produced by ST2 cells in response to 1alpha,25(OH)(2) Vitamin D(3). 3MC dose-dependently inhibited the formation of mono- and multinuclear osteoclast-like cells. However, 3MC did not inhibit the formation of osteoclast-like cells from mouse spleen cells which was supported by the exogenous soluble RANKL and M-CSF. 3MC did not affect the formation of an actin ring and pits on slices of dentine by osteoclast-like cells, both of which are typical indices of osteoclast activity. These results suggest that 3MC affects osteoclast-supporting cells such as ST2 cells but not osteoclast precursor cells and mature osteoclastic cells. When we measured the expression levels of RANKL mRNA in ST2 cells, 3MC dose-dependently decreased the level of this mRNA. However, 3MC did not affect levels of mRNAs for osteoprotegerin (OPG), M-CSF, and the receptor of 1alpha,25(OH)(2) Vitamin D(3) in ST2 cells. Furthermore, soluble RANKL was able to counteract the inhibitory effect of 3MC on the formation of osteoclast-like cells. Our findings indicate that 3MC inhibits osteoclastogenesis via the inhibition of RANKL expression in osteoblastic cells.     

Involvement of hydrogen peroxide in the differentiation and apoptosis of preosteoclastic cells exposed to arsenite

Long-term exposure to sodium arsenite (AsO₂) promotes the development of various cancers. Paradoxically, arsenic also induces pro-myelomonocytic leukemia cell differentiation, and at higher concentrations, apoptosis. The present study investigated the effects of AsO₂ on preosteoclasts. When treated with 2.5-5 μM AsO₂, RAW264.7 cells underwent osteoclast differentiation as evidenced by an increase in the number of multinucleate cells expressing tartrate resistant acid phosphatase (TRAP). The appearance of these phenotypic markers was preceded by a low level increase in extracellular production of H₂O₂ and was prevented by the addition of catalase (4.5 μg/ml), an enzyme that removes H₂O₂. Only at high concentrations (10-25 μM) of AsO₂ was a significant loss of cell viability and a high level increase in H₂O₂ production (1.5 μM) observed. Apoptosis was blocked by pretreatment with diphenylene iodonium chloride (2 μM), a NAD(P)H-flavoprotein inhibitor, suggesting the involvement of NADPH-oxidase. The data show that AsO₂, dose-dependently, stimulates increasing amounts of H₂O₂ production. Moreover, at concentrations found in tissues of individuals exposed to geochemical AsO₂, osteoclasts underwent an H₂O₂-dependent differentiation. Therefore, chronic exposure to low-level amounts of AsO₂ could result in increased bone resorption and contribute to bone related pathologies.     

Inhibitory effects of mevastatin and a geranylgeranyl transferase I inhibitor (GGTI-2166) on mononuclear osteoclast formation induced by receptor activator of NF kappa B ligand (RANKL) or tumor necrosis factor-alpha (TNF-alpha)

We have previously reported that the statin mevastatin (compactin) reversibly inhibits the fusion of TRAP-positive mononuclear preosteoclasts (pOCs) into multinucleated osteoclasts and disrupts the actin ring in mature osteoclasts through the inhibition of protein prenylation. Protein geranylgeranylation, specifically, is known to be required for pOC fusion and for the function and survival of mature osteoclasts. However, it has not been determined whether protein geranylgeranylation is involved in early differentiation of osteoclasts (pOC formation). The current study shows that statins and the geranylgeranyl transferase I inhibitor GGTI-2166 inhibit the pOC formation induced by RANKL or TNF-alpha in cultures of both mouse marrow-derived macrophage-colony-stimulating factor (M-CSF) dependent monocytes (MD cells) and the mouse monocyte cell line RAW 264.7 (RAW cells). Mevastatin, 0.1-0.6 microM, inhibited the formation of pOCs induced by receptor activator of nuclear factor-kappaB ligand (RANKL) or tumor necrosis factor (TNF-alpha) in both cell cultures. The inhibitory effects of mevastatin were overcome by the addition of mevalonate, farnesyl pyrophosphate or geranylgeranyl pyrophosphate. GGTI-2166 inhibited TRAP activity induced by RANKL or TNF-alpha in both cell cultures and prevented the incorporation of [3H]all-trans geranylgeraniol into prenylated proteins in RAW cells. However, the farnesyl transferase inhibitor FTI-2153 did not inhibit TRAP activity although FTI prevented the incorporation of [14C]mevalonate into farnesylated proteins in RAW cells. Clostridium difficile cytotoxin B (toxin B) inhibited pOC formation induced by RANKL or TNF-alpha in both cell cultures. The inhibitory effects of statins and GGTI-2166 on pOC formation may result from the inhibition of the geranylgeranylation of G-proteins, such as Rho or Rac, suggesting that the geranylgeranylation of these proteins is involved in the early differentiation of progenitor cells into pOCs.     

Evidence that genetic deletion of the TNF receptor p60 or p80 inhibits Fas mediated apoptosis in macrophages

Almost 19 members of the tumor necrosis factor (TNF) superfamily have been identified that interact with 29 different receptors. Whether these receptors communicate with each other is not understood. Recently, we have shown that receptor activator of NF-kappaB ligand signaling is modulated by genetic deletion of the TNF receptor. In the current report, we investigated the possibility of a cross-talk between Fas and TNF-alpha signaling pathway in macrophage cell lines derived from wild-type (WT) mice and from mice with genetic deletion of the type 1 TNF receptor (p60(-/-)), the type 2 TNF receptor (p80(-/-)), or both receptors (p60(-/-)p80(-/-)). We found that the macrophages expressing TNF receptors were highly sensitive to apoptosis induced by anti-Fas. The genetic deletion of TNF receptors, however, made the cells resistance to anti-Fas-induced apoptosis. Anti-Fas induced activation of caspase-3 and PARP cleavage in WT cells but not in TNF receptor-deleted cells. This difference was found to be independent of the expression of Fas, Fas-associated protein with death domain (FADD) or TNF receptor-associated death domain (TRADD). We found that anti-Fas induced recruitment of TNFR1 into Fas-complex. We also found that TRADD, which mediates TNF signaling, was constitutively bound to Fas receptor in TNF receptor-deleted cells but not in wild-type cells. Transient transfection of TNFR1 in TNFR1-deleted cells sensitized them to anti-Fas-induced apoptosis. Overall our results demonstrate that Fas signaling is modulated by the TNF receptors and thus provide the evidence of cross-talk between the receptors of two cytokines.     

Dose-dependent effects of genistein on bone homeostasis in rats' mandibular subchondral bone

Aim:

To investigate the effect of genistein on bone homeostasis in mandibular subchondral bone of rats.

Methods:

Female SD rats were administered with genistein (10 and 50 mg/kg) or placebo by oral gavage for 6 weeks. Then the animals were sacrificed, and histomorphology and micro-structure of mandibular condyle were examined using HE staining and micro-CT analysis, respectively. The expression levels of alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin (OPG), the receptor activator of nuclear factor κB ligand (RANKL) and estrogen receptors (ERs) in mandibular condyle were detected using real-time PCR. Cultured osteoblasts were prepared from rat mandibular condyle for in in vitro study. The cells were treated with genistein (10⁻⁷ or 10⁻⁴ mol/L) for 48 h. The expression of the bone homeostasis-associated factors and estrogen receptors (ERs) was detected using real-time PCR, and ER silencing was performed.

Results:

At both the low- and high-doses, genistein significantly increased the bone mineral density (BMD) and bone volume, and resulted in thicker subchondral trabecular bone in vivo. In both in vivo and in vitro study, the low-dose genistein significantly increased the expression of ALP, OC and OPG, but decreased the expression of RANKL and the RANKL/OPG ratio. The high-dose genistein decreased the expression of all these bone homeostasis-associated factors. Both the low and high doses of genistein significantly increased the expression of ERβ, while ERα expression was increased by the low dose genistein and decreased by the high dose genistein. ERβ silencing abrogated most of the effects of genistein treatment.

Conclusion:

In rat mandibular condylar subchondral bone, low-dose genistein increases bone formation and inhibit bone resorption, while excess genistein inhibits both bone formation and resorption. The effects of genistein were predominantly mediated through ERβ.     

Identification of a signaling cascade for interleukin-8 production by Helicobacter pylori in human gastric epithelial cells

Infecting gastric epithelial cells with Helicobacter pylori (H. pylori) has been shown to induce interleukin-8 (IL-8) production, but the signal transduction mechanism leading to IL-8 production is not defined clearly. In the present study, we investigated the molecular mechanism responsible for H. pylori-induced IL-8 release in human gastric epithelial cells. IL-8 levels in culture supernatants were determined by an enzyme linked-immunosorbent assay. Extracellular signal-regulated kinase (ERK) activity was tested using an in vitro kinase assay, which measured the incorporation of [gamma-33P]ATP into a synthetic peptide that is a specific ERK substrate. ERK phosphorylation and IkappaBalpha degradation by H. pylori infection were assessed by western blotting. In MKN45 cells, H. pylori-induced IL-8 release in a time-dependent manner. This IL-8 release was abolished by treatment with intracellular Ca2+ chelators (BAPTA-AM and TMB-8) but not by EGTA or nifedipine. The Ca2+ ionophore A23187 also induced IL-8 release to an extent similar to that of H. pylori infection. Calmodulin inhibitors (W7 and calmidazolium) and tyrosine kinase inhibitors (genistein and ST638) completely blocked IL-8 release by H. pylori and A23187. PD98059, an ERK pathway inhibitor, completely abolished H. pylori-induced IL-8 release. Moreover, BAPTA-AM, calmidazolium, and genistein, but not nifedipine, suppressed the ERK activation induced by H. pylori infection. PD98059 as well as MG132, an NF-kappaB pathway inhibitor, blocked both IL-8 production and degradation of IkappaBalpha induced by H. pylori infection, whereas only PD98059 inhibited ERK activity in response to H. pylori. There was no significant difference between IL-8 production induced by the cagA positive wild-type strain and the cagA negative isogenic mutant strain of H. pylori; therefore, CagA is not involved in the IL-8 production pathway. H. pylori-induced IL-8 production is dominantly regulated by Ca2+/calmodulin signaling, and ERK plays an important role in signal transmission for the efficient activation of H. pylori-induced NF-kappaB activity, resulting in IL-8 production.     

α-Tomatine inactivates PI3K/Akt and ERK signaling pathways in human lung adenocarcinoma A549 cells: Effect on metastasis

This study first investigates the anti-metastastic effect of α-tomatine in the human lung adenocarcinoma cell line: A549. In this study, we first noted α-tomatine inhibited A549 cells invasion and migration by wound-healing assay and Boyden chamber assay. The data also showed α-tomatine could inhibit phosphorylation of Akt and extracellular signal-regulated kinase 1 and 2 (ERK1/2), which is involved in the up-regulating matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) or urokinase-type plasminogen activator (u-PA), whereas it did not affect phosphorylation of c-Jun N-terminal kinase (JNK) and p38. Next, α-tomatine significantly decreased the nuclear levels of nuclear factor kappa B (NF-κB), c-Fos, and c-Jun. Also, treating A549 cells with α-tomatine also leads to a dose-dependent inhibition on the binding abilities of NF-κB and activator protein-1 (AP-1). Further, the treatment of inhibitors specific for PI3K (Wortmannin) or ERK (U0126) to A549 cells could cause reduced activities of MMP-2, MMP-9, and u-PA. These results showed α-tomatine could inhibit the metastatic ability of A549 cells by reducing MMP-2, MMP-9, and u-PA activities through suppressing phosphoinositide 3-kinase/Akt (PI3K/Akt) or ERK1/2 signaling pathway and inhibition NF-κB or AP-1 binding activities. These findings proved α-tomatine might be an anti-metastastic agent against human lung adenocarcinoma.     

Antithrombotic and profibrinolytic activities of phloroglucinol

Phloroglucinol is the monomeric units of phlorotannins abundant in brown algae. Several biological effects of phloroglucinol have been reported, however, antithrombotic and profibrinolytic activities of phloroglucinol have not been studied yet. In this study, the anticoagulant properties of phloroglucinol were determined by assays of activated partial thromboplastin time (aPTT), prothrombin time (PT) and cell based thrombin and activated factor X (FXa) generation activities. And the effects of phloroglucinol on the expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were tested in tumor necrosis factor-α (TNF-α) activated human endothelial cells (HUVECs). I found that phloroglucinol prolonged aPTT and PT significantly and inhibited thrombin and FXa generation in HUVECs. Furthermore, phloroglucinol inhibited TNF-α induced PAI-1 production. I then used pathway inhibitors to investigate which step of the TNF-α induced signaling pathway was targeted by phloroglucinol. I observed that the c-Jun N-terminal kinase (JNK) inhibitor increased the inhibitory effects of phloroglucinol, whereas the nuclear factor factor-κB (NF-κB) and the extracellular signal-regulated kinase (ERK) inhibitor did not. Therefore these results suggest that phloroglucinol possess antithrombotic and profibrinolytic activities and that NF-κB and ERK pathways are possible targets of phloroglucinol in the regulation of TNF-α stimulated PAI-1 production in HUVECs.