Green tea polyphenol (−)‐epigallocatechin gallate suppressed the differentiation of murine osteoblastic MC3T3‐E1 cells

Recently, various physiological effects of the tea polyphenol catechin for alleviating diseases such as cancer, arteriosclerosis, hyperlipidaemia and osteoporosis have been reported. However, the physiological effect of catechin on bone metabolism remains unclear. We examined the physiological effect of EGCG [(?)‐epigallocatechin‐3‐gallate], which is the main component of green tea catechin, on osteoblast development using the precursor cell line of osteoblasts, MC3T3‐E1, and co‐culture of the osteoblasts from mouse newborn calvaria and mouse bone marrow cells. Although EGCG did not affect the viability and proliferation of MC3T3‐E1 cells, EGCG inhibited the osteoblast differentiation. Furthermore, EGCG did not affect the mineralization of differentiated MC3T3‐E1 cells, and reduced osteoclast formation in co‐culture. These results suggest that EGCG can effectively suppress bone resorption, and can be used as an effective medicine in the treatment of the symptoms of osteoporosis.     

The small GTPase RhoA is crucial for MC3T3‐E1 osteoblastic cell survival

Prolongation of cell survival through prevention of apoptosis is considered to be a significant factor leading to anabolic responses in bone. The current studies were carried out to determine the role of the small GTPase, RhoA, in osteoblast apoptosis, since RhoA has been found to be critical for cell survival in other tissues. We investigated the effects of inhibitors and activators of RhoA signaling on osteoblast apoptosis. In addition, we assessed the relationship of this pathway to parathyroid hormone (PTH) effects on apoptotic signaling and cell survival. RhoA is activated by geranylgeranylation, which promotes its membrane anchoring. In serum‐starved MC3T3‐E1 osteoblastic cells, inhibition of geranylgeranylation with geranylgeranyl transferase I inhibitors increased activity of caspase‐3, a component step in the apoptosis cascade, and increased cell death. Dominant negative RhoA and Y27632, an inhibitor of the RhoA effector Rho kinase, also increased caspase‐3 activity. A geranylgeranyl group donor, geranylgeraniol, antagonized the effect of the geranylgeranyl tranferase I inhibitor GGTI‐2166, but could not overcome the effect of the Rho kinase inhibitor. PTH 1‐34, a potent anti‐apoptotic agent, completely antagonized the stimulatory effects of GGTI‐2166, dominant negative RhoA, and Y27632, on caspase‐3 activity. The results suggest that RhoA signaling is essential for osteoblastic cell survival but that the survival effects of PTH 1‐34 are independent of this pathway. J. Cell. Biochem. 106: 896–902, 2009. © 2009 Wiley‐Liss, Inc.     

Glucosamine inhibits IL‐1β‐mediated IL‐8 production in prostate cancer cells by MAPK attenuation

Inflammation is a complex process involving cytokine production to regulate host defense cascades. In contrast to the therapeutic significance of acute inflammation, a pathogenic impact of chronic inflammation on cancer development has been proposed. Upregulation of inflammatory cytokines, such as IL‐1β and IL‐8, has been noted in prostate cancer patients and IL‐8 has been shown to promote prostate cancer cell proliferation and migration; however, it is not clear whether IL‐1β regulates IL‐8 expression in prostate cancer cells. Glucosamine is widely regarded as an anti‐inflammatory agent and thus we hypothesized that if IL‐1β activated IL‐8 production in prostate cancer cells, then glucosamine ought to blunt such an effect. Three prostate cancer cell lines, DU‐145, PC‐3, and LNCaP, were used to evaluate the effects of IL‐1β and glucosamine on IL‐8 expression using ELISA and RT‐PCR analyses. IL‐1β elevated IL‐8 mRNA expression and subsequent IL‐8 secretion. Glucosamine significantly inhibited IL‐1β‐induced IL‐8 secretion. IL‐8 appeared to induce LNCaP cell proliferation by MTT assay; involvement of IL‐8 in IL‐1β‐dependent PC‐3 cell migration was demonstrated by wound‐healing and transwell migration assays. Inhibitors of MAPKs and NFκB were used to pinpoint MAPKs but not NFκB being involved in IL‐1β‐mediated IL‐8 production. IL‐1β‐provoked phosphorylation of all MAPKs was notably suppressed by glucosamine. We suggest that IL‐1β can activate the MAPK pathways resulting in an induction of IL‐8 production, which promotes prostate cancer cell proliferation and migration. In this context, glucosamine appears to inhibit IL‐1β‐mediated activation of MAPKs and therefore reduces IL‐8 production; this, in turn, attenuates cell proliferation/migration. J. Cell. Biochem. 108: 489–498, 2009. © 2009 Wiley‐Liss, Inc.     

Fluid shear stress induces Runx‐2 expression via upregulation of PIEZO1 in MC3T3‐E1 cells

Mechanically induced biological responses in bone cells involve a complex biophysical process. Although various mechanosensors have been identified, the precise mechanotransduction pathway remains poorly understood. PIEZO1 is a newly discovered mechanically activated ion channel in bone cells. This study aimed to explore the involvement of PIEZO1 in mechanical loading (fluid shear stress)‐induced signaling cascades that control osteogenesis. The results showed that fluid shear stress increased PIEZO1 expression in MC3T3‐E1 cells. The fluid shear stress elicited the key osteoblastic gene Runx‐2 expression; however, PIEZO1 silencing using small interference RNA blocked these effects. The AKT/GSK‐3β/β‐catenin pathway was activated in this process. PIEZO1 silencing impaired mechanically induced activation of the AKT/GSK‐3β/β‐catenin pathway. Therefore, the results demonstrated that MC3T3‐E1 osteoblasts required PIEZO1 to adapt to the external mechanical fluid shear stress, thereby inducing osteoblastic Runx‐2 gene expression, partly through the AKT/GSK‐3β/β‐catenin pathway.     

rIL‐10 enhances IL‐10 signalling proteins in foetal alveolar type II cells exposed to hyperoxia

Although the mechanisms by which hyperoxia promotes bronchopulmonary dysplasia are not fully defined, the inability to maintain optimal interleukin (IL)‐10 levels in response to injury secondary to hyperoxia seems to play an important role. We previously defined that hyperoxia decreased IL‐10 production and pre‐treatment with recombinant IL‐10 (rIL‐10) protected these cells from injury. The objectives of these studies were to investigate the responses of IL‐10 receptors (IL‐10Rs) and IL‐10 signalling proteins (IL‐10SPs) in hyperoxic foetal alveolar type II cells (FATIICs) with and without rIL‐10. FATIICs were isolated on embryonic day 19 and exposed to 65%‐oxygen for 24 hrs. Cells in room air were used as controls. IL‐10Rs protein and mRNA were analysed by ELISA and qRT‐PCR, respectively. IL‐10SPs were assessed by Western blot using phospho‐specific antibodies. IL‐10Rs protein and mRNA increased significantly in FATIICs during hyperoxia, but JAK1 and TYK2 phosphorylation showed the opposite pattern. To evaluate the impact of IL‐8 (shown previously to be increased) and the role of IL‐10Rs, IL‐10SPs were reanalysed in IL‐8‐added normoxic cells and in the IL‐10Rs’ siRNA‐treated hyperoxic cells. The IL‐10Rs’ siRNA‐treated hyperoxic cells and IL‐8‐added normoxic cells showed the same pattern in IL10SPs with the hyproxic cells. And pre‐treatment with rIL‐10 prior to hyperoxia exposure increased phosphorylated IL‐10SPs, compared to the rIL‐10‐untreated hyperoxic cells. These studies suggest that JAK1 and TYK2 were significantly suppressed during hyperoxia, where IL‐8 may play a role, and rIL‐10 may have an effect on reverting the suppressed JAK1 and TYK2 in FATIICs exposed to hyperoxia.     

IL‐1α and IL‐1β have different effects on formation and activity of large osteoclasts

Interleukin 1 (IL‐1) is a proinflammatory cytokine upregulated in conditions such as rheumatoid arthritis and periodontal disease. Both isoforms, IL‐1α and IL‐1β, have been shown to activate osteoclasts (OCs), the cells responsible for resorbing bone. Inflammatory conditions are also characterized by increased bone loss and by the presence of large OCs (10+ nuclei). We and others have previously shown that large OCs are more likely to be resorbing compared to small OCs (2–5 nuclei). Moreover, large OCs express higher levels of the IL‐1 activating receptor IL‐1RI, integrins αv and β3, RANK, and TNFR1, while small OCs have higher levels of the decoy receptor IL‐1RII. We hypothesized that IL‐1 would have different effects on large and small OCs due to these distinct receptor expression patterns. To test this hypothesis, RAW 264.7 cells were differentiated into populations of small and large OCs and treated with IL‐1α or IL‐1β (1 and 10 ng/ml). In the presence of sRANKL, both IL‐1α and IL‐1β increased total OC number and resorptive activity of large OCs. IL‐1α stimulated formation of large OCs and increased the number of resorption pits, while IL‐1β changed the morphology of large OCs and integrin‐β3 phosphorylation. No effects were seen in small OCs in response to either IL‐1 isoform. These results demonstrate that IL‐1 predominantly affects large OCs. The dissimilarity of responses to IL‐1α and IL‐1β suggests that these isoforms activate different signaling pathways within the two OC populations. J. Cell. Biochem. 109: 975–982, 2010. © 2010 Wiley‐Liss, Inc.     

Dexamethasone induces caspase activation in murine osteoblastic MC3T3-E1 cells

Glucocorticoids are widely used as anti-inflammatory and chemotherapeutic agents. However, prolonged use of glucocorticoids leads to osteoporosis. This study was designed to examine the mechanism of dexamethasone (DEX)-induced apoptosis in murine osteoblastic MC3T3-E1 cells. Total RNA was extracted from MC3T3-E1 cells treated with 10(-7) M DEX for 6 h. DEX exerted a variety of effects on apoptotic gene expression in osteoblasts. Ribonuclease protection assays (RPA) revealed that DEX upregulated mRNA levels of caspases-1, -3, -6, -8, -11, -12, and bcl-XL. Western blot analysis showed enhanced processing of these caspases, with the appearance of their activated enzymes 8 h after DEX treatment. In addition, DEX also induced the activation of caspase-9. DEX elevated the levels of cleaved poly(ADP-ribose) polymerase and lamin A, a caspase-3 and a caspase-6 substrate, respectively. Expression of bcl-XL protein level was upregulated by DEX. Cytochrome c release was detected in the cytosol of DEX-treated cells. Furthermore, caspase-3 enzyme activity was elevated by 2-fold after DEX treatment for 7 h. Finally, early apoptotic cells were detected in cells treated with DEX for 3 h. Our results demonstrate that DEX-induced apoptosis involves gene activation of a number of caspases.     

Effects of insulin and insulin‐like growth factor 1 on osteoblast proliferation and differentiation: differential signalling via Akt and ERK

Insulin and insulin‐like growth factor 1 (IGF‐1) are evolutionarily conserved hormonal signalling molecules, which influence a wide array of physiological functions including metabolism, growth and development. Using genetic mouse studies, both insulin and IGF‐1 have been shown to be anabolic agents in osteoblasts and bone development primarily through the activation of Akt and ERK signalling pathways. In this study, we examined the temporal signalling actions of insulin and IGF‐1 on primary calvarial osteoblast growth and differentiation. First, we observed that the IGF‐1 receptor expression decreases whereas insulin receptor expression increases during osteoblast differentiation. Subsequently, we show that although both insulin and IGF‐1 promote osteoblast differentiation and mineralization in vitro, IGF‐1, but not insulin, can induce osteoblast proliferation. The IGF‐1‐induced osteoblast proliferation was mediated via both MAPK and Akt pathways because the IGF‐1‐mediated cell proliferation was blocked by U0126, an MEK/MAPK inhibitor, or LY294002, a PI3‐kinase inhibitor. Osteocalcin, an osteoblast‐specific protein whose expression corresponds with osteoblast differentiation, was increased in a dose‐ and time‐dependent manner after insulin treatment, whereas it was decreased with IGF‐1 treatment. Moreover, insulin treatment dramatically induced osteocalcin promoter activity, whereas IGF‐1 treatment significantly inhibited it, indicating direct effect of insulin on osteocalcin synthesis. Copyright © 2012 John Wiley & Sons, Ltd.     

Fluorofenidone attenuates pulmonary inflammation and fibrosis via inhibiting the activation of NALP3 inflammasome and IL‐1β/IL‐1R1/MyD88/NF‐κB pathway

Interleukin (IL)‐1β plays an important role in the pathogenesis of idiopathic pulmonary fibrosis. The production of IL‐1β is dependent upon caspase‐1‐containing multiprotein complexes called inflammasomes and IL‐1R1/MyD88/NF‐κB pathway. In this study, we explored whether a potential anti‐fibrotic agent fluorofenidone (FD) exerts its anti‐inflammatory and anti‐fibrotic effects through suppressing activation of NACHT, LRR and PYD domains‐containing protein 3 (NALP3) inflammasome and the IL‐1β/IL‐1R1/MyD88/NF‐κB pathway in vivo and in vitro. Male C57BL/6J mice were intratracheally injected with Bleomycin (BLM) or saline. Fluorofenidone was administered throughout the course of the experiment. Lung tissue sections were stained with haemotoxylin and eosin and Masson's trichrome. Cytokines were measured by ELISA, and α‐smooth muscle actin (α‐SMA), fibronectin, collagen I, caspase‐1, IL‐1R1, MyD88 were measured by Western blot and/or RT‐PCR. The human actue monocytic leukaemia cell line (THP‐1) were incubated with monosodium urate (MSU), with or without FD pre‐treatment. The expression of caspase‐1, IL‐1β, NALP3, apoptosis‐associated speck‐like protein containing (ASC) and pro‐caspase‐1 were measured by Western blot, the reactive oxygen species (ROS) generation was detected using the Flow Cytometry, and the interaction of NALP3 inflammasome‐associated molecules were measured by Co‐immunoprecipitation. RLE‐6TN (rat lung epithelial‐T‐antigen negative) cells were incubated with IL‐1β, with or without FD pre‐treatment. The expression of nuclear protein p65 was measured by Western blot. Results showed that FD markedly reduced the expressions of IL‐1β, IL‐6, monocyte chemotactic protein‐1 (MCP‐1), myeloperoxidase (MPO), α‐SMA, fibronectin, collagen I, caspase‐1, IL‐1R1 and MyD88 in mice lung tissues. And FD inhibited MSU‐induced the accumulation of ROS, blocked the interaction of NALP3 inflammasome‐associated molecules, decreased the level of caspase‐1 and IL‐1β in THP‐1 cells. Besides, FD inhibited IL‐1β‐induced the expression of nuclear protein p65. This study demonstrated that FD, attenuates BLM‐induced pulmonary inflammation and fibrosis in mice via inhibiting the activation of NALP3 inflammasome and the IL‐1β/IL‐1R1/MyD88/ NF‐κB pathway.     

Transplant of insulin‐like growth factor‐1 expressing bone marrow stem cells improves functional regeneration of injured rat uterus by NF‐κB pathway

To investigate the potential beneficial effect of insulin‐like growth factor‐1 (IGF‐1) in BMSC transplantation therapy of uterus injury and the underlying molecular mechanisms, rat BMSCs were isolated and cultured. The relative expressions of IGF‐1 and IL‐10 were determined by RT‐PCR and immunoblotting. The secretory IL‐10 and released E2 were measured using ELISA kits. The relative vWF and α‐SMA expressions were determined by immunohistochemistry. The direct binding of NF‐κB subunit p50 with IL‐10 promoter was analysed by chromatin immunoprecipitation assay. The regulation of IL‐10 expression by p50 was interrogated by luciferase reporter assay. Our data demonstrated that IGF‐1 expression in BMSCs induced IL‐10 expression and secretion, which was further enhanced by E2‐PLGA. IGF‐1 overexpression improved BMSCs transplantation therapy in rat uterus injury. We further demonstrated that both inhibition and knockdown of p50 abolished IGF‐1‐induced expression and secretion of IL‐10 in BMSCs, which consequently compromised the IGF‐1 conferred therapeutic benefits against uterus injury. Furthermore, we elucidated that p50 regulated IL‐10 expression via direct association with its promoter. Our data suggested that transplantation of IGF‐1 overexpressing BMSCs improved functional regeneration of injured uterus by inducing IL‐10 expression and secretion via activation of NF‐κB signalling.