Regulatory effects of IL-1ß in the interaction of GBM and tumor-associated monocyte through VCAM-1 and ICAM-1.

Glioblastoma (GBM) is the most common and lethal brain tumor with high inflammation. GBM cells infiltrate microglia and macrophages and are surrounded by pro-inflammatory cytokines. Interleukin (IL)-1ß, which is abundantly expressed in the tumor microenvironment, is involved in tumor progression. Intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 mediate cell-cell interactions, and these cell adhesion molecules (CAMs) can be regulated by cytokines in immune cells or cancer cells in the inflammatory tumor microenvironment. In this study, we found that ICAM-1 and VCAM-1 expression was induced when GBM cells were treated with IL-1ß, and that adhesive interaction between monocytes and GBM cells increased accordingly. The levels of soluble CAMs (sICAM-1 and sVCAM-1) were also increased in the supernatants induced by IL-1ß. Furthermore, the conditioned media contained sICAM-1 and sVCAM-1, which further promoted IL-6 and CCL2 expression in differentiated macrophages. IL-1ß downregulated Src homology 1 domain-containing protein tyrosine phosphatase (SHP-1) in GBM. The expression of ICAM-1 and VCAM-1 was regulated by p38, AKT, and NF-κB signaling pathways, which were modulated by SHP-1 signaling. The present study suggests that IL-1ß-induced protein expression of ICAM-1 and VCAM-1 in GBM may modulate the adhesive interaction between GBM and monocytes. In addition, IL-1ß also induced the soluble form of ICAM-1 and VCAM-1 in GBM, which plays a key role in the regulation of tumor-associated monocyte/macrophage polarization.

Hypoxia induced mitogenic factor (HIMF) triggers angiogenesis by increasing interleukin-18 production in myoblasts.

Inflammatory myopathy is a rare autoimmune muscle disorder. Treatment typically focuses on skeletal muscle weakness or inflammation within muscle, as well as complications of respiratory failure secondary to respiratory muscle weakness. Impaired respiratory muscle function contributes to increased dyspnea and reduced exercise capacity in pulmonary hypertension (PH), a debilitating condition that has few treatment options. The initiation and progression of PH is associated with inflammation and inflammatory cell recruitment and it is established that hypoxia-induced mitogenic factor (HIMF, also known as resistin-like molecule α), activates macrophages in PH. However, the relationship between HIMF and inflammatory myoblasts remains unclear. This study investigated the signaling pathway involved in interleukin-18 (IL-18) expression and its relationship with HIMF in cultured myoblasts. We found that HIMF increased IL-18 production in myoblasts and that secreted IL-18 promoted tube formation of the endothelial progenitor cells. We used the mouse xenograft model and the chick chorioallantoic membrane assay to further explore the role of HIMF in inflammatory myoblasts and angiogenesis in vivo. Thus, our study focused on the mechanism by which HIMF mediates IL-18 expression in myoblasts through angiogenesis in vitro and in vivo. Our findings provide an insight into HIMF functioning in inflammatory myoblasts.

Effects of paeonol on anti-neuroinflammatory responses in microglial cells.

Increasing studies suggest that inflammatory processes in the central nervous system mediated by microglial activation plays an important role in numerous neurodegenerative diseases. Development of planning for microglial suppression is considered a key strategy in the search for neuroprotection. Paeonol is a major phenolic component of Moutan Cortex, widely used as a nutrient supplement in Chinese medicine. In this study, we investigated the effects of paeonol on microglial cells stimulated by inflammagens. Paeonol significantly inhibited the release of nitric oxide (NO) and the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Treatment with paeonol also reduced reactive oxygen species (ROS) production and inhibited an ATP-induced increased cell migratory activity. Furthermore, the inhibitory effects of neuroinflammation by paeonol were found to be regulated by phosphorylated adenosine monophosphate-activated protein kinase-α (AMPK-α) and glycogen synthase kinase 3 α/ß (GSK 3α/ß). Treatment with AMPK or GSK3 inhibitors reverse the inhibitory effect of neuroinflammation by paeonol in microglial cells. Furthermore, paeonol treatment also showed significant improvement in the rotarod performance and microglial activation in the mouse model as well. The present study is the first to report a novel inhibitory role of paeonol on neuroinflammation, and presents a new candidate agent for the development of therapies for inflammation-related neurodegenerative diseases.

Interaction of inflammatory and anti-inflammatory responses in microglia by Staphylococcus aureus-derived lipoteichoic acid.

We investigated the interaction between proinflammatory and inflammatory responses caused by Staphylococcus aureus-derived lipoteichoic acid (LTA) in primary cultured microglial cells and BV-2 microglia. LTA induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein levels increase in a concentration- and time-dependent manner. Meanwhile, LTA also increased nitric oxide (NO) and PGE2 production in microglia. Administration of TLR2 antagonist effectively inhibited LTA-induced NO, iNOS, and COX-2 expression. Moreover, treatment of cells with LTA caused a time-dependent activation of ERK, p38, JNK, as well as AKT. We also found that LTA-induced iNOS and COX-2 up-regulation were attenuated by p38, JNK, and PI3-kinase inhibitors. On the other hand, LTA-enhanced HO-1 expression was attenuated by p38 and PI3-kinase inhibitors. Treatment of cells with NF-κB and AP-1 inhibitors antagonized LTA-induced iNOS and COX-2 expression. However, only NF-κB inhibitors reduced LTA-induced HO-1 expression in microglia. Furthermore, stimulation of cells with LTA also activated IκBα phosphorylation, p65 phosphorylation at Ser5³6, and c-Jun phosphorylation. Moreover, LTA-induced increases of κB-DNA and AP-1-DNA binding activity were inhibited by p38, JNK, and PI3-kinase inhibitors. HO-1 activator CoPP IX dramatically reversed LTA-induced iNOS expression. Our results provided mechanisms linking LTA and inflammation/anti-inflammation, and indicated that LTA plays a regulatory role in microglia activation.

Hypoxia induces BMP-2 expression via ILK, Akt, mTOR, and HIF-1 pathways in osteoblasts.

It has been shown that hypoxia stimulation regulates bone formation, maintenance, and repair. Bone morphogenetic protein (BMP) plays important roles in osteoblastic differentiation and bone formation. However, the effects of hypoxia exposure on BMP-2 expression in cultured osteoblasts are largely unknown. Here we found that hypoxia stimulation increased mRNA and protein levels of BMP-2 by qPCR, Western blot and ELISA assay in osteoblastic cells MG-63, hFOB and bone marrow stromal cells M2-10B4. Integrin-linked kinase (ILK) inhibitor (KP-392), Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol-2-[(R)-2-O-methyl-3-O-octadecylcarbonate]) or mammalian target of rapamycin (mTOR) inhibitor (rapamycin) inhibited the potentiating action of hypoxia. Exposure to hypoxia increased the kinase activity of ILK and phosphorylation of Akt and mTOR. Furthermore, hypoxia also increased the stability and activity of HIF-1 protein. The binding of HIF-1alpha to the HRE elements after exposure to hypoxia was measured by EMSA assay. Moreover, the use of pharmacological inhibitors or genetic inhibition revealed that both ILK/Akt and mTOR signaling pathway were potentially required for hypoxia-induced HIF-1alpha activation and subsequent BMP-2 up-regulation. Taken together, our results provide evidence that hypoxia enhances BMP-2 expression in osteoblasts by an HIF-1alpha-dependent mechanism involving the activation of ILK/Akt and mTOR pathways.

FAK activation is required for TNF-alpha-induced IL-6 production in myoblasts.

Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine produced by activated macrophages. IL-6 is a multifunctional cytokine that plays a central role in both innate and acquired immune responses. We investigated the signaling pathway involved in IL-6 production stimulated by TNF-alpha in cultured myoblasts. TNF-alpha caused concentration-dependent increases in IL-6 production. TNF-alpha-mediated IL-6 production was attenuated by focal adhesion kinase (FAK) mutant and siRNA. Pretreatment with phosphatidylinositol 3-kinase inhibitor (PI3K; Ly294002 and wortmannin), Akt inhibitor, NF-kappaB inhibitor (pyrrolidine dithiocarbamate, PDTC), and IkappaB protease inhibitor (L-1-tosylamido-2-phenyl phenylethyl chloromethyl ketone, TPCK) also inhibited the potentiating action of TNF-alpha. TNF-alpha increased the FAK, PI3K, and Akt phosphorylation. Stimulation of myoblasts with TNF-alpha activated IkappaB kinase alpha/beta (IKKalpha/beta), IkappaBalpha phosphorylation, p65 phosphorylation, and kappaB-luciferase activity. TNF-alpha mediated an increase of kappaB-luciferase activity which was inhibited by Ly294002, wortmannin, Akt inhibitor, PDTC and TPCK or FAK, PI3K, and Akt mutant. Our results suggest that TNF-alpha increased IL-6 production in myoblasts via the FAK/PI3K/Akt and NF-kappaB signaling pathway.

Glial cell-derived neurotrophic factor increases migration of human chondrosarcoma cells via ERK and NF-kappaB pathways.

Invasion of tumor cells is the primary cause of therapeutic failure in the treatment of malignant chondrosarcomas. Glial cell-derived neurotrophic factor (GDNF) plays a crucial role in migration and metastasis of human cancer cells. Integrins are the major adhesive molecules in mammalian cells. Here we found that GDNF directed the migration and increased cell surface expression of alphav and beta3 integrin in human chondrosarcoma cells. Pretreated of JJ012 cells with MAPK kinase (MEK) inhibitors PD98059 or U0126 inhibited the GDNF-mediated migration and integrin expression. Stimulation of cells with GDNF increased the phosphorylation of MEK and extracellular signal-regulating kinase (ERK). In addition, NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also inhibited GDNF-mediated cells migration and integrin up-regulation. Stimulation of cells with GDNF induced IkappaB kinase (IKKalpha/beta) phosphorylation, IkappaB phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. Furthermore, the GDNF-mediated increasing of kappaB-luciferase activity was inhibited by PD98059, U0126, PDTC and TPCK or MEK, ERK, IKKalpha, and IKKbeta mutants. Taken together, these results suggest that the GDNF acts through MEK/ERK, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activations of alphavbeta3 integrin and contributing the migration of human chondrosarcoma cells.

Leptin enhances cell migration in human chondrosarcoma cells through OBRl leptin receptor.

Leptin, an adipocyte-derived cytokine that is closely associated with obesity, has recently been shown to be involved in carcinogenesis and cancer progression. Integrins are the major adhesive molecules in mammalian cells and have been associated with metastasis of cancer cells. In this study, we found that leptin increased the migration and the expression of alphavbeta3 integrin in human chondrosarcoma cells. We also found that human chondrosarcoma tissues and chondrosarcoma cell lines had significant expression of the long form (OBRl) leptin receptor, which was higher than that in normal cartilage and human primary chondrocyte. Leptin-mediated migration and integrin upregulation were attenuated by OBRl receptor antisense oligonucleotide. Activations of insulin receptor substrate (IRS)-1, phosphatidylinositol 3-kinase (PI3K), Akt and nuclear factor-kappaB (NF-kappaB) pathways after leptin treatment were demonstrated, and leptin-induced expression of integrin and migration activity was inhibited by the specific inhibitor, small-interfering RNA and mutant of IRS-1, PI3K, Akt and NF-kappaB cascades. Taken together, our results indicated that leptin enhances the migration of chondrosarcoma cells by increasing alphavbeta3 integrin expression through the OBR1/IRS-1/PI3K/Akt/NF-kappaB signal transduction pathway.

Suramin inhibits beta-bungarotoxin-induced activation of N-methyl-D-aspartate receptors and cytotoxicity in primary neurons.

We demonstrated that beta-bungarotoxin (beta-BuTX), a snake presynaptic neurotoxin, exhibited a potent cytotoxic effect on cultured cerebellar granule neurons. The mechanism of action of beta-BuTX and the cytoprotective agents against beta-BuTX were studied. The neuronal death of cerebellar granule neurons induced by beta-BuTX was manifested with apoptosis and necrosis processes as revealed by neurite fragmentation, morphological alterations, and staining apoptotic bodies with the fluorescent dye Hoechst 33258. By means of microspectrofluorimetry and fura-2, we measured intracellular Ca2+ concentration, [Ca2+]i and found that [Ca2+]i was increased markedly prior to the morphological changes and cytotoxicity. The downstream pathway of the increased [Ca2+]i was investigated: there was increased production of free radicals, decreased mitochondrial membrane potential, and depleted cellular ATP content. MK801 and suramin effectively suppressed these detrimental effects of beta-BuTX. Furthermore, the [3H]MK801 binding was reduced by unlabeled MK801, beta-BuTX, and suramin. Thus, activation of N-methyl-D-aspartate (NMDA) receptors appeared to play a crucial role in the cytotoxic effects following betaBuTX exposure. In conclusion, the novel finding of this study was that a polypeptide beta-BuTX exerted a potent cytotoxic effect through sequential events, including activating NMDA receptors followed by increasing [Ca2+]i, ROS production, and impaired mitochondrial energy metabolism. Suramin, clinically used as a trypanocidal agent, was an effective antagonist against beta-BuTX. Data suggest that suramin might have value to detect the possible pathway of certain neuropathological disorders.

Neuronal death signaling by beta-bungarotoxin through the activation of the N-methyl-D-aspartate (NMDA) receptor and L-type calcium channel.

The aim of this study was to elucidate the mechanism of the neurotoxic effect of beta-bungarotoxin (beta-BuTX, a snake presynaptic neurotoxin isolated from the venom of Bungarus multicinctus) on cultured cerebellar granule neurons. beta-BuTX exerted a potent, time-dependent, neurotoxic effect on mature granule neurons. Mature neurons, with an abundance of neurite outgrowths, were obtained after 7-8 days in culture. By means of microspectrofluorimetry and fura-2, we measured the intracellular Ca(2+) concentration ([Ca(2+)](i)) and found it to be increased markedly. BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tertrakis(acetoxymethyl ester)], EGTA, MK801 (dizocilpine maleate), and diltiazem prevented not only the elevation of [Ca(2+)](i), but also the beta-BuTX-induced neurotoxic effect. The signaling pathway involved in the elevation of [Ca(2+)](i) in beta-BuTX-induced neurotoxicity was studied. The results obtained indicated that beta-BuTX initially increased the production of reactive oxygen species and subsequently reduced mitochondrial membrane potential and depleted ATP. All of these events in the signaling pathway were blocked by MK801, diltiazem, EGTA, and BAPTA-AM. These findings suggest that the neurotoxic effect of beta-BuTX is mediated, at least in part, by a cascade of events that include the direct or indirect activation of N-methyl-D-aspartate (NMDA) receptors and L-type calcium channels that, in turn, lead to Ca(2+) influx, oxidative stress, mitochondrial dysfunction, and ATP depletion. Therefore, we suggest that this polypeptide neurotoxin, as a result of its high potency and irreversible properties, is a useful tool to elucidate the mechanisms of neurodegenerative diseases.

Long-term lithium treatment prevents neurotoxic effects of beta-bungarotoxin in primary cultured neurons.

Lithium is the most commonly used drug for the treatment of manic-depressive illness. The precise mechanisms underlying its clinical efficacy remain unknown. In this study, we found that long-term exposure to lithium chloride protected cultured cerebellar granule neurons (CGNs) against beta-bungarotoxin (beta-BuTX)-induced neurotoxicity. This neuroprotection was exhibited at the therapeutically relevant concentration of 1.2 mM lithium. Pretreatments for 3-5 days (long-term) were required for protection to occur; but a 3 hr treatment (short-term) was ineffective. In contrast, a longer treatment for 6-7 days or a higher concentration of 3 mM lithium led not only to loss of the neuroprotective effect but also to a neurotoxic effect. These findings suggest that lithium protection is limited to its narrow window of concentration and apparently relevant to its narrow therapeutic index in clinical application. Measurement of intracellular calcium [Ca(2+)](i) revealed that neurotoxic concentrations of beta-BuTX markedly increased [Ca(2+)](i), which could be attenuated by long-term, but not short-term, lithium treatment. Thus, the protection induced by lithium in CGNs was attributed to its inhibition of calcium overload. In addition, the Ca(2+) signaling pathway, including reactive oxygen species production and mitochondrial membrane potential reduction, along with the neurotoxic effect of beta-BuTX was blocked by long-term, but not short-term, lithium treatment. All of these results indicate that a crucial step for lithium protection is modulation of [Ca(2+)](i) homeostasis and that lithium neurotoxicity possibly, at least in part, is due to calcium overload. In conclusion, our results suggest that lithium, in addition to its use in treatment of bipolar depressive illness, may have an expanded use in intervention for neurotoxicity.