Ganglioside GD1a regulation of caveolin-1 and Stim1 expression in mouse FBJ cells:Augmented expression of caveolin-1 and Stim1 in cells with increased GD1a content

GD1a was previously shown responsible for regulating cell motility, cellular adhesiveness to vitronectin, phosphorylation of c-Met and metastatic ability of mouse FBJ osteosarcoma cells. To determine the particular molecules regulated by GD1a, FBJ cells were assessed for tumor-related gene expression by semi-quantitative RT-PCR. Caveolin-1 and stromal interaction molecule 1 (Stim1) expression in FBJ-S1 cells, rich in GD1a, were found to be 6 and 4 times as much, respectively, than in FBJ-LL cells devoid of GD1a. Enhanced production of caveolin-1 in protein was confirmed by Western blotting. A low-metastatic FBJ-LL cell variant, having high GD1a expression through β1-4GalNAcT-1 (GM2/GD2 synthase) cDNA transfection (Hyuga S, et al, Int J Cancer 83: 685-91, 1999), showed enhanced production of caveolin-1 and Stim1 in mRNA and protein, compared to mock-transfectant M5. Incubation of FBJ-M5 cells with exogenous GD1a augmented the expression of caveolin-1 in mRNA and protein and Stim1 in mRNA as well. Treatment of FBJ-S1 with fumonisin B1, an inhibitor of N-acylsphinganine synthesis, for 15 days caused the complete depletion of gangliosides and suppressed the expression of caveolin-1 and Stim1. St3gal5 siRNA transfected cells showed decreased expression of caveolin-1 and Stim1 mRNA, as well as St3gal5 mRNA. These findings clearly indicate ganglioside GD1a to be involved in the regulation of the transformation suppressor genes, caveolin-1 and Stim1. Moreover, treatment with GD1a of mouse melanoma B16 cells and human hepatoma HepG2 cells brought about elevated expression of caveolin-1 and Stim1. Li Wang and Shizuka Takaku are equal contributors to the present work     

Involvement of Ext1 and heparanase in migration of mouse FBJ osteosarcoma cells

To know the involvement of glycosaminoglycans (GAGs) in the metastasis of mouse FBJ osteosarcoma cells, N ^α -lauroyl-O-(β-d-xylopyranosyl)-l-serinamide (Xyl-Ser-C12), which initiates elongation of GAG chains using the glycan biosynthesis system in cells, was administered to FBJ cells with different metastatic capacities. Production of glycosylated products derived from Xyl-Ser-C12, especially heparan sulfate (HS) GAG-type oligosaccharides such as GalNAc-GlcA-GlcNAc-GlcA-Gal-Gal-Xyl-Ser-C12, was indicated in poorly metastatic FBJ-S1 cells more than in highly metastatic FBJ-LL cells by LC–MS. The results of RT-PCR revealed that HS synthases, Ext1 and Ext2, were expressed in FBJ-S1 cells more than in FBJ-LL cells. Furthermore, siRNA against Ext1 suppressed the expression of HS and enhanced the motility of FBJ-S1 cells. In addition, the expression of heparanase (HPSE) was enhanced in Ext-1-knockdown FBJ-S1 cells, and responsible for the increase in cell motility caused by the down-regulation of Ext1 expression. Our data provide the first evidence that Ext1 regulates the expression of HPSE and also indicated that levels of Ext1 and HPSE influenced the motility of FBJ cells.     

AMP-activated protein kinase suppresses matrix metalloproteinase-9 expression in mouse embryonic fibroblasts

Matrix metalloproteinase-9 (MMP-9) plays a critical role in tissue remodeling under both physiological and pathological conditions. Although MMP-9 expression is low in most cells and is tightly controlled, the mechanism of its regulation is poorly understood. We utilized mouse embryonic fibroblasts (MEFs) that were nullizygous for the catalytic α subunit of AMP-activated protein kinase (AMPK), which is a key regulator of energy homeostasis, to identify AMPK as a suppressor of MMP-9 expression. Total AMPKα deletion significantly elevated MMP-9 expression compared with wild-type (WT) MEFs, whereas single knock-out of the isoforms AMPKα1 and AMPKα2 caused minimal change in the level of MMP-9 expression. The suppressive role of AMPK on MMP-9 expression was mediated through both its activity and presence. The AMPK activators 5-amino-4-imidazole carboxamide riboside and A769662 suppressed MMP-9 expression in WT MEFs, and AMPK inhibition by the overexpression of dominant negative (DN) AMPKα elevated MMP-9 expression. However, in AMPKα(-/-) MEFs transduced with DN AMPKα, MMP-9 expression was suppressed. AMPKα(-/-) MEFs showed increased phosphorylation of IκBα, expression of IκBα mRNA, nuclear localization of nuclear factor-κB (NF-κB), and DNA-binding activity of NF-κB compared with WT. Consistently, selective NF-κB inhibitors BMS345541 and SM7368 decreased MMP-9 expression in AMPKα(-/-) MEFs. Overall, our results suggest that both AMPKα isoforms suppress MMP-9 expression and that both the activity and presence of AMPKα contribute to its function as a regulator of MMP-9 expression by inhibiting the NF-κB pathway.     

Liver X receptor α (LXRα) promoted invasion and EMT of gastric cancer cells by regulation of NF-κB activity

Aberrant expression of Liver X receptor α (LXRα) has been frequently reported in various types of cancers excluding gastric cancer (GC). Moreover, the role of LXRα in human GC has not been previously reported. In this study, we investigated the effect of LXRα down-regulation on invasion and EMT of GC. The expression of LXRα in GC cell lines was detected by real-time PCR. The LXRα siRNA was transiently transfected into GC cells using Lipofectamine? 2000 reagent. Subsequently, cell invasive ability was evaluated by Transwell assays. Western blot and real-time PCR were used to determined the expressions of matrix metalloproteinase-2 and -9 (MMP-2 and -9), E-cadherin, N-cadherin, Vimentin, Snail, Slug, and Twist in GC cells. In addition, the effect of LXRα down-regulation on the phosphoinositide 3-kinase (PI3K)/Akt/nuclear factor (NF)-κB signaling pathway was explored by Western blot. From our results, we found that the expression of LXRα was significantly increased in GC tissues and cell lines. Knockdown of LXRα suppressed the invasive ability of GC cells. The levels of MMP-2 and -9 were dramatically decreased by down-regulating LXRα. In addition, we found a decrease of N-cadherin, Twist, and Slug expressions and an increase of E-cadherin expression, but no influence on the expression levels of Vimentin and Snail. We also found that LXRα down-regulation might suppress the phosphorylation of Akt, NF-κB, and IκB. Collectively, our results indicated that down-regulation of LXRα was shown to suppress invasion and EMT of GC cells by decreasing the expressions of related proteins through inhibiting the PI3K/Akt/NF-κB signaling pathway.     

TNFα-induced up-regulation of miR-155 inhibits adipogenesis by down-regulating early adipogenic transcription factors

Tumor necrosis factor α (TNFα) is known to inhibit adipogenesis, but the molecular mechanism of this inhibition remains elusive. In the present study, we found that TNFα-induced inhibition of adipogenesis mainly occurs when 3T3-L1 preadipocytes are treated with TNFα within 2 h induction of adipogenesis. We revealed that TNFα treatment results in the up-regulation of miR-155 through the NFκB pathway in 3T3-L1 cells. This overexpression of miR-155 may suppress the expression of C/EBPβ and CREB by directly targeting their 3′ untranslated regions (3′ UTRs). Importantly, anti-miR-155 reduces the TNFα-induced inhibition of adipogenesis, whereas exogenous expression of mir-155 inhibits adipogenesis. Taken together, these findings reveal a novel role for TNFα in the regulation of anti-adipogenic miRNAs.     

Phosphatidic acid as a regulator of matrix metalloproteinase-9 expression via the TNF-alpha signaling pathway

Phosphatidic acid (PA) is implicated in pathophysiological processes associated with cellular signaling events and inflammation, which include the expressional regulation of numerous genes. Here, we show that PA stimulation increases matrix metalloproteinase-9 (MMP-9) expression in macrophages through tumor necrosis factor (TNF)-alpha signaling. We performed antibody array analysis on proteins from macrophages stimulated with PA. PA was found to induce the production of TNF-alpha, but not of TNF receptor (TNFR)1 and TNFR2 in a time-dependent manner and stimulated significant, though delayed, MMP-9 expression. PA induced the phosphorylations of both ERK1/2 and p38, but not of c-jun amino-terminal kinase. Moreover, only ERK1/2 inhibition by U0126 suppressed PA-induced TNF-alpha production and MMP-9 expression. Neutralizing TNF-alpha, TNFR1 or TNFR2 antibodies significantly suppressed PA-induced MMP-9 expression, suggesting that the production of TNF-alpha in response to PA preceded the expression of MMP-9. Moreover, lipopolysaccharide-induced PA also led to TNF-alpha release and resulted in MMP-9 expression. Taken together, these observations suggest that PA may play a role in MMP-9 regulation through ERKs/TNF-alpha/TNFRs-dependent signaling pathway.     

Apigenin inhibits the TNFα-induced expression of eNOS and MMP-9 via modulating Akt signalling through oestrogen receptor engagement

Apigenin is a naturally occurring plant flavone with strong anti-oxidant and anti-inflammatory activity. While the anticancer properties of Apigenin have been extensively studied, little is known about its effects on endothelial dysfunction. We investigated the effects of Apigenin in EAhy926 endothelial cells exposed to TNFα by evaluating the expression of eNOS and MMP-9, two key molecules in endothelial dysfunction. MMP-9 activity was measured by gel zymography. Western blot analysis was performed to analyze eNOS expression and signal transduction. Treatment with Apigenin (50?μM) counteracted the TNFα-induced expression of eNOS and MMP-9 and the TNFα- triggered activation of Akt, p38MAPK and JNK signalling suggesting that multiple signalling pathways are involved in mediating the protective effects of Apigenin on endothelial function. To better understand the molecular mechanisms underlying the protective effects of Apigenin, we used a pharmacological approach with specific inhibitors. The use of an Akt inhibitor mimicked the inhibitory effects of Apigenin on eNOS and MMP-9 expression, suggesting that eNOS and MMP-9 induction by TNFα depends on Akt activation. The TNFα-induced expression of MMP-9 was also affected by the JNK inhibitor SP600125. No effect on eNOS and MMP-9 expression was observed in the presence of the p38MAPK inhibitor SB203580 or the ERK 1/2 inhibitor PD98059. Pretreatment with ‘classic’ (ERα and ERβ) or ‘non classic’ (GPR30) oestrogen receptor (ER) inhibitors (ICI182,780 and PTX, respectively) counteracted the ability of Apigenin to decrease the TNFα-triggered activation of the Akt pathway. Consistently, the use of both ER inhibitors reversed the inhibitory effects of Apigenin on the TNFα-induced expression of eNOS and, to a lesser extent, MMP-9. We can conclude that Apigenin exerts its inhibitory effect on the TNFα-induced expression of eNOS and MMP-9 through the Akt signalling inhibition generated by ER activation. Oestrogen signalling has been implicated in protection from cardiovascular disease. Therefore, having regard to its ability to bind to ERs, Apigenin may be considered an oestrogen-like molecule to potentially be used against the onset and progression of vascular diseases associated with endothelial dysfunction.     

Inductive effect of phytoglycoprotein (38 kDa) on G0/G1 arrest and apoptosis in diethylnitrosamine-treated ICR mice

Preeclampsia complicates 5–10 % of pregnancies and is a leading cause of maternal/fetal morbidity and mortality. Although the cause is unknown, the reduced migration/invasion of extravillous trophoblasts is generally regarded as a key feature of preeclampsia genesis. The present study examined the expression of activator protein-2α (AP-2α), tissue inhibitor of metalloproteinase 2 (TIMP-2), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and E-cadherin in severe preeclamptic placentas and normal placentas using real-time PCR and immunohistochemistry. The expression levels of AP-2α, TIMP-2, and E-cadherin were elevated, while MMP-2 and MMP-9 levels were decreased in severe preeclamptic placentas when compared with normal placentas. To explore the underlying molecular mechanisms, BeWo cells were transfected with an AP-2α-expression construct as well as a siRNA against AP-2α. The over-expression of AP-2α decreased the invasive abilities of BeWo cells. AP-2α induction was followed by the induction of TIMP-2 and E-cadherin and a significant reduction of MMP-2 and MMP-9. Whereas in AP-2α-silencing BeWo cells, we observed the decreased expression of TIMP-2 and E-cadherin and the increased expression of MMP-2 and MMP-9. We presume that AP-2α may suppress trophoblast invasion by repression of MMP-2 and MMP-9 and up-regulation of E-cadherin, thus leading to shallow placentation in severe preeclampsia.