Oxidized LDL induces transcription factor activator protein-1 in rat mesangial cells

It has been shown that oxidized low-density lipoprotein (ox-LDL), through the activation of glomerular cells, stimulates pathobiological processes involved in monocyte infiltration into the mesangium. The underlying molecular mechanisms are not fully understood. The present study showed that ox-LDL strongly induced AP-1 binding activity in rat mesangial cells (RMCs) in a dose- and time-dependent manner, reaching the maximal activation at 250 microg ml(-1) within 24 h. The results from mobility shift assays and Western blotting analysis revealed that this AP-1 binding increase involved c-Jun, but not c-Fos. Moreover, this ox-LDL-increased AP-1 binding was inhibited by several protein kinase (PK) inhibitors: the protein kinase C (PKC) inhibitor Bisindolylmaleimide I, the cAMP-dependent PK (PKA) inhibitor H89, and the tyrosine PK (PTK) inhibitor genistein. Protein phosphorylation represents mitogen-activated protein kinase (MAPK) activity. Therefore, we examined the role of ox-LDL on the activation of mesangial cell JNK/SAPK, the only recognized protein kinase that catalyses phosphorylation of c-Jun. The incubation of mesangial cells with ox-LDL induced phosphorylation of JNK1/SAPK dose dependently, with the maximal response at 150 microg ml(-1). This study demonstrates that multiple kinase activities are involved in the mechanism of ox-LDL-induced AP-1 activation in mesangial cells, and ox-LDL stimulates AP-1 through JNK-c-Jun other than MEK-c-Fos signalling pathway.     

Epidermal growth factor induces tumour marker AKR1B10 expression through activator protein-1 signalling in hepatocellular carcinoma cells

AKR1B10 (aldo-keto reductase 1B10) is overexpressed in liver and lung cancer, and plays a critical role in tumour development and progression through promoting lipogenesis and eliminating cytotoxic carbonyls. AKR1B10 is a secretory protein and potential tumour marker; however, little is known about the regulatory mechanism of AKR1B10 expression. The present study showed that AKR1B10 is induced by mitogen EGF (epidermal growth factor) and insulin through the AP-1 (activator protein-1) signalling pathway. In human HCC (hepatocellular carcinoma) cells (HepG2 and Hep3B), EGF (50?ng/ml) and insulin (10?nM) stimulated endogenous AKR1B10 expression and promoter activity. In the AKR1B10 promoter, a putative AP-1 element was found at bp -222 to -212. Deletion or mutation of this AP-1 element abrogated the basal promoter activity and response to EGF and AP-1 proteins. This AP-1 element bound to nuclear proteins extracted from HepG2 cells, and this binding was stimulated by EGF and insulin in a dose-dependent manner. Chromatin immunoprecipitation showed that the AP-1 proteins c-Fos and c-Jun were the predominant factors bound to the AP-1 consensus sequence, followed by JunD and then JunB. The same order was followed in the stimulation of endogenous AKR1B10 expression by AP-1 proteins. Furthermore, c-Fos shRNA (short hairpin RNA) and AP-1 inhibitors/antagonists (U0126 and Tanshinone IIA) inhibited endogenous AKR1B10 expression and promoter activity in HepG2 cells cultured in vitro or inoculated subcutaneously in nude mice. U0126 also inhibited AKR1B10 expression induced by EGF. Taken together, these results suggest that AKR1B10 is up-regulated by EGF and insulin through AP-1 mitogenic signalling and may be implicated in hepatocarcinogenesis.     

Hepatocyte growth factor induces delayed STAT3 phosphorylation through interleukin-6 expression

Met receptor tyrosine kinase mediates pleiotropic cellular responses following its activation by hepatocyte growth factor or scatter factor (HGF/SF). STAT3 was reported to be one of direct downstream molecules in HGF/SF-Met signaling. In the present study, however, we observed that Tyr705 of STAT3 was phosphorylated from 2 h or 6 h in NIH3T3 and Chang liver cells, respectively, after HGF/SF treatment. Blocking of the phosphorylation by cycloheximide or actinomycin D and the rapid STAT3 phosphorylation with the conditioned medium from HGF/SF-treated NIH3T3 cells suggested that a newly synthesized secretory protein was responsible for the delayed STAT3 phosphorylation. Among the known mediators to induce STAT3 phosphorylation, interleukin-6 (IL-6) mRNA and protein were induced by HGF/SF, and the released IL-6 was accumulated in the conditioned medium after HGF/SF treatment. Furthermore, the neutralizing IL-6 antibody abolished the STAT3 phosphorylation. Treatment with LY294002, a PI3 kinase inhibitor, but not with other signal inhibitors, resulted in the loss of delayed STAT3 phosphorylation by HGF/SF, showing the involvement of PI3 kinase pathway. Collectively, these results demonstrate that HGF/SF-Met signal cascade stimulates IL-6 production via PI3 kinase pathway, leading to STAT3 phosphorylation as a secondary effect.     

Platelet-derived growth factor induces interleukin-1 receptor gene expression in Balb/c 3T3 fibroblasts

Our previous studies have demonstrated that platelet-derived growth factor (PDGF) modulated cellular responses to interleukin-1 (IL-1). In this communication, we show that PDGF regulates expression of IL-1 receptor (IL-1 R) gene. Treatment of quiescent cultures of Balb/c 3T3 fibroblasts with PDGF produced 20-30-fold stimulation of IL-1 R mRNA with a concomitant increase in cell surface 125I-binding. IL-1 R mRNA accumulation occurred after an initial lag period and with a time course preceding the increase in 125I-IL-1 binding to cells. Induction of IL-1 R mRNA was blocked by inhibitors of protein synthesis, suggesting that a product of a gene expressed immediately after PDGF addition is required for IL-1 R gene expression. These latter data provide evidence for an ordered sequence of expression of PDGF-inducible "immediate early" gene(s) and IL-1 R gene. These results suggest that in connective tissues, PDGF may be an important determinant in initiating and maintaining cellular responses to IL-1. Such responses may have important consequences in the actions of IL-1 under normal and pathological conditions such as arthritis and atherosclerosis.     

Extracellular sphingomyelinase induces interleukin-6 synthesis in osteoblasts

In osteoblast-like MC3T3-E1 cells, we have recently reported that sphingosine 1-phosphate among sphingomyelin metabolites acts as a second messenger for tumor necrosis factor-alpha (TNF)-induced interleukin-6 (IL-6) synthesis. In the present study, we investigated the effect of extracellular sphingomyelinase on IL-6 synthesis in MC3T3-E1 cells. Sphingomyelinase stimulated IL-6 synthesis in a time-dependent manner for up to 24 h. This stimulative effect was dose dependent in the range between 1 and 300 mU/ml. Calphostin C, a highly and potent inhibitor of protein kinase C, enhanced sphingomyelinase-induced IL-6 synthesis. DL-Threo-dihydrosphingosine, an inhibitor of sphingosine kinase, significantly inhibited the IL-6 synthesis induced by sphingomyelinase. Sphingomyelinase markedly elicited sphingomyelin hydrolysis. In addition, the effect of a combination of sphingomyelinase and TNF on IL-6 synthesis was synergistic. These results strongly suggest that extracellular sphingomyelinase induces sphingomyelin hydrolysis in osteoblasts, resulting in IL-6 synthesis, and that protein kinase C acts as a negative controller of the IL-6 synthesis.     

Hypoxia induces transcription factor ETS-1 via the activity of hypoxia-inducible factor-1.

ETS-1 plays an important role in angiogenesis and cancer invasion, and hypoxia is a common feature in these phenomena. We examined whether hypoxia influenced ETS-1 expression. Hypoxia induced ETS-1 in a human bladder cancer cell line, T24, and promoter analysis revealed that the deletion of -424 to -279 bp from the human ETS-1 promoter decreased the hypoxia-mediated inducibility. This region contained a hypoxia responsive element-like sequence, and HIF-1 bound to it under the hypoxic condition. Double-stranded synthetic oligonucleotides of this sequence as a decoy inhibited the hypoxia-mediated inducibility. These results indicate that hypoxia induces ETS-1 via the activity of HIF-1.     

Interleukin-1beta induces interleukin-6 production through the production of prostaglandin E(2) in human osteoblasts, MG-63 cells.

This study was conducted to investigate the mechanism of interleukin-1beta (IL-1beta)-induced IL-6 production in human osteoblasts (MG-63 cells). Stimulation with IL-1beta resulted in the production of IL-6 and prostaglandin E(2) (PGE(2)). IL-6 production gradually increased and peaked 96 h after stimulation. IL-6 mRNA was detected between 4 and 72 h after IL-1beta stimulation. The patterns of PGE(2) production and the expression of cyclooxygenase-2 (COX-2) mRNA were biphasic after stimulation. Actinomycin D, cycloheximide, indomethacin, and NS-398 (COX-2 inhibitor) suppressed the production of IL-6 and PGE(2). Anti-PGE(2) antibody markedly reduced the production of IL-6. In addition, stimulation with 17-phenyl-PGE(2), a PGE receptor-1 (EP-1 receptor) agonist, led to the expression of IL-6 mRNA after pretreatment with IL-1beta. These findings indicate that IL-1beta-induced IL-6 production in MG-63 cells involves the following sequence of steps: IL-1beta-induced COX-2 activation, PGE(2) production, and EP-1 receptor signaling prior to IL-6 production.