Apigenin and LY294002 prolong EGF-stimulated ERK1/2 activation in PC12 cells but are unable to induce full differentiation

In rat pheochromocytoma cell line (PC12) cells, initial epidermal growth factor (EGF)-stimulated extracellular signal-regulated protein kinases 1/2 (ERK1/2) phosphorylation was similar to that promoted by nerve growth factor (NGF), but declined rapidly. Pre-treatment with apigenin or LY294002 sustained EGF-stimulated ERK1/2 phosphorylation whereas wortmannin partially blocked initial ERK1/2 phosphorylation. Changes in ERK1/2 phosphorylation correlated with alterations in p90 ribosomal S6 kinase activity. Wortmannin, LY294002 and apigenin totally blocked growth factor-induced protein kinase B phosphorylation. However, none of them potentiated Raf activation, which was in fact decreased by LY290042 and wortmannin. The sustained EGF-induced ERK1/2 activation promoted by apigenin was not sufficient to commit PC12 cells to differentiate, which was achieved by stimulation with NGF, either alone or in the presence of apigenin.     

Interaction of the receptor FGFRL1 with the negative regulator Spred1

FGFRL1 is a member of the fibroblast growth factor receptor family. It plays an essential role during branching morphogenesis of the metanephric kidneys, as mice with a targeted deletion of the Fgfrl1 gene show severe kidney dysplasia. Here we used the yeast two-hybrid system to demonstrate that FGFRL1 binds with its C-terminal, histidine-rich domain to Spred1 and to other proteins of the Sprouty/Spred family. Members of this family are known to act as negative regulators of the Ras/Raf/Erk signaling pathway. Truncation experiments further showed that FGFRL1 interacts with the SPR domain of Spred1, a domain that is shared by all members of the Sprouty/Spred family. The interaction could be verified by coprecipitation of the interaction partners from solution and by codistribution at the cell membrane of COS1 and HEK293 cells. Interestingly, Spred1 increased the retention time of FGFRL1 at the plasma membrane where the receptor might interact with ligands. FGFRL1 and members of the Sprouty/Spred family belong to the FGF synexpression group, which also includes FGF3, FGF8, Sef and Isthmin. It is conceivable that FGFRL1, Sef and some Sprouty/Spred proteins work in concert to control growth factor signaling during branching morphogenesis of the kidneys and other organs.     

Mechanisms for HIV Tat upregulation of IL-10 and other cytokine expression: kinase signaling and PKR-mediated immune response

HIV Tat has been known to have multiple regulatory roles including replication of HIV and modulation of cellular kinases. We investigated whether signaling kinase PKR plays a critical role in mediating Tat-induced cytokine dysregulation. We showed Tat induction of IL-10 dysregulation is associated with PKR activation. To examine the mechanism involved, inhibition of PKR activity abrogated the Tat-induced cytokine induction. We next identified that the MAP kinases including ERK-1/2 and p38 are downstream of PKR in these Tat-induced pathways. Thus, PKR may play a critical role in mediating the subversive effects of HIV Tat resulting in IL-10 induction.     

ERK5 Protein Promotes, whereas MEK1 Protein Differentially Regulates, the Toll-like Receptor 2 Protein-dependent Activation of Human Endothelial Cells and Monocytes

Endothelial cell (EC) Toll-like receptor 2 (TLR2) activation up-regulates the expression of inflammatory mediators and of TLR2 itself and modulates important endothelial functions, including coagulation and permeability. We defined TLR2 signaling pathways in EC and tested the hypothesis that TLR2 signaling differs in EC and monocytes. We found that ERK5, heretofore unrecognized as mediating TLR2 activation in any cell type, is a central mediator of TLR2-dependent inflammatory signaling in human umbilical vein endothelial cells, primary human lung microvascular EC, and human monocytes. Additionally, we observed that, although MEK1 negatively regulates TLR2 signaling in EC, MEK1 promotes TLR2 signaling in monocytes. We also noted that activation of TLR2 led to the up-regulation of intracellularly expressed TLR2 and inflammatory mediators via NF-κB, JNK, and p38-MAPK. Finally, we found that p38-MAPK, JNK, ERK5, and NF-κB promote the attachment of human neutrophils to lung microvascular EC that were pretreated with TLR2 agonists. This study newly identifies ERK5 as a key regulator of TLR2 signaling in EC and monocytes and indicates that there are fundamental differences in TLR signaling pathways between EC and monocytes.     

NTS2 modulates the intracellular distribution and trafficking of NTS1 via heterodimerization

Neurotensin (NT) receptors NTS1 and NTS2 are known to display considerable distributional overlap in mammalian central nervous system (CNS). Using co-immunoprecipitation approaches, we demonstrated here that NTS1 forms constitutive heterodimers with NTS2 in transfected COS-7 cells. We also showed that co-expression of NTS2 with NTS1 markedly decreases the cell surface density of NTS1 without affecting ERK1/2 MAPK activity or NT-induced NTS1 internalization. However, radioligand-binding studies indicated that upon prolonged NT stimulation, cell surface NTS1 receptors are more resistant to down-regulation in cells co-expressing NTS1 and NTS2 than in cells expressing NTS1 alone. Taken together, these data suggest that NTS1/NTS2 heterodimerization affects the intracellular distribution and trafficking of NTS1 by making it more similar to that of NTS2 as witnessed in cells expressing NTS2 alone. NTS1/NTS2 heterodimerization might therefore represent an additional mechanism in the regulation of NT-triggered responses mediated by NTS1 and NTS2 receptors.     

Inhibition of ERK1/2 and Activation of Liver X Receptor Synergistically Induce Macrophage ABCA1 Expression and Cholesterol Efflux

ATP-binding cassette transporter A1 (ABCA1), a molecule mediating free cholesterol efflux from peripheral tissues to apoAI and high density lipoprotein (HDL), inhibits the formation of lipid-laden macrophage/foam cells and the development of atherosclerosis. ERK1/2 are important signaling molecules regulating cellular growth and differentiation. The ERK1/2 signaling pathway is implicated in cardiac development and hypertrophy. However, the role of ERK1/2 in the development of atherosclerosis, particularly in macrophage cholesterol homeostasis, is unknown. In this study, we investigated the effects of ERK1/2 activity on macrophage ABCA1 expression and cholesterol efflux. Compared with a minor effect by inhibition of other kinases, inhibition of ERK1/2 significantly increased macrophage cholesterol efflux to apoAI and HDL. In contrast, activation of ERK1/2 reduced macrophage cholesterol efflux and ABCA1 expression. The increased cholesterol efflux by ERK1/2 inhibitors was associated with the increased ABCA1 levels and the binding of apoAI to cells. The increased ABCA1 by ERK1/2 inhibitors was due to increased ABCA1 mRNA and protein stability. The induction of ABCA1 expression and cholesterol efflux by ERK1/2 inhibitors was concentration-dependent. The mechanism study indicated that activation of liver X receptor (LXR) had little effect on ERK1/2 expression and activation. ERK1/2 inhibitors had no effect on macrophage LXRα/β expression, whereas they did not influence the activation or the inhibition of the ABCA1 promoter by LXR or sterol regulatory element-binding protein (SREBP). However, inhibition of ERK1/2 and activation of LXR synergistically induced macrophage cholesterol efflux and ABCA1 expression. Our data suggest that ERK1/2 activity can play an important role in macrophage cholesterol trafficking.     

Dipyrithione inhibits lipopolysaccharide-induced iNOS and COX-2 up-regulation in macrophages and protects against endotoxic shock in mice

Dipyrithione (PTS2) possesses anti-bacterial and anti-fungal activity. In the present study, we found that PTS2 dose-dependently inhibited the LPS-induced up-regulation of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein level in RAW264.7 cells. RT-PCR experiments showed that PTS2 suppressed LPS-induced iNOS but not COX-2 expression at the mRNA level. As expected, PTS2 prevented NO secretion in RAW264.7 cells. Furthermore, PTS2 administration significantly decreased LPS-induced mortality in mice. Mechanistically, PTS2 decreased expression and phosphorylation of STAT1, but did not interfere with the MAPK and NF-kappaB pathways. In conclusion, PTS2 protects mice against endotoxic shock and inhibits LPS-induced production of pro-inflammatory mediators, suggesting that PTS2 could play an anti-inflammatory role in response to LPS.     

The production of macrophage inflammatory protein-2 induced by soluble intercellular adhesion molecule-1 in mouse astrocytes is mediated by src tyrosine kinases and p42/44 mitogen-activated protein kinase

Severe traumatic brain injury stimulates the release of soluble intercellular adhesion molecule-1 (sICAM-1) into CSF. Studies in cultured mouse astrocytes suggest that sICAM-1 induces the production of macrophage inflammatory protein-2 (MIP-2). In the present study, we investigated the underlying mechanisms for MIP-2 induction. sICAM-1 induced MIP-2 in astrocytes lacking membrane-bound ICAM-1, indicating that its action is due to heterophilic binding to an undescribed receptor rather than homophilic binding to surface ICAM-1. Signal transduction may be mediated by src tyrosine kinases, as the src tyrosine kinase inhibitors herbimycin A and PP2 abolished MIP-2 induction by sICAM-1. Phosphorylation of p42/44 mitogen-activated protein kinase (MAPK), but not of p38 MAPK, occurred further downstream, as evidenced by western blot analysis combined with the use of herbimycin A and specific MAPK inhibitors. By contrast, induction of MIP-2 by tumour necrosis factor-alpha (TNF-alpha) involved both p42/44 MAPK and p38 MAPK. Following stimulation with either sICAM-1 or TNF-alpha, astrocyte supernatants promoted chemotaxis of human neutrophils and incubation of these supernatants with anti-MIP-2 antibodies more efficiently suppressed the migration induced by sICAM-1 than by TNF-alpha. These results show that sICAM-1 induces the production of biologically active MIP-2 in astrocytes by heterophilic binding to an undefined receptor and activation of src tyrosine kinases and p42/44 MAPK.     

Soluble FGFR4 extracellular domain inhibits FGF19-induced activation of FGFR4 signaling and prevents nonalcoholic fatty liver disease

Fibroblast growth factor receptor 4 (FGFR4) is a transmembrane tyrosine kinase receptor that plays a crucial role in the regulation of hepatic bile acid and lipid metabolism. FGFR4 underlies high-fat diet-induced hepatic steatosis, suggesting that inhibition of FGFR4 activation may be an effective way to prevent or treat nonalcoholic fatty liver disease (NAFLD). To determine whether neutralization of FGFR4 ligands by soluble FGFR4 extracellular domain (FGFR4-ECD) can inhibit the activation of FGFR4, we constructed FGFR4-ECD expression vector and showed that FGFR4-ECD was effectively expressed in cells and secreted into culture medium. FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling and reduced steatosis of HepG2 induced by palmitic acid in vitro. Furthermore, in a tetracycline-induced fatty liver model, expression of FGFR4-ECD in mouse liver reduced the accumulation of hepatic lipids and partially restored the expression of peroxisome proliferator-activated receptor α (PPARα), which promotes the mitochondrial fatty acid beta-oxidation but is repressed by tetracycline. Taken together, these results demonstrate that FGFR4-ECD can block FGFR4 signaling and prevent hepatic steatosis, highlighting the potential value of inhibition of FGFR4 signaling as a method for therapeutic intervention against NAFLD.     

The stability of eukaryotic initiation factor 2-associated glycoprotein, p67, increases during skeletal muscle differentiation and that inhibits the phosphorylation of extracellular signal-regulated kinases 1 and 2

Eukaryotic initiation factor 2-associated glycoprotein, p67, protects eIF2 from phosphorylation by its kinases. To understand the roles of p67 during skeletal muscle differentiation of mouse C2C12 myoblasts, we measured the level of p67 during myotube formation. We noticed that the level of p67 increases during myoblast differentiation and this increased level is controlled at the translational stage. The stability of p67 in the myotubes is due to its low turnover rate. The phosphorylation of the extracellular signal-regulated kinases (ERKs 1 and 2) is high in growth-factor-mediated cycling of C2C12 myoblasts and this phosphorylation decreases at 96 h when these myoblasts are grown in differentiation medium. At this time of differentiation, the level of p67 is higher compared to 0 h of differentiation. p67 binds to ERK2 and inhibits its activity in vitro. Taken together, these results suggest that the stability of p67 increases during myotube formation while inhibiting the phosphorylation of ERKs 1 and 2.     

Proliferation and motility of HaCaT keratinocyte derivatives is enhanced by fibroblast nemosis

The role of paracrine tumor-stroma regulation in the progression of cancer is under intense investigation. Activated fibroblasts are key components of the tumor microenvironment providing the soluble factors mediating the regulation. Nemosis is an experimental model to study these parameters: formation of a multicellular spheroid activates fibroblasts and leads to increased production of soluble factors involved in the promotion of growth and motility. Role of nemosis was investigated in the tumorigenesis of HaCaT derivatives representing skin carcinoma progression. Conditioned medium from fibroblast spheroids increased proliferation rate of HaCaT derivatives. Expression of proliferation marker Ki-67 increased significantly in benign A5 and low-grade malignant II-4 cells, but did not further increase in the metastatic RT3 cells. Expression of p63, keratinocyte stem cell marker linked to cancer progression, was augmented by medium from nemotic fibroblasts; this increase was also seen in RT3 cells. Scratch-wound healing of the keratinocytes was enhanced in response to fibroblast nemosis. Neutralizing antibodies against growth factors inhibited wound healing to some extent; the response varied between benign and malignant keratinocytes. Migration and invasion were enhanced by conditioned medium from nemotic fibroblasts in benign and low-grade malignant cells. RT3 keratinocyte migration was further augmented, but invasion was not, indicating their intrinsic capacity to invade. Our data demonstrate that fibroblast nemosis increases proliferation and motility of HaCaT keratinocyte derivatives, and thus nemosis can be used as a model to study the role of soluble factors secreted by fibroblasts in tumor progression.     

Modulation of mitochondrial function and morphology by interaction of Omi/HtrA2 with the mitochondrial fusion factor OPA1

Loss of Omi/HtrA2 function leads to nerve cell loss in mouse models and has been linked to neurodegeneration in Parkinson's and Huntington's disease. Omi/HtrA2 is a serine protease released as a pro-apoptotic factor from the mitochondrial intermembrane space into the cytosol. Under physiological conditions, Omi/HtrA2 is thought to be involved in protection against cellular stress, but the cytological and molecular mechanisms are not clear. Omi/HtrA2 deficiency caused an accumulation of reactive oxygen species and reduced mitochondrial membrane potential. In Omi/HtrA2 knockout mouse embryonic fibroblasts, as well as in Omi/HtrA2 silenced human HeLa cells and Drosophila S2R+ cells, we found elongated mitochondria by live cell imaging. Electron microscopy confirmed the mitochondrial morphology alterations and showed abnormal cristae structure. Examining the levels of proteins involved in mitochondrial fusion, we found a selective up-regulation of more soluble OPA1 protein. Complementation of knockout cells with wild-type Omi/HtrA2 but not with the protease mutant [S306A]Omi/HtrA2 reversed the mitochondrial elongation phenotype and OPA1 alterations. Finally, co-immunoprecipitation showed direct interaction of Omi/HtrA2 with endogenous OPA1. Thus, we show for the first time a direct effect of loss of Omi/HtrA2 on mitochondrial morphology and demonstrate a novel role of this mitochondrial serine protease in the modulation of OPA1. Our results underscore a critical role of impaired mitochondrial dynamics in neurodegenerative disorders.     

Interleukin-1ß is a positive regulator of TIARP/STAMP2 gene and protein expression in adipocytes in vitro

The impact of interleukin (IL)-1ß on tumor necrosis factor α-induced adipose-related protein (TIARP)/six-transmembrane protein of prostate 2 (STAMP2) was determined in adipocytes. TIARP/STAMP2 mRNA synthesis was significantly stimulated by IL-1ß in a dose- and time-dependent fashion in 3T3-L1 adipocytes. Signaling studies suggested that janus kinase 2, nuclear factor κB, and p44/42 mitogen-activated protein kinase are involved in IL-1ß-induced TIARP/STAMP2 mRNA expression. Furthermore, IL-1ß, TNFα, and IL-6 showed synergistic stimulatory effects on TIARP/STAMP2 gene expression. Moreover, both TIARP/STAMP2 mRNA synthesis and protein expression were induced by IL-1ß in fully differentiated human mesenchymal stem cell-derived adipocytes (hMSC-Ad). Taken together, TIARP/STAMP2 is highly upregulated in 3T3-L1 cells and hMSC-Ad by IL-1ß and might, therefore, modulate proinflammatory and insulin resistance-inducing effects of IL-1ß.     

CDK1, not ERK1/2 or ERK5, is required for mitotic phosphorylation of BIMEL

The pro-apoptotic BH3 only protein BIM_EL is phosphorylated by ERK1/2 and this targets it for proteasome-dependent degradation. A recent study has shown that ERK5, an ERK1/2-related MAPK, is activated during mitosis and phosphorylates BIM_EL to promote cell survival. Here we show that treatment of cells with nocodazole or paclitaxel does cause phosphorylation of BIM_EL, which is independent of ERK1/2. However, this was not due to ERK5-catalysed phosphorylation, since it was not reversed by the MEK5 inhibitor BIX02189 and proceeded normally in ERK5−/− fibroblasts. Indeed, although ERK5 is phosphorylated at multiple sites in the C-terminal transactivation region during mitosis, these do not include the activation-loop and ERK5 kinase activity does not increase. Mitotic phosphorylation of BIM_EL occurred at proline-directed phospho-acceptor sites and was abolished by selective inhibition of CDK1. Furthermore, cyclin B1 was able to interact with BIM and cyclin B1/CDK1 complexes could phosphorylate BIM in vitro. Finally, we show that CDK1-dependent phosphorylation of BIM_EL drives its polyubiquitylation and proteasome-dependent degradation to protect cells during mitotic arrest. These results provide new insights into the regulation of BIM_EL and may be relevant to the therapeutic use of agents such as paclitaxel.     

Acquisition of chemoresistance in intrahepatic cholangiocarcinoma cells by activation of AKT and extracellular signal-regulated kinase (ERK)1/2

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignant tumor and is refractory to conventional chemotherapy. The aim of this study is therefore to elucidate the mechanism of chemoresistance in ICC which is not fully understood. We generated cisplatin resistant ICC cells via long term exposure to cisplatin and found that these cells are also resistant to 5-fluorouracil (5-FU) and gemcitabine. The chemoresistant cells showed enhanced Bcl-2 expression and reduced Bax expression compared to parental ICC cells. In addition, the resistant cells showed enhanced activation of AKT and extracellular signal-regulated kinase (ERK) 1/2. Inhibition of AKT activation by phosphoinocitide 3-kinase (PI3K) inhibitor LY294002 resulted in reduced Bcl-2 expression and enhanced Bax expression and thus induced apoptosis in the resistant cells, whereas inhibition of ERK1/2 activation by mitogen-activated protein kinase (MEK) inhibitor U0126 did not induce apoptosis without affecting the expression of Bcl-2 and Bax but decreased cell growth. Moreover, the inhibition of AKT or ERK1/2 sensitized the resistant cells to cisplatin and therefore resulted in greatly enhanced cisplatin-induced apoptosis and growth inhibition in the cells. The results indicate that AKT and ERK1/2 signaling mediate chemoresistance in the cells and could be important therapeutic targets for overcoming chemoresistance in ICC.     

Activation of ERK by sodium tungstate induces protein synthesis and prevents protein degradation in rat L6 myotubes

The balance between the rates of protein synthesis and degradation in muscle is regulated by PI3K/Akt signaling. Here we addressed the effect of ERK activation by sodium tungstate on protein turnover in rat L6 myotubes. Phosphorylation of ERK by this compound increased protein synthesis by activating MTOR and prevented dexamethasone-induced protein degradation by blocking FoxO3a activity, but it did not alter Akt phosphorylation. Thus, activation of ERK by tungstate improves protein turnover in dexamethasone-treated cells. On the basis of our results, we propose that tungstate be considered an alternative to IGF-I and its analogs in the prevention of skeletal muscle atrophy.