NHERF2 increases platelet-derived growth factor-induced proliferation through PI-3-kinase/Akt-, ERK-, and Src family kinase-dependent pathway

Platelet-derived growth factor (PDGF) has multiple functions including inhibition of apoptosis and promotion of cell proliferation. In this study, we show that Na(+)/H(+) exchanger regulatory factor 2 (NHERF2) binds to the carboxyl-terminal PDZ domain-binding motif of the PDGF receptor through a PDZ domain-mediated interaction, and evaluate the consequence on PDGF-induced proliferation. Stable transfection with NHERF2 increased the PDGF-induced phosphorylation of ERK and Akt in Rat1 embryonic fibroblasts. The phosphorylation of Akt was blocked by pretreatment with LY294002, a PI-3-kinase inhibitor, in both Rat1/NHERF2 and Rat1/vector cells. In Rat1/vector cells, PDGF-induced phosphorylation of ERK was completely inhibited by pretreatment with PD98059, a MEK inhibitor. In contrast, the NHERF2-dependent increase of ERK phosphorylation was not affected by pretreatment with PD98059 in Rat1/NHERF2 cells. Thus, the NHERF2-dependent increase of ERK phosphorylation occurs in a MEK-independent fashion. Pretreatment with PP2, a specific inhibitor of Src family tyrosine kinase, completely blocked the NHERF2-dependent increase of the phosphorylation of ERK and Akt, suggesting that NHERF2 up-regulates Erk phosphorylation through a Src family kinase-dependent pathway. Consistent with these results, the PDGF-induced thymidine incorporation was increased in Rat1/NHERF2 cells, and the NHERF2-dependent increase of thymidine incorporation was prevented by treatment with LY294002 and PP2 but not with PD98059. These results suggest that NHERF2 stimulates PDGF-induced proliferation by increasing PI-3-kinase/Akt, MEKindependent ERK, and Src family kinase-mediated signaling pathways.     

Involvement of ERK1/2 pathway in TGF-beta1-induced VEGF secretion in normal human cytotrophoblast cells

Transforming growth factor-beta1 (TGF-beta1) plays a pivotal role in the angiogenesis during the development of placenta, but the intracellular signaling mechanism by which TGF-beta1 stimulates this process remains poorly understood. In this article, we demonstrated that exposure of normal human cytotrophoblast cells to TGF-beta1 stimulated the secretion of the VEGF gene encoding vascular endothelial growth factor, which is a key factor in angiogenesis. Meanwhile, treatment of normal human cytotrophoblast cells with TGF-beta1-induced expression of HIF-1a, the regulated subunit of hypoxia-inducible factor 1, a known transactivator of the VEGF gene. Our data indicated that TGF-beta1 induced extracellular signal- regulated kinase (ERK) 1/2 phosphorylation in normal human cytotrophoblast cells. Moreover, treating cells with PD98059, an inhibitor of ERK1/2 signaling, inhibited TGF-beta1 stimulation of VEGF secretion and HIF-1a protein expression. These data indicated that in normal human cytotrophoblast cells, TGF-beta1 induced HIF-1a-mediated VEGF secretion, and TGF-beta1-stimulated-ERK1/2 activation may be involved in this process.     

Metalloproteinases stimulate ErbB-dependent ERK signaling in human skin organ culture

To investigate the role of ERK signaling in human skin responses to wounding, organ cultures of human skin were maintained for 0.5-24 h in the presence of various inhibitors, followed by measurement of ERK phosphorylation or mRNA levels. The MEK inhibitor PD98059 produced near-complete (97-98%) inhibition of ERK phosphorylation, whereas inhibition of c-Fos, c-Jun, HB-EGF, AR, and VEGF mRNA by this compound was incomplete (41-65%). PD98059 was significantly more effective than either PD158780 or BB2516 as an inhibitor of ERK phosphorylation and of the rapid rise in c-Fos and c-Jun mRNA expression. In contrast, all three compounds inhibited the more delayed rise in HB-EGF mRNA to the same extent. Exogenous epidermal growth factor abrogated the inhibition of ERK phosphorylation caused by BB2516. These data indicate that one or more metalloproteinases activate ErbB signaling in skin organ culture, that ErbB signaling plays an important but not exclusive role in the activation of ERK, and that non-ERK pathways contribute to gene expression in this system. Because metalloproteinase-mediated cleavage of the HB-EGF transmembrane precursor is known to be ERK-dependent, our data suggest that ERK activation resulting from initial trauma leads to metalloproteinase-mediated cleavage of HB-EGF, thereby triggering the ErbB signaling cascade.     

Urokinase-type plasminogen activator receptor crosslinking in an NK cell line increases integrin surface expression by the MAP kinase/ERK 1/2 signaling pathway

Urokinase-type plasminogen activator receptor (uPAR) is attached to cell membranes by a glycosylphosphatidylinositol (GPI) anchor, and as such is devoid of an intracellular domain, but is nevertheless able to initiate signal transduction. Herein, we report a relationship between integrins and uPAR on the surface of the human NK cell line, YT. Our data reveals that crosslinking uPAR, which mimics uPAR clustering at focal adhesion sites, causes increases in expression of the alpha(M), alpha(V), and beta(2) integrins on the surface of YT cells. Activation of the MEK/ERK signaling cascade occurs following uPAR crosslinking, as phosphorylation of both MEK 1/2 and ERK 1/2 results from receptor clustering. The MEK-specific inhibitors PD98059 and U0126 blocked MAP kinase phosphorylation; furthermore, PD98059 inhibited the increase in integrin expression induced by uPAR clustering. This study suggests that uPAR is a signaling receptor and regulator of integrins in NK cells and may impact NK cell function, including the potential for their accumulation within tumor metastases following adoptive transfer.     

ERK-dependent threonine phosphorylation of EGF receptor modulates receptor downregulation and signaling

Epidermal growth factor (EGF) signaling is critical in normal and aberrant cellular behavior. Extracellular signal-regulated kinase (ERK) mediates important downstream aspects of EGF signaling. Additionally, EGFR undergoes MEK1-dependent ERK consensus site phosphorylation in response to EGF or cytokines such as growth hormone (GH) and prolactin (PRL). GH- or PRL-induced EGFR phosphorylation alters subsequent EGF-induced EGFR downregulation and signal characteristics in an ERK-dependent fashion. We now use reconstitution to study mutation of the sole EGFR ERK phosphorylation consensus residue, (669)T. CHO-GHR cells, which lack EGFR and express GHR, were stably transfected to express human wild-type or T669A ((669)T changed to alanine) EGFRs at similar abundance. Treatment of cells with GH or EGF caused phosphorylation of WT, but not T669A EGFR, in an ERK activity-dependent fashion that was detected with an antibody that recognizes phosphorylation of ERK consensus sites, indicating that (669)T is required for this phosphorylation. Notably, EGF-induced downregulation of EGFR abundance was much more rapid in cells expressing EGFR T669A vs. WT EGFR. Further, pretreatment with the MEK1/ERK inhibitor PD98059 enhanced EGF-induced EGFR loss in cells expressing WT EGFR, but not EGFR T669A, suggesting that the ERK-dependent effects on EGFR downregulation required phosphorylation of (669)T. In signaling experiments, EGFR T669A displayed enhanced acute (15 min) EGFR tyrosine phosphorylation (reflecting EGFR kinase activity) compared to WT EGFR. Further, acute EGF-induced ubiquitination of WT EGFR was markedly enhanced by PD98059 pretreatment and was increased in EGFR T669A-expressing cells independent of PD98059. These signaling data suggest that ERK-mediated (669)T phosphorylation negatively modulates EGF-induced EGFR kinase activity. We furthered these investigations using a human fibrosarcoma cell line that endogenously expresses EGFR and ErbB-2 and also harbors an activating Ras mutation. In these cells, EGFR was constitutively detected with the ERK consensus site phosphorylation-specific antibody and EGF-induced EGFR downregulation was modest, but was substantially enhanced by pretreatment with MEK1/ERK inhibitor. Collectively, these data indicate that ERK activity, by phosphorylation of a threonine residue in the EGFR juxtamembrane cytoplasmic domain, modulates EGFR trafficking and signaling.     

HGF triggers activation of the COX-2 gene in rat gastric epithelial cells: action mediated through the ERK2 signaling pathway.

Although it is established that growth factors and prostaglandins function in the maintenance of gastric mucosal integrity and in the healing of gastric mucosal injury and ulceration, the regulatory relationship between growth factors and prostaglandins in the gastric mucosa is not well characterized. Therefore, we investigated whether hepatocyte growth factor (HGF) affects expression of COX-2 (the inducible form of the prostaglandin synthesizing enzyme, cyclooxygenase) in gastric epithelial cells and whether this action is mediated through the MAP (ERK) kinase signaling pathway. In RGM1 cells (an epithelial cell line derived from normal rat gastric mucosa), HGF caused an increase in COX-2 mRNA and protein by 236% and 175%, respectively (both P<0.05). This induction of COX-2 expression was abolished by pretreatment with the MAPK kinase (MEK) inhibitor PD98059. HGF also triggered a 13-fold increase in c-Met/HGF receptor phosphorylation (P<0.005) and increased ERK2 activity by 684% (P<0.01). Pretreatment with PD98059 abolished the HGF-induced increase in ERK2 activity, but not c-Met/HGF receptor phosphorylation. The specific inhibitor of p38 MAP kinase, SB203580, had no effect on HGF-induced COX-2 expression. Thus, HGF triggers activation of the COX-2 gene in gastric epithelial cells through phosphorylation of c-Met/HGF receptor and activation of the ERK2 signaling pathway.-Jones, M. K., Sasaki, E., Halter, F., Pai, R., Nakamura, T., Arakawa, T., Kuroki, T., Tarnawski, A. S. HGF triggers activation of the COX-2 gene in rat gastric epithelial cells: action mediated through the ERK2 signaling pathway.     

H4R activation utilizes distinct signaling pathways for the production of RANTES and IL-13 in human mast cells

Context: The histamine H4 receptor functionally expressed on human mast cells and their signaling pathways for the production of IL-13 and RANTES have never been analyzed side by side in a directly comparable manner.

Objective: Therefore, the aim of the study was to investigate signaling transduction pathways of H4R via ERK1/2, Akt and NFκB leading to the induction of inflammatory cytokine expression.

Materials and methods: In the present study, HMC-1 cells and CBMCs were pretreated individually with H4R antagonist JNJ7777120, H1R antagonist mepyramine and signaling molecule inhibitors PD 98059, LY294002, Bay 117082 followed by stimulation was done with or without histamine or 4-MH. Furthermore, the siRNA mediated H4R gene silencing effects are studied at the H4R protein expression level and also signal transduction level.

Results: We found that the pretreatment with JNJ7777120 and H4R gene silencing decreased histamine, 4-MH induced phosphorylation of ERK1/2, Akt and NFκB-p65. Moreover, PD 98059, LY294002 and Bay 117082, which respectively inhibited the histamine and 4-methylhistamine induced phosphorylation of ERK1/2, Akt and NFκB-p65 respectively. We also found that the activation of H4R caused the release of IL-13 and RANTES on human mast cells. The MEK inhibitor PD98059 blocked H4R mediated RANTES/CCL5 production by 20.33?pg/ml and inhibited IL-13 generation by 95.71?pg/ml. In contrast, PI3 kinase inhibitor LY294002 had no effect on 4-MH induced RANTES/CCL5 production but blocked IL-13 generation by 117.58?pg/ml.

Discussion and conclusion: These data demonstrate that the H4R activates divergent signaling pathways to induce cytokine and chemokine production in human mast cells.     

Forskolin increases angiogenesis through the coordinated cross-talk of PKA-dependent VEGF expression and Epac-mediated PI3K/Akt/eNOS signaling

Forskolin, a potent activator of adenylyl cyclases, has been implicated in modulating angiogenesis, but the underlying mechanism has not been clearly elucidated. We investigated the signal mechanism by which forskolin regulates angiogenesis. Forskolin stimulated angiogenesis of human endothelial cells and in vivo neovascularization, which was accompanied by phosphorylation of CREB, ERK, Akt, and endothelial nitric oxide synthase (eNOS) as well as NO production and VEGF expression. Forskolin-induced CREB phosphorylation, VEGF promoter activity, and VEGF expression were blocked by the PKA inhibitor PKI. Moreover, phosphorylation of ERK by forskolin was inhibited by the MEK inhibitor PD98059, but not PKI. The forskolin-induced Akt/eNOS/NO pathway was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, but not significantly suppressed by PKI. These inhibitors and a NOS inhibitor partially inhibited forskolin-induced angiogenesis. The exchange protein directly activated by cAMP (Epac) activator, 8CPT-2Me-cAMP, promoted the Akt/eNOS/NO pathway and ERK phosphorylation, but did not induce CREB phosphorylation and VEGF expression. The angiogenic effect of the Epac activator was diminished by the inhibition of PI3K and MEK, but not by the PKA inhibitor. Small interfering RNA-mediated knockdown of Epac1 suppressed forskolin-induced angiogenesis and phosphorylation of ERK, Akt, and eNOS, but not CREB phosphorylation and VEGF expression. These results suggest that forskolin stimulates angiogenesis through coordinated cross-talk between two distinct pathways, PKA-dependent VEGF expression and Epac-dependent ERK activation and PI3K/Akt/eNOS/NO signaling.     

Activation of VEGFR-2 signaling in response to moderate dose of ultraviolet B promotes survival of normal human keratinocytes

Mounting evidence indicates that signaling via VEGF receptors (VEGFRs) extends beyond blood vessel formation. Recently, VEGFRs are also found to be constitutively expressed in keratinocytes and epidermal appendages. Here, we show that the expression of VEGFRs (including VEGFR-1, VEGFR-2, and NRP-1) was significantly enhanced by moderate dose of ultraviolet B (UVB) in normal human keratinocytes and epidermis. The elevated expression of VEGFRs by UVB was independent of autocrine stimulation by their natural ligand, VEGF, but mainly mediated through hypoxia and oxidative stress. Moderate dose UVB also promoted tyrosine phosphorylation of VEGFR-1 and VEGFR-2, this effect was again VEGF independent. Both α and δ isoforms of protein kinase C (PKC) were required for UVB-induced phosphorylation of VEGFR-1, but only the δ isoform was required for VEGFR-2 phosphorylation. The phosphorylation of VEGFRs or isoforms of PKC was completely inhibited by PP2, a specific inhibitor for Src family kinases (SFKs), indicating that SFKs are upstream of PKC and VEGFRs. Moderate dose UVB-induced VEGF exerted an anti-apoptotic effect for keratinocytes, whereas high dose UVB-induced VEGF played as an inflammatory factor. Of note, neutralization of VEGFR-2 but not VEGFR-1 exacerbated UVB-induced cell death and reduced survival of keratinocytes. Furthermore, VEGFR-2 neutralization inhibited the activation of ERK1/2 and Akt by UVB, suggesting that VEGFR-2 signaling was involved in the pro-survival mechanism via ERK1/2 and PI3-K/Akt pathway. Taken together, we demonstrate for the first time that VEGFR-2 signaling is activated and promotes survival of keratinocytes under moderate dose of UVB irradiation.     

Interleukin-6 triggers human cerebral endothelial cells proliferation and migration: the role for KDR and MMP-9

Interleukin-6 (IL-6) is involved in angiogenesis. However, the underlying mechanisms are unknown. Using human cerebral endothelial cell (HCEC), we report for the first time that IL-6 triggers HCEC proliferation and migration in a dose-dependent manner, specifically associated with enhancement of VEGF expression, up-regulated and phosphorylated VEGF receptor-2 (KDR), and stimulated MMP-9 secretion. We investigated the signal pathway of IL-6/IL-6R responsible for KDR's regulation. Pharmacological inhibitor of PI3K failed to inhibit IL-6-mediated VEGF overexpression, while blocking ERK1/2 with PD98059 could abolish IL-6-induced KDR overexpression. Further, neutralizing endogenous VEGF attenuated KDR expression and phosphorylation, suggesting that IL-6-induced KDR activation is independent of VEGF stimulation. MMP-9 inhibitor GM6001 significantly decreases HCEC proliferation and migration (p<0.05), indicating the crucial function of MMP-9 in promoting angiogenic changes in HCECs. We conclude that IL-6 triggers VEGF-induced angiogenic activity through increasing VEGF release, up-regulates KDR expression and phosphorylation through activating ERK1/2 signaling, and stimulates MMP-9 overexpression.     

Calcitonin gene-related peptide increases proliferation of human HaCaT keratinocytes by activation of MAP kinases

Psoriasis is a chronic disease characterized by keratinocyte hyperproliferation and inflammation. It has been demonstrated that the expression of calcitonin gene-related peptide (CGRP) is elevated in psoriasis lesions and CGRP-containing neuropeptide nerve fibers are denser in the psoriatic epidermis. CGRP has been previously described to influence proliferation of several cell types, such as Schwann cell, tracheal epithelial cells, and human gingival fibroblasts. In the present study, we determined the effect of CGRP on HaCaT keratinocyte proliferation and the role of mitogen-activated protein kinases (MAPKs) in CGRP induced keratinocyte proliferation. Our data indicate CGRP increased [³H]-thymidine incorporation and MTT activity of HaCaT in a concentration-dependent manner. CGRP also enhanced serum-induced HaCaT cell proliferation. HaCaT cells cultured with CGRP had a significant increase in phosphorylated ERK1/2, p38 and JNK, and CGRP induced DNA synthesis was inhibited by PD 98059 or SB 203580, selective inhibitors of MAP kinase kinase (MEK, which is upstream from ERK) and p38, respectively. These findings suggest that HaCaT cell proliferate in response to CGRP, which is mediated by phosphorylation of ERK1/2 and p38 MAPK.     

Retinoic acid increases amount of phosphorylated raf; Ectopic expression of cFMS reveals that retinoic acid-induced differentiation is more strongly dependent on ERK2 signaling than induced go arrest is

Summary Retinoic acid is known to cause the myeloid differentiation and G1/0 cell cycle arrest of HL-60 cells in a process that requires mitogen-activated protein/extracellular signal regulated kinase (MEK)-dependent extracellular signal regulated kinase (ERK)2 activation. It has also been shown that ectopic expression of cFMS, a platelet-derived growth factor (PDGF)-family transmembrane tyrosine kinase receptor, enhances retinoic acid-induced differentiation and G1/0 arrest. The mechanism of how the retinoic acid and cFMS signaling pathways intersect is not known. The present data show that the ectopic expression of cFMS results in the differential loss of sensitivity of retinoic acid-induced differentiation or G1/0 arrest to inhibition of ERK2 activation. PD98059 was used to inhibit MEK and consequently ERK2. In wild-type HL-60 cells, PD98059 blocked retinoic acid-induced differentiation; but in cFMS stable transfectants, PD98059 only attenuated the induced differentiation, with the resulting response resembling that of retinoic acid-treated wild-type HL-60. In wild-type HL-60, PD98059 greatly attenuated the retinoic acid-induced G1/0 arrest allied with retinoblastoma (RB) hypophosphorylation; but in cFMS stable transfectants, PD98059 had no inhibitory effect on RB hypophosphorylation and G1/0 arrest. This differential sensitivity to PD98059 and uncoupling of retinoic acid-induced differentiation and G1/0 arrest in cFMS transfectants is associated with changes in mitogen-activated protein kinase signaling molecules. The cFMS transfectants had more activated ERK2 than did the wild-type cells, which surprisingly was not attributable to enhanced mitogen-activated protein-kinase-kinase-kinase (RAF) phosphorylation. Retinoic acid increased the amount of activated ERK2 and phosphorylated RAF in both cell lines. But PD98059 eliminated detectable ERK2 activation, as well as inhibited RAF phosphorylation, in untreated and retinoic acid-treated wild-type HL-60 and cFMS transfectants, consistent with MEK or ERK feedback-regulation of RAF, in all four cases. Since PD98059 blocks the cFMS-conferred enhancement of the retinoic acid-induced differentiation, but not growth arrest, the data indicate that cFMS-enhanced differentiation acts primarily through MEK and ERK2, but cFMS-enhanced G1/0 arrest allied with RB hypophosphorylation depends on another cFMS signal route, which by itself can effect G1/0 arrest without activated ERK2. Ectopic expression of cFMS and differential sensitivity to ERK2 inhibition thus reveal that retinoic acid-induced HL-60 cell differentiation and G1/0 arrest are differentially dependent on ERK2 and can be uncoupled. A significant unanticipated finding was that retinoic acid caused a MEK-dependent increase in the amount of phosphorylated RAF. This increase may help sustain prolonged ERK2 activation.     

Blockade of focal clustering and active conformation in beta 2-integrin-mediated adhesion of eosinophils to intercellular adhesion molecule-1 caused by transduction of HIV TAT-dominant negative Ras

We transduced dominant negative (dn) HIV TAT-Ras protein into mature human eosinophils to determine the signaling pathways and mechanism involved in integrin-mediated adhesion caused by cytokine, chemokine, and chemoattractant stimulation. Transduction of TAT-dnRas into nondividing eosinophils inhibited endogenous Ras activation and extracellular signal-regulated kinase (ERK) phosphorylation caused by IL-5, eotaxin-1, and fMLP. IL-5, eotaxin-1, or fMLP caused 1) change of Mac-1 to its active conformation and 2) focal clustering of Mac-1 on the eosinophil surface. TAT-dnRas or PD98059, a pharmacological mitogen-activated protein/ERK kinase inhibitor, blocked both focal surface clustering of Mac-1 and the change to active conformational structure of this integrin assessed by the mAb CBRM1/5, which binds the activation epitope. Eosinophil adhesion to the endothelial ligand ICAM-1 was correspondingly blocked by TAT-dnRas and PD98059. As a further control, we used PMA, which activates ERK phosphorylation by postmembrane receptor induction of protein kinase C, a mechanism which bypasses Ras. Neither TAT-dnRas nor PD98059 blocked eosinophil adhesion to ICAM-1, up-regulation of CBRM1/5, or focal surface clustering of Mac-1 caused by PMA. In contrast to beta(2)-integrin adhesion, neither TAT-dnRas nor PD98059 blocked the eosinophil adhesion to VCAM-1. Thus, a substantially different signaling mechanism was identified for beta(1)-integrin adhesion. We conclude that H-Ras-mediated activation of ERK is critical for beta(2)-integrin adhesion and that Ras-protein functions as the common regulator for cytokine-, chemokine-, and G-protein-coupled receptors in human eosinophils.     

Normoxic induction of the hypoxic-inducible factor-1 alpha by interleukin-1 beta involves the extracellular signal-regulated kinase 1/2 pathway in normal human cytotrophoblast cells

During early pregnancy, an environment of relative low oxygen tension is essential for normal embryonic and placental vasculature. In low-oxygen conditions, the hypoxic-inducible factor-1 (HIF-1), composed of alpha and beta subunits, controls the expression of a number of genes such as vascular endothelial growth factor (VEGF), a key angiogenic factor. The recent studies in some tumor cells have found that the labile component, HIF-1 alpha, is not only activated by hypoxia but also by peptides such as interleukin-1 (IL-1) in normoxia. In this article, we demonstrated that exposure of normal human cytotrophoblast cells to IL-1 beta stimulated the expression of HIF-1 alpha protein. Meanwhile, IL-1 beta also induced the secretion of VEGF in normal human cytotrophoblast cells. Our data indicated that IL-1 beta induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. Moreover, treatment of cells with PD98059, an inhibitor of ERK1/2 signaling, inhibited the stimulation of HIF-1 alpha protein expression and VEGF secretion by IL-1 beta. These data indicate that, in normal human cytotrophoblast cells, IL-1 beta induces HIF- 1 alpha-mediated VEGF secretion and that IL-1 beta-stimulated ERK1/2 activation may be involved in this process.     

Atorvastatin suppresses LPS-induced rapid upregulation of Toll-like receptor 4 and its signaling pathway in endothelial cells

In the present study, we tested our hypothesis that atorvastatin exerts its anti-inflammation effect via suppressing LPS-induced rapid upregulation of Toll-like receptor 4 (TLR4) mRNA and its downstream p38, ERK, and NF-κB signaling pathways in human umbilical-vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs). TLR4 mRNA expression and its downstream kinase activities induced by LPS alone or atorvastatin + LPS in endothelial cells were quantified using quantitative real-time PCR and enzyme-linked immunosorbent assay. Preincubation of LPS-stimulated endothelial cells with TLR4 siRNA was conducted to identify the target of the anti-inflammatory effects of atorvastatin. Atorvastatin incubation resulted in the reduction of LPS-induced TLR4 mRNA expression, ERK1/2 and P38 MAPK phosphorylation, and NF-κB binding activity. Pretreatment with MEK/ERK1/2 inhibitor PD98059 attenuated atorvastatin + LPS-induced NF-κB activity but had no effect on P38 MAPK phosphorylation. In contrast, pretreatment with P38 MAPK inhibitor SB203580 resulted in upregulation of atorvastatin + LPS-induced ERK1/2 phosphorylation but had no significant effects on NF-κB activity. On the other hand, blocking NF-κB with SN50 produced no effects on atorvastatin + LPS-induced ERK1/2 and P38 MAPK phosphorylation. Moreover, TLR4 gene silencing produced the same effects as the atorvastatin treatment. In conclusion, atorvastatin downregulated TLR4 mRNA expression by two distinct signaling pathways. First, atorvastatin stabilized Iκ-Bα, which directly inhibited NF-κB activation. Second, atorvastatin inactivated ERK phosphorylation, which indirectly inhibited NF-κB activation. Suppression of p38 MAPK by atorvastatin upregulates ERK but exerts no effect on NF-κB.     

Exaggerated inflammatory responses mediated by Burkholderia cenocepacia in human macrophages derived from Cystic fibrosis patients

Glioblastoma (GBM) is a highly aggressive cancer type characterized by intense neovascularization. Several lines of evidence indicate that blood clotting enzymes play an important role in the tumor microenvironment, mainly through the activation of protease-activated receptors (PAR). In particular, PAR1 and PAR2 isoforms may activate signal transduction pathways that promote a number of pro-tumoral responses. However, little is known concerning the role of PAR1/PAR2 in GBM progression. In this study, we investigated the expression and function of PAR1 and PAR2 in the human GBM cell lines A172 and U87-MG. We also evaluated the effect of agonist peptides for PAR1 (PAR1-AP) and PAR2 (PAR2-AP) on signaling pathways and the expression of vascular endothelial growth factor (VEGF). Immunoblotting assays showed that A172 and U87-MG constitutively express PAR1 and PAR2. Treatment of GBM cells with PAR1-AP or PAR2-AP enhanced Akt (protein kinase B) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in a time-dependent manner. LY29042 and PD98059, inhibitors of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, decreased PAR-mediated activation of Akt and ERK1/2, respectively. In addition, we observed that PAR2, but not PAR1, activation increased VEGF secretion in U87-MG and A172 cells. Notably, only PD98059 reduced PAR2-mediated VEGF production by GBM cells. Our results suggest that PAR2 modulates VEGF production through the MAPK/ERK1/2 pathway, and not the PI3K/Akt pathway, in human GBM cell lines. Therefore, the PAR2/MAPK signaling axis might be regarded as a relevant target for adjuvant treatment of GBM with a possible impact on tumor angiogenesis.     

Pref-1 (preadipocyte factor 1) activates the MEK/extracellular signal-regulated kinase pathway to inhibit adipocyte differentiation

Preadipocyte factor 1 (Pref-1) is found in preadipocytes but is absent in adipocytes. Pref-1 is made as a transmembrane protein but is cleaved to generate a biologically active soluble form. Although Pref-1 inhibition of adipogenesis has been well studied in vitro and in vivo, the signaling pathway for Pref-1 is not known. Here, by using purified soluble Pref-1 in Pref-1 null mouse embryo fibroblasts (MEF), we show that Pref-1 increases MEK/extracellular signal-regulated kinase (ERK) phosphorylation in a time- and dose-dependent manner. Compared to wild-type MEF, differentiation of Pref-1 null MEF into adipocytes is enhanced, as judged by lipid accumulation and adipocyte marker expression. Both wild-type and Pref-1 null MEF show a transient burst of ERK phosphorylation upon addition of adipogenic agents. Wild-type MEF show a significant, albeit lower, second increase in ERK phosphorylation peaking at day 2. This ERK phosphorylation, corresponding to Pref-1 abundance, is absent during differentiation of Pref-1 null MEF. Prevention of this second increase in ERK1/2 phosphorylation in wild-type MEF by the MEK inhibitor PD98059 or by transient depletion of ERK1/2 via small interfering RNA-enhanced adipocyte differentiation. Furthermore, treatment of Pref-1 null MEF with Pref-1 restores this ERK phosphorylation, resulting in inhibition of adipocyte differentiation primarily by preventing peroxisome proliferator-activated receptor gamma2 induction. However, in the presence of PD98059 or depletion of ERK1/2, exogenous Pref-1 cannot inhibit adipocyte differentiation in Pref-1 null MEF. We conclude that Pref-1 activates MEK/ERK signaling, which is required for Pref-1 inhibition of adipogenesis.     

ERK/PP1a/PLB/SERCA2a and JNK Pathways Are Involved in Luteolin-Mediated Protection of Rat Hearts and Cardiomyocytes following Ischemia/Reperfusion

Luteolin has long been used in traditional Chinese medicine for treatment of various diseases. Recent studies have suggested that administration of luteolin yields cardioprotective effects during ischemia/reperfusion (I/R) in rats. However, the precise mechanisms of this action remain unclear. The aim of this study is to confirm that luteolin-mediated extracellular signal regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways are responsible for their cardioprotective effects during I/R. Wistar rats were divided into the following groups: (i) DMSO group (DMSO); (ii) I/R group (I/R); (iii) luteolin+I/R group (Lut+I/R); (iv) ERK1/2 inhibitor PD98059+I/R group (PD+I/R); (v) PD98059+luteolin+I/R group (PD+Lut+I/R); and (vi) JNK inhibitor SP600125+I/R group (SP+I/R). The following properties were measured: contractile function of isolated heart and cardiomyocytes; infarct size; the release of lactate dehydrogenase (LDH); the percentage of apoptotic cells; the expression levels of Bcl-2 and Bax; and phosphorylation status of ERK1/2, JNK, type 1 protein phosphatase (PP1a), phospholamban (PLB) and sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a). Our data showed that pretreatment with luteolin or SP600125 significantly improved the contraction of the isolated heart and cardiomyocytes, reduced infarct size and LDH activity, decreased the rate of apoptosis and increased the Bcl-2/Bax ratio. However, pretreatment with PD98059 alone before I/R had no effect on the above indexes. Further, these consequences of luteolin pretreatment were abrogated by co-administration of PD98059. We also found that pretreatment with PD98059 caused a significant increase in JNK expression, and SP600125 could cause ERK1/2 activation during I/R. In addition, we are the first to demonstrate that luteolin affects PP1a expression, which results in the up-regulation of the PLB, thereby relieving its inhibition of SERCA2a. These results showed that luteolin improves cardiomyocyte contractile function after I/R injury by an ERK1/2-PP1a-PLB-SERCA2a-mediated mechanism independent of JNK signaling pathway.