Signal-regulated Kinase Mitogen-activated Protein Kinase Research Articles

Angiotensin II–Induced MMP-2 Activity and MMP-14 and Basigin Protein Expression Are Mediated via the Angiotensin II Receptor Type 1–Mitogen-Activated Protein Kinase 1 Pathway in Retinal Pigment Epithelium: Implications for Age-Related Macular Degeneration

Accumulation of various lipid-rich extracellular matrix (ECM) deposits under the retinal pigment epithelium (RPE) has been observed in eyes with age-related macular degeneration (AMD). RPE-derived matrix metalloproteinase (MMP)-2, MMP-14, and basigin (BSG) are major enzymes involved in the maintenance of ECM turnover. Hypertension (HTN) is a systemic risk factor for AMD. It has previously been reported that angiotensin II (Ang II), one of the most important hormones associated with HTN, increases MMP-2 activity and its key regulator, MMP-14, in RPE, inducing breakdown of the RPE basement membrane, which may lead to progression of sub-RPE deposits. Ang II exerts most of its actions by activating the mitogen-activated protein kinase (MAPK) signaling pathway. Herein is explored the MAPK signaling pathway as a potential key intracellular modulator of Ang II-induced increase in MMP-2 activity and MMP-14 and BSG protein expression. It was observed that Ang II stimulates phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK in RPE cells and ERK/p38 and Jun N-terminal kinase (JNK) in mice. These effects were mediated by Ang II type 1 receptors. Blockade of ERK or p38 MAPK abrogated the increase in MMP-2 activity and MMP-14 and BSG proteins in ARPE-19 cells. A better understanding of the molecular events by which Ang II induces ECM dysregulation is of critical importance to further define its contribution to the progression of sub-RPE deposits in AMD patients with HTN.

Chloroform extract of garlic (Allium sativum) stimulates production of superoxide anion and CXCL8 in human neutrophils

Garlic has garnered attention as a useful material which contains many bioactive molecules for the prevention and/or treatment of various human ailments, including infectious diseases. In this study we tested several extracts of garlic on cellular activity in human neutrophils. Stimulation of human neutrophils with chloroform extract of garlic (GCE) elicited intracellular calcium ([Ca2+]i) increases in a phospholipase C independent manner. GCE also stimulated superoxide anion production, which was completely blocked by a calcium (Ca2+) chelator. We also observed that stimulation of human neutrophils with GCE caused production of the chemokine CXCL8 in a concentration-dependent manner. GCE also stimulated activity of two important mitogen activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK) and p38 MAPK. Moreover, GCE-induced CXCL8 production was inhibited by a p38 MAPK inhibitor (SB203580) but not by an ERK inhibitor (PD98059). Taken together, our results demonstrate that GCE contains bioactive natural compounds that stimulate [Ca2+]i signaling, superoxide anion production, and CXCL8 production. Thus, GCE may be useful for the development of immune-modulating agents against infectious diseases.

Pheophorbide a: A photosensitizer with immunostimulating activities on mouse macrophage RAW 264.7 cells in the absence of irradiation

Pheophorbide a (Pa) has been proposed to be a potential photosensitizer for the photodynamic therapy of human cancer. However, the immunomodulatory effect of Pa, in the absence of irradiation, has not yet been investigated. The present study revealed that Pa possessed immunostimulating effect on a murine macrophages cell line RAW 264.7. Pa could significantly stimulate the growth of RAW 264.7 cells with the maximum effect at 1.0 μM after 24, 48 and 72 h of treatment (all p < 0.05). Besides, intracellular mitogen activated protein kinases (MAPK) including extracellular signal-regulated kinase (ERK) and p38 MAPK were activated by Pa treatment in a dose-dependent manner. The activation of ERK and p38 MAPK was found to be related to the Pa-induced intracellular reactive oxygen species. Furthermore, Pa could significantly induce the release of interleukine-6 and tumour necrosis factor-α, and enhance the phagocytic activity of RAW 264.7 cells (all p < 0.05). The present work is the first report to demonstrate the potential immunomodulatory effect of Pa on macrophages, apart from its well-studied anti-tumour activity.

Differential activation of mitogen-activated protein kinases and glial cells in the trigeminal sensory nuclear complex following lingual nerve injury

Mitogen-activated protein kinases (MAPKs) play a pivotal role in the mediation of cellular responses to a variety of signaling molecules. The current study demonstrates phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK in each subdivision of the trigeminal sensory nuclear complex (TSNC) following lingual nerve injury. Immunohistochemical labeling for phosphorylated ERK (p-ERK) or phosphorylated p38 (p-p38) MAPK was performed in histological sections of the brainstem. A transient increase in the immunoreactivity for p-ERK was found in each subdivision of the TSNC followed by a prolonged increase in the immunoreactivity for p-p38 MAPK after nerve injury. Double immunofluorescence labeling with cell-specific markers revealed that ERK and p38 MAPK were phosphorylated predominantly by OX-42-positive microglia or GFAP-positive astrocytes. Increased immunofluorescence labeling for OX-42 and GFAP indicated that microglia and astrocytes were activated by nerve injury in the TSNC. Activation of MAPKs and glial cells in the rostral subdivisions of the TSNC was comparable with that in the subnucleus caudalis of the trigeminal spinal tract nucleus (Vc). We conclude that differential activation of MAPKs and glial cells in the rostral subdivisions of the TSNC as well as the Vc may have a substantial role in the pathogenesis of neuropathic pain following trigeminal nerve injury.

Tetrahydropalmatine Inhibits Pro-Inflammatory Mediators in Lipopolysaccharide-Stimulated THP-1 Cells

For centuries, Corydalis Rhizoma has been used in Korean traditional medicine. Tetrahydropalmatine is an alkaloid compound and a prominent anti-inflammatory agent found in plants, including Corydalis yanhusuo. However, the therapeutic mechanisms of tetrahydropalmatine are still not well understood. To provide insight into the biological effects of tetrahydropalmatine, we examined its influence on lipopolysaccharide (LPS)-induced interleukin (IL)-8 production in the human monocytic cell line THP-1. In the present study, IL-8 production was measured using enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction, and mitogen-activated protein kinase (MAPK) activation, as determined by western blot analysis. Tetrahydropalmatine inhibited LPS-induced IL-8 production in a dose-dependent manner. Furthermore, tetrahydropalmatine inhibited extracellular signal-regulated kinase and p38 MAPK phosphorylation, which suggests that tetrahydropalmatine inhibits IL-8 secretion by blocking MAPK phosphorylation. Taken together, these findings may help elucidate the mechanism by which tetrahydropalmatine modulates THP-1 cell activation under inflammatory conditions.

Effect of penehyclidine hydrochloride on expressions of MAPK in mice with CLP-induced acute lung injury

Penehyclidine hydrochloride (PHC) is a new anticholinergic drug. PHC has been shown to have a good curative effect for sepsis. Mitogen-activated protein kinase (MAPK) has recently been considered to play an important role in sepsis. In this study, the role of MAPK signal pathways in protective effects of PHC preconditioning on acute lung injury in cecal ligation and puncture (CLP)-induced sepsis was investigated. Healthy female mice were randomly divided into 4 groups: sham control, CLP, and 0.3 or 0.45 mg/kg PHC. At 12 h after surgery, arterial blood was drawn for blood gas analysis, and lung tissue samples were collected to examine pulmonary microvascular permeability, IL-6 levels and myeloperoxidase (MPO) activity. MAPK protein expressions were measured using western blot technique. Compared with sham control mice, acute lung injury was induced in CLP group, which was indicated by decreased PaO(2)/FiO(2), increased pulmonary microvascular permeability, IL-6 levels and MPO activity. Furthermore, mice' exposure to CLP induced the increased protein levels of MAPK. Treatment of 0.45 mg/kg PHC markedly improved PaO(2)/FiO(2), decreased pulmonary microvascular permeability, IL-6 levels and MPO activity, and inhibited expressions of extracellular signal-regulated kinase (ERK1/2) and p38 MAPK. Taken together, these results suggest that PHC ameliorated acute lung injury through the inhibition of extracellular signal-regulated kinase (ERK1/2) and p38 MAPK activation in septic mice.

Toll-Like Receptor 4-Mediated Activation of p38 Mitogen-Activated Protein Kinase Is a Determinant of Respiratory Virus Entry and Tropism

Respiratory viruses exert a heavy toll of morbidity and mortality worldwide. Despite this burden there are few specific treatments available for respiratory virus infections. Since many viruses utilize host cell enzymatic machinery such as protein kinases for replication, we determined whether pharmacological inhibition of kinases could, in principle, be used as a broad antiviral strategy for common human respiratory virus infections. A panel of green fluorescent protein (GFP)-expressing recombinant respiratory viruses, including an isolate of H1N1 influenza virus (H1N1/Weiss/43), was used to represent a broad range of virus families responsible for common respiratory infections (Adenoviridae, Paramyxoviridae, Picornaviridae, and Orthomyxoviridae). Kinase inhibitors were screened in a high-throughput assay that detected virus infection in human airway epithelial cells (1HAEo-) using a fluorescent plate reader. Inhibition of p38 mitogen-activated protein kinase (MAPK) signaling was able to significantly inhibit replication by all viruses tested. Therefore, the pathways involved in virus-mediated p38 and extracellular signal-regulated kinase (ERK) MAPK activation were investigated using bronchial epithelial cells and primary fibroblasts derived from MyD88 knockout mouse lungs. Influenza virus, which activated p38 MAPK to approximately 10-fold-greater levels than did respiratory syncytial virus (RSV) in 1HAEo- cells, was internalized about 8-fold faster and more completely than RSV. We show for the first time that p38 MAPK is a determinant of virus infection that is dependent upon MyD88 expression and Toll-like receptor 4 (TLR4) ligation. Imaging of virus-TLR4 interactions showed significant clustering of TLR4 at the site of virus-cell interaction, triggering phosphorylation of downstream targets of p38 MAPK, suggesting the need for a signaling receptor to activate virus internalization.

Facilitation of central imidazoline I1‐site/extracellular signal‐regulated kinase/p38 mitogen‐activated protein kinase signalling mediates the hypotensive effect of ethanol in rats with acute renal failure

This study investigated the role of central sympathetic activity and related mitogen-activated protein kinase (MAPK) signalling in the cardiovascular effects of ethanol in a model of acute renal failure (ARF). The effects of pharmacological interventions that inhibit peripheral or central sympathetic activity or MAPK on the cardiovascular actions of ethanol in rats with ARF induced by glycerol were evaluated. Glycerol (50%, 10 mL.kg(-1), i.m.) caused progressive increases and decreases in blood pressure (BP) and heart rate (HR) respectively. Subsequent i.v. ethanol (0.25 or 1 g.kg(-1)) elicited dose-related changes in BP (decreases) and HR (increases). These effects were replicated after intracisternal (i.c.) administration of ethanol. Blockade of nicotinic cholinoceptors (nAChR, hexamethonium, 20 mg.kg(-1)) or alpha(1)-adrenoceptors (prazosin, 1 mg.kg(-1)) attenuated cardiovascular effects of ethanol. Ethanol hypotension was also attenuated after the centrally acting sympatholytic drug moxonidine (selective I(1)-site agonist, 100 microg.kg(-1) i.v.), but not guanabenz (selective alpha(2)-receptor agonist, 30 microg.kg(-1), i.v.), suggesting involvement of central circuits of I(1) sites in ethanol-evoked hypotension. Selective blockade I(1) sites (efaroxan) but not alpha(2) (yohimbine) adrenoceptors abolished the hypotensive response to ethanol. Intracisternal administration of PD98059 or SB203580, inhibitors of extracellular signal-regulated kinase (ERK 1/2) and p38 MAPK, respectively, reduced the hypotensive action of moxonidine or ethanol. When used simultaneously, the two MAPK inhibitors produced additive attenuation of ethanol hypotension. Sympathoinhibitory pathways of central I(1)-sites and downstream ERK/p38 MAPK signalling were involved in the hypotensive action of ethanol in ARF.

Krüppel-like Factor 4 Inhibits Proliferation by Platelet-derived Growth Factor Receptor β-mediated, Not by Retinoic Acid Receptor α-mediated, Phosphatidylinositol 3-Kinase and ERK Signaling in Vascular Smooth Muscle Cells

Proliferation inhibition of vascular smooth muscle cells (VSMCs) is governed by the activity of a transcription factor network. Krüppel-like factor 4 (Klf4), retinoic acid receptor (RAR alpha), and platelet-derived growth factor receptor (PDGFR) are expressed in VSMCs and are components of such a network. However, the relationship among them in the regulation of VSMC proliferation remains unknown. Here, we investigated the mechanisms whereby Klf4 mediates the growth inhibitory effects in VSMCs through RAR alpha and PDGFR beta. We demonstrated that Klf4 directly binds to the 5' regulatory region of RAR alpha, down-regulates RAR alpha expression, and specifically inhibits RAR alpha-mediated phosphatidylinositol 3-kinase (PI3K) and ERK signaling in cultured VSMCs induced by the synthetic retinoid Am80. Of particular interest, Klf4 inhibits RAR alpha and PDGFR beta expression while blocking PI3K and ERK signaling induced by Am80 and PDGF-BB, respectively. The anti-proliferative effects of Klf4 on neointimal formation depend largely on PDGFR-mediated PI3K signaling without involvement of the RAR alpha-activated signaling pathways. These findings provide a novel mechanism for signal suppression and growth inhibitory effects of Klf4 in VSMCs. Moreover, the results of this study suggest that Klf4 is one of the key mediators of retinoid actions in VSMCs.

Effect of TLR3 and TLR7 activation in uterine NK cells from non-obese diabetic (NOD) mice

Toll-like receptor (TLR)-TLR cross talk is thought to be important in TLR signaling. Herein, we investigated the effect of specific TLR3 and TLR7 agonists, poly (I:C) and R837, individually and in combination, on uterine immune cell function and their subsequent effects on pregnancy outcome. Allogeneic pregnancies in the non-obese diabetic (NOD) mousexC57BL/6 and wild-type BALB/cxC57BL/6 model were used. An additive increase in embryo resorption was observed after induction with both poly (I:C) and R837, and was associated with elevated numbers of both TNF-alpha- and IFN-gamma-producing CD45(+) cells in the uterus. Further examination showed that while cytokine expression was detected in both CD3(+) cells and CD49b(+) cells in BALB/c mice, NOD mouse cells behaved differently. In NOD mice, elevated cytokine expression was attributed to CD3(+) T cells, with no response detected in the CD49b(+) NK cells. The additive effect of combined agonists was partially inhibited by the Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) inhibitor SP600125 and almost completely abrogated by the extracellular signal-regulated kinase (ERK) MAPK inhibitor PD98059. These results suggest that increased TLR3 and TLR7 signals are transmitted via Th1-type T cells, rather than NK cells, in NOD mice. Furthermore, the ERK MAPK pathway may be critical in TLR3 and TLR7 signaling.

Synergistic effect of combined double-stranded RNA and imiquimod stimulation

Objective To investigate the effect of combined double-stranded RNA (dsRNA) and imiquimod stimulation on uterine immune cells. Methods In BALB/c × C57BL/6 mice and non-obese dia-betic (NOD) × C57BI/6 mice, embryo resorption rate was detected in the presence or absence of Toll-like receptor 3 (TLR3) agonist dsRNA [poly( 1: C)], TLR7 agonist imiquimod ( R837), or their combination, respectively. In in vivo system, the status of intracellular cytokine production in uterine CD45 + cells was de-tected by flow cytometry. To identify the CD45 + cells, uterine CD3+ T cells and CD49b + NK cells derived from placenta and decidua basalis were stimulated with dsRNA and imiquimod in in vitro systems, and the status of intracellular cytokine production was detected. Mitogen-activated protein kinase (MAPK) antago- nists SP600125 and PD98059 were used to block the increase of cytokine production. Results A synergistic increase of embryo resorption was observed after the induction of dsRNA and imiquimod combination. Mean-while, a synergistic increase of TNF-α and IFN-γ production was detected after the induction in CD45 + cells. Further study found that although synergistic effect can be detected in both CD3 + cells and CD49b + cells in BALB/c mice, the status was different in NOD mice. The cytokine increase should mainly be attrib-uted to CD3 + T cells, since no such increase was detected among the CD49b + NK cells in the NOD mice. The synergistic effect of combined agonists was partially inhibited by Jun N-terminal kinase (JNK) MAPK inhibitor SP600125 and almost completely abrogated by extracellular signal-regulated kinase (ERK) MAPK inhibitor PD98059. Conclusion Boosted TLR3 and TLR7 signal may be transmitted via Thl-type T cells, rather than NK cells in NOD mice. ERK MAPK pathway may be critical in TLR3 and TLR7 involved signa- ling. Key words: Animal model; Immunodeficieney; Immunomodulation; Toll-like receptor

Soluble vascular cell adhesion molecule‐1 induces human eosinophil migration

Tissue eosinophilia is one of the hallmarks of allergic diseases and Th2-type immune responses including asthma. Adhesion molecules are known to play an important role in the accumulation of eosinophils in allergic inflammatory foci, and they contribute to eosinophil activation. Elevated levels of the soluble forms of adhesion molecules in the body fluid of asthmatic patients have been observed, although their pathophysiological significance remains to be fully elucidated. Peripheral blood eosinophils were purified, and the effect of soluble vascular cell adhesion molecule-1 (sVCAM-1) on eosinophil migration was investigated using in vitro systems. We found that sVCAM-1 (1 to 10 mug/ml) induced eosinophil chemotaxis, rather than chemokinesis, in a concentration-dependent fashion. In addition, sVCAM-1 induced cell shape change and actin polymerization, which are necessary for cell movement. Manipulations with very late antigen (VLA)-4-neutralizing antibody and signal inhibitors indicated that the sVCAM-1-induced chemotaxis was mediated through ligand-dependent activation of tyrosine kinase Src, p38 mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase (ERK) MAPK. Rapid phosphorylation of these signaling molecules was observed using a bead-based multiplex assay. Our results raise the possibility of sVCAM-1 in the fluid phase as a significant contributor to the heightened eosinophilic inflammatory response.

Cilostazol Protects Endothelial Cells Against Lipopolysaccharide-Induced Apoptosis Through ERK1/2- and P38 MAPK-Dependent Pathways

Background/AimsWe examined the effects of cilostazol on mitogen-activated protein kinase (MAPK) activity and its relationship with cilostazol-mediated protection against apoptosis in lipopolysaccharide (LPS)-treated endothelial cells.MethodsHuman umbilical vein endothelial cells (HUVECs) were exposed to LPS and cilostazol with and without specific inhibitors of MAPKs; changes in MAPK activity in association with cell viability and apoptotic signaling were investigated.ResultsCilostazol protected HUVECs against LPS-induced apoptosis by suppressing the mitochondrial permeability transition, cytosolic release of cytochrome c, and subsequent activation of caspases, stimulating extracellullar signal-regulated kinase (ERK1/2) and p38 MAPK signaling, and increasing phosphorylated cAMP-responsive element-binding protein (CREB) and Bcl-2 expression, while suppressing Bax expression. These cilostazol-mediated cellular events were effectively blocked by MAPK/ERK kinase (MEK1/2) and p38 MAPK inhibitors.ConclusionsCilostazol protects HUVECs against LPS-induced apoptosis by suppressing mitochondria-dependent apoptotic signaling. Activation of ERK1/2 and p38 MAPKs, and subsequent stimulation of CREB phosphorylation and Bcl-2 expression, may be responsible for the cellular signaling mechanism of cilostazol-mediated protection.

Activator protein-1 transcription factors are associated with progression and recurrence of prostate cancer: Ouyang X, Jessen WJ, Al-Ahmadie H, Serio AM, Lin Y, Shih WJ, Reuter VE, Scardino PT, Shen MM, Aronow BJ, Vickers AJ, Gerald WL, Abate-Shen C, Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry, New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ

To identify biomarkers that discriminate the aggressive forms of prostate cancer, we performed gene expression profiling of prostate tumors using a genetically engineered mouse model that recapitulates the stages of human prostate cancer, namely Nkx3.1; Pten mutant mice. We observed a significant deregulation of the epidermal growth factor and mitogen-activated protein kinase (MAPK) signaling pathways, as well as their major downstream effectors–the activator protein-1 transcription factors c-Fos and c-Jun. Forced expression of c-Fos and c-Jun in prostate cancer cells promotes tumorigenicity and results in activation of extracellular signal-regulated kinase (Erk) MAPK signaling. In human prostate cancer, up-regulation of c-Fos and c-Jun proteins occurs in advanced disease and is correlated with Erk MAPK pathway activation, whereas high levels of c-Jun expression are associated with disease recurrence. Our analyses reveal a hitherto unappreciated role for AP-1 transcription factors in prostate cancer progression and identify c-Jun as a marker of high-risk prostate cancer. This study provides a striking example of how accurate mouse models can provide insights on molecular processes involved in progression and recurrence of human cancer.

Hemin inhibits cyclooxygenase-2 expression through nuclear factor-kappa B activation and ornithine decarboxylase expression in 12- O-tetradecanoylphorbol-13-acetate-treated mouse skin

Inflammation induced by various stimuli has been found to be associated with increased risk for most types of human cancer. Inflammation facilitates the initiation of normal cells, as well as the growth of initiated cells and their progression to malignancy through production of proinflammatory cytokines and diverse reactive oxygen/nitrogen species. These also activate the signaling molecules that are involved in inflammation and carcinogenesis. Our previous studies have demonstrated that hemin inhibited 7,12-dimethylbenz[ a]anthracene (DMBA)-induced bacterial mutagenesis and oxidative DNA damage, reduced the level of DNA-DMBA adduct and 12- O-tetradecanoylphorobl-13-acetate (TPA)-induced tumor formation in DMBA-initiated ICR mouse skin, and inhibited myeloperoxidase and ornithine decarboxylase (ODC) activity and H 2O 2 formation in TPA-treated mouse skin. In the present study, to further elucidate the molecular mechanisms underlying the chemopreventive activity of hemin, its effect on the expression of ODC and cyclooxygenase (COX)-2, and the activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) regulating these proteins were explored in mouse skin with TPA-induced inflammation. Topically applied hemin inhibited ear edema and epidermal thickness in mice treated with TPA. Pretreatment with hemin reduced the expression of ODC and COX-2, and also reduced NF-κB activation in TPA-stimulated mouse skin. In addition, hemin suppressed the TPA-induced activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK in a dose-dependent manner. Taken together, hemin inhibited TPA-induced COX-2 expression by altering NF-κB signaling pathway via ERK and p38 MAPK, as well as TPA-induced ODC expression in mouse skin. Thereby, hemin may be an attractive candidate for a chemopreventive agent.

Activator protein-1 transcription factors are associated with progression and recurrence of prostate cancer.

To identify biomarkers that discriminate the aggressive forms of prostate cancer, we performed gene expression profiling of prostate tumors using a genetically engineered mouse model that recapitulates the stages of human prostate cancer, namely Nkx3.1; Pten mutant mice. We observed a significant deregulation of the epidermal growth factor and mitogen-activated protein kinase (MAPK) signaling pathways, as well as their major downstream effectors--the activator protein-1 transcription factors c-Fos and c-Jun. Forced expression of c-Fos and c-Jun in prostate cancer cells promotes tumorigenicity and results in activation of extracellular signal-regulated kinase (Erk) MAPK signaling. In human prostate cancer, up-regulation of c-Fos and c-Jun proteins occurs in advanced disease and is correlated with Erk MAPK pathway activation, whereas high levels of c-Jun expression are associated with disease recurrence. Our analyses reveal a hitherto unappreciated role for AP-1 transcription factors in prostate cancer progression and identify c-Jun as a marker of high-risk prostate cancer. This study provides a striking example of how accurate mouse models can provide insights on molecular processes involved in progression and recurrence of human cancer.

Involvement of PI3K/Akt Pathway in TGF-β2-Mediated Epithelial Mesenchymal Transition in Human Lens Epithelial Cells

Background: Epithelial mesenchymal transition (EMT) of postoperative remnants of lens epithelial cells (LECs) can lead to posterior capsule opacification. This study was designed to determine the effect of signaling pathways that contribute to TGF-β₂-mediated EMT in human lens epithelial B-3 cells (HLEB-3 cells). Methods: The HLEB-3 cells were cultured and stimulated with TGF-β₂ at different concentrations for an indicated time. The effect of TGF-β₂ on cell cycle distribution was measured by flow cytometry. Western blot and immunofluorescence were used to analyze changes in connexin 43, fibronectin, desmin and integrin β₁ protein expression associated with EMT in HLEB-3 cells. Activation of phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways was also detected by Western blot. Results: The cell cycle progression of HLEB-3 cells was limited, and the cells underwent morphological alteration after treatment with TGF-β₂. Stimulation of HLEB-3 cells with TGF-β₂ suppressed connexin 43 protein expression, increased fibronectin, desmin and integrin β₁ protein expression. TGF-β₂ activated PI3K/Akt in a time-dependent manner, but not extracellular signal-regulated kinase and p38 MAPK. The activation of PI3K/Akt was necessary for the TGF-β₂-stimulated downregulation of connexin 43, which in turn was necessary for TGF-β₂-induced EMT in HLEB-3 cells. Conclusions: TGF-β₂ is a potent growth factor for LEC EMT. TGF-β₂-induced EMT in LECs is mediated by the downregulation of connexin 43, which is regulated through the PI3K/Akt pathway.

Differential Contribution of Spinal Mitogen-Activated Protein Kinases to the Phase of Long-Lasting Allodynia Evoked by Intrathecal Administration of ATP in Rats

Several lines of evidence suggest that activation of spinal mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK) and p38 MAPK, contributes to the induction and maintenance of chronic pain. We recently reported that an intrathecal (i.t.) administration of ATP evoked tactile allodynia, which lasted more than 1 week in rats. The long-lasting allodynia was induced by activation of spinal P2X 2/3-receptors, and the induction and early phase of maintenance, but not the late phase, was mediated, at least in part, by the activation of spinal glial cells. In this study, we examined the involvement of spinal ERK and p38 MAPK in each phase of i.t. ATP-evoked long-lasting allodynia. I.t. administration of ATP (100 nmol) markedly increased phosphorylated ERK, which peaked at 1-8 h before gradually decreasing to a level that was sustained until 7 d after administration. In contrast, only a slight increase in phosphorylated p38 MAPK was observed. Consistent with the increased phosphorylation of MAPKs, the ERK kinase MEK inhibitor, U0126 (3 nmol), attenuated the induction phase (co-administration with ATP) and early maintenance phase (1-d post-ATP administration) of the i.t. ATP-evoked allodynia, but not the late maintenance phase (7-d post-ATP administration), while the p38 MAPK inhibitor, SB203580 (10 microg), had little effect. These results suggest that the induction phase and early maintenance phase, but not the late maintenance phase of long-lasting allodynia is mediated by the activation of ERK, rather than by the activation of p38 MAPK, in the spinal cord. These findings are informative for elucidating the mechanisms underlying the pathogenesis of chronic pain.

Mume Fructus Water Extract Inhibits Pro-Inflammatory Mediators in Lipopolysaccharide-Stimulated Macrophages

Mume Fructus (Family Rosaceae) is used as a traditional drug and health food in Asian countries. However, its therapeutic mechanisms and effects on macrophage-mediated inflammation remain unknown. In this study we examined the effect of Mume Fructus water extract (MFWE) on pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The investigation focused on whether MFWE inhibited nitric oxide (NO) and prostaglandin (PG) E2 productions, as well as the expressions of inducible NO synthase (iNOS), cyclooxygenase (COX)-2, interleukin (IL)-6, nuclear factor-kappaB (NF-kappaB), and mitogen-activated protein kinases (MAPKs) in LPS-stimulated RAW 264.7 cells. We found that MFWE inhibited LPS-induced NO, PGE(2), and IL-6 productions as well as the expressions of iNOS and COX-2. Furthermore, MFWE suppressed the LPS-induced phosphorylations of p38 MAPK and extracellular signal-regulated kinase MAPK, as well as IkappaBalpha degradation and NF-kappaB activation. These results suggest that MFWE has inhibitory effects on LPS-induced PGE2, NO, and IL-6 production, as well as the expressions of iNOS and COX-2 in the murine macrophage. These inhibitory effects occur through blockades on the phosphorylation of MAPKs following IkappaBalpha degradation and NF-kappaB activation.

Capacitative Calcium Entry Contributes to the Differential Transactivation of the Epidermal Growth Factor Receptor in Response to Thiazolidinediones

Thiazolidinediones (TZDs) are synthetic ligands for the peroxisome proliferator-activated receptor gamma (PPARgamma) but also elicit PPARgamma-independent effects, most notably activation of mitogen-activated protein kinases (MAPKs). Ciglitazone rapidly activates extracellular signal-regulated kinase (Erk) MAPK, an event requiring c-Src kinase-dependent epidermal growth factor receptor (EGFR) transactivation, whereas troglitazone only weakly activates Erk and does not induce EGFR transactivation; the mechanism underlying this difference remains unclear. In this study, both ciglitazone and troglitazone increased Src activation. Similar effects were observed with Delta2-derivatives of each TZD, compounds that bind PPARgamma but do not lead to its activation, further indicating a PPARgamma-independent mechanism. Neither EGFR kinase nor Pyk2 inhibition prevented Src activation; however, inhibition of Src kinase activity prevented Pyk2 activation. Intracellular calcium chelation blocks TZD-induced Pyk2 activation; here, Src activation by both TZDs and ciglitazone-induced EGFR transactivation were prevented by calcium chelation. Accordingly, both TZDs increased calcium concentrations from intracellular stores; however, only ciglitazone produced a secondary calcium influx in the presence of extracellular calcium. Removal of extracellular calcium or inhibition of capacitative calcium entry by 2-APB prevented ciglitazone-induced EGFR transactivation and Erk activation but did not affect upstream kinase signaling pathways. These results demonstrate that upstream kinases (i.e., Src and Pyk2) are required but not sufficient for EGFR transactivation by TZDs. Moreover, influx of extracellular calcium through capacitative calcium entry may be an unrecognized component that provides a mechanism for the differential induction of EGFR transactivation by these compounds.