Fibronectin promotes proplatelet formation in the human megakaryocytic cell line UT-7/TPO

We investigated PPF (proplatelet formation) in the human megakaryocytic cell line UT-7/TPO in vitro and signal transduction pathways responsible for PPF. The megakaryocytic cell lines are useful for studying megakaryocyte biology, although PPF is induced only in the presence of phorbol ester. TPO (thrombopoietin) stimulates megakaryocyte proliferation and differentiation; however, no PPF occurred in the megakaryocytic cell lines, even after the addition of TPO. Therefore, factors other than TPO may play an important role in the process of PPF. As PPF occurs in the bone marrow in vivo, we noted extracellular matrix proteins and found that soluble FN (fibronectin) induced potent PPF in UT-7/TPO without phorbol ester. A Western blot analysis showed that the expression of integrins was not increased by FN treatment. Anti-β1 antibody and the RGD (arginine-glycine-aspartate) peptide inhibited FN-induced PPF. This result indicates that the signal originated from integrin β1, which is essential to inducing PPF in UT-7/TPO. Results of the experiments using several inhibitors suggest that activation of the MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]-ERK and PI3K (phosphoinositide 3-kinase) pathways are necessary for PPF. The phosphorylation of ERK gradually increased for 2 h after the addition of soluble FN, which suggests that activation of ERK is essential for the initial induction of FN-induced PPF in UT-7/TPO. UT-7/TPO is a useful cell line that enables us to study the signals of PPF without effects of chemical compounds.     

Mitogen-activated protein kinase activity may not be necessary for the neuropathology of Niemann-Pick type C mice

Hyperphosphorylation of neurofilament and tau, and formation of cytoskeletal lesions, are notable features of several human neurodegenerative diseases, including Niemann-Pick Disease Type C (NPC). Previous studies suggested that the MAPKs, extracellular signal regulated kinase 1 and 2 (ERK1/2) may play a significant role in this aspect of NPC. To test this idea, we treated npc mice with PD98059, a specific and potent inhibitor of MAPK activation. Although activity of ERK1/2 was inhibited by 40%, a 2-week intracerebroventricular infusion of PD98059 just prior to onset of cytoskeletal pathology and symptoms in npc mice did not delay or inhibit prominent hallmarks of NPC. Unexpectedly, ERK1/2 inhibition led to aggravation of tau hyperphosphorylation, particularly in oligodendroctyes, in a manner similar to that of certain human tauopathies. Our results suggest that ERK1/2 does not play a major role in NPC neuropathology, and therefore, that MAPK inhibition is unlikely to be a useful strategy for managing the disease.     

Luteolin induces apoptotic cell death through AIF nuclear translocation mediated by activation of ERK and p38 in human breast cancer cell lines

The flavonoid, luteolin, has been shown to have anticancer activity in various cancer cells; however, the precise molecular mechanism of its action is not completely understood, and studies were conducted to find out how it induces apoptosis in breast cancer cells. Luteolin induced a reduction of viability in a dose- and time-dependent manner. The pro-apoptotic effect of luteolin was demonstrated by cell cycle measurement and Hoechst 3325 staining. Western blot analysis showed that luteolin activates ERK (extracellular-signal-regulated kinase) and p38. Pharmacological inhibition or knockdown of ERK and p38 protected against luteolin-induced cell death; however, the caspase-3-specific inhibitor had no effect. Immunocytochemical examination indicated that luteolin induced nuclear translocation of AIF (apoptosis-inducing factor), which was mediated by activation of ERK and p38. Transfection of a vector expressing the miRNA (microRNA) of AIF prevented luteolin-induced apoptosis. The data suggest that luteolin induces a caspase-dependent and -independent apoptosis involving AIF nuclear translocation mediated by activation of ERK and p38 in breast cancer cells.     

Naringenin targets ERK2 and suppresses UVB‐induced photoaging

A number of natural phytochemicals have anti‐photoaging properties that appear to be mediated through the inhibition of matrix metalloproteinase‐1 (MMP‐1) expression, but their direct target molecule(s) and mechanism(s) remain unclear. We investigated the effect of naringenin, a major flavonoid found in citrus, on UVB‐induced MMP‐1 expression and identified its direct target. The HaCaT human skin keratinocyte cell line and 3‐dimensional (3‐D) human skin equivalent cultures were treated or not treated with naringenin for 1 hr before exposure to UVB. The mechanism and target(s) of naringenin were analysed by kinase assay and multiplex molecular assays. Dorsal skins of hairless mice were exposed to UVB 3 times per week, with a dose of irradiation that was increased weekly by 1 minimal erythema dose (MED; 45 mJ/cm²) to 4 MED over 15 weeks. Wrinkle formation, water loss and water content were then assessed. Naringenin suppressed UVB‐induced MMP‐1 expression and AP‐1 activity, and strongly suppressed UVB‐induced phosphorylation of Fos‐related antigen (FRA)‐1 at Ser265. Importantly, UVB irradiation‐induced FRA1 protein stability was reduced by treatment with naringenin, as well as with a mitogen‐activated protein kinase (MEK) inhibitor. Naringenin significantly suppressed UVB‐induced extracellular signal‐regulated kinase 2 (ERK2) activity and subsequently attenuated UVB‐induced phosphorylation of p90^RSK by competitively binding with ATP. Constitutively active MEK (CA‐MEK) increased FRA1 phosphorylation and expression and also induced MMP‐1 expression, whereas dominant‐negative ERK2 (DN‐ERK2) had opposite effects. U0126, a MEK inhibitor, also decreased FRA1 phosphorylation and expression as well as MMP‐1 expression. The photoaging data obtained from mice clearly demonstrated that naringenin significantly inhibited UVB‐induced wrinkle formation, trans‐epidermal water loss and MMP‐13 expression. Naringenin exerts potent anti‐photoaging effects by suppressing ERK2 activity and decreasing FRA1 stability, followed by down‐regulation of AP‐1 transactivation and MMP‐1 expression.     

Differential activation of ERK1,2 MAP kinase signaling pathway in mesenchymal stem cell from control and osteoporotic postmenopausal women

Osteoblasts, the cells responsible for bone formation, derive from mesenchymal stem cells (MSCs) in bone marrow. To acquire a new cell phenotype, uncommitted MSCs must undergo several proliferation and differentiation changes. Although, it is known that extracellular signal-regulated protein kinases (ERKs) mitogen-activated protein (MAP) kinase pathway signaling is involved in the proliferation and differentiation processes, the role of ERKs in osteogenic differentiation it is controversial, at present. In addition, the function that ERK could play in MSCs derived from osteoporotic patients it is not well documented. In this study, we analyze whether previously observed differences in the dynamic response of MSCs from normal and osteoporotic postmenopausal women can be explained by changes in the activation of this signal transduction pathway. Levels of ERK phosphorylation and their correlation with osteogenic differentiation were evaluated in cultures of MSCs derived from osteoporotic postmenopausal women and "healthy" controls. The results show that, under basal conditions, MSCs derived from osteoporotic donors show a level of ERK phosphorylation 2.5 times higher than MSCs derived from control donors. The addition of the osteogenic stimulus only slightly increases the p-ERK level in cells derived from osteoporotic donors, and is higher in cells derived from control women. Important differences in the ability of PD98059 to inhibit phosphorylation of ERK in both types of cells were also observed, as well as the effect that this inhibition produced on calcium deposition. We conclude that the MAP kinase pathway signaling is differentially activated in MSCs derived from osteoporotic postmenopausal women. The high p-ERK levels in MSC derived from osteoporotic donors could determine the unresponsiveness of these cells to the osteogenic differentiation stimulus.     

Anti‐invasion effect of rosmarinic acid via the extracellular signal‐regulated kinase and oxidation–reduction pathway in Ls174‐T cells

Rosmarinic acid is a major phenylpropanoid isolated from Prunella vulgaris L., which is a composition of herbal tea for centuries in China. However, the anti‐invasion activity on Ls174‐T human colon carcinoma cells has not been studied. In this study, we investigated the anti‐metastasis functions according to wound healing assay, adhesion assay, and Transwell assay and found that rosmarinic acid could inhibit migration, adhesion, and invasion dose‐dependently. Rosmarinic acid also could decrease the level of reactive oxygen species by enhancing the level of reduced glutathione hormone. In addition, rosmarinic acid repressed the activity and expression of matrix metalloproteinase‐2,9. According to Western blot and quantitative real‐time PCR assay, rosmarinic acid may inhibit metastasis from colorectal carcinoma mainly via the pathway of extracellular signal‐regulated kinase. In animal experiment, intraperitoneal administration of 2 mg of rosmarinic acid reduced weight of tumors and the number of lung nodules significantly compared with those of control group. Therefore, these results demonstrated that rosmarinic acid can effectively inhibit tumor metastasis in vitro and in vivo. J. Cell. Biochem. 111: 370–379, 2010. © 2010 Wiley‐Liss, Inc.     

Role of ROS and MAPK in TPA-induced ICAM-1 expression in the myeloid ML-1 cell line

Intercellular adhesion molecule 1 (ICAM-1) has been implicated in playing a key role in the mechanism of inflammatory process initiated in response to environmental agents, and during normal hematopoietic cell differentiation. Though induction of ICAM-1 by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in myeloid cells has been reported, the molecular mechanism by which TPA upregulates ICAM-1 expression remains unclear. In the present study, we investigated the signaling mechanism associated with TPA-induced ICAM-1 expression in ML-1 cells. Herein, our microarray, flow cytometry, and Western blot analysis indicated that ICAM-1 was constitutively expressed at a low level in ML-1 cells, but its expression was further upregulated at both the mRNA and protein levels in response to TPA. ICAM-1 expression in response to TPA was inhibited by pretreatment with GF109203X [a specific inhibitor of protein kinase C (PKC)], or with PD98059 and U0126 (specific inhibitors of MEK), suggesting the importance of PKC, and Erk1/2 signaling cascades in this response. Interestingly, ICAM-1 expression in response to TPA-induced PKC activation was linked to the generation of reactive oxygen species (ROS), as pretreatment with NAC (an ROS scavenger) blocked both ErK1/2 activation and ICAM-1 expression induced by TPA. In addition, TPA-induced ICAM-1 expression was blocked by inhibition of nuclear factor-kappaB (NF-kappaB) activation following pretreatment with BAY11-7085 (a specific inhibitor of NF-kappaB activation). TPA-induced NF-kappaB activation was shown by increased degradation of IkB (NF-kappaB specific inhibitory protein). Together, these observations demonstrated that TPA, a potent activator of PKC, induces ICAM-1 expression via a ROS- and ERK1/2-dependent signaling mechanism in ML-1 cells.     

Interferon‐γ induces leucine‐rich repeat kinase LRRK2 via extracellular signal‐regulated kinase ERK5 in macrophages

The gene encoding leucine‐rich repeat kinase 2 (LRRK2) comprises a major risk factor for Parkinson's disease. Recently, it has emerged that LRRK2 plays important roles in the immune system. LRRK2 is induced by interferon‐γ (IFN‐γ) in monocytes, but the signaling pathway is not known. Here, we show that IFN‐γ‐mediated induction of LRRK2 was suppressed by pharmacological inhibition and RNA interference of the extracellular signal‐regulated kinase 5 (ERK5). This was confirmed by LRRK2 immunostaining, which also revealed that the morphological responses to IFN‐γ were suppressed by ERK5 inhibitor treatment. Both human acute monocytic leukemia THP‐1 cells and human peripheral blood monocytes stimulated the ERK5‐LRRK2 pathway after differentiation into macrophages. Thus, LRRK2 is induced via a novel, ERK5‐dependent IFN‐γ signal transduction pathway, pointing to new functions of ERK5 and LRRK2 in human macrophages.

     

Ferulic acid inhibits endothelial cell proliferation through NO down-regulating ERK1/2 pathway

The aim of this study was to determine the antiproliferative mechanism of ferulic acid (FA) on serum induced ECV304 cell, a human umbilical vein endothelial line. The results suggest that FA significantly suppressed ECV304 cells proliferation and blocked the cell cycle in G0/G1 phase. Treatment of the cells with FA increased nitric oxide (NO) production and inactivated the extracellular signal-regulated kinase (EERK1/2), and the NO donor, sodium nitroprusside, inhibited both ECV304 cells proliferation and phosphorylation of ERK1/2. However, the NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester, caused ECV304 cells proliferation. PD 98059, the inhibitor of ERK1/2, had no effect on the NO production. These results indicate that NO suppressed ECV304 cells proliferation through down-regulating ERK1/2 pathway. Moreover, the inhibition of cell cycle progression was associated with the decrement of cyclin D1 expression and phosphorylation of retinoblastoma protein (pRb) by increment of p21 level. The findings not only present the first evidence that FA is a potent inhibitor on ECV304 cells proliferation, but also reveal the potential signaling molecules involved in its action.     

Secretory phospholipase A2 promotes MMP‐9‐mediated cell death by degrading type I collagen via the ERK pathway at an early stage of chondrogenesis

Background information. sPLA₂ (secretory phospholipase A₂) has been implicated in a wide range of cellular responses, including cell proliferation and ECM (extracellular matrix) remodelling. Even though ECM remodelling is an essential step for chondrogenesis, the expression and functions of sPLA₂ during chondrogenesis have not been studied. Results. In the present study, for the first time, we detect the secretion of sPLA₂ during limb development and suggest that sPLA₂ influences the proliferation and/or survival of limb mesenchymal cells. Treatment of wing bud mesenchymal cells with exogenous sPLA₂ promoted cell death by activating MMP‐9 (matrix metalloproteinase‐9) and increasing type I collagen degradation. The additive chondro‐inhibitory actions were induced by co‐treatment of mp‐BSA (p‐aminophenyl‐mannopyranoside‐BSA), a known ligand of the mannose receptor. Chondro‐inhibitory actions by sPLA₂ were prevented by functional blocking of FcRY (chicken yolk sac IgY receptor), a mannose receptor family member that is the orthologue of the mammalian PLA₂ (phospholipase A₂) receptor and by inhibition of ERK (extracellular‐signal‐regulated kinase) activity. Conclusions. Taken together, our results suggest that elevated levels of sPLA₂ secreted by wing bud mesenchymal cells promote type I collagen degradation by MMP‐9 in a manner typical of receptor‐mediated signalling and that these events lead to cell death.     

Neuroprotection of immortalized hippocampal neurones by brain-derived neurotrophic factor and Raf-1 protein kinase: role of extracellular signal-regulated protein kinase and phosphatidylinositol 3-kinase

We have investigated the molecular mechanisms of neurotrophin-mediated cell survival in HT22 cells, a murine cell line of hippocampal origin, expressing the brain-derived neurotrophic factor (BDNF) receptor TrkB as well as the TrkB.T1 splice variant. Stimulation with BDNF protected HT22-TrkB cells, but not HT22-TrkB.T1 cells, against programmed cell death induced by serum deprivation. BDNF did not, however, provide protection against oxidative glutamate toxicity, indicating that serum deprivation-induced cell death differs substantially from glutamate-induced cell death. Using a pharmacological strategy to block either the extracellular signal-regulated protein kinase (ERK) or the phosphatidylinositol 3-kinase (PI3) pathway, we show that activation of PI3 kinase is required for the neuroprotective activity of BDNF in HT22 cells. To further analyse the role of ERK in neuroprotection we expressed an inducible deltaRaf-1:ER fusion protein in HT22 cells. Activation of this conditionally active form of Raf-1 induced a sustained phosphorylation of ERK, and protected the cells from serum withdrawal-induced cell death. Inhibition of ERK activation at different time points revealed that a prolonged activation of ERK is essential to protect HT22 cells from cell death triggered by the withdrawal of serum, indicating that the duration of ERK activation is of major importance for its neuroprotective biological function.     

Coordinated activation of AMP‐activated protein kinase,extracellular signal‐regulated kinase,and autophagy regulates phorbol myristate acetate‐induced differentiation of SH‐SY5Y neuroblastoma cells

We explored the interplay between the intracellular energy sensor AMP‐activated protein kinase (AMPK), extracellular signal‐regulated kinase (ERK), and autophagy in phorbol myristate acetate (PMA)‐induced neuronal differentiation of SH‐SY5Y human neuroblastoma cells. PMA‐triggered expression of neuronal markers (dopamine transporter, microtubule‐associated protein 2, β‐tubulin) was associated with an autophagic response, measured by the conversion of microtubule‐associated protein light chain 3 (LC3)‐I to autophagosome‐bound LC3‐II, increase in autophagic flux, and expression of autophagy‐related (Atg) proteins Atg7 and beclin‐1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference‐mediated silencing of AMPK suppressed PMA‐induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA‐induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA‐induced differentiation of SH‐SY5Y cells. Therefore, PMA‐induced neuronal differentiation of SH‐SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response.

     

Sanguinarine inhibits growth and invasion of gastric cancer cells via regulation of the DUSP4/ERK pathway

Sanguinarine, a bioactive benzophenanthridine alkaloid extracted from plants of the Papaveraceae family, has shown antitumour effects in multiple cancer cells. But the therapeutic effects and regulatory mechanisms of sanguinatine in gastric cancer (GC) remain elusive. This study was aimed to investigate the correlation of dual‐specificity phosphatase 4 (DUSP4) expression with clinicopathologic features and overall survival in patients with GC and explore the effects of sanguinarine on tumour growth and invasion in GC cells (SGC‐7901 and HGC‐27) and underlying molecular mechanisms. Immunohistochemical analysis showed that decreased DUSP4 expression was associated with the sex, tumour size, depth of invasion and distant metastasis in patients with GC. Functional experiments including CCK‐8, Transwell and flow cytometry analysis indicated that sanguinarine or DUSP4 overexpression inhibited GC cell viability and invasive potential, and induced cell apoptosis and cycle arrest in S phase, but DUSP4 knockdown attenuated the antitumour activity of sanguinarine. Further observation demonstrated that sanguinarine up‐regulated the expression of DUSP4 and Bcl‐2‐associated X protein (Bax), but down‐regulated phosphorylated extracellular signal‐regulated kinase (p‐ERK), proliferating cell nuclear antigen (PCNA), matrix metalloproteinase 2 (MMP‐2) and B‐cell lymphoma 2 (Bcl‐2) expression. Taken together, our findings indicate that sanguinarine inhibits growth and invasion of GC cells through regulation of the DUSP4/ERK pathway, suggesting that sanguinarine may have potential for use in GC treatment.     

Lidocaine inhibits melanoma cell proliferation by regulating ERK phosphorylation

The melanoma is responsible for the majority of all skin cancer–related deaths worldwide. Evidence suggests that local anesthetics provide some benefit in the treatment of cancer via inhibition of cellular proliferation, invasion and migration. However, the potential antiproliferative effects of local anesthetics in the treatment of melanoma remain to be elucidated. In this study, we investigated the antiproliferative effects and underlying mechanism of the commonly used local anesthetic (lidocaine) on melanoma cells. A375 melanoma cells were treated by lidocaine or vemurafenib. Cell Counting Kit-8, histological staining, flow cytometric analysis, immunohistochemical staining, and Western blot analyses were carried out to test the effects of lidocaine and vemurafenib on A375 cells. BALB/C-nu/nu mice intraperitoneally injected with A375 cells were treated by lidocaine, and then tumor volume and weight were calculated. Lidocaine exhibited vemurafenib-like effects totally. Lidocaine inhibited A375 melanoma cell proliferation in a dose- and time-dependent manner and colony formation also showed a dose-dependent inhibition. Lidocaine treatment resulted in the arrest of cell-cycle progression in the G1 phase and inhibited Ki-67 expression in a dose-dependent manner. This effect was associated with inhibited extracellular signal–regulated kinase (ERK) phosphorylation. In vivo experiments revealed that intravenous injections of lidocaine suppressed tumor volume and weight. Lidocaine inhibits melanoma cell proliferation in a dose- and time-dependent manner via a mechanism that may involve inhibition of the ERK signaling pathway. Thus, lidocaine may provide some benefit for the treatment of melanoma.     

Myelin-associated glycoprotein modulates expression and phosphorylation of neuronal cytoskeletal elements and their associated kinases

Decreased phosphorylation of neurofilaments in mice lacking myelin-associated glycoprotein (MAG) was shown to be associated with decreased activities of extracellular-signal regulated kinases (ERK1/2) and cyclin-dependent kinase-5 (cdk5). These in vivo changes could be caused directly by the absence of a MAG-mediated signaling pathway or secondary to a general disruption of the Schwann cell-axon junction that prevents signaling by other molecules. Therefore, in vitro experimental paradigms of MAG interaction with neurons were used to determine if MAG directly influences expression and phosphorylation of cytoskeletal proteins and their associated kinases. COS-7 cells stably transfected with MAG or with empty vector were co-cultured with primary dorsal root ganglion (DRG) neurons. Total amounts of the middle molecular weight neurofilament subunit (NF-M), microtubule-associated protein 1B (MAP1B), MAP2, and tau were up-regulated significantly in DRG neurons in the presence of MAG. There was also increased expression of phosphorylated high molecular weight neurofilament subunit (NF-H), NF-M, and MAP1B. Additionally, in similar in vitro paradigms, total and phosphorylated NF-M were increased significantly in PC12 neurons co-cultured with MAG-expressing COS cells or treated with a soluble MAG Fc-chimera. The increased expression of phosphorylated cytoskeletal proteins in the presence of MAG in vitro was associated with increased activities of ERK 1/2 and cdk5. We propose that interaction of MAG with an axonal receptor(s) induces a signal transduction cascade that regulates expression of cytoskeletal proteins and their phosphorylation by these proline-directed protein kinases.