Extracellular Regulated Kinase Inhibitor Research Articles

Participation of estrogen receptors in the enhancement of osteoblast differentiation by TAK-778

TAK-778 has been shown to stimulate osteogenesis both in vitro and in vivo. However, the mechanism by which TAK-778 exerts its effects is still unclear. There is evidence that TAK-778 acts via estrogen-receptor (ER)-mediated signaling; this study therefore aimed to investigate the roles that ERalpha, ERbeta, and membrane ER play in the osteogenic effect of TAK-778. To this end, human bone marrow mesenchymal cells were cultured with TAK-778 in the presence of either ICI182,780 (ERalpha and ERbeta antagonist) or MPP (ERalpha antagonist) or PD98059 (an extracellular-regulated kinase inhibitor that acts on the membrane ER pathway). The following parameters were evaluated: cell proliferation, collagen content, alkaline phosphatase (ALP) activity and bone-like formation. Data were compared using ANOVA. The effect of TAK-778 on expression of ERalpha and ERbeta was investigated by immunolabeling. In order to investigate whether TAK-778 binds to ER, an ER binding assay was performed. Both immunolabeling and binding assays were conducted using cells from human alveolar bone. The osteogenic effect of TAK-778 was inhibited by ICI182,780 and MPP; however, it was not affected by PD98059. The expression of both ERalpha and ERbeta was not affected by TAK-778. The competition curve obtained from the binding assay using TAK-778 showed maximal displacement when 10(-5) M TAK-778 was used. This study's results show that TAK-778 enhances osteoblast differentiation through an ERalpha-dependent pathway by binding to this receptor and not by increasing the expression of ER.

JNK and ERK MAP kinases mediate induction of IL-1β, TNF-α and IL-8 following hyperosmolar stress in human limbal epithelial cells

Hyperosmolarity has been recognized to be a pro-inflammatory stress to the corneal epithelium. The cell signalling pathways linking hyperosmolar stress and inflammation have not been well elucidated. This study investigated whether exposure of human limbal epithelial cells to hyperosmotic stress activates the mitogen-activated protein kinase (MAPK) pathways and induces production of pro-inflammatory cytokines, interleukin (IL) -1β, tumor necrosis factor (TNF) α, and the C-X-C chemokine IL-8. Primary human limbal epithelial cultures in normal osmolar media (312 mOsM) were exposed to media with higher osmolarity (400–500 mOsM) by adding 50–90 mM NaCl, with or without SB202190, an inhibitor of c-Jun N-terminal kinases (JNK) pathway, PD 98059, an inhibitor of extracellular-regulated kinase (ERK) pathway, dexamethasone or doxycycline for different lengths of time. The conditioned media were collected after 24 hr of treatment for ELISA. Total RNA was extracted from cultures treated for 6 hr for semi-quantitative RT-PCR. Cells treated for 15–60 min were lysed in RIPA buffer and subjected to Western blot with phospho (p)-specific antibodies against p-JNK and p-ERK. The concentrations of IL-1β, TNF-α and IL-8 proteins in 24 hr conditioned media of limbal epithelial cells progressively increased as the media osmolarity increased from 312 to 500 mOsM. Active p-JNK-1/p-JNK-2 and p-ERK-1/p-ERK-2 were detected by Western blot and peaked at 60 min in cells exposed to hyperosmolar media. The levels of p-JNK-1/p-JNK-2 and p-ERK1/p-ERK2 were positively correlated with the medium osmolarity. SB202190, PD98059 and doxycycline markedly suppressed the levels of p-JNK-1/p-JNK-2 and/or p-ERK1/p-ERK2, as well as IL-1β, TNF-α and IL-8 mRNAs and proteins stimulated by hyperosmolar media. These findings provide direct evidence that hyperosmolarity induces inflammation in human limbal epithelial cells by increasing expression and production of pro-inflammatory cytokines and chemokines, a process that appears to be mediated through activation of the JNK and ERK MAPK signalling pathways. The efficacy of doxycycline in treating ocular surface diseases may be due to its ability to suppress JNK and ERK signalling activation and inflammatory mediator production in the limbal epithelium.

Involvement of extracellular regulated kinase and p38 kinase in hippocampal seizure tolerance

The mechanisms underlying brain seizure tolerance, a phenomenon in which brief periods of seizures protect brain against the lethal effects of subsequent sustained seizures, are poorly understood. Because brain seizure tolerance and brain ischemia tolerance likely share certain common mechanisms, the recent evidence that activation of extracellular regulated kinase (ERK) and p38 kinase pathways plays a critical role in ischemic preconditioning suggests that a similar mechanism may underlie brain seizure tolerance. We investigated the hypothesis in a rat kainic acid preparation of seizure preconditioning and tolerance, which was established by induction of one episode of priming epileptic status lasting for 20 min on the first day and another episode of sustained epileptic status lasting for 2 hr on the second day. We observed that acute seizures lead to a rapid activation of ERK and p38 in the hippocampal CA3 area, the brain region most susceptible to the lethal effects of epileptic status. Pretreatment with the ERK inhibitor PD98059 and the p38 inhibitor SB203580 selectively reduces seizure-elicited activation of ERK and p38, respectively, and significantly reduces priming seizure-induced protection of CA3 neurons. These findings indicate that, similar to brain ischemia tolerance, brain seizure tolerance also involves the ERK and p38 signaling pathways.

Inhibitory Action of Daunorubicin on Lipopolysaccharide-Stimulated Inducible-Type Nitric Oxide Synthase Induction in Alveolar Macrophages

The purpose of this study was to investigate the effects of daunorubicin on lipopolysaccharide (LPS)-stimulated inducible-type nitric oxide synthase (iNOS) expression in macrophages. LPS-stimulated iNOS expression and NO production were significantly inhibited in alveolar macrophages from rats administrated daunorubicin (4 mg/kg body weight per day) for 5 consecutive days. Incubation of macrophages with daunorubicin at 1 µM but not at 0.1 and 0.5 µM significantly inhibited LPS-stimulated NO production and iNOS induction. Activation of extracellular regulated kinase (ERK) by LPS was markedly attenuated in both macrophages isolated from in vivo daunorubicin-treated rats and those incubated in vitro with daunorubicin at 1 µM. ERK activation, iNOS induction, and NO production following LPS stimulation were all markedly inhibited in the presence of U0126, an ERK inhibitor. The viability of macrophages was decreased by incubation with daunorubicin at 0.5 and 1 µM, while treatment of rats with daunorubicin did not affect viability of macrophages isolated from the rats. These results suggest that in vivo treatment of rats with daunorubicin attenuates LPS-induced iNOS expression of macrophages through inhibition of ERK activation, while inhibition of iNOS induction by in vitro incubation with daunorubicin may be mainly due to its cytotoxicity.

Protease-activated Receptor 2 is Associated with Activation of Human Macrophage Cell Line THP-1

Protease-activated receptor 2 (PAR2) belongs to a family of G protein coupled receptors activated by proteolytic cleavage. Trypsin-like serine proteases interact with PAR2 expressed by a variety of tissues and immune cells. The aim of our study was to investigate whether PAR2 stimulation can lead to the activation of human mac rophages. Methods: PAR2-mediated proliferation of human macrophage cell line THP-1 was measured with MTT assay. We also examined the extracellular regulated kinase (ERK) phosphorylation and cytokine production induced by trypsin and PAR2-agonist using western blot and enzyme-linked immunosorbent assay (ELISA), respectively. Results: Treatment of trypsin or PAR2-activating peptide increased cell proliferation in a dose-dependent manner, and induced the activation of ERK1/2 in THP-1 cells. In addition, trypsin-induced cell proliferation was inhibited by pretreatment of an ERK inhibitor (pD98059) or trypsin inhibitor (SBTI). Moreover, PAR2 activation by trypsin increased the secretion of TNF-α in THP-1 cells. Conclusion: There results suggest that P AR2 activation by trypsin-like serine proteases can induce cell proliferation through the activation of ERK in human macrophage and that PAR2 may playa crucial role in the cell proliferation and cytokine secretion induced by trypsin-like serine proteases.

Inhibition of acetylcholine-induced activation of extracellular regulated protein kinase prevents the encoding of an inhibitory avoidance response in the rat

It has been demonstrated that the forebrain cholinergic system and the extracellular regulated kinase signal transduction pathway are involved in the mechanisms of learning, encoding, and storage of information. We investigated the involvement of the cholinergic and glutamatergic systems projecting to the medial prefrontal cortex and ventral hippocampus and of the extracellular regulated kinase signal transduction pathway in the acquisition and recall of the step-down inhibitory avoidance response in the rat, a relatively simple behavioral test acquired in a one-trial session. To this aim we studied by microdialysis the release of acetylcholine and glutamate, and by immunohistochemistry the activation of extracellular regulated kinase during acquisition, encoding and recall of the behavior. Cholinergic, but not glutamatergic, neurons projecting to the medial prefrontal cortex and ventral hippocampus were activated during acquisition of the task, as shown by increase in cortical and hippocampal acetylcholine release. Released acetylcholine in turn activated extracellular regulated kinase in neurons located in the target structures, since the muscarinic receptor antagonist scopolamine blocked extracellular regulated kinase activation. Both increased acetylcholine release and extracellular regulated kinase activation were necessary for memory formation, as administration of scopolamine and of extracellular regulated kinase inhibitors was followed by blockade of extracellular regulated kinase activation and amnesia. Our data indicate that a critical function of the learning-associated increase in acetylcholine release is to promote the activation of the extracellular regulated kinase signal transduction pathway and help understanding the role of these systems in the encoding of an inhibitory avoidance memory.

German Cockroach Proteases Regulate Interleukin-8 Expression via Nuclear Factor for Interleukin-6 in Human Bronchial Epithelial Cells

German cockroach extract synergistically regulates tumor necrosis factor-alpha (TNF-alpha)-induced interleukin (IL)-8 expression in human airway epithelial cells. The IL-8 promoter contains nuclear factor (NF)-kappaB, activating protein (AP)-1, and NF for IL-6 (NF-IL6) transcription factor binding regions. Because cockroach extract activates extracellular regulated kinase (ERK), a known activator of AP-1 and NF-IL6, we focused on the regulation of these transcription factors. Although TNF-alpha and cockroach extract both increased AP-1 translocation, mutation of the AP-1 site in the context of the wild-type promoter had no effect on cockroach extract-induced synergy. Mutation of the NF-IL6 site in the context of the wild-type IL-8 promoter, or overexpression of a dominant-negative NF-IL6 mutant, each abolished cockroach extract-induced synergy. Cockroach extract induced NF-IL6 translocation and DNA binding, an effect that was further increased in the presence of TNF-alpha. Cockroach extract-induced regulation of NF-IL6 was due to active serine proteases in the extract as well as activation of protease activated receptor (PAR)-2, but not PAR-1. Chemical inhibition of ERK also attenuated cockroach extract-induced NF-IL6-DNA binding. We conclude that proteases in German cockroach extract regulate PAR-2 and ERK to increase NF-IL6 activity and synergistically regulate TNF-alpha-induced IL-8 promoter activity in human airway epithelium.

Erythropoietin Signaling in Tumor Cells: Differential Activation of Mitogen Activated Protein Kineses and Their Effect upon Cell Survival.

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid progenitors via its receptor, EPO-R and through various Mitogen activated protein kinase (MAPK) pathways. The role of EPO on other cell types is still unkown. In the present study we have elected to examine this aspect in a transformed pancreatic cell line, AR42J cells. We investigated the activation of two MAPKs, namely extracellular regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK) after exposure of AR42J cells to 1 U/ml EPO using the Western blot analysis. We also examined cell viability during EPO exposure by high sensitivity fluoremetric method using CCK8. We found a rapid activation of ERK-1/2 in AR42J cells reaching the maximum of 3.3 fold in 5 min, while it took 30 min for JNK-1/2 to reach the maximum. In the absence of EPO, addition of specific JNK inhibitor, SP600125, reduced cell viability to 50% at a dose of 20 μM while with ERK inhibitor, UO126, cell viability was not reduced even up to 60 μM of the drug. To examine the effect of induction of MAPK by EPO on AR42J cell survival, cells were treated with inhibitors to ERK or JNK 1h prior to EPO addition and the cumulative cell survival were calculated from day 1 through 4. EPO addition to AR42J cells resulted in significantly higher cumulative cell survival of 1.00 ± 0.04 unit absorption compared to the value of 0.35 ± 0.02 unit absorption seen in controls without EPO. When cells were treated with EPO and ERK inhibitor a significantly higher cumulative cell survival of 1.50 ± 0.04 unit absorption was observed (p < 0.01) indicating ERK to be less effective in their survival. On the other hand, samples treated with EPO and JNK inhibitor had significantly lower cell survival (0.55 ± 0.06 unit absorption, p < 0.01) than the EPO control indicating an essential role of JNK in their survival. These results indicate that in EPO mediated survival of AR42J cells, activation of JNK appears to be more important than ERK activation thus indicating a key role of JNK in cell viability. It is very important to understand the role of EPO signaling in the viability of transformed cells since EPO is often supplemented after bone marrow transplantation to enhance the recovery after chemotherapy.

Cerebral activation of mitogen-activated protein kinases after circulatory arrest and low flow cardiopulmonary bypass*1

Mitogen-activated protein kinases (MAPK) are important intermediates in the signal transduction pathways involved in neuronal dysfunction following cerebral ischemia-reperfusion injury. One subfamily, extracellular regulated kinase 1/2, has been heavily implicated in the pathogenesis of post-ischemic neuronal damage. However, the contribution of extracellular regulated kinase 1/2 to neuronal damage following deep hypothermic circulatory arrest and low flow cardiopulmonary bypass is unknown. We attempted to correlate the extent of neuronal damage present following deep hypothermic circulatory arrest and low flow cardiopulmonary bypass with phosphorylated extracellular regulated kinase 1/2 expression in the cerebral vascular endothelium. Piglets underwent normal flow cardiopulmonary bypass (n=4) deep hypothermic circulatory arrest (n=6) and low flow cardiopulmonary bypass (n=5). Brains were harvested following 24 h of post-cardiopulmonary bypass recovery. Cerebral cortical watershed zones, hippocampus, basal ganglia, thalamus, cerebellum, mesencephalon, pons and medulla were evaluated using hematoxylin and eosin staining. A section of ischemic cortex was evaluated by immunohistochemistry with rabbit polyclonal antibodies against phosphorylated extracellular regulated kinase 1/2. Compared to cardiopulmonary bypass controls, the deep hypothermic circulatory arrest and low flow cardiopulmonary bypass piglets exhibited diffuse ischemic changes with overlapping severity and distribution. Significant neuronal damage occurred in the frontal watershed zones and basal ganglia of the deep hypothermic circulatory arrest group (P<0.05). No detectable phosphorylated extracellular regulated kinase 1/2 immunoreactivity was found in the cardiopulmonary bypass controls; however, ERK 1/2 immunoreactivity was present in the cerebral vascular endothelium of the deep hypothermic circulatory arrest and low flow cardiopulmonary bypass groups. Our results indicate that phosphorylated extracellular regulated kinase 1/2 may play a prominent role in early cerebral ischemia-reperfusion injury and endothelial dysfunction. The pharmacologic inhibition of extracellular regulated kinase 1/2 represents a new and exciting opportunity for the modulation of cerebral tolerance to low flow cardiopulmonary bypass and deep hypothermic circulatory arrest.

ERK couples chronic survival of NK cells to constitutively activated Ras in lymphoproliferative disease of granular lymphocytes (LDGL)

Chronic NK lymphoproliferative disease of large granular lymphocytes (LDGL) is characterized by the expansion of activated CD3-, CD16+ or CD56+ lymphocytes. The mechanism of survival of NK cells from LDGL patients is unknown but may be related to antigenic stimulation. There is currently no standard effective therapy for LDGL, and the disease is characteristically resistant to standard forms of chemotherapy. We found evidence of constitutive activation of extracellular-regulated kinase (ERK) in NK cells from 13/13 patients with NK-LDGL (one patient with aggressive and 12 patients with chronic disease). Ablation of ERK activity by inhibitors or a dominant-negative form of MEK, the upstream activator of ERK, reduced the survival of patient NK cells. Ras was also constitutively active in patient NK cells, and exposure of cells to the Ras inhibitor FTI2153 or to dominant-negative-Ras resulted not only in ERK inhibition but also in enhanced apoptosis in both the presence and absence of anti-Fas. Therefore, we conclude that a constitutively active Ras/MEK/ERK pathway contributes to the accumulation of NK cells in patients with NK-LDGL. These findings suggest that strategies to inhibit this signaling pathway may be useful for the treatment of the NK type of LDGL.

Pancreatic cancer stimulates pancreatic stellate cell proliferation and TIMP-1 production through the MAP kinase pathway

Pancreatic adenocarcinoma is characterized by an intense desmoplastic reaction that surrounds the tumor. Pancreatic stellate cells (PSCs) are thought to be responsible for production of this extracellular matrix. When activated, PSCs have a myofibroblast phenotype and produce not only components of the extracellular matrix including collagen, fibronectin, and laminin, but also matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). Since PSCs are found in the stroma surrounding human pancreatic adenocarcinoma, we postulate that pancreatic cancer could impact PSC proliferation and TIMP-1 production. Rat PSCs were isolated and cultured. Isolated PSCs were exposed to PANC-1 conditioned medium (CM) and proliferation, activation of the mitogen-activated protein (MAP) kinase pathway, and TIMP-1 gene induction were determined. Exposure to PANC-1 CM increased PSC DNA synthesis, cell number, and TIMP-1 mRNA (real-time PCR) as well as activating the extracellular-regulated kinase (ERK) 1/2. Inhibition of ERK 1/2 phosphorylation (U0126) prevented the increases in growth and TIMP-1 expression. PANC-1 CM stimulates PSC proliferation and TIMP-1 through the MAP kinase (ERK 1/2) pathway.

Role of the Pyk2–MAP Kinase–cPLA2Signaling Pathway in Shear-Dependent Platelet Aggregation

Mechanisms of shear-induced platelet aggregation are not established. Data that ristocetin-induced von Willebrand factor (VWF) binding to glycoprotein (Gp) Ibalpha activates proline-rich tyrosine kinase 2 (Pyk2) and extracellular-regulated kinase (ERK) has led to speculation that these events are coupled and that a MAP kinase may activate cytosolic phospholipase A2 (cPLA2)-mediated arachidonic acid (AA) release. To test this hypothesis and clarify the role of AA metabolism in shear-induced VWF-dependent platelet aggregation, we examined Pyk2, ERK1/2, and p38 phosphorylation, and arachidonic acid release and metabolism in platelets subjected to pathological shear stress in vitro. We observe tyrosine phosphorylation of Pyk2, p38, and ERK1/2 but no measurable increase in free AA, 12-hydroxyeicosatetraenoic acid, or thromboxane A2. Inhibitors of ERK, p38, or cyclooxygenase activation fail to affect shear-induced platelet aggregation. When washed platelets are aspirin-pretreated, arachidonic acid release becomes measurable and aggregation at 60 and 120 s is attenuated. These data indicate that shear-induced VWF binding to platelet GpIb-IX-V activates Pyk2, ERK1/2, p38, and cPLA2, but that the magnitude of these responses is below the threshold needed to enhance shear-induced VWF-dependent platelet aggregation in the presence of plasma. These results provide a mechanistic basis for the long-standing observation that shear-dependent platelet aggregation is unaffected by the antiplatelet drug aspirin.

P38 MAP kinase mediates endotoxin-induced expression of cyclooxygenase-2 in enterocytes

BackgroundNecrotizing enterocolitis (NEC) occurs only after bacterial colonization of the intestine, suggesting that bacterial products, including lipopolysaccharide (endotoxin,) interact with enterocytes in the pathogenesis of this disease. Inflammatory molecules such as cyclooxygenase-2 (COX-2) are important mediators of the septic response leading to NEC. We therefore hypothesized that endotoxin activates production of COX-2 in enterocytes and explored the relative contributions of known mitogen-activated protein kinases (MAPK) pathways in this process. MethodsIEC-6 enterocytes were treated with 5 μg/mL endotoxin, or various stresses, or media alone, and COX-2 protein levels were assayed by immunoblots with anti-COX-2 antibodies. Activation of MAPK was examined by immunoblots with phospho-MAPK antibodies. MAPK activity was blocked by treatment with pharmacologic inhibitors or transfection with dominant-negative MAPK constructs. ResultsEndotoxin treatment caused increased expression of the COX-2 protein 24 hours after treatment. This was preceded by rapid and transient activation of the 3 major MAPKs: extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. SB203580, a specific inhibitor of p38, but not U0126 (ERK inhibitor) or SP600125 (JNK inhibitor), blocked endotoxin-induced accumulation of COX-2 protein. This response was also blocked by expression of dominant-negative p38 but not by the dominant-negative ERK construct. Genotoxic stress that activated p38 but not ERK was an effective inducer of COX-2, whereas stresses that activated both p38 and ERK were not effective. ERK inhibition by U1026 enhanced endotoxin-induced production of COX-2, consistent with negative regulation of COX-2 by ERK. These data point to p38 as the MAPK that mediates endotoxin-induced production of COX-2 in enterocytes. ConclusionsEndotoxin may be capable of inducing the production of COX-2 in enterocytes via the p38 MAPK pathway, which may be relevant to the development of NEC.

PI3K mediates protection against TRAIL-induced apoptosis in primary human melanocytes.

Melanocytes are cells of the epidermis that synthesize melanin, which is responsible for skin pigmentation. Transformation of melanocytes leads to melanoma, a highly aggressive neoplasm, which displays resistance to apoptosis. In this report, we demonstrate that TNF-related apoptosis-inducing ligand (TRAIL), which was thought to kill only transformed cells, promotes very efficiently apoptosis of primary human melanocytes, leading to activation of caspases 8, 9 and 3, and the cleavage of vital proteins. Further, we show that stem cell factor (SCF), a physiologic melanocyte growth factor that activates both the phosphatidyl-inositol-3 kinase (PI3K) and the extracellular regulated kinase (ERK) pathways, strongly protects melanocytes from TRAIL and staurosporine killing. Interestingly, inhibition of PI3K or its downstream target AKT completely blocks the antiapoptotic effect of SCF, while inhibition of ERK has only a moderate effect. Our data indicate that protection evoked by SCF/PI3K/AKT cascade is not mediated by an increase in the intracellular level of FLIP. Further, only a sustained PI3K activity can protect melanocytes from apoptosis, thereby indicating that the PI3K/AKT pathway plays a pivotal role in melanocyte survival. The results gathered in this report bring new information on the molecular mechanisms involved in primary melanocyte apoptosis and survival that would help to better understand the process by which melanomas acquire their resistance to apoptosis.

Nitric oxide increases IL-8 gene transcription and mRNA stability to enhance IL-8 gene expression in lung epithelial cells.

Interleukin (IL)-8, a C-X-C chemokine, is a potent chemoattractant and an activator for neutrophils, T cells, and other immune cells. The airway and respiratory epithelia play important roles in the initiation and modulation of inflammatory responses via production of cytokines and surfactant. The association between elevated levels of nitric oxide (NO) and IL-8 in acute lung injury associated with sepsis, acute respiratory distress syndrome, respiratory syncytial virus infection in infants, and other inflammatory diseases suggested that NO may play important roles in the control of IL-8 gene expression in the lung. We investigated the role of NO in the control of IL-8 gene expression in H441 lung epithelial cells. We found that a variety of NO donors significantly induced IL-8 mRNA levels, and the increase in IL-8 mRNA was associated with an increase in IL-8 protein. NO induction of IL-8 mRNA was due to increases in IL-8 gene transcription and mRNA stability. NO induction of IL-8 mRNA levels was not inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and KT-5823, inhibitors of soluble guanylate cyclase and protein kinase G, respectively, and 8-bromo-cGMP did not increase IL-8 mRNA levels. This indicated that NO induces IL-8 mRNA levels independently of changes in the intracellular cGMP levels. NO induction of IL-8 mRNA was significantly reduced by inhibitors of extracellular regulated kinase and protein kinase C. IL-8 induction by NO was also reduced by hydroxyl radical scavengers such as dimethyl sulfoxide and dimethylthiourea, indicating the involvement of hydroxyl radicals in the induction process. NO induction of IL-8 gene expression could be a significant contributing factor in the initiation and induction of inflammatory response in the respiratory epithelium.

Tissue inhibitor of metalloproteinase-1 stimulates proliferation of human cancer cells by inhibiting a metalloproteinase.

TIMP-1, an ∼30 kDa glycosylated protein found predominantly in extracellular compartments, is involved in the regulation of a variety of developmental, remodelling, and pathological processes. One function of TIMP-1 is to inhibit certain members of a group of extracellular and cell surface enzymes known collectively as metalloproteinases (MP). These include the matrix metalloproteinases and the adamalysin-like disintegrin and metalloproteinases (ADAMs). Additional activities of TIMP-1 include potentiating the activity of erythroid precursors and stimulating proliferation of certain cancer cell lines. Published evidence suggests that the apparent proliferative action of TIMP-1 is independent of its MP-inhibitory activity; however, reports of a cell surface receptor for TIMP-1 have not been confirmed. We have utilised a baculovirus-based system to produce TIMP-1. Data presented here show that TIMP-1 and synthetic hydroxamate (GM6001) MP inhibitors stimulate proliferation and metabolic activity of MDA-MB-435 cancer cells with similar kinetics. An inactive hydroxamate derivative was ineffective. The TIMP-1-induced increase in proliferation and metabolic activity was not the consequence of the inhibition of apoptosis by TIMP-1 in the serum-free medium. These data taken together imply that the mechanism by which TIMP-1 enhances cell growth depends on its ability to inhibit a metalloproteinase, rather than to stimulate a cell surface receptor by a process independent of its MP-inhibitory activity. Inhibitors of extracellular regulated kinase (U0126) and p38 (SB203580), and to a lesser extent the phosphatidylinositol-3-kinase inhibitor LY294002, suppressed the action of TIMP-1. Assays for ERK1/2 and p38 showed that both were activated by TIMP-1 and GM6001. Mechanisms by which TIMP-1 might act to stimulate cell proliferation are described.

Sphingosine 1-phosphate antagonizes human neutrophil apoptosis via p38 mitogen-activated protein kinase.

Sphingosine 1-phosphate (SPP) is associated with the regulation of apoptosis, although its role in neutrophil apoptosis remains poorly investigated. Here, we show that exogenous SPP antagonizes spontaneous and anti-Fas-induced apoptosis in neutrophils. Pre-treatment with pertussis toxin clearly reduced the apoptosis-inhibiting capacity of SPP. Consequently, we investigated the involvement of potential modulators of apoptosis that are activated downstream of Gi/G0-coupled receptors. Neither Akt activity nor change in basal activity of c-Jun N-terminal kinases was detected during apoptosis or after adding SPP. In contrast, there was a transient decrease in phosphorylation of both extracellular-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) during both spontaneous and anti-Fas-induced apoptosis. Although exogenous SPP reversed these reductions in kinase activity, experiments with inhibitors of ERK (PD98059) and p38 MAPK (SB203580) revealed that only SB203580 counteracted the effect of SPP. Thus, SPP counteracts neutrophil apoptosis via a Gi/G0 protein survival-signalling pathway that includes modulation of p38 MAPK activity.

Na(+)/K(+)/Cl(-) cotransporter activates mitogen-activated protein kinase in fibroblasts and lymphocytes.

In a previous work, we have shown that overexpression of the Na(+)/K(+)/Cl(-) cotransporter (NKCC1) induces cell proliferation and transformation. We investigate in the present study the role of the NKCC1 in the mitogenic signal transduction. We show that overexpression of the cotransporter gene (NKCC1) in stablely transfected cells (Balb/c-NKCC1), resulted in enhanced phosphorylation of the extracellular regulated kinase (ERK) to produce double phosphorylated ERK (DP-ERK). Furthermore, the level of DP-ERK was reduced by 50-80% following the addition of bumetanide, a specific inhibitor of the Na(+)/K(+)/Cl(-) cotransporter, in quiescent as well as in proliferating cultures of the Balb/c-NKCC1 clone. In order to explore further the role of the Na(+)/K(+)/Cl(-) cotransporter in mitogenic signal transduction, we measured the effect of the two specific inhibitors of the cotransporter; bumetanide and furosemide, on DP-ERK level in immortalized non-transformed cells. In Balb/c 3T3 fibroblasts stimulated with FGF, bumetanide, and furosemide inhibited 50-60% of the ERK 1/2 phosphorylation. The inhibitor concentration needed for maximal inhibition of ERK 1/2 phosphorylation was similar to the concentration needed to block the K(+) influx mediated by the Na(+)/K(+)/Cl(-) cotransporter in these cells. To analyze whether the Na(+)/K(+)/Cl(-) cotransporter has a role in the mitogenic signal of normal cells, we measured the effect of bumetanide on ERK phosphorylation in human peripheral blood lymphocytes. The phosphorylation of ERK 1/2 in resting human lymphocytes, as well as in lymphocytes stimulated with phytohemagglutinin (PHA) was inhibited by bumetanide. The effect of bumetanide on ERK 2 phosphorylation was much lower than that of ERK 1 phosphorylation. The finding that the Na(+)/K(+)/Cl(-) cotransporter controls the ERK/MAPK (mitogen-activated protein kinase) signal transduction pathway, support our hypothesis that Na(+) and K(+) influxes mediated by this transporter plays a central role in the control of normal cell proliferation. Exploring the cellular ionic currents and levels, mediated by the Na(+)/K(+)/Cl(-) cotransporter, should lead to a better comprehension of cell proliferation and transformation machinery.

Delineating Integrin Activation and Migration

Monocyte chemoattractant protein-1 (MCP-1) is important for both integrin activation and cell migration during the chemotactic response of monocytes. Although these two processes are closely connected, the signaling pathways associated with each have not been well defined. Ashida et al. show that different mitogen-activated protein kinase (MAPK) pathways are required in each process. Inhibition of p38 MAPK inhibited chemotaxis, whereas ERK (extracellular-regulated kinase) inhibition greatly reduced integrin activation, as measured by adhesion to fibronectin. Additionally, inhibiting the function of Rho or Rho kinase also blocked migration but not integrin activation. Although the biochemical signaling point at which both MCP-1-dependent pathways diverge has not been identified, the elucidation of specific proteins involved in migration and integrin activation should help clarify these signaling pathways. N. Ashida, H. Arai, M. Yamasaki, T. Kita, Distinct signaling pathways for MCP-1-dependent integrin activation and chemotaxis. J. Biol. Chem. 276 , 16555-16560 (2001). [Abstract] [Full Text]

N-Methyl- d-aspartate receptors and p38 mitogen-activated protein kinase are required for cAMP-dependent cyclase response element binding protein and Elk-1 phosphorylation in the striatum

In vivo cyclic adenosine monophosphate (cAMP)-induced N-methyl- d-aspartate receptor and mitogen-activated protein kinase activation was investigated in the dorsal striatum by semiquantitative immunocytochemistry. Intracerebroventricular infusion of 8-bromo-adenosine 3′,5′-cyclic monophosphorothioate, Sp isomer (Sp-8-Br-cAMPS), increased phosphorylated cAMP-responsive element binding protein, phosphorylated Elk-1 and Fos immunoreactivity in a dose-dependent manner. Intracerebroventricular infusion of the N-methyl- d-aspartate antagonist, MK801, decreased, but tetrodotoxin or the mitogen-activated extracellular-regulated kinase inhibitor, PD98059, did not affect Sp-8-Br-cAMPS-induced phosphorylated c-AMP-responsive element binding protein, phosphorylated Elk-1, phosphorylated extracellular-signal-regulated kinase and Fos immunoreactivity. The p38 mitogen-activated protein kinase inhibitor, SB203580, decreased the Sp-8-Br-cAMPS-induced increase in all markers, except phosphorylated extracellular-signal-regulated kinase, in a dose-dependent manner. We suggest that N-methyl- d-aspartate receptors couple c-AMP to phosphorylation events and immediate early gene induction in the nucleus of striatal medium spiny neurons. These events are mediated by crosstalk between protein kinase A and mitogen-activated protein kinase cascades in vivo.