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MEK1 and MEK2, the only known activators of ERK, are attractive therapeutic candidates for both cancer and autoimmune diseases. However, how MEK signaling finely regulates immune cell activation is only partially understood. To address this question, we specifically delete Mek1 in hematopoietic cells in the Mek2 null background. Characterization of an allelic series of Mek mutants reveals the presence of distinct degrees of spontaneous B cell activation, which are inversely proportional to the levels of MEK proteins and ERK activation. While Mek1 and Mek2 null mutants have a normal lifespan, 1Mek1 and 1Mek2 mutants retaining only one functional Mek1 or Mek2 allele in hematopoietic cell lineages die from glomerulonephritis and lymphoproliferative disorders, respectively. This establishes that the fine-tuning of the ERK/MAPK pathway is critical to regulate B and T cell activation and function and that each MEK isoform plays distinct roles during lymphocyte activation and disease development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Interference from endogenous signaling enzymes represents a major hurdle for building orthogonal signaling cascades inside cells, particularly among closely related isoforms within an enzyme family. Here, we employed a genetically encoded chemical decaging strategy to build orthogonally activated kinase isoforms, with the endogenous counterparts temporally disabled by an extracellularly delivered bacterial effector. This approach eliminated any potential interference from other kinase isoforms as well as endogenous kinases, which allowed the specific, gain-of-function report of mitogen-activated protein kinase kinase 1 (MEK1) activity as opposed to MEK2 with high temporal resolution. Our study dissected the distinct enzymatic activity, feedback regulation and signal outputs between these closely related kinase isoforms., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: To observe effect of acupuncture combined with hypothermia therapy on MAPK/ERK pathway and apoptosis related factorsin rats suffered cerebral ischemia reperfusion and to explore underlying mechanisms.
 Methods: Middle cerebral artery ischemia model were established.Ninety SD rats were randomly assigned into a blank group, a control group, a model group, an acupuncture group, a mild hypothermia group, and an acupuncture with hypothermia group. After 72 h treatment, nerve function defect scores were observed, and infarction area percent was detected by 2, 3, 5-triphenyl-2H-tetrazolium chloride (TTC) staining; expressions of Bcl-2 and Bax were examined by immunohistochemistry; apoptotic cells were detected by TUNEL assay; and expression levels of phospho-mitogen-activated protein kinase(p-MEK2) and phospho-extracellular signal regulated kinase 1/2 (p-ERK1/2) in the rats' hippocampus ischemic side were determined by Western blot.
 Results: In the rats of the model group, the neural function defect scores, the infarction area percent, the expression level of Bax, and apoptotic cells increased, while the level of Bcl-2 decreased significantly. The level of p-MEK2 and p-ERK1/2 increased obviously compared with the blank and control groups (P<0.05 or P<0.01). After treatment with acupuncture and hypothermia, the neural function defect scores, infarction area percent, and the level of Bax, apoptotic cells and the levels of p-MEK2 and p-ERK1/2 were significantly decreased, while the level of Bcl-2 in the treatment group was significantly elevated (P<0.05 or P<0.01) compared with the model group. Compared with the acupuncture group or the hypothermia group, the neural function defect scores and the levels of p-MEK2 and p-ERK1/2 in the acupuncture combined with hypothermia group were significantly reduced (P<0.05 or P<0.01).
 Conclusion: Acupuncture and hypothermia therapy can improve cerebral function, and reduce the cerebral injury through down-regulation of Bax level, and up-regulation of Bcl-2 level, which is related to reducing the levels of p-MEK2 and p-ERK1/2. The therapeutic effects on cerebral ischemia reperfusion injury for combination of acupuncture with hypothermia are better than those with single application of acupuncture or hypothermia.

It has been reported that alkaloids derived from Coptis chinensis exert anti-adipogenic activity on 3T3-L1 adipocytes by downregulating peroxisome proliferation-activity receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein-α (C/EBP-α). However, the signaling-based mechanism of the inhibitory role of epiberberine in the early stages of 3T3-L1 adipocyte differentiation is uncharacterized. Here, we show that epiberberine had inhibitory effects on adipocyte differentiation and significantly decreased lipid accumulation by downregulating an adipocyte-specific transcription factor, sterol regulatory element-binding protein-1 (SREBP-1). Furthermore, we observed that epiberberine markedly suppressed the differentiation-mediated phosphorylation of components of both the Raf/mitogen-activated protein kinase 1 (MEK1)/extracellular signal-regulated protein kinase 1/2 (ERK1/2) and AMP-activated protein kinase-α1 (AMPKα)/Akt pathways. In addition, gene expression of fatty acid synthase (FAS) was significantly inhibited by treatment with epiberberine during adipogenesis. These results indicate that the anti-adipogenic mechanism of epiberberine is associated with inhibition of phosphorylation of Raf/MEK1/ERK1/2 and AMPKα/Akt, followed by downregulation of the major transcription factors of adipogenesis, such as PPAR-γ, C/EBP-α, and SREBP-1, and FAS. Taken together, this study suggests that the anti-adipogenic effect of epiberberine is mediated by downregulation of the Raf/MEK1/ERK1/2 and AMPKα/Akt pathways during 3T3-L1 adipocyte differentiation. Moreover, the anti-adipogenic effects of epiberberine were not accompanied by modulation of β-catenin.

Background: Polo-like kinase 1 (Plk1) is a serine/threonine protein kinase that has been implicated in the regulation of mitosis. In addition, the activation of mitogen-activated protein kinase (MAPK) is a key event in the early stage of the growth factor response. The role of Plk1 in MAPK phosphorylation in cells has not been investigated., Methods: Immunoblot analysis was used to evaluate Plk1 and MAPK phosphorylation in cells upon stimulation with platelet-derived growth factor (PDGF). We also generated stable Plk1 knockdown (KD) cells to assess the role of Plk1 in MAPK activation and cell proliferation. Furthermore, we used a non-phosphorylatable Plk1 mutant to determine the function of Plk1 phosphorylation in these processes., Results: Treatment with PDGF increased Plk1 phosphorylation at Thr-210 (an indication of Plk1 activation) in human airway smooth muscle cells. Plk1 KD attenuated the PDGF-induced phosphorylation of MEK1/2 and ERK1/2 as well as cell proliferation. However, phosphorylation of Raf-1 and AKT upon stimulation with PDGF was not reduced in Plk1 KD cells. Furthermore, the expression of T210A Plk1 (alanine substitution at Thr-210) inhibited the PDGF-stimulated MEK1/2 phosphorylation, ERK1/2 phosphorylation and cell proliferation., Conclusions: Together, these findings suggest that Plk1 is activated upon growth factor stimulation, which may control the activation of MEK1/2 and ERK1/2, and smooth muscle cell proliferation.

The mammalian genome contains two ERK/MAP kinase genes, Mek1 and Mek2, which encode dual-specificity kinases responsible for ERK/MAP kinase activation. In order to define the function of the ERK/MAPK pathway in the lung development in mice, we performed tissue-specific deletions of Mek1 function on a Mek2 null background. Inactivation of both Mek genes in mesenchyme resulted in several phenotypes, including giant omphalocele, kyphosis, pulmonary hypoplasia, defective tracheal cartilage and death at birth. The absence of tracheal cartilage rings establishes the crucial role of intracellular signaling molecules in tracheal chondrogenesis and provides a putative mouse model for tracheomalacia. In vitro, the loss of Mek function in lung mesenchyme did not interfere with lung growth and branching, suggesting that both the reduced intrathoracic space due to the dysmorphic rib cage and the omphalocele impaired lung development in vivo. Conversely, Mek mutation in the respiratory epithelium caused lung agenesis, a phenotype resulting from the direct impact of the ERK/MAPK pathway on cell proliferation and survival. No tracheal epithelial cell differentiation occurred and no SOX2-positive progenitor cells were detected in mutants, implying a role for the ERK/MAPK pathway in trachea progenitor cell maintenance and differentiation. Moreover, these anomalies were phenocopied when the Erk1 and Erk2 genes were mutated in airway epithelium. Thus, the ERK/MAPK pathway is required for the integration of mesenchymal and epithelial signals essential for the development of the entire respiratory tract., (© 2014. Published by The Company of Biologists Ltd.)

Mitogen-activated protein kinases (MAPKs) are a family of ubiquitous eukaryotic signal transduction enzymes which link extracellular stimuli to intracellular gene expression pathways. While several three-tiered MAPK cascades have been elucidated in mammals, the prototypical pathway involves a network of proteins and kinases including the Rat sarcoma protein (Ras), mitogen-activated protein kinase kinase kinase (Raf or MAP3K), mitogen-activated protein kinase kinase (MEK or MAP2K), and extracellular signal regulated protein kinase (ERK or MAPK). This MAPK cascade (the Ras/Raf/MEK/ERK pathway) is a receptor tyrosine kinase mediated signaling pathway that regulates cell proliferation, cell cycle progression, and cell migration. There are multiple molecular mechanisms of interaction and activation between the upstream nodes of the Ras/Raf/MEK/ERK cascade and other cell signaling pathways, all ultimately leading to the activation of the nuclear transcription factor ERK. Important downstream targets include MEK1/2, which comprise the final step leading to ERK transcription factor activation. While multiple conduits exist to activate ERK upstream of MEK, there is little redundancy downstream. Located at this pivotal intersection between a limited number of upstream activators and its exclusive downstream targets, MEK is an appealing molecular target of novel cancer therapies. MEK inhibitors are small molecules that inhibit MEK phosphorylation by binding to a pocket adjacent to the ATP binding site, decreasing both the amount of MEK activity, and the quantity of activated ERK in the cell. Unique allosteric noncompetitive binding sites of MEK inhibitors allow specific targeting of MEK enzymes and prevent cross-activation of other serine/threonine protein kinases through the conserved ATP binding site. This paper reviews the translational evidence in favor of MEK inhibitors in cancer, their role in gynecologic malignancies, and details regarding the status of the fourteen MEK inhibitors currently being clinically tested: trametinib, selumetinib, pimasertib, refametinib, PD-0325901, MEK162, TAK733, RO5126766, WX-554, RO4987655, cobimetinib, AZD8330, MSC2015103B, and ARRY-300., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background & Aims: Kras signaling via mitogen-activated protein kinase (MAPK) is highly up-regulated in pancreatic cancer cells. We investigated whether MAPK signaling is required for the initiation and maintenance of pancreatic carcinogenesis in mice., Methods: We studied the formation and maintenance of pancreatic intraepithelial neoplasia (PanINs) in p48Cre; TetO-KrasG12D; Rosa26(rtTa-IRES-EGFP) (iKras*) mice and LSL-KrasG12D mice bred with p48Cre mice (KC). Mice were given oral PD325901, a small-molecule inhibitor of MEK1 and MEK2 (factors in the MAPK signaling pathway), along with injections of cerulein to induce pancreatitis. Other mice were given PD325901 only after PanINs developed. Pancreatic tissues were collected and evaluated using histologic, immunohistochemical, immunofluorescence, and electron microscopy analyses. Acinar cells were isolated from the tissues and the effects of MEK1 and 2 inhibitors were assessed., Results: PD325901 prevented PanIN formation, but not pancreatitis, in iKras* and KC mice. In iKras* or KC mice given PD325901 at 5 weeks after PanINs developed, PanINs regressed and acinar tissue regenerated. The regression occurred through differentiation of the PanIN cells to acini, accompanied by re-expression of the acinar transcription factor Mist1., Conclusions: In iKras* and KC mice, MAPK signaling is required for the initiation and maintenance of pancreatic cancer precursor lesions. MAPK signaling promotes formation of PanINs by enabling dedifferentiation of acinar cells into duct-like cells that are susceptible to transformation., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

We investigated whether combinatorial post-injury treatment with progesterone (P4) and vitamin D hormone (VDH) would reduce ischemic injury more effectively than P4 alone in an oxygen glucose deprivation (OGD) model in primary cortical neurons and in a transient middle cerebral artery occlusion (tMCAO) model in rats. In the OGD model, P4 and VDH each showed neuroprotection individually, but combination of the "best" doses did not show substantial efficacy; instead, the lower dose of VDH in combination with P4 was the most effective. In the tMCAO model, P4 and VDH were given alone or in combination at different times post-occlusion for 7 days. In vivo data confirmed the in vitro findings and showed better infarct reduction at day 7 and functional outcomes (at 3, 5 and 7 days post-occlusion) after combinatorial treatment than when either agent was given alone. VDH, but not P4, upregulated heme oxygenase-1, suggesting a pathway for the neuroprotective effects of VDH differing from that of P4. The combination of P4 and VDH activated brain-derived neurotrophic factor and its specific receptor, tyrosine kinase receptor-B. Under specific conditions VDH potentiates P4's neuroprotective efficacy and should be considered as a potential partner of P4 in a low-cost, safe and effective combinatorial treatment for stroke., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Interaction between the immune system and cancer cells allows for the use of biological response modifiers, like OK-432, in cancer therapy. We have studied the involvement of monocytes (MOs) in the immune response to OK-432 by examining MCP-1, MIP-1α and MIP-1β secretion, in vitro. OK-432-induced IL-6/TNF-α secretion has previously been shown to depend on mitogen-activated protein kinases (MAPKs) ERK1/2 and p38, and we therefore investigated the role of these MAPKs in OK-432-induced chemokine secretion. Here we demonstrate that pharmacological MEK1/2 kinase inhibition generally impaired chemokine secretion from MOs, whereas p38 MAPK inhibition in particular reduced MIP-1α production. Furthermore, simultaneous inhibition of MEK1/2 and Syk kinase was seen to have an additive impact on reduced MCP-1, MIP-1α and MIP-1β secretion. Based on single cell flow cytometry analyses, OK-432, lipoteichoic acid (LTA) and lipopolysaccharide (LPS) were seen to induce p38 MAPK and NF-κB phosphorylation in MOs with different time kinetics. LTA and LPS have been shown to induce ERK1/2 phosphorylation, whereas the levels of phosphorylated ERK1/2 remained constant following OK-432 treatment at the time points tested. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, and we demonstrate increased TLR2 cell surface levels on the MO population, most profoundly following stimulation with LTA and OK-432. Together these results indicate that modulation of MEK1/2 and p38 MAPK signalling could affect the response to OK-432 treatment, having the potential to improve its therapeutic potential within cancer and lymphangioma treatment., (© 2012 The Authors APMIS © 2012 APMIS.)

The p53R2 protein, a newly identified member of the ribonucleotide reductase family that provides nucleotides for DNA damage repair, is directly regulated by p53. We show that p53R2 is also regulated by a MEK2 (ERK kinase 2/MAP kinase kinase 2)-dependent pathway. Increased MEK1/2 phosphorylation by serum stimulation coincided with an increase in the RNR activity in U2OS and H1299 cells. The inhibition of MEK2 activity, either by treatment with a MEK inhibitor or by transfection with MEK2 siRNA, dramatically decreased the serum-stimulated RNR activity. Moreover, p53R2 siRNA, but not R2 siRNA, significantly inhibits serum-stimulated RNR activity, indicating that p53R2 is specifically regulated by a MEK2-dependent pathway. Co-immunoprecipitation analyses revealed that the MEK2 segment comprising amino acids 65-171 is critical for p53R2-MEK2 interaction, and the binding domain of MEK2 is required for MEK2-mediated increased RNR activity. Phosphorylation of MEK1/2 was greatly augmented by ionizing radiation, and RNR activity was concurrently increased. Ionizing radiation-induced RNR activity was markedly attenuated by transfection of MEK2 or p53R2 siRNA, but not R2 siRNA. These data show that MEK2 is an endogenous regulator of p53R2 and suggest that MEK2 may associate with p53R2 and upregulate its activity.

The ERK/MAPK signaling pathway is involved in several cellular functions. Inactivation in mice of genes encoding members of this pathway is often associated with embryonic death resulting from abnormal placental development. The placenta is essential for nutritional and gaseous exchanges between maternal and embryonic circulations, as well as for the removal of metabolic wastes. These exchanges take place without direct contact between the two circulations. In mice, the hematoplacental barrier consists in a triple layer of trophoblast cells and endothelial cells of the embryo. MEK1 and MEK2 are double specificity serine-threonine/tyrosine kinases responsible for the activation of ERK1 and ERK2. Mek1 inactivation results in placental anomalies due to trophoblast cell proliferation and differentiation defects leading to severe delays in the development of placenta and causing the death of the embryo. Although Mek2(-/-) mutant mice survived without any apparent phenotype, double heterozygous Mek1(+/-)Mek2(+/-) mutants die during gestation from placental malformations. Together, these data emphasize the crucial role of the ERK/MAPK cascade in the formation of extraembryonic structures., (© 2012 médecine/sciences – Inserm / SRMS.)

Caspase plays an important role in apoptosis and physiological processes such as synaptic plasticity. However, the caspase substrate at the synapse is still unknown. Here we used an in vitro cleavage assay with a small-pool human brain cDNA library. We identified the presynaptic protein Caytaxin as a substrate of caspase-3 and caspase-7. Deficiency in Caytaxin causes Cayman ataxia, a disorder characterized by cerebellar dysfunction and mental retardation. Caytaxin cleavage in cerebellar granule neurons is dependent on caspase-3 activation. The cleavage site is upstream of the cellular retinal and the TRIO guanine exchange factor domain, producing a C-terminal fragment that may play an alternative role in inhibiting MEK2 signaling. Thus, we concluded that Caytaxin is a novel substrate of caspase-3 at the presynapse.

Background and Objective: The antimicrobial peptide LL-37, derived from human neutrophils, can directly chemoattract leukocytes and up-regulate the expression of several immune-related genes in various cell types. In this study, we wanted to determine the immunoregulatory effect of LL-37 on interleukin-8 (IL-8) expression in human gingival fibroblasts (HGFs) and to characterize intracellular signaling pathway(s) and receptor(s) involved in IL-8 induction., Material and Methods: Cultured fibroblasts were treated with different concentrations of LL-37 or interleukin-1β (IL-1β), as a positive control, for specific periods of time in the presence or absence of various inhibitors. RT-PCR and real-time PCR were conducted to analyze the expression of IL-8 mRNA, and the IL-8 levels in cell-free culture media were measured using ELISAs. The MTT assay was performed to determine the cytotoxicity of LL-37., Results: Nontoxic concentrations of LL-37 (up to 10 μm) and IL-1β significantly up-regulated the expression of IL-8 mRNA in a dose-dependent manner (p < 0.05). The IL-8 protein levels were consistently significantly elevated in conditioned media of LL-37-treated HGFs (p < 0.05). IL-8 up-regulation by LL-37 was completely abrogated by 20 μm U0126, consistent with transient phosphorylation of p44/42 MAP kinases. Moreover, pretreatment with Brilliant Blue G (a selective antagonist of the P2X(7) receptor) and the neutralizing antibody against P2X(7) blocked IL-8 up-regulation in a dose-dependent manner, consistent with expression of the P2X(7) receptor in HGFs., Conclusion: These findings indicate that LL-37 induces IL-8 expression via the P2X(7) receptor and the MEK1/2-dependent p44/42 MAP kinases in HGFs, suggesting both direct and indirect involvement of LL-37 in neutrophil recruitment into an inflammatory site within diseased periodontal tissues., (© 2011 John Wiley & Sons A/S.)

Mitogen-activated protein kinase kinases (MKK or MEK) 1 and 2 are usually treated as redundant kinases. However, in assessing their relative contribution towards ERK-mediated biologic response investigators have relied on tests of necessity, not sufficiency. In response we developed a novel experimental model using lethal toxin (LeTx), an anthrax toxin-derived pan-MKK protease, and genetically engineered protease resistant MKK mutants (MKKcr) to test the sufficiency of MEK signaling in melanoma SK-MEL-28 cells. Surprisingly, ERK activity persisted in LeTx-treated cells expressing MEK2cr but not MEK1cr. Microarray analysis revealed non-overlapping downstream transcriptional targets of MEK1 and MEK2, and indicated a substantial rescue effect of MEK2cr on proliferation pathways. Furthermore, LeTx efficiently inhibited the cell proliferation and anchorage-independent growth of SK-MEL-28 cells expressing MKK1cr but not MEK2cr. These results indicate in SK-MEL-28 cells MEK1 and MEK2 signaling pathways are not redundant and interchangeable for cell proliferation. We conclude that in the absence of other MKK, MEK2 is sufficient for SK-MEL-28 cell proliferation. MEK1 conditionally compensates for loss of MEK2 only in the presence of other MKK.

IFN-β and sIL-1Ra play crucial roles in the regulation of innate immunity and inflammation. IFN-β, which is widely used to improve the course of relapsing, remitting multiple sclerosis, induces the production of sIL-1Ra in human monocytes through mechanisms that remain largely unknown. In this study, we identified PI3Kδ and MEK2 as key elements that control sIL-1Ra production in isolated human monocytes activated by IFN-β. Blockade of MEK2, but not of MEK1, by inhibitors and siRNA prevented IFN-β-induced PI3Kδ recruitment to the membrane, Akt phosphorylation, and sIL-1Ra production, suggesting that MEK2 acted upstream of PI3Kδ. Furthermore, ERK1/2, the only identified substrates of MEK1/2 to date, are dispensable for sIL-1Ra production in response to IFN-β stimulation. Upon IFN-β activation, MEK2 and PI3Kδ are translocated to monocyte membranes. These data suggest that MEK1 and MEK2 display different, nonredundant functions in IFN-β signaling. That neither MEK1 nor ERK1/2 play a part in this mechanism is also an unexpected finding that gives rise to a better understanding of the MAPK signaling network. Together, these findings demonstrate that IFN-β triggers an atypical MEK2/PI3Kδ signaling cascade to regulate sIL-1Ra expression in monocytes. The premise that MEK1 and MEK2 play a part in the induction of the proinflammatory cytokine, IL-1β in human monocytes provides a rationale for an alternative, IFN-β-mediated pathway to induce/enhance sIL-1Ra production and thus, to dampen inflammation.

The mitogen-activated protein kinase kinase 1/2 (MEK1/2) signalling pathway plays a central role in tumour progression. Small molecules that inhibit MEK1/2 are therefore considered attractive candidates for anti-cancer drugs. However, the exact contributions of MEK1 and MEK2 to the development of pancreatic cancer remain to be established. To differentiate the functions of MEK1 and MEK2 in a cultured pancreatic cancer cell line, we utilised shRNA-mediated knockdown of their two mRNAs individually. We studied the effects of MEK1 and MEK2 knockdown on cell morphology, proliferation, mitotic arrest, and in vitro invasion capability in PC-1.0 cells. The results showed that inhibition of MEK1 expression was an effective and specific approach to inhibit cell proliferation and induce G0/G1 arrest. On the other hand, MEK2 knockdown specially altered cell morphology and inhibited the invasive ability of pancreatic cancer cells. Therefore, MEK1 and MEK2 mediate different biological responses in cultured pancreatic cancer cells. These proteins could become distinct targets for the inhibition of specific cellular functions in the treatment of pancreatic cancer.

Translocator protein TSPO is an 18 kDa protein implicated in numerous cell functions and is highly expressed in secretory and glandular tissues, especially in steroidogenic cells. TSPO expression is altered in pathological conditions such as certain cancers and neurological diseases. In search of the factors regulating Tspo expression, we recently showed that high levels of TSPO in steroidogenic cells may be due to high constitutive expression of protein kinase Cepsilon (PKCepsilon), while phorbol 12-myristate 13-acetate (PMA) activation of PKCepsilon drives inducible TSPO expression in nonsteroidogenic cells, likely through activator protein 1 (AP1). In this study, we aimed to identify the signal transduction pathway through which PKCepsilon regulates Tspo gene expression. The MEK1/2 specific inhibitor U0126, but not NFkappaB inhibitors, reduced basal Tspo promoter activity in TSPO-rich steroidogenic cells (MA-10 Leydig), as well as basal and PMA-induced Tspo promoter levels in TSPO-poor nonsteroidogenic cells (NIH-3T3 fibroblasts). AP1 and signal transducer and activation of transcription 3 (STAT3) have binding sites in the Tspo promoter and are downstream targets of PKCepsilon and MAPK (Raf-1-ERK1/2) pathways. PKCepsilon overexpression induced STAT3 phosphorylation in NIH-3T3 cells, while PKCepsilon knockdown reduced STAT3 and c-Jun phosphorylation in Leydig cells. MEK1/2, ERK2, c-Jun, and STAT3 knockdown reduced Tspo mRNA and protein levels in Leydig cells. Additionally, Raf-1 reduced Tspo mRNA levels in the same cells. MEK1/2, c-Jun, and STAT3 knockdown also reduced basal as well as PMA-induced Tspo mRNA levels in NIH-3T3 cells. Together, these results demonstrate that PKCepsilon regulates Tspo gene expression through a MAPK (Raf-1-MEK1/2-ERK1/2) signal transduction pathway, acting at least in part through c-Jun and STAT3 transcription factors.

Purpose: Obesity is considered to be a risk factor for prostate cancer. Mitogenic actions of leptin, an adipocyte-derived hormone in a variety of cancer cell types have been identified. We have investigated the proliferative effects of leptin on human prostate cancer cells and assessed the role of tyrosine kinase signalling in mediating these actions., Materials and Methods: Two human androgen-resistant prostate cancer cell lines and one androgen-sensitive human prostate adenocarcinoma cell line were treated with leptin (5-100 ng / mL) for up to 48 hours. Under serum-free conditions, cell proliferation was measured using an enzyme-linked colorimetric assay. Furthermore, phosphorylation of a downstream component of MAPK (ERK1 / 2) was detected by Western blotting and a specific inhibitor of MAPK (PD98059; 40 microM) was used to evaluate the role of this signalling pathway., Results: Leptin dose-dependently increased the cell number in both androgen-resistant cell lines after 24 h and 48 h of incubation (percent of control: DU145 = 194.6 +/- 5.9 %, PC-3 = 177.9 +/- 6.8 %; 100 ng / mL leptin; 48 h; p < 0.001). Conversely, leptin's proliferative effect on the androgen-sensitive cell line was less pronounced (percent of control: LNCaP = 112.3 +/- 6.1 %; 100 ng / mL leptin; 48 h). Leptin also caused dose-dependent ERK1 / 2 phosphorylation in both androgen-resistant cell lines. In addition, pre-treatment with PD98059 inhibited these responses and attenuated leptin's mitogenic action., Conclusions: Data from this in vitro study suggest an association between obesity-associated hyperleptinemia and an increased risk for prostate cancer. Further investigations are necessary to clarify whether these data have clinical relevance regarding the use as a prognostic marker for predicting the timing of the occurrence of androgen resistency., (Copyright Georg Thieme Verlag Stuttgart . New York.)

The HER-2 oncogene, a member of the erythroblastosis oncogene B (ERBB)-like oncogene family, has been shown to be amplified in many types of cancer, including breast cancer. However, the molecular mechanism of HER-2 overexpression is not completely understood. The phosphorylation of proteins on the serine or threonine residues that immediately precede proline (pSer/Thr-Pro) is specifically catalyzed by the prolyl isomerase Pin1 and is a key signaling mechanism in cell proliferation and transformation. Here, we found that Pin1 interacts with mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) protein kinase 1, resulting in the induction of HER-2 expression. Pin1(-/-) mouse embryonic fibroblasts exhibited a decrease in epidermal growth factor (EGF)-induced MEK1/2 phosphorylation compared with Pin1(+/+) mouse embryonic fibroblast. In addition, a knockdown of Pin1 resulted in the inhibition of MEK1/2 phosphorylation induced by EGF in MCF-7 cells. Furthermore, PD98059, a specific inhibitor of MEK1/2, and Juglone, a potent Pin1 inhibitor, markedly suppressed the expression of activator protein-2alpha and the HER-2 promoter activity induced by EGF or 12-O-tetradecanoylphorbol-13-acetate in MCF-7 cells. Importantly, these inhibitors inhibited the neoplastic cell transformation induced by EGF in Pin1-overexpressing JB6 Cl41 cells, which showed enhanced cellular formation compared with the control cells. Therefore, Juglone and PD98059 inhibited the colony formation of MCF-7 breast cancer cells in soft agar. These results indicate that Pin1 amplifies EGF signaling in breast cancer cells through its interaction with MEK1 and then enhances HER-2 expression, suggesting that Pin1 plays an important role in the overexpression of HER-2 through Pin1-MEK1-activator protein-2alpha signaling in breast cancer.

Extreme body mass indexes may impair reproductive outcome in assisted reproductive technologies. Leptin reflects the amount of body fat and could act as a modulator of oocyte quality through activation of specific transcription factors. The aim of this work was to establish whether: 1) leptin influences meiotic and cytoplasmic oocyte maturation; 2) STAT3 and MAPK mediate the effects of leptin and 3) leptin modulates steroid secretion by cumulus-oocyte complexes (COC) during in vitro maturation (IVM). We confirmed immunolocalisation of leptin receptor in oocytes, cumulus/granulosa cells during the peri-ovulatory period. The confocal study showed that COC supplemented with 1, 10 and 100 ng/ml leptin had a significantly higher metaphase II (MII) percentage than those IVM without leptin (P<0.05) and a similar MII index compared to the group supplemented with 10% FCS. Leptin did not increase the percentage of cytoplasmically matured oocytes in terms of cortical granule migration rate, whereas a significantly higher index was found in the FCS group (P<0.001). Oestradiol concentrations in spent media were higher in the FCS group compared to other treatments (P<0.001). Leptin-stimulated nuclear oocyte maturation was significantly impaired when leptin-induced JAK2/STAT3 and MEK 1/2 activation was suppressed by the inhibitors (P<0.001). Steroid secretion of COC was not affected by leptin activation of JAK2/STAT3 or MEK 1/2 pathways. In conclusion, JAK2/STAT3 and MEK 1/2 pathways mediate the enhancement of nuclear oocyte maturation by leptin; however, neither cytoplasmic oocyte maturation nor steroidogenic response of COC were improved in the present rabbit model.

The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1beta and TNFalpha. In this work, we have demonstrated that treatment of HepG2 human hepatoma cells with chemical inhibitors for JNK, p38 protein kinases, and NFkappaB transcription factor abolishes the TNFalpha-mediated inhibition of human apoA-I gene expression in HepG2 cells. In addition, we have shown that TNFalpha decreases also the rate of secretion of apoA-I protein by HepG2 cells, and this effect depends on JNK and p38, but not on NFkappaB and MEK1/2 signaling pathways. The inhibitory effect of TNFalpha has been found to be mediated by the hepatic enhancer of the apoA-I gene. The decrease in the level of human apoA-I gene expression under the impact of TNFalpha appears to be partly mediated by the inhibition of HNF4alpha and PPARalpha gene expression. Treatment of HepG2 cells with PPARalpha antagonist (MK886) or LXR agonist (TO901317) abolishes the TNFalpha-mediated decrease in the level of apoA-I gene expression. PPARalpha agonist (WY-14643) abolishes the negative effect of TNFalpha on apoA-I gene expression in the case of simultaneous inhibition of MEK1/2, although neither inhibition of MEK1/2 nor addition of WY-14643 leads to the blocking of the TNFalpha-mediated decrease in the level of apoA-I gene expression individually. The ligand-dependent regulation of apoA-I gene expression by PPARalpha appears to be affected by the TNFalpha-mediated activation of MEK1/2 kinases, probably through PPARalpha phosphorylation. Treatment of HepG2 cells with PPARalpha and LXR synthetic agonists also blocks the inhibition of apoA-I protein secretion in HepG2 cells under the impact of TNFalpha. A chromatin immunoprecipitation assay demonstrates that TNFalpha leads to a 2-fold decrease in the level of PPARalpha binding with the apoA-I gene hepatic enhancer. At the same time, the level of LXRbeta binding with the apoA-I gene hepatic enhancer is increased 3-fold under the impact of TNFalpha. These results suggest that nuclear receptors HNF4alpha, PPARalpha, and LXRs are involved in the TNFalpha-mediated downregulation of human apoA-I gene expression and apoA-I protein secretion in HepG2 cells.

Bromocriptine (Bc) produces pituitary tumoral mass regression which induces the cellular death that was classically described as apoptosis. However, recent works have related that other mechanisms of cell death could also be involved in the maintenance of physiological and pathological pituitary homeostasis. The aim of this study was to evaluate and characterize the different types of cell death in the involution induced by Bc in experimental rat pituitary tumors. The current study demonstrated that Bc induced an effective regression of estrogen induced pituitary tumors by a mechanism identified as parapoptosis. This alternative cell death was ultrastructurally recognized by extensive cytoplasmic vacuolization and an increased cell electron density, represented around 25% of the total pituitary cells counted. Furthermore, the results obtained from biochemical assays did not correspond to the criteria of apoptosis or necrosis. We also investigated the participation of p38, ERK1/2 and PKC delta in the parapoptotic pathway. An important observation was the significant increase in phosphorylated forms of these MAPKs, the holoenzyme and catalytic fragments of PKC delta in nuclear fractions after Bc administration compared to control and estrogen treated rats. Furthermore, the immunolocalization at ultrastructural level of these kinases showed a similar distribution pattern, with a prevalent localization at nuclear level in lactotrophs from Bc treated rats. In summary, we determined that parapoptosis is the predominant cell death type involved in the regression of pituitary tumors in response to Bc treatment, and may cause the activation of PKC delta, ERK1/2 and p38.

The extracellular signal-regulated kinase cascade is a central signaling pathway that is stimulated by various extracellular stimuli. The signals of these stimuli are then transferred by the cascade's components to a large number of targets at distinct subcellular compartments, which in turn induce and regulate a large number of cellular processes. To achieve these functions, the cascade exhibits versatile and dynamic subcellular distribution that allows proper temporal and spatial modulation of the appropriate processes. In this review, we discuss the intracellular localizations of different components of the ERK cascade, and the impact of these localizations on their activation and specificity., (Copyright 2009 International Union of Biochemistry and Molecular Biology, Inc.)

Background/aims: Based on the observation of biphasic induction of SGK1 expression in the regenerating liver, we investigated the role of SGK1 in the regulation of MEK/ERK signaling pathway which plays a crucial role in regulating growth and survival signaling., Methods: To determine the role of SGK1 in the activation of MEK/ERK signaling cascade, we infected primary hepatocytes with recombinant adenoviral vector encoding SGK1, and assessed its effect on the MEK/ERK signaling pathway., Results: Partial hepatectomy resulted in the biphasic transcriptional induction of SGK1 in regenerating liver tissues. Infection of primary hepatocytes with an adenoviral vector encoding SGK1 enhanced the ERK phosphorylation under serum-starved conditions and this was blocked by the expression of kinase-dead SGK1. SGK1 was found to physically interact with ERK1/2 as well as MEK1/2. Furthermore, SGK1 mediated the phosphorylation of ERK2 on Ser(29) in a serum-dependent manner. Replacement of Ser(29) to aspartic acid, which mimics the phosphorylation of Ser(29), enhanced the ERK2 activity as well as the MEK/ERK complexes formation., Conclusions: SGK1 expression during liver regeneration is a part of a signaling pathway that is necessary for enhancing ERK signaling activation through modulating the MEK/ERK complex formation.

Ras genes are commonly mutated in human cancers of the skin and other tissues. Oncogenic Ras signals through multiple effector pathways, including the Erk1/2 mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3K) and the Ral guanine nucleotide exchange factor (RalGEF) cascades. In epidermis, the activation of oncogenic Ras induces hyperplasia and inhibits differentiation, features characteristic of squamous cell carcinoma. The downstream effector pathways required for oncogenic Ras effects in epidermis, however, are undefined. In this study, we investigated the direct contribution of Mek1 and Mek2 MAPKKs to oncogenic Ras signaling. The response of murine epidermis to conditionally active oncogenic Ras was unimpaired by deletion of either Mek1 or Mek2 MAPKKs individually. In contrast, Ras effects were entirely abolished by combined deletion of all Mek1/2 alleles, whereas epidermis retaining only one allele of either Mek1 or Mek2 showed intermediate responsiveness. Thus, the effects of oncogenic Ras on proliferation and differentiation in skin show a gene dosage-dependent requirement for the Erk1/2 MAPK cascade at the level of Mek1/2 MAPKKs.

Ischemic postconditioning (IPoC) reduces infarct size following ischemia/reperfusion. Whether or not phosphorylation of RISK (reperfusion injury salvage kinases) (AKT, ERK1/2, P70S6K, GSK3beta) is causal for protection by IPoC is controversial. We therefore studied the impact of RISK on IPoC in anesthetized pigs subjected to 90 minutes of left anterior descending coronary artery hypoperfusion and 120 minutes of reperfusion. In protocol 1, IPoC, by 6 cycles of 20/20 seconds of reperfusion/reocclusion (n=13), was compared with immediate full reperfusion (IFR) (n=15). In protocol 2, IPoC (n=4) or IFR (n=4) was performed with pharmacological RISK blockade by IC coinfusion of Wortmannin and U0126. Infarct size was determined by TTC staining, and the expression of phosphorylated RISK proteins by Western blot analysis in biopsies. In protocol 1, infarct size was 20+/-3% (percentage of area at risk; mean+/-SEM) with IPoC and 33+/-4% (P<0.05) with IFR. RISK phosphorylation increased with reperfusion but was not different between IPoC and IFR. In protocol 2, Wortmannin and U0126 blocked the increases in RISK phosphorylation during reperfusion, but infarct size was still smaller with IPoC (15+/-7%) than with IFR (35+/-6%; P<0.05).

Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2(-/-) mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2(-/-) mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV(T) MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.

Extracellular signal-regulated kinase (ERK) signal transduction pathway is widely implicated in multiple physiological processes. However, it remains to be determined whether ERK pathway plays roles in anxiety. Here we investigated the changes of phosphorylated ERK1/2 (pERK1/2) and c-Fos expression by immunostaining in the medial prefrontal cortex (mPFC) of anxious rats. The results indicated that the levels of pERK and c-Fos were significantly increased during anxiety. Inhibition of ERK phosphorylation blocked the anxiety-induced c-Fos expression. In the animal behavioral tests, the PD98059-treated anxious rats had a significant increase in the numbers of the open-arm entries, the time spent in the open-arms and the numbers of head-dipping in EPM test, and increase the inner locomotion in the open field test compared with the anxious rats. The results suggested that the ERK signal transduction pathway might play an important role in anxiety, and inhibition of the ERK pathway in the mPFC could produce anxiolysis effect.

Neuropeptide Y (NPY) is a 36 amino acid peptide widely present in the CNS, including the retina. Previous studies have demonstrated that NPY promotes cell proliferation of rat post-natal hippocampal and olfactory epithelium precursor cells. The aim of this work was to investigate the role of NPY on cell proliferation of rat retinal neural cells. For this purpose, primary retinal cell cultures expressing NPY, and NPY Y(1), Y(2), Y(4) and Y(5) receptors [Alvaro et al., (2007) Neurochem. Int., 50, 757] were used. NPY (10-1000 nM) stimulated cell proliferation through the activation of NPY Y(1), Y(2) and Y(5) receptors. NPY also increased the number of proliferating neuronal progenitor cells (BrdU(+)/nestin(+) cells). The intracellular mechanisms coupled to NPY receptors activation that mediate the increase in cell proliferation were also investigated. The stimulatory effect of NPY on cell proliferation was reduced by L-nitroarginine-methyl-esther (L-NAME; 500 microM), a nitric oxide synthase inhibitor, 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ; 20 microM), a soluble guanylyl cyclase inhibitor or U0126 (1 microM), an inhibitor of the extracellular signal-regulated kinase 1/2 (ERK 1/2). In conclusion, NPY stimulates retinal neural cell proliferation, and this effect is mediated through nitric oxide-cyclic GMP and ERK 1/2 pathways.

Plant defense against pathogens often includes rapid programmed cell death known as the hypersensitive response (HR). Recent genetic studies have demonstrated the involvement of a specific mitogen-activated protein kinase (MAPK) cascade consisting of three tobacco MAPKs, SIPK, Ntf4 and WIPK, and their common upstream MAPK kinase (MAPKK or MEK), NtMEK2. Potential upstream MAPKK kinases (MAPKKKs or MEKKs) in this cascade include the orthologs of Arabidopsis MEKK1 and tomato MAPKKKalpha. Activation of the SIPK/Ntf4/WIPK pathway induces cell death with phenotypes identical to pathogen-induced HR at macroscopic, microscopic and physiological levels, including loss of membrane potential, electrolyte leakage and rapid dehydration. Loss of membrane potential in NtMEK2(DD) plants is associated with the generation of reactive oxygen species (ROS), which is preceded by disruption of metabolic activities in chloroplasts and mitochondria. We observed rapid shutdown of carbon fixation in chloroplasts after SIPK/Ntf4/WIPK activation, which can lead to the generation of ROS in chloroplasts under illumination. Consistent with a role of chloroplast-generated ROS in MAPK-mediated cell death, plants kept in the dark do not accumulate H(2)O(2) in chloroplasts after MAPK activation, and cell death is significantly delayed. Similar light dependency was observed in HR cell death induced by tobacco mosaic virus, which is known to activate the same MAPK pathway in an N-gene-dependent manner. These results suggest that activation of the SIPK/Ntf4/WIPK cascade by pathogens actively promotes the generation of ROS in chloroplasts, which plays an important role in the signaling for and/or execution of HR cell death in plants.

Aim: To evaluate the expression of C-X-C motif chemokine receptor 4 (CXCR4) and its signaling cascades, which were previously identified as a key factor for cancer cell progression and metastasis, in cholangiocarcinoma cell lines., Methods: The expression of CXCR4 and its signaling cascades were determined in the cholangiocarcinoma cell lines (RMCCA1 and KKU100) by Western blotting. The invasion assays and the detection of actin polymerization were tested in these cholangiocarcinoma cells treated with CXC chemokine ligand -12 (CXCL12)., Results: Expression of CXCR4 was detected in both cholangiocarcinoma cell lines and activation of CXCR4 with CXCL12 triggered the signaling via the extracellular signal-regulated kinase-1/2 (ERK1/2) and phosphoinositide 3-kinase (PI3K) and induction of cholangiocarcinoma cell invasion, and displayed high levels of actin polymerization. Addition of CXCR4 inhibitor (AMD3100) abrogated CXCL12-induced phosphorylation of MEK1/2 and Akt in these cells. Moreover, treatment with MEK1/2 inhibitor (U0126) or PI3K inhibitor (LY294002) also attenuated the effect of CXCL12-induced cholangiocarcinoma cell invasion., Conclusion: These results indicated that the activation of CXCR4 and its signaling pathways (MEK1/2 and Akt) are essential for CXCL12-induced cholangiocarcinoma cell invasion. This rises Implications on a potential role for the inhibition of CXCR4 or its signal cascades in the treatment of cholangiocarcinoma.

ERK1/2 is an important subfamily of mitogen-activated protein kinases that control a broad range of cellular activities and physiological processes. ERK1/2 can be activated transiently or persistently by MEK1/2 and upstream MAP3Ks in conjunction with regulation and involvement of scaffolding proteins and phosphatases. Activation of ERK1/2 generally promotes cell survival; but under certain conditions, ERK1/2 can have pro-apoptotic functions.

Zinc is an essential nutrient that can also be toxic. We have previously reported that zinc-related renal toxicity is due, in part, to free radical generation in the renal epithelial cell line, LLC-PK(1) cells. We have also shown that an MEK1/2 inhibitor, U0126, markedly inhibits zinc-induced renal cell injury. In this study, we investigated the role of an upstream MEK/ERK pathway, Raf-1 kinase pathway, and the transcription factor and ERK substrate Elk-1, in rat renal cortical slices exposed to zinc. Immediately after preparing slices from rat renal cortex, the slices were incubated in medium containing Raf-1 and MEK inhibitors. ERK1/2 and Elk-1 activation were determined by Western blot analysis for phosphorylated ERK (pERK) 1/2 and phosphorylated Elk-1 (pElk-1) in nuclear fractions prepared from slices exposed to zinc. Zinc caused not only increases in 4-hydroxynonenal (4-HNE) modified protein and lipid peroxidation, as an index of oxidant stress, and decreases in PAH accumulation, as that of renal cell injury in the slices. Zinc also induced a rapid increase in ERK/Elk-1 activity accompanied by increased expressions of pERK and pElk-1 in the nuclear fraction. A Raf-1 kinase inhibitor and an MEK1/2 inhibitor U0126 significantly attenuated zinc-induced decreases PAH accumulation in the slices. The Raf-1 kinase inhibitor and U0126 also suppressed ERK1/2 activation in nuclear fractions prepared from slices treated with zinc. The present results suggest that a Raf-1/MEK/ERK1/2 pathway and the ERK substrate Elk-1 are involved in free radical-induced injury in rat renal cortical slices exposed to zinc.

Interferon-gamma (IFN-gamma) exerts potent antiviral activity in the hepatitis C virus (HCV) replicon systems. However, the mechanisms underlying the direct antiviral effect have not been determined. We found that the type II transcriptional response to IFN-gamma could be suppressed by inhibition of MEK1/2 kinase activity by MEK1/2 inhibitor U0126 in the hepatoma cell line Huh-7. Using a bicistronic HCV replicon system expressing a luciferase reporter gene in Huh-7 cells (RLuc-replicon), we showed that inhibition of MEK1/2 kinase activity is sufficient to counteract the antiviral activity of IFN-gamma. Expression of a constitutive active form of Ras inhibited the luciferase activity of RLuc-replicon, whereas a dominant-negative mutant of Ras enhanced the reporter activity, indicating that the Ras-MAPK pathway has a role in limiting replication of the viral RNA. Consistent with the involvement of the Ras-MAPK pathway, treatment with epidermal growth factor suppressed HCV protein expression in the RLuc-replicon cells, an effect that could be abolished by U0126. Inhibition of MEK1/2 kinase activity correlated with reduced phosphorylation of the HCV NS5A protein and enhanced RLuc-replicon luciferase reporter activity, in line with recent reports that phosphorylation of NS5A negatively modulates HCV RNA replication. Finally, genetic deletion analysis in yeast supported the role of a MEK-like kinase(s) in the regulation of NS5A phosphorylation. In conclusion, the direct anti-HCV effect of IFN-gamma in cell culture is, at least in part, mediated through the Ras-MAPK signaling pathway, which possibly involves a direct or indirect modulation of NS5A protein phosphorylation.

Supramaximal stimulation of the rat pancreas with CCK, or its analog caerulein, triggers acute pancreatitis and a number of pancreatitis-associated acinar cell changes including intracellular activation of digestive enzyme zymogens and acinar cell injury. It is generally believed that some of these various acinar cell responses to supramaximal secretagogue stimulation are interrelated and interdependent. In a recent report, Lu et al. showed that secretin, by causing generation of cAMP and activation of PKA, sensitizes acinar cells to secretagogue-induced zymogen activation, and, as a result, submaximally stimulating concentrations of caerulein can, in the presence of secretin, trigger intracellular zymogen activation. We found that secretin also sensitizes acinar cells to secretagogue-induced cell injury and to subapical F-actin redistribution but that it did not alter the caerulein concentration dependence of other pancreatitis-associated changes such as the induction of a peak plateau intracellular [Ca(2+)] rise, inhibition of secretion, activation of ERK1/2, and activation of NF-kappaB. The finding that secretin sensitizes acinar cells to both intracellular zymogen activation and cell injury is consistent with the concept that these two early events in pancreatitis are closely interrelated and, possibly, interdependent. On the other hand, the finding that, in the presence of secretin, caerulein can trigger subapical F-actin redistribution without inhibiting secretion challenges the concept that disruption of the subapical F-actin web is causally related to high-dose secretagogue-induced inhibition of secretion in pancreatic acinar cells.

Prions represent a unique class of infectious agents in which the normal cellular prion protein (PrPC) is converted to an abnormal isoform (PrPSc), which accumulates in the brain and constitutes the major, if not the only, component of the infectious particle. Factors that still remain to be identified may facilitate the conversion of PrPC to PrPSc. In the present study, we first demonstrated that a growth factor of the neurotrophin family, brain-derived neurotrophic factor (BDNF), stimulates the formation of PrPSc in a gonadotropin-releasing hormone-secreting neuronal cell line (GT1-1 cells) infected with the Rocky Mountain Laboratory (RML) strain of scrapie as determined by Western blot analysis. We then observed that the prion-infected cells can be cleared from PrPSc by treatment with three inhibitors of mitogen-activated protein kinase kinase 1/2 (MEK1/2) [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene and 2-(2-amino-3-methyoxyphenyl)-4H-1-benzopyran-4-one, as well as alpha-[amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl) benzeneacetonitrile, which passes the blood-brain barrier], a component of one of the intracellular signaling pathways activated by BDNF. The MEK1/2 inhibitors were also efficient in clearing PrPSc from prion-infected GT1-1 cells stimulated to accumulate high levels of PrPSc by enhanced serum concentrations in the medium or by the use of a serum-free neuron-specific neurobasal medium. PrPSc did not reappear in the cultures within 5 weeks after completion of treatment. We conclude that inhibitors of the MEK1/2 pathway can efficiently and probably irreversibly clear PrP(Sc) from prion-infected cells. The MEK pathway may therefore be a suitable target for therapeutic intervention in prion diseases.

Atherosclerosis is considered to be an inflammatory disease. Tissue factor (TF), a prothrombotic molecule expressed by various cell types within atherosclerotic plaques, is thought to play an essential role in thrombus formation after atherosclerotic plaque rupture. Recent studies suggest that the antiinflammatory cytokine interleukin-10 (IL-10) has many antiatherosclerotic properties. Therefore, the effects of IL-10 on TF expression in response to inflammation were investigated. Mouse macrophages were stimulated with lipopolysaccharide (LPS) in the presence or absence of IL-10. Pretreatment with IL-10 resulted in a 50% decrease in TF mRNA expression and TF promoter activity. Binding of early growth response gene-1 (Egr-1) to the consensus DNA sequence, a key transcriptional activator of TF expression in response to inflammation, and the expression of Egr-1 mRNA were also inhibited by IL-10. This inhibition was independent of the induction of suppressor of cytokine signaling protein-3 by IL-10. Macrophages that had been transfected with luciferase reporter constructs containing the murine Egr-1 5'-flanking sequence exhibited reduced reporter gene activity in response to LPS stimulation with IL-10 pretreatment. Studies with deletion constructs of the Egr-1 promoter identified the proximal serum response element SRE3 as a potential regulatory site for the IL-10 mediated suppression of Egr-1 expression. Furthermore, activation of the upstream signal-transduction elements, such as mitogen-activated protein kinase kinase (MEK) 1/2, extracellular signal-regulated kinase 1/2, and Elk-1 were also inhibited by IL-10 pretreatment. Taken together, these results demonstrate a pathway for the IL-10 mediated inhibition of TF expression during inflammation and may explain the antiatherosclerotic effects of IL-10.

Somatostatin is a neuropeptide that acts as an endogenous inhibitor of various cellular functions including endocrine and exocrine secretions and the proliferation of normal and tumour cells. Its action is mediated by a family of G-protein-coupled receptors (sst1-sst5) that are widely distributed in normal and tumour cells. Gastroenteropancreatic endocrine tumours express multiple somatostatin receptors, sst2 being clearly predominant. These receptors represent the molecular basis for the clinical use of somatostatin analogues in the treatment of endocrine tumours and their in vivo localisation. This review covers current knowledge in somatostatin receptor biology and signalling.

Kaposi's sarcoma-associated herpesvirus (KSHV) in vitro target cell infection is characterized by the expression of the latency-associated genes ORF 73 (LANA-1), ORF 72, and K13 and by the transient expression of a very limited number of lytic genes such as lytic cycle switch gene ORF 50 (RTA) and the immediate early (IE) lytic K5, K8, and v-IRF2 genes. During the early stages of infection, several overlapping multistep complex events precede the initiation of viral gene expression. KSHV envelope glycoprotein gB induces the FAK-Src-PI3K-RhoGTPase (where FAK is focal adhesion kinase) signaling pathway. As early as 5 min postinfection (p.i.), KSHV induced the extracellular signal-regulated kinase 1 and 2 (ERK1/2) via the PI3K-PKCzeta-MEK pathway. In addition, KSHV modulated the transcription of several host genes of primary human dermal microvascular endothelial cells (HMVEC-d) and fibroblast (HFF) cells by 2 h and 4 h p.i. Neutralization of virus entry and infection by PI-3K and other cellular tyrosine kinase inhibitors suggested a critical role for signaling molecules in KSHV infection of target cells. Here we investigated the induction of ERK1/2 by KSHV and KSHV envelope glycoproteins gB and gpK8.1A and the role of induced ERK in viral and host gene expression. Early during infection, significant ERK1/2 induction was observed even with low multiplicity of infection of live and UV-inactivated KSHV in serum-starved cells as well as in the presence of serum. Entry of UV-inactivated virus and the absence of viral gene expression suggested that ERK1/2 induction is mediated by the initial signal cascade induced by KSHV binding and entry. Purified soluble gpK8.1A induced the MEK1/2 dependent ERK1/2 but not ERK5 and p38 mitogen-activated protein kinase (MAPK) in HMVEC-d and HFF. Moderate ERK induction with soluble gB was seen only in HMVEC-d. Preincubation of gpK8.1A with heparin or anti-gpK8.1A antibodies inhibited the ERK induction. U0126, a selective inhibitor for MEK/ERK blocked the gpK8.1A- and KSHV-induced ERK activation. ERK1/2 inhibition did not block viral DNA internalization and had no significant effect on nuclear delivery of KSHV DNA during de novo infection. Analyses of viral gene expression by quantitative real-time reverse transcriptase PCR revealed that pretreatment of cells with U0126 for 1 h and during the 2-h infection with KSHV significantly inhibited the expression of ORF 73, ORF 50 (RTA), and the IE-K8 and v-IRF2 genes. However, the expression of lytic IE-K5 gene was not affected significantly. Expression of ORF 73 in BCBL-1 cells was also significantly inhibited by preincubation with U0126. Inhibition of ERK1/2 also inhibited the transcription of some of the vital host genes such as DUSP5 (dual specificity phosphatase 5), ICAM-1 (intercellular adhesion molecule 1), heparin binding epidermal growth factor, and vascular endothelial growth factor that were up-regulated early during KSHV infection. Several MAPK-regulated host transcription factors such as c-Jun, STAT1alpha, MEF2, c-Myc, ATF-2 and c-Fos were induced early during infection, and ERK inhibition significantly blocked the c-Fos, c-Jun, c-Myc, and STAT1alpha activation in the infected cells. AP1 transcription factors binding to the RTA promoter in electrophoretic mobility shift assays were readily detected in the infected cell nuclear extracts which were significantly reduced by ERK inhibition. Together, these results suggest that very early during de novo infection, KSHV induces the ERK1/2 to modulate the initiation of viral gene expression and host cell genes, which further supports our hypothesis that beside the conduit for viral DNA delivery into the cytoplasm, KSHV interactions with host cell receptor(s) create an appropriate intracellular environment facilitating infection.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a transmissible lung cancer of sheep. The virus can induce tumors rapidly, and we previously found that the JSRV envelope protein (Env) functions as an oncogene, because it can transform mammalian and avian fibroblast cell lines. (N. Maeda, Proc. Natl. Acad. Sci. USA 98:4449-4454, 2001). The molecular mechanisms of JSRV Env transformation are of considerable interest. Several reports suggested that the phosphatidylinositol 3-kinase/Akt pathway is important for transformation of mammalian fibroblasts but not for chicken fibroblasts. In this study, we found that Akt/mTOR is involved in JSRV transformation of mouse NIH 3T3 fibroblasts, because treatment with the mTOR inhibitor rapamycin reduced transformation. We also found that H/N-Ras inhibitor FTI-277 and MEK1/2 inhibitors PD98059 and U0126 strongly inhibited JSRV transformation of NIH 3T3 fibroblasts, suggesting that the H/N-Ras-MEK-mitogen-activated protein kinase (MAPK) p44/42 pathway is necessary for the transformation. In RK3E epithelial cells, the MEK1/2 inhibitors also eliminated transformation, but FTI-277 only partially inhibited transformation. It was noteworthy that p38 MAPK inhibitors enhanced JSRV transformation in both fibroblasts and epithelial cells. Treatment of transformed cells with p38 inhibitors both increased levels of phospho-MEK1/2 and phospho-p44/42 and induced rapid enhancement of the transformed phenotype. Immunohistochemical staining of tumor tissues from naturally and experimentally induced OPA and naturally occurring enzootic nasal adenocarcinoma revealed strong activation of MAPK p44/42 in all cases examined. However, p38 activation was not generally observed. These results indicate that signaling through two pathways (in particular, H/N-Ras-MEK-MAPK and, to a lesser extent, Akt-mTOR) is important for JSRV-induced transformation and that p38 MAPK has a negative regulatory effect on transformation, perhaps via MEK1/2 and p44/42.

Stimulation of alpha1-adrenoceptors induces proliferation of vascular smooth muscle cells (SMCs) and contributes to arterial remodeling. Although activation of NAD(P)H oxidase and generation of reactive oxygen species (ROS) are required, little is known about this pathway. In this study, we examined the hypothesis that epidermal growth factor receptor (EGFR) transactivation and extracellular regulated kinases (ERK) are involved in alpha1-adrenoceptor-mediated SMC growth. Phenylephrine increased protein synthesis in association with a rapid (< or =5 minutes) and sustained (> or =60 minutes) doubling of phosphorylation of EGFR and ERK1/2, but not p38 or JNK in the media of rat aorta maintained in organ culture. Antagonists of EGFR phosphotyrosine activity (AG-1478) and ERK phosphorylation (PD-98059, U-0126) abolished phenylephrine-induced protein synthesis, whereas antagonists of p38 or JNK phosphorylation had no specific effect. A competitive antagonist (P22) for heparin binding EGF-like growth factor (HB-EGF) blocked phenylephrine-induced protein synthesis, as did downregulation of pro-HB-EGF (CRM197). Phenylephrine-induced protein synthesis was inhibited by neutralizing antibody to HB-EGF and absent in HB-EGF-/- SMCs. Inhibitors of metalloproteinases (BiPS, KB-R7785) also blocked adrenergic growth. The neutralizing antibody against HB-EGF had no effect on the two-fold increase in ROS generation induced by phenylephrine (DCF fluorescence), suggesting that stimulation of NAD(P)H oxidase by alpha1-adrenoceptor occupation precedes HB-EGF release. Cell culture studies confirmed and extended these findings. These data suggest that alpha1-adrenoceptor-mediated SMC growth requires ROS-dependent shedding of HB-EGF, transactivation of EGFR, and activation of the MEK1/2-dependent MAP kinase pathway. This trophic pathway may link sympathetic activity to arterial wall growth in adaptive remodeling and hypertrophic disease.

To investigate the role for initiation factor phosphorylation in de novo translation, we have studied the recovery of human kidney cells from hypertonic stress. Previously, we have demonstrated that hypertonic shock causes a rapid inhibition of protein synthesis, the disaggregation of polysomes, the dephosphorylation of eukaryotic translation initiation factor (eIF)4E, 4E-BP1, and ribosomal protein S6, and increased association of 4E-BP1 with eIF4E. The return of cells to isotonic medium promotes a transient activation of Erk1/2 and the phosphorylation of initiation factors, promoting an increase in protein synthesis that is independent of a requirement for eIF4E phosphorylation. As de novo translation is associated with the phosphorylation of 4E-BP1, we have investigated the role of the signaling pathways required for this event by the use of cell-permeable inhibitors. Surprisingly, although rapamycin, RAD001, wortmannin, and LY294002 inhibited the phosphorylation of 4E-BP1 and its release from eIF4E, they did not prevent the recovery of translation rates. These data suggest that only a small proportion of the available eIF4F complex is required for maximal translation rates under these conditions. Similarly, prevention of Erk1/2 activity alone with low concentrations of PD184352 did not impinge upon de novo translation until later times of recovery from salt shock. However, U0126, which prevented the phosphorylation of Erk1/2, ribosomal protein S6, TSC2, and 4E-BP1, attenuated de novo protein synthesis in recovering cells. These results indicate that the phosphorylation of 4E-BP1 is mediated by both phosphatidylinositol 3-kinase-dependent rapamycin-sensitive and Erk1/2-dependent signaling pathways and that activation of either pathway in isolation is sufficient to promote de novo translation.