Phosphorylation Levels Of MAPK Research Articles

Anti-inflammatory activity of mulberry leaf extract through inhibition of NF-κB

Mulberry leaf has been traditionally used to treat chronic diseases such as diabetes and cancer. The effect and mechanism of mulberry leaf extract (MLE) in LPS-induced activation of macrophage was investigated by the levels of production of proinflammatory mediators and cytokines, and their transcriptions. MLE effectively decreased proinflammatory mediators and cytokines production. Their expression levels were decreased at both transcript and protein levels by the pretreatment of MLE. Since all these genes are transcribed by NF-κB and MAPK, their activities were also examined. NF-κB activity was proved to be reduced by the preincubation with MLE through inhibition of IκBα phosphorylation. However, MLE-treatment had no effect on phosphorylation levels of MAPK. There results suggest that MLE can be used as an anti-inflammatory agent to inhibit NF-κB-mediated inflammatory response. In addition, our data support the idea that MLE can inhibit the activities of proinflammatory mediators and cytokines to ameliorate the disease conditions.

Isolation and Functional Characterization of Peptide Agonists of PTPRJ, a Tyrosine Phosphatase Receptor Endowed with Tumor Suppressor Activity

PTPRJ is a receptor-type protein tyrosine phosphatase whose expression is strongly reduced in the majority of investigated cancer cell lines and tumor specimens. PTPRJ negatively interferes with mitogenic signals originating from several oncogenic receptor tyrosine kinases, including HGFR, PDGFR, RET, and VEGFR-2. Here we report the isolation and characterization of peptides from a random peptide phage display library that bind and activate PTPRJ. These agonist peptides, which are able to both circularize and form dimers in acqueous solution, were assayed for their biochemical and biological activity on both human cancer cells and primary endothelial cells (HeLa and HUVEC, respectively). Our results demonstrate that binding of PTPRJ-interacting peptides to cell cultures dramatically reduces the extent of both MAPK phosphorylation and total phosphotyrosine levels; conversely, they induce a significant increase of the cell cycle inhibitor p27(Kip1). Moreover, PTPRJ agonist peptides both reduce proliferation and trigger apoptosis of treated cells. Our data indicate that peptide agonists of PTPRJ positively modulate the PTPRJ activity and may lead to novel targeted anticancer therapies.

Different effects of omega-3 fatty acids on the cell cycle in C2C12 myoblast proliferation

Polyunsaturated fatty acids (PUFAs) are important molecules for human health. We investigated the effects of three major omega-3 PUFAs on C2C12 myoblast proliferation. Both docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids decreased cell growth, whereas linolenic (ALA) acid did not, compared with the control. Cell cycle analysis showed that G(1) phase duration was increased markedly and S-phase duration was decreased by DHA and EPA. In contrast, there was no change in the G(1) or S-phase duration when the cells were treated with linolenic acid. To determine how DHA and EPA affected the cell cycle, cyclins and MAPK proteins were investigated. Western blotting and real-time quantitative PCR showed that DHA and EPA decreased cyclin E and CDK2 levels at both the protein and mRNA level. Also, MAPK phosphorylation levels were decreased by treatment with DHA and EPA. Our results indicated that different kinds of n-3 PUFA differentially affected myoblast cell proliferation. DHA and EPA decreased skeletal muscle cell proliferation through a mechanism involving MAPK-ERK.

17β-Estradiol Rapidly Activates Calcium Release from Intracellular Stores via the GPR30 Pathway and MAPK Phosphorylation in Osteocyte-Like MLO-Y4 Cells

Estrogen regulates critical cellular functions, and its deficiency initiates bone turnover and the development of bone mass loss in menopausal females. Recent studies have demonstrated that 17β-estradiol (E(2)) induces rapid non-genomic responses that activate downstream signaling molecules, thus providing a new perspective to understand the relationship between estrogen and bone metabolism. In this study, we investigated rapid estrogen responses, including calcium release and MAPK phosphorylation, in osteocyte-like MLO-Y4 cells. E(2) elevated [Ca(2+)]( i ) and increased Ca(2+) oscillation frequency in a dose-dependent manner. Immunolabeling confirmed the expression of three estrogen receptors (ERα, ERβ, and G protein-coupled receptor 30 [GPR30]) in MLO-Y4 cells and localized GPR30 predominantly to the plasma membrane. E(2) mobilized calcium from intracellular stores, and the use of selective agonist(s) for each ER showed that this was mediated mainly through the GPR30 pathway. MAPK phosphorylation increased in a biphasic manner, with peaks occurring after 7 and 60min. GPR30 and classical ERs showed different temporal effects on MAPK phosphorylation and contributed to MAPK phosphorylation sequentially. ICI182,780 inhibited E(2) activation of MAPK at 7min, while the GPR30 agonist G-1 and antagonist G-15 failed to affect MAPK phosphorylation levels. G-1-mediated MAPK phosphorylation at 60min was prevented by prior depletion of calcium stores. Our data suggest that E(2) induces the non-genomic responses Ca(2+) release and MAPK phosphorylation to regulate osteocyte function and indicate that multiple receptors mediate rapid E(2) responses.

A Group III histidine kinase (mhk1) upstream of high‐osmolarity glycerol pathway regulates sporulation, multi‐stress tolerance and virulence of Metarhizium robertsii, a fungal entomopathogen

The role of Metarhizium robertsii Group III histidine kinase (mhk1) in regulating various phenotypes of the fungal entomopathogen and the transcripts of 25 downstream genes likely associated with the phenotypes were probed by constructing Δmhk1 and Δmhk1/mhk1 mutants. All examined Δmhk1 phenotypes except unchanged sensitivity to fungicide (dimethachlon) differed significantly from those of wild type and Δmhk1/mhk1, which were similar to each other. Significant phenotypic changes in Δmhk1 included increased conidial yields on two media, increased tolerance to H(2)O(2) , decreased tolerance to menadione, increased tolerance to hyperosmolarity, increased conidial thermotolerance, decreased conidial UV-B resistance and reduced virulence to Tenebrio molitor larvae. The mhk1 disruption elevated the transcripts of nine genes, including two associated with conidiation (flbC and hymA) and three encoding catalases but decreased seven other gene transcripts, including three for superoxide dismultases, under normal conditions. The high-osmolarity glycerol pathway MAPK phosphorylation level in Δmhk1 culture was increased 1.0- to 1.8-fold by KCl, sucrose and menadione stresses but reduced drastically by H(2)O(2) or heat (40°C) stress, accompanied with different transcript patterns of all examined genes under the stresses. Our results confirmed the crucial role of mhk1 in regulating the expression of the downstream genes and associated phenotypes important for the fungal biocontrol potential.

Octa-Arginine Mediated Delivery of Wild-Type Lnk Protein Inhibits TPO-Induced M-MOK Megakaryoblastic Leukemic Cell Growth by Promoting Apoptosis

BackgroundLnk plays a non-redundant role by negatively regulating cytokine signaling of TPO, SCF or EPO. Retroviral expression of Lnk has been shown to suppress hematopoietic leukemic cell proliferation indicating its therapeutic value in cancer therapy. However, retroviral gene delivery carries risks of insertional mutagenesis. To circumvent this undesired consequence, we fused a cell permeable peptide octa-arginine to Lnk and evaluated the efficacy of inhibition of leukemic cell proliferation in vitro.Methodology/Principal FindingsIn this study, proliferation assays, flow cytometry, Western Blot analyses were performed on wild-type (WT), mutant Lnk R8 or BSA treated M-MOK cells. We found that delivered WT, but not mutant Lnk R8 blocked TPO-induced M-MOK megakaryoblastic leukemic cell proliferation. In contrast, WT Lnk R8 showed no growth inhibitive effect on non-hematopoietic HELA or COS-7 cell. Moreover, we demonstrated that TPO-induced M-MOK cell growth inhibition by WT Lnk R8 was dose-dependent. Penetrated WT Lnk R8 induced cell cycle arrest and apoptosis. Immunoprecipitation and Western blots data indicated WT Lnk R8 interacted with endogeneous Jak2 and downregulated Jak-Stat and MAPK phosphorylation level in M-MOK cells after TPO stimulation. Treatment with specific inhibitors (TG101348 and PD98059) indicated Jak-Stat and MAPK pathways were crucial for TPO-induced proliferation of M-MOK cells. Further analyses using TF-1 and HEL leukemic cell-lines showed that WT Lnk R8 inhibited Jak2-dependent cell proliferation. Using cord blood-derived CD34+ stem cells, we found that delivered WT Lnk R8 blocked TPO-induced megakaryopoiesis in vitro.Conclusions/SignificanceIntracellular delivery of WT Lnk R8 fusion protein efficiently inhibited TPO-induced M-MOK leukemic cell growth by promoting apoptosis. WT Lnk R8 protein delivery may provide a safer and more practical approach to inhibit leukemic cell growth worthy of further development.

Is sprint exercise a leptin signaling mimetic in human skeletal muscle?

This study was designed to determine whether sprint exercise activates signaling cascades linked to leptin actions in human skeletal muscle and how this pattern of activation may be interfered by glucose ingestion. Muscle biopsies were obtained in 15 young healthy men in response to a 30-s sprint exercise (Wingate test) randomly distributed into two groups: the fasting (n = 7, C) and the glucose group (n = 8, G), who ingested 75 g of glucose 1 h before the Wingate test. Exercise elicited different patterns of JAK2, STAT3, STAT5, ERK1/2, p38 MAPK phosphorylation, and SOCS3 protein expression during the recovery period after glucose ingestion. Thirty minutes after the control sprint, STAT3 and ERK1/2 phosphorylation levels were augmented (both, P < 0.05). SOCS3 protein expression was increased 120 min after the control sprint but PTP1B protein expression was unaffected. Thirty and 120 min after the control sprint, STAT5 phosphorylation was augmented (P < 0.05). Glucose abolished the 30 min STAT3 and ERK1/2 phosphorylation and the 120 min SOCS3 protein expression increase while retarding the STAT5 phosphorylation response to sprint. Activation of these signaling cascades occurred despite a reduction of circulating leptin concentration after the sprint. Basal JAK2 and p38 MAPK phosphorylation levels were reduced and increased (both P < 0.05), respectively, by glucose ingestion prior to exercise. During recovery, JAK2 phosphorylation was unchanged and p38 MAPK phosphorylation was transiently reduced when the exercise was preceded by glucose ingestion. In conclusion, sprint exercise performed under fasting conditions is a leptin signaling mimetic in human skeletal muscle.

Estrogen receptor-β activation in combination with letrozole blocks the growth of breast cancer tumors resistant to letrozole therapy

Treatment with anti-estrogens or aromatase inhibitors (AI) is the main therapeutic strategy used against estrogen receptor ERα-positive breast cancer. Resistance to these therapies presents a major challenge in the management of breast cancer. Little is known about ERβ in breast carcinogenesis. Our aim in this study is to examine potential novel strategies utilizing ERβ activity to overcome AI resistance. We provide evidence that ERβ agonist can reduce the growth of AI-resistant breast cancer cells. Our data further confirm that therapeutic activation of ERβ by DPN, an ERβ agonist, blocks letrozole-resistant tumor growth in a xenograft model. Interestingly, DPN exerted tumor growth inhibition only in the presence of the AI letrozole, suggesting that combination therapy including ERβ activators and AI may be used in the clinical setting treating AI resistant breast cancer. An increase in ERβ levels, with diminished ERα/ERβ ratio, was observed in the tumors from mice treated with DPN/letrozole combination compared to single agents and control. Decreased Cyclin D1 and increased CyclinD1/CDK inhibitors p21 and p27 levels in DPN/letrozole treated tumors were observed, suggesting that the combination treatment may inhibit tumor growth by blocking G1/S phase cell cycle progression. Our data show a decrease in MAPK phosphorylation levels without affecting total levels. In addition to providing evidence suggesting the potential use of ERβ agonists in combination with letrozole in treating AI resistant breast cancer and prolonging sensitivity to AI, we also provide mechanistic evidence supporting the role of ERβ in altering the expression profile associated with resistance.

Abstract 1658: Fenretinide induces lethal autophagy in pancreatic cancer cells

Abstract Background: Recent studies show that the synthetic vitamin A analog fenretinide (4-HPR) is cytotoxic in a number of human tumor cell lines and is a chemopreventative agent that is well-tolerated clinically. 4-HPR has been shown to augment intracellular levels of reactive oxygen species (ROS) and ceramides, and to activate the MAPK family of kinases. Pancreatic cancer cells are sensitive to increased levels of ROS, ceramides, and MAPK phosphorylation, suggesting 4-HPR will be an effective agent against this disease. Results: 4-HPR exposure markedly decreased viability of MIA PaCa-2 and PANC-1 cells, as shown by a dose-dependent decrease in absorbance from an MTS assay and a 3-fold increase in propidium iodide staining. A 50% reduction in [3H]thymidine incorporation showed that cell proliferation was halted as well. 4-HPR treatment caused a 3-fold increase in de novo ceramide production at 24 h and a time- and dose-dependent generation of ROS. 4-HPR led to a minor increase in caspase 3/7 activity and in FITC-Annexin V-positive cells. However, other measures of apoptosis were lacking, suggesting apoptosis was not the major death pathway elicited by 4-HPR. Further investigation of the mechanism of cell death suggests that the dominant pathway triggered by 4-HPR is lethal autophagy. After 4-HPR treatment, microscopic analysis of cells exposed to acridine orange showed bright red autophagic vesicles, and Western blot showed a robust increase in LC3 II protein expression. Pretreatment with the autophagy inhibitor 3-methyladenine (3-MA) or the ROS scavenger vitamin E (VE) abrogated the increase in LC3 II expression caused by 4-HPR exposure, and both 3-MA and VE partially preserved viability. A sustained, dose-dependent increase in phosphorylation of the stress kinases JNK and p38 was observed following incubation with 4-HPR, whereas phosphorylation of the mitogenic kinase ERK 1/2 decreased. Pretreatment with myriocin (an inhibitor of de novo ceramide production), 3-MA, or VE attenuated the increase in JNK and p38 phosphorylation caused by 4-HPR. Additionally, VE pretreatment blocked the decrease in ERK 1/2 phosphorylation elicited by 4-HPR. Conclusions: These data suggest that 4-HPR induces lethal autophagy in human pancreatic cancer cells by a mechanism that involves generation of ROS and engagement of the MAPK and sphingolipid signaling pathways. 4-HPR used alone or in combination with current therapies or signaling pathway modulators may be a promising avenue for future pancreatic cancer therapies. [Support: NIH/NIGMS grant no. GM077391] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1658.

Characterization of Synergistic Induction of CX3CL1/Fractalkine by TNF-α and IFN-γ in Vascular Endothelial Cells: An Essential Role for TNF-α in Post-Transcriptional Regulation of CX3CL1

CX3CL1/fractalkine, a chemokine specific to monocytes and NK cells, is induced synergistically by TNF-alpha and IFN-gamma in vascular endothelial cells. However, the mechanism for this synergism remains unclear. This study explored the hypothesis that the CX3CL1 expression is regulated at a posttranscriptional level, which may responsible for the synergism between TNF-alpha and IFN-gamma. Brief exposure of HUVECs to TNF-alpha led to a robust increase in IFN-gamma-induced CX3CL1 production. We found that TNF-alpha stabilized CX3CL1 mRNA in HUVECs stimulated with IFN-gamma. Cloning of 3' untranslated region (UTR) of CX3CL1 mRNA revealed the presence of a single copy of nonametric AU-rich element in its 3'UTR, and a luciferase reporter assay showed that a single AU-rich element is a crucial cis-element in the posttranscriptional regulation of CX3CL1. TNF-alpha treatment resulted in the phosphorylation of p38 MAPK and its downstream target, MAPK-activated protein kinase-2, but IFN-gamma did not affect the levels of MAPK and MAPK-activated protein kinase-2 phosphorylation induced by TNF-alpha. Treatment of the cells with an inhibitor of p38 MAPK accelerated the decay of CX3CL1 mRNA induced by TNF-alpha or the combination of TNF-alpha and IFN-gamma. Immunoprecipitation assay revealed that mRNA stabilizer HuR directly binds to 3'UTR of CX3CL1 mRNA. CX3CL1 expression is under control of posttranscriptional regulation, which is involved in the synergistic induction of CX3CL1 in response to the combined stimulation with TNF-alpha and IFN-gamma.

Molecular mechanism underlying the synergistic interaction between trifluorothymidine and the epidermal growth factor receptor inhibitor erlotinib in human colorectal cancer cell lines

The pyrimidine trifluorothymidine (TFT) inhibits thymidylate synthase (TS) and can be incorporated into the DNA. TFT, as part of TAS-102, is clinically evaluated in phase II studies as an oral chemotherapeutic agent. Erlotinib is a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR) that is often deregulated in colorectal cancer. This study investigated molecular mechanisms underlying the cytotoxic actions of the combination of an EGFR-tyrosine kinase inhibitor with TFT in colorectal cancer cells Caco2, WiDR, Lovo92, and Colo320. Drug interactions were examined by the sulforhodamine B assay and subsequent combination index (CI) analyses, cell cycle effects by FACS analysis of propidium iodide stained cells, Akt, MAPK and EGFR phosphorylation and expression levels by Western blotting and TS activity by the TS in situ assay. All combination schedules were synergistic in wt-EGFR expressing (but with mutated downstream pathways) WiDR and Lovo92 (CI 0.4-0.8) and very synergistic in Caco2 cells (with wt-EGFR and functional downstream pathways; CI 0.1-0.3), but in EGFR-lacking Colo320 cells, no additional activity was found (CI 1.0-1.2). Synergism was mostly related to the induction of cell cycle arrest and an erlotinib-mediated inhibition of the pro-survival signaling through Akt and MAPK that was activated (phosphorylated) by TFT. Erlotinib inhibited TS activity in EGFR-expressing cell lines, probably due to cell cycle arrest in the G(1) phase. TS activity was slightly lower in the combinations, probably due to cell cycle interference. Taken together, the combination of erlotinib with TFT seems to present a potential strategy in the field of molecular therapeutics.

Effects of Angiotensin Type I Receptor Blockade on the Cardiac Raf/MEK/ERK Cascade Activated via Adrenergic Receptors

A close interaction between adrenergic nerves and angiotensin systems has been documented. The present study was designed to investigate the mechanisms of angiotensin-receptor blocker (ARB) suppression of β-adrenergic receptor stimulation–induced cardiac hypertrophy. Chronic isoproterenol (ISO)–induced cardiac hypertrophy was inhibited in wild-type mice and AT1aR−/− mice treated with the ARB Candesartan (CV11974). Acute ISO–induced increase in phosphorylation levels of ERK MAPK was completely inhibited and increases in phosphorylation levels of p38 and JNK MAPKs were partially suppressed in both types of mice. Analysis of the activity of the small GTPase–regulating protein Raf indicated that the mechanisms by which ARB inhibits the Raf/MEK/ERK pathway under β-adrenergic receptor stimulation basically depended on changes in the binding activities of Ras (stimulatory to Raf cascade) and Rap-1 (inhibitory to Raf cascade). Binding activities of Ras and Rap-1 in the heart were markedly augmented by ISO, whereas ARB suppressed only Ras, but not Rap-1, binding activity. Raf immunoprecipitation results confirmed that ISO-induced increases in its association with total and phosphorylated forms of MEK were completely normalized by ARB. These results might provide a molecular basis for the beneficial effects of AT1-receptor antagonists on cardiac remodeling and functions in patients with sympatho-excitatory heart failure.

PTPRR Protein Tyrosine Phosphatase Isoforms and Locomotion of Vesicles and Mice

Protein tyrosine phosphatases (PTPs) are central players in many different cellular processes and their aberrant activity is associated with multiple human pathologies. In this review, we present current knowledge on the PTPRR subfamily of classical PTPs that is expressed in neuronal cells and comprises receptor-type (PTPBR7, PTP-SL) as well as cytosolic (PTPPBSγ-37, PTPPBSγ-42) isoforms. The two receptor-type isoforms PTPBR7 and PTP-SL both localize in late endosomes and the Golgi area. PTPBR7, however, is additionally localized at the cell surface and on early endosomes. During cerebellar maturation, PTPBR7 expression in developing Purkinje cells ceases and is replaced by PTP-SL expression in the mature Purkinje cells. All PTPRR isoforms contain a kinase interacting motif that makes them mitogen-activated protein kinase phosphatases. The distinct subcellular localization of the different PTPRR isoforms may reflect differential roles in growth-factor-induced MAPK-mediated retrograde signaling cascades. Studies in PTPRR-deficient mice established that PTPRR isoforms are physiological regulators of MAPK phosphorylation levels. Surprisingly, PTPRR-deficient mice display defects in motor coordination and balancing skills, while cerebellar morphological abnormalities, which are often encountered in ataxic mouse models, are absent. This is reminiscent of the phenotype observed in a handful of mouse mutants that have alterations in cerebellar calcium ion homeostasis. Elucidation of the molecular mechanisms by which PTPRR deficiency imposes impairment of cerebellar neurons and motor coordination may provide candidate molecules for hereditary cerebellar ataxias that still await identification of the corresponding disease genes.Electronic supplementary materialThe online version of this article (doi:10.1007/s12311-008-0088-y) contains supplementary material, which is available to authorized users.

Decreased p44/42 Mitogen-Activated Protein Kinase Phosphorylation in Gender- or Hormone-Related But Not during Age-Related Adrenal Gland Growth in Mice

Postnatal growth of the mouse adrenal gland shows a characteristic gender-dependent pattern, resulting in an almost 2-fold higher adrenal weight in 11-wk-old female vs. male mice. We demonstrated that the higher weight of the adrenal glands in female mice is due to a significantly (P < 0.05) increased growth rate in female mice and a shorter growth phase of the adrenal glands in male mice (P < 0.05). To address the signaling mechanisms underlying these differential growth patterns, we evaluated the phosphorylation levels of p44/42 and p38 MAPK. In female mice, age-dependent reductions of p38 MAPK phosphorylation were found between wk 3 and 9 (47% reduction; P < 0.05). At the age of 11 wk, the p38 MAPK phosphorylation level in female adrenal glands was about 60% lower than in the male counterparts (P < 0.01). Similarly, the phosphorylation level of p44/42 MAPK was 50% lower in female adrenal glands (P < 0.001). Reduced activation of p44/42 MAPK was also observed after growth stimulation of the adrenal glands in male mice after ACTH treatment (-36%; P < 0.001) or by expression of a GH transgene (-34%; P < 0.001), whereas p38 MAPK, JNK, or PDK1 activation was unaffected. From our findings in three independent mouse models where partial deactivation of p44/42 MAPK was observed under conditions of elevated growth, we suggest a function of p44/42 MAPK for adrenal growth and a role of p44/42 MAPK for the integration of different endocrine stimuli.

P-64 Decreased p44/42 MAPK phosphorylation in gender- or hormone-related but not during age-related adrenal gland growth in mice

Postnatal growth of the mouse adrenal gland shows a characteristic gender-dependent pattern, resulting in an almost two-fold higher adrenal weight in 11-week-old female versus male mice. We demonstrated that the higher weight of the adrenal glands in female mice is due to a significantly (p<0.05) increased growth rate in female mice and a shorter growth phase of the adrenal glands in male mice (p<0.05). To address the signaling mechanisms underlying these differential growth patterns, we evaluated the phosphorylation levels of p44/42 and p38 MAPK. In female mice, age dependent reductions of p38 MAPK phosphorylation were found between week 3 and week 9 (47% reduction; p<0.05). At the age of 11 weeks the p38 MAPK phosphorylation level in female adrenal glands was about 60% lower than in the male counterparts (p<0.01). Similarly the phosphorylation level of p44/42 MAPK was 50% lower in female adrenal glands (p<0.001). Reduced activation of p44/42 MAPK was also observed after growth stimulation of the adrenal glands in male mice after ACTH treatment (-36%; p<0.001) or by expression of a growth hormone transgene (-34%; p<0.001), while p38 MAPK, JNK or PDK1 activation were unaffected. From our findings in three independent mouse models where partial deactivation of p44/42 MAPK was observed under conditions of elevated growth we suggest a function of p44/42 MAPK for adrenal growth and a role of p44/42 MAPK for the integration of different endocrine stimuli.

The role of p38 mitogen-activated kinase (MAPK) in the mechanism regulating cyclooxygenase gene expression in equine leukocytes

The goal of this study was to define the role for p38 mitogen-activated kinase (MAPK) in the signaling mechanism regulating pro-inflammatory cyclooxygenase (COX) gene expression in lipopolysaccharide (LPS)-activated equine leukocytes for the purposes of identifying novel targets for anti-inflammatory therapy in endotoxemic horses. The p38 MAPK has been shown to positively regulate inflammatory gene expression in human leukocytes and can be activated by a variety of stimuli including LPS, TNF-α, and IL-1. Activation-associated phosphorylated p38 MAPK has been implicated in the up-regulation of several inflammatory genes, including COX-2 which ultimately results in the production of prostanoids that are responsible for the pathophysiology associated with endotoxemia. Our hypothesis is that activation of p38 MAPK is essential for LPS-induced COX-2 expression in equine peripheral blood leukocytes. We tested our hypothesis by investigating the effects of the specific p38 MAPK inhibitors SB203580 and SB202190 on LPS-induced COX-2 protein expression and PGE 2 production in equine leukocytes. LPS stimulation activated p38 MAPK and increased COX-2 expression in a dose-dependent manner with maximal activation observed after 30 min and 4 h, respectively, at a concentration of 10 ng/ml LPS. In contrast, LPS stimulation did not affect COX-1 protein expression. Pretreatment with SB203580 or SB202190 significantly inhibited LPS-induced activation-associated p38 MAPK phosphorylation, COX-2 mRNA and protein levels, and PGE 2 production in equine leukocytes. Maximal inhibition of LPS-induced COX-2 protein expression was achieved at a concentration of 10 μM SB203580. We concluded that p38 MAPK is essential for LPS-induced COX-2 expression suggesting that p38 MAPK is a potential target for anti-inflammatory therapy during equine endotoxemia.

Oncogenic KRAS Activates Hedgehog Signaling Pathway in Pancreatic Cancer Cells

Hedgehog (Hh) signaling is deregulated in multiple human cancers including pancreatic ductal adenocarcinoma (PDA). Because KRAS mutation represents one of the earliest genetic alterations and occurs almost universally in PDA, we hypothesized that oncogenic KRAS promotes pancreatic tumorigenesis in part through activation of the Hh pathway. Here, we report that oncogenic KRAS activates hedgehog signaling in PDA cells, utilizing a downstream effector pathway mediated by RAF/MEK/MAPK but not phosphatidylinositol 3-kinase (PI3K)/AKT. Oncogenic KRAS transformation of human pancreatic ductal epithelial cells increases GLI transcriptional activity, an effect that is inhibited by the MEK-specific inhibitors U0126 and PD98059, but not by the PI3K-specific inhibitor wortmannin. Inactivation of KRAS activity by a small interfering RNA specific for oncogenic KRAS inhibits GLI activity and GLI1 expression in PDA cell lines with activating KRAS mutation; the MEK inhibitors U0126 and PD98059 elicit a similar response. In addition, expression of the constitutively active form of BRAF(E600), but not myr-AKT, blocks the inhibitory effects of KRAS knockdown on Hh signaling. Finally, suppressing GLI activity leads to a selective attenuation of the oncogenic transformation activity of mutant KRAS-expressing PDA cells. These results demonstrate that oncogenic KRAS, through RAF/MEK/MAPK signaling, is directly involved in the activation of the hedgehog pathway in PDA cells and that collaboration between these two signaling pathways may play an important role in PDA progression.

キナーゼプロファイリングのための新しいリン酸アフィニティー電気泳動法

We developed a novel type of phosphate-affinity polyacrylamide gel electrophoresis using an alkoxide-bridged dinuclear manganese(II) complex as a phosphate-binding tag, Phos-tag. The phosphate-affinity site is a polyacrylamide-bound Mn2+-Phos-tag that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart in an SDS-PAGE gel and the quantitative analysis of kinase reactions. Herein, we describe three applications of protein kinase profiling using the phosphate-affinity electrophoresis (Mn2+-Phos-tag SDS-PAGE). The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein status. The activity profiles of six kinds of kinases were determined using a substrate, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time-course changes in the EGF-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting analysis. The final application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl, was determined using the substrate Abltide-GST and the approved inhibitor Glivec. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the monophosphorylated and nonphosphorylated Abltide-GST bands.

Influences of Lovastatin on membrane ion flow and intracellular signaling in breast cancer cells

Lovastatin, an inhibitor of cellular cholesterol synthesis, has an apparent anti-cancer property, but the detailed mechanisms of its anti-cancer effects remain poorly understood. We investigated the molecular mechanism of Lovastatin anti-tumor function through the study of its effect on membrane ion flow, gap junctional intercellular communication (GJIC), and the pathways of related signals in MCF-7 mammary cancer cells. After treatment for 24–72 h with 4, 8 or 16 μmol/L Lovastatin, cellular proliferation was examined via the MTT assay, and changes in membrane potential and cellular [Ca2+]i were monitored using confocal laser microscopy. In addition, the expression of plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA was analyzed via RT-PCR, the GJIC function was examined using the scrape-loading dye transfer (SLDT) technique, and MAPK phosphorylation levels were tested with the kinase activity assay. The results showed that Lovastatin treatment significantly inhibited the growth of MCF-7 breast cancer cells. It also increased the negative value of the membrane potential, leading to the hyperpolarization of cells. Moreover, Lovastatin treatment continuously enhanced [Ca2+]i, although the levels of PMCA1 mRNA were unchanged. GJIC was also upregulated in MCF-7 cells, with transfer of LY Fluorescence reaching 4 to 5 rows of cells from the scraped line after treatment with 16 μmol/L Lovastatin for 72 h. Finally, downregulation of ERK1 and p38MAPK phosphorylation were found in Lovastatin-treated MCF-7 cells. It could be deduced that Lovastatin can induce changes in cellular hyperpolarization and intracellular Ca2+ distributions, and increase GJIC function. These effects may result in changes in the downstream signal cascade, inhibiting the growth of MCF-7 cells.

VEGF-C promotes survival in podocytes

Vascular endothelial growth factor (VEGF)-A is an autocrine survival factor for podocytes, which express two VEGF receptors, VEGF-R1 and VEGF-R3. As VEGF-A is not a known ligand for VEGF-R3, the aim of this investigation was to examine whether VEGF-C, a known ligand for VEGF-R3, served a function in podocyte biology and whether this was VEGF-R3 dependent. VEGF-C protein expression was localized to podocytes in contrast to VEGF-D, which was expressed in parietal epithelial cells. Intracellular calcium ([Ca2+]i) experiments demonstrated that VEGF-C induced a 0.74+/-0.09-fold reduction in [Ca2+]i compared with baseline in human conditionally immortalized podocytes (hCIPs; P<0.05, one sample t-test, n=8). Cytotoxicity experiments revealed that in hCIPs VEGF-C reduced cytotoxicity to 81.4+/-1.9% of serum-starved conditions (P<0.001, paired t-test, n=16), similar to VEGF-A (82.8+/-4.5% of serum-starved conditions, P<0.05, paired t-test). MAZ51 (a VEGF-R3 kinase inhibitor) inhibited the VEGF-C-induced reduction in cytotoxicity (106.2+/-2.1% of serum-starved conditions), whereas MAZ51 by itself had no cytotoxic effects on hCIPs. VEGF-C was also shown to induce a 0.5+/-0.13-fold reduction in levels of MAPK phosphorylation compared with VEGF-A and VEGF-A-Mab treatment (P<0.05, ANOVA, n=4), yet had no effect on Akt phosphorylation. Surprisingly, immunoprecipitation studies detected no VEGF-C-induced autophosphorylation of VEGF-R3 in hCIPs but did so in HMVECs. Moreover, SU-5416, a tyrosine kinase inhibitor, blocked the VEGF-C-induced reduction in cytotoxicity (106+/-2.8% of serum-starved conditions) at concentrations specific for VEGF-R1. Together, these results suggest for the first time that VEGF-C acts in an autocrine manner in cultured podocytes to promote survival, although the receptor or receptor complex activated has yet to be elucidated.