Down-regulation Of ERK Phosphorylation Research Articles

Tumor-Promoting Role of GNA14 in Colon Cancer Development.

Recent studies have shown that mutations in members of the G-protein α family contribute to the onset and progression of cancer. However, the role of GNA14 in CRC remains unknown. In this study, we examined the effect of GNA14 on CRC through genetic approaches in vitro and in vivo. We found that GNA14 knockdown by small interfering RNA (siRNA) inhibited the proliferation of CRC cells SW403 and HT29. Gna14 knockout mice developed normally without obvious abnormalities. However, the number of polyps in the small intestine was significantly reduced in Gna14 knockout mice compared to control mice after mating with ApcMin mice, a representative CRC mouse model. In particular, deletion of the Gna14 inhibited polyp growth, especially in the distal end of the small intestine. Histological examination showed that Gna14 knockout mice suppressed malignant tumor progression due to decreased proliferation and increased apoptosis in polyps compared to controls. In addition, GNA14 knockdown in CRC cells resulted in downregulation of ERK phosphorylation and β-catenin and β-catenin phosphorylation at S675. Similarly, ERK phosphorylation and phospho-β-catenin phosphorylation at S675 were decreased in polyps of Gna14 knockout mice. Collectively, these analyses show that GNA14 may accelerate CRC cell proliferation and malignant tumor progression through ERK and β-catenin pathways.

PGC7 Regulates Genome-Wide DNA Methylation by Regulating ERK-Mediated Subcellular Localization of DNMT1.

DNA methylation is an epigenetic modification that plays a vital role in a variety of biological processes, including the regulation of gene expression, cell differentiation, early embryonic development, genomic imprinting, and X chromosome inactivation. PGC7 is a maternal factor that maintains DNA methylation during early embryonic development. One mechanism of action has been identified by analyzing the interactions between PGC7 and UHRF1, H3K9 me2, or TET2/TET3, which reveals how PGC7 regulates DNA methylation in oocytes or fertilized embryos. However, the mechanism by which PGC7 regulates the post-translational modification of methylation-related enzymes remains to be elucidated. This study focused on F9 cells (embryonic cancer cells), which display high levels of PGC7 expression. We found that both knockdown of Pgc7 and inhibition of ERK activity resulted in increased genome-wide DNA methylation levels. Mechanistic experiments confirmed that inhibition of ERK activity led to the accumulation of DNMT1 in the nucleus, ERK phosphorylated DNMT1 at ser717, and DNMT1 Ser717-Ala mutation promoted the nuclear localization of DNMT1. Moreover, knockdown of Pgc7 also caused downregulation of ERK phosphorylation and promoted the accumulation of DNMT1 in the nucleus. In conclusion, we reveal a new mechanism by which PGC7 regulates genome-wide DNA methylation via phosphorylation of DNMT1 at ser717 by ERK. These findings may provide new insights into treatments for DNA methylation-related diseases.

Topical application of imatinib mesylate suppresses vitamin D3 analog-induced dermatitis in Balb/c mice.

Atopic dermatitis (AD) is an allergic disease mediated by Th2 cells. In AD, externally stimulated keratinocytes release inflammatory cytokines, such as IL-33 and TSLP. Inflammatory cells infiltrate skin tissue and increase vascular permeability. Therefore, we hypothesized that imatinib mesylate (IMT), which suppresses vascular permeability, may be a candidate therapeutic agent for AD. A vitamin D3 analog (MC903) was administered daily to both ears of Balb/c mice to create a murine AD model to which IMT was applied. The skin lesions were evaluated histopathologically and by immunostaining. Cytokine expression in the skin was assessed by using real-time polymerase chain reaction (PCR) and immunostaining and was investigated using Evans Blue to determine whether IMT suppressed vascular permeability due to histamine. The suppressive effect of TNF-α/IL-4-induced TSLP expression in primary mouse keratinocytes (MKCs) treated with IMT was then investigated. Tslp gene and protein expression in the lesion was measured using real-time PCR and ELISA. The activation of signal transduction was analysed by western blotting. Topical application of IMT significantly reduced ear thickness, Evans blue leakage, and scratch onset. IMT suppressed the number of infiltrating cells (CD4+ T cells, eosinophils, and basophils), and the expression of IL-13, IL-33, and TSLP in a MC903-induced, murine AD model and inhibited TNF-α/IL-4-induced TSLP expression via downregulation of ERK phosphorylation in MKCs. IMT reduced the skin symptoms in a MC903-induced, murine AD model, suggesting that it may have potential as a new treatment for AD.

Bilirubin represents a negative regulator of ILC2 in allergic airway inflammation.

Group 2 innate lymphoid cells (ILC2s) play an important role in allergic airway inflammation. Despite recent advances in defining molecular mechanisms that control ILC2 development and function, the role of endogenous metabolites in the regulation of ILC2s remains poorly understood. Herein, we demonstrated that bilirubin, an end product of heme catabolism, was a potent negative regulator of ILC2s. Bilirubin metabolism was found to be significantly induced during airway inflammation in mouse models. The administration of unconjugated bilirubin (UCB) dramatically suppressed ILC2 responses to interleukin (IL)-33 in mice, including cell proliferation and the production of effector cytokines. Furthermore, UCB significantly alleviated ILC2-driven airway inflammation, which was aggravated upon clearance of endogenous UCB. Mechanistic studies showed that the effects of bilirubin on ILC2s were associated with downregulation of ERK phosphorylation and GATA3 expression. Clinically, newborns with hyperbilirubinemia displayed significantly lower levels of ILC2 with impaired function and suppressed ERK signaling. Together, these findings indicate that bilirubin serves as an endogenous suppressor of ILC2s and might have potential therapeutic value in the treatment of allergic airway inflammation.

Glycolytic inhibitor 2-deoxyglucose suppresses inflammatory response in innate immune cells and experimental staphylococcal endophthalmitis

Previously, we have shown that Staphylococcus (S) aureus induces a glycolytic response in retinal residential (microglia) and infiltrated cells (neutrophils and macrophages) during endophthalmitis. In this study, we sought to investigate the physiological role of glycolysis in bacterial endophthalmitis using a glycolytic inhibitor, 2-deoxyglucose (2DG). Our data showed that 2DG treatment attenuated the inflammatory responses of mouse bone marrow-derived macrophages (BMDM) and neutrophils (BMDN) when challenged with either live or heat-killed S. aureus (HKSA). Among the inflammatory mediators, 2DG caused a significant reduction in levels of cytokines (TNF-α, IL-1β, IL-6) and chemokines (CXCL1 and CXCL2). Western blot analysis of 2DG treated cells showed downregulation of bacterial-induced MEK/ERK pathways. In vivo, intravitreal administration of 2DG both pre- and post-bacterial infection resulted in a significant reduction in intraocular inflammation in C57BL/6 mouse eyes and downregulation of ERK phosphorylation in retinal tissue. Collectively, our study demonstrates that 2DG attenuates inflammatory response in bacterial endophthalmitis and cultured innate immune cells via inhibition of ERK signaling. Thus glycolytic inhibitors in combination with antibiotics could mitigate inflammation-mediated tissue damage in ocular infections.

Abstract 2351: Characterization of Neutrophil elastase uptake in breast cancer: implications for immunotherapy

Abstract Neutrophil Elastase (NE)_a serine protease released by neutrophils in the tumor microenvironment_induces invasion and metastasis. We demonstrated that while NE is not endogenously expressed in breast cancer cells, breast cancer cells (BrCA) take up exogenous NE derived from tumor-associated neutrophils. NE uptake results in the expression of the HLA-A2-restricted peptides CCNE1 and PR1, derived from cyclin E and NE, respectively, on the surface of triple-negative (TN) BrCA cells (MDA-MB-231). Expression of these peptides/HLA-A2 molecules induces BrCA cell susceptibility to cytolysis by CCNE1- and PR1-specific cytotoxic T lymphocytes (CTLs), and to 8F4, a monoclonal antibody that binds specifically to the PR1/HLA-A2 complex. We hypothesize that NE uptake is a receptor-mediated process that results in cross-presentation of NE-derived peptides on HLA molecules of BrCA cells. In this study, we found that NE uptake in BrCA is specific, time and dose-dependent, and become saturated, suggesting a receptor-mediated uptake mechanism. We showed that MDA-MB-231 did not take up cathepsin G, a related serine protease, suggesting specificity of NE receptor uptake. NE internalization was partially blocked by chlorpromazine and by wortmannin, implicating clathrin-dependent uptake and PI3Kinase-dependence, respectively. Confocal microscopy showed that NE was co-localized with the early endosome marker, EEA-1, as early as 10 minutes after uptake. Flow cytometry indicated that surface-bound NE on MDA-MB-231 cells decreased after five minutes, and Western blot showed a simultaneous decrease of MAPKinases phosphorylation (p-Erk, p-p38) and loss of IRS-1 signaling. Inhibition of NE enzyme activity by serine proteases inhibitors, such as elafin or PMSF, potentiated NE uptake in BrCA cells, indicating that enzyme activity is not required for uptake. Conclusion: The results support a novel mechanism of rapid receptor-mediated uptake of soluble exogenous NE by TN BrCA. NE uptake is efficient, PI3 kinase-dependent, sensitive to clathrin inhibition, and associated with down-regulation of Erk phosphorylation and IRS-1. In addition, uptake does not require NE enzyme activity. Following uptake, NE co-localizes to an early endosomal compartment, an organelle associated with HLA class I peptide loading. Since we previously linked NE uptake to CCNE1 and PR1 peptide presentation on MDA-MB-231 cells, which enhanced BrCA susceptibility to immunotherapies that target CCNE1 and PR1, our results here strongly suggest receptor-mediated NE uptake that could regulate HLA class I peptide presentation. Understanding the mechanisms that regulate NE uptake and peptide presentation on the surface of BrCA will help us develop new strategies to enhance peptide presentation by BrCA, potentially improving the response of BrCA and other tumor types to antigen-specific immunotherapies. Citation Format: Celine Kerros, Satyendra C. Tripathi, Anne V. Philips, Gheath Al-Atrash, Kathryn E. Ruisaard, Karen C. Dwyer, Elizabeth A. Mittendorf, Samir Hanash, Jeffrey J. Molldrem. Characterization of Neutrophil elastase uptake in breast cancer: implications for immunotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2351. doi:10.1158/1538-7445.AM2015-2351

Abstract P5-04-09: Characterization of neutrophil elastase receptor in breast cancer: Implication for immunotherapy

Abstract Neutrophil Elastase (NE), a serine protease released by tumor-associated neutrophils in the tumor microenvironment induces invasion and metastasis. We have demonstrated that NE is not endogenously expressed in breast cancer (BrCA), but is taken up by BrCA cells. NE uptake results in the expression of the HLA-A2-bound peptides CCNE1 and PR1, derived from cyclin E and NE, respectively, on the surface of triple-negative (TN) BrCA cells (MDA-MB-231). Expression of these peptide/HLA-A2 molecules induces BrCA cell susceptibility to cytolysis by CCNE1- and PR1-specific cytotoxic T lymphocytes (CTLs), and to 8F4, a monoclonal antibody that binds specifically to the PR1/HLA-A2 complex. We hypothesize that NE uptake is a receptor-mediated process that results in cross-presentation of NE-derived peptides on HLA molecules of BrCA cells. Here, we found that NE uptake is specific, time- and dose-dependent, and saturable, suggesting a receptor-mediated uptake mechanism. We showed that MDA-MB-231 did not take up cathepsin G, a related serine protease, suggesting specificity of receptor uptake. NE internalization was partially blocked by chlorpromazine and by wortmannin, suggesting clathrin-dependent uptake and PI3Kinase-dependence, respectively. Confocal microscopy showed that NE was colocalized with the early endosome marker, EEA-1, as early as 10 min after uptake. Flow cytometry indicated that surface-bound NE on MDA-MB-231 cells decreased after 5 minutes, and both flow cytometry and Western blot showed a simultaneous decrease of phospho-Erk and loss of IRS-1 signaling. Inhibition of NE enzyme activity by elafin or PMSF potentiated NE uptake in BrCA cells, thus enzyme activity is not required for uptake. Conclusion: The results support a novel mechanism of rapid receptor-mediated uptake of soluble exogenous NE by TN BrCA. NE uptake is efficient, PI3 kinase-dependent, sensitive to clathrin inhibition, and associated with down-regulation of Erk phosphorylation and IRS-1. In addition, uptake does not require NE enzyme activity. Following uptake, NE colocalizes to an early endosomal compartment, an organelle associated with peptide loading of MHC-I molecules for expression of the cell surface. We previously showed that the NE-derived peptide PR1 is cross-presented on MDA-MB-231 cells after NE uptake, leading to susceptibility to immunotherapies that target PR1/HLA-A2. Taken together, our results demonstrate receptor-mediated NE uptake, which is a potentially novel paradigm that could vastly expand the number of tumor-associated antigens that could be targeted on BrCA. An understanding of the mechanism that mediates NE uptake will help us develop strategies to increase expression of immunogenic peptides on cancer cells and to guide the design of targeted immunotherapies against CCNE1 and PR1 as new clinical therapies for BrCA. Citation Format: Celine Kerros, Satyendra C Tripathi, Anne V Philips, Gheath Al-Atrash, Kathryn E Ruisaard, Karen C Dwyer, Elizabeth A Mittendorf, Samir M Hanash, Jeffrey J Molldrem. Characterization of neutrophil elastase receptor in breast cancer: Implication for immunotherapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-04-09.

MiRNA-130b is required for the ERK/FOXM1 pathway activation-mediated protective effects of isosorbide dinitrate against mesenchymal stem cell senescence induced by high glucose

The present study was carried out to investigate the hypothesis that organic nitrates can attenuate the senescence of mesenchymal stem cells (MSCs), a superior cell source involved in the regeneration and repair of damaged tissue. MSCs were treated with high glucose (HG) in order to induce senescence, which was markedly attenuated by pre-treatment with isosorbide dinitrate (ISDN), a commonly used nitrate, as indicated by senescence-associated galactosidase (SA-β-gal) activity, p21 expression, as well as by the mRNA levels of DNA methyltransferase 1 (DNMT1) and differentiated embryo chondrocyte expressed gene 1 (DEC1), which are senescence-related biomarkers. It was also found that the senescent MSCs (induced by HG glucose) exhibited a marked downregulation in ERK activity and forkhead box M1 (FOXM1) expression, which was reversed by ISDN preconditioning. Of note, the inhibition of ERK phosphorylation or the downregulation of FOXM1 statistically abolished the favourable effects of ISDN. In addition, the investigation of the senescence-associated miR-130 family suggested that miR-130b mediates the beneficial effects of ISDN; it was found that the protective effects of ISDN against the senescence of MSCs were prominently reversed by the knockdown of miR-130b. Furthermore, the downregulation of ERK phosphorylation or FOXM1 expression decreased the miR-130b expression level; however, the suppression of miR-130b demonstrated no significant impact on ERK phosphorylation or FOXM1 expression. Taken together, to the best of our knowledge, the present study is the first to demonstrate the favourable effects of ISDN against HG-induced MSC senescence, which are mediated through the activation of the ERK/FOXM1 pathway and the upregulation of miR-130b.

Design and Synthesis of Active Site Inhibitors of the Human Farnesyl Pyrophosphate Synthase: Apoptosis and Inhibition of ERK Phosphorylation in Multiple Myeloma Cells

Human farnesyl pyrophosphate synthase (hFPPS) controls intracellular levels of FPP and post-translational prenylation of small GTPase proteins, which are essential for cell signaling and cell proliferation. Clinical investigations provide evidence that N-BP inhibitors of hFPPS are disease modifying agents that improve survival of multiple myeloma (MM) patients via mechanisms unrelated to their skeletal effects. A new series of N-BPs was designed that interact with a larger portion of the GPP subpocket, as compared to the current therapeutic drugs, and rigidify the (364)KRRK(367) tail of hFPPS in the closed conformation in the absence of IPP. An analogue of this series was used to demonstrate inhibition of the intended biological target, resulting in apoptosis and down-regulation of ERK phosphorylation in human MM cell lines.

Abstract 684: The difference between gefitinib and serum deprivation in cancer cells

Abstract Gefitinib is a selective inhibitor of epidermal growth factor receptor tyrosine kinase. The serum free condition could resemble to gefitinib exposure since it blocks EGF stimuli. In this study, we examined distinction between gefitinib exposure and serum deprivation in cultured cancer cells. At first, we verified that ERK phosphorylation was downregulated by gefitinib exposure in A431 and A549 cells. We examined the phosphorylation status of ERK under the serum deprivation condition. The phosphorylation of ERK in A431 cells was downregulated after 1hr culture in serum free medium, while after 3-6hr ERK was phosphorylated again. We hypothesized that an auto- or paracrine mechanism would work to phosphorylate ERK. In the cells cultured with the conditioned serum free medium from the dish in which A431 cultured for 24hr, the downregulation of ERK phosphorylation was weaker than with the fresh serum free medium. We confirmed that gefitinib could inhibit the ERK phosphorylation under the serum free condition. Exogenous EGF attenuated the inhibitory effect on ERK phosphorylation with serum free medium. These results suggest EGF may be one of the auto- or paracrine substance that induces ERK phosphorylation. Since the ERK phosphorylation status affects the stability of c-MYC, we also examined whether “gefitinib” and “serum deprivation” affect the expression of c-MYC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 684. doi:10.1158/1538-7445.AM2011-684

Human p38δ MAP kinase mediates UV irradiation induced up-regulation of the gene expression of chemokine BRAK/CXCL14

The mitogen-activated protein kinase (MAPK) family comprises ERK, JNK, p38 and ERK5 (big-MAPK, BMK1). UV irradiation of squamous cell carcinoma cells induced up-regulation of gene expression of chemokine BRAK/CXCL14, stimulated p38 phosphorylation, and down-regulated the phosphorylation of ERK. Human p38 MAPKs exist in 4 isoforms: p38α, β, γ and δ. The UV stimulation of p38 phosphorylation was not inhibited by the presence of SB203580 or PD169316, inhibitors of p38α and β, suggesting p38 phosphorylation was not dependent on these 2 isoforms and that p38γ and/or δ was responsible for the phosphorylation. In fact, inhibition of each of these 4 p38 isoforms by the introduction of short hairpin (sh) RNAs for respective isoforms revealed that only shRNA for p38δ attenuated the UV-induced up-regulation of BRAK/CXCL14 gene expression. In addition, over-expression of p38 isoforms in the cells showed the association of p38δ with ERK1 and 2, concomitant with down-regulation of ERK phosphorylation. The usage of p38δ isoform by UV irradiation is not merely due to the abundance of this p38 isoform in the cells. Because serum deprivation of the cells also induced an increase in BRAK/CXCL14 gene expression, and in this case p38α and/or β isoform is responsible for up-regulation of BRAK/CXCL14 gene expression. Taken together, the data indicate that the respective stress-dependent action of p38 isoforms is responsible for the up-regulation of the gene expression of the chemokine BRAK/CXCL14.

Abstract 3974: TIAF1 blocks p53 suppression of SMAD promoter activation

Abstract p53 participates in the TGF-β signaling. How p53 regulates SMAD gene transcription is largely unknown. TIAF1 is a TGF-β1-induced factor and a downstream effector of the TGF-β signaling. TIAF1 is known to control nuclear localization of Ser15-phosphorylated p53. Ectopic TIAF1 induces growth inhibition and apoptosis of monocytic U937 and many non-fibroblasts, via upregulation of p53, p21, and Smad2/4, but downregulation of ERK phosphorylation. Wild type p53 suppresses the transcriptional activation of SMAD gene, whereas isoform p53γ induces the gene activation. TGF-β1 increases the physical association of TIAF1 with Smad4, and when overexpressed, TIAF1 inhibits Smad4-dependent promoter activation. In combination, TIAF1 and p53 support the promoter activation regulated by endogenous or ectopic Smad4, indicating that TIAF1 blocks p53 suppression of the SMAD promoter activation, and vice versa. Without Ser46 phosphorylation in p53 (p53ΔS46), p53ΔS46 alone, or in the co-presence of TIAF1, abolishes the Smad4-regulated promoter activation. These observations suggest a critical role of Ser46 in p53 in controlling SMAD transcriptional activation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 3974.

GRK2-Dependent Desensitization Downstream of G Proteins

G protein-coupled receptor kinases (GRKs) are serine/threonine kinases first discovered by its role in receptor desensitization. Phosphorylation of the C-terminal tail of GPCRs by GRKs triggers the docking of β-arrestins and the functional uncoupling of G proteins and receptors. In addition, we and others have uncovered new direct ways by which GRKs could impinge into intracellular signalling pathways independently of receptor phosphorylation. In particular, we have characterized that elevated GRK2 levels can reduce CCR2-mediated activation of the ERK MAPK route in a manner that is independent of kinase activity and also of G proteins. This inhibition of ERK occurred in the absence of any reduction on MEK phosphorylation, what implicates that GRK2 is acting at the level of MEK or at the MEK-ERK interface to achieve a downregulation of ERK phosphorylation. In fact, we describe here that a direct association between GRK2 and MEK proteins can be detected in vitro. p38 MAPK pathway also appears to be regulated directly by GRK2 in a receptor-independent manner. p38 can be phosphorylated by GRK2 in threonine 123, a residue sitting at the entrance of a docking groove by which this MAPK associates to substrates and upstream activators. The T123phospho-mimetic mutant of p38 shows a reduced ability to bind to MKK6, concomitant with an impaired p38 activation, and a decreased phosphorylation of downstream substrates such as MEF2, MK2 and ATF2. Elevated levels of GRK2 downregulate p38-dependent cellular responses, such as differentiation of preadipocytic cells, while LPS-induced cytokine release is enhanced in macrophages from GRK2 (+/−) mice. In sum, we describe in this article different ways by which GRK2 directly regulates MAPK-mediated cellular events. This regulation of the MAPK modules by GRK2 could be relevant in pathological situations where the levels of this kinase are altered, such as during inflammatory diseases or cardiovascular pathologies.

TIAF1 and p53 functionally interact in mediating apoptosis and silencing of TIAF1 abolishes nuclear translocation of serine 15-phosphorylated p53.

TIAF1 is a TGF-beta 1-induced factor that protects L929 fibroblasts from TNF-mediated apoptosis. In contrast, overexpressed TIAF1 induces growth inhibition and apoptosis of monocytic U937 and various nonfibroblast cells. TIAF1-mediated apoptosis of U937 cells involves upregulation of p53, p21, and Smad2/4, but downregulation of ERK phosphorylation. To determine whether p53 and TIAF1 functionally interact in regulating cell death, ectopic TIAF1 and p53 were shown to induce apoptosis of U937 cells in both synergistic and antagonistic manners. At optimal levels both TIAF1 and p53 mediated apoptosis cooperatively. Also, both proteins suppressed adherence-independent growth of L929 cells. In contrast, initiation of apoptosis by overexpressed TIAF1 was blocked by low doses of p53, and vice versa. Furthermore, ectopic p53 blocked an ongoing apoptosis in U937 cells stably expressing TIAF1. Yeast two-hybrid analyses failed to demonstrate the binding of p53 with TIAF1, suggesting an unidentified protein that links the p53/TIFA1 interaction. Suppression of TIAF1 expression by siRNA could not inhibit Ser15 phosphorylation in p53 in response to UV and etoposide. However, nuclear translocation of these Ser15-phosphorylated p53 was significantly reduced in TIAF1-silenced cells. Taken together, TIAF1 and p53 functionally interact in regulating apoptosis, and TIAF1 is likely to participate in the nuclear translocation of activated p53.