Mitogen-associated Protein Kinase Pathway Research Articles

SVCAM1 in the Hippocampus Contributes to Postoperative Cognitive Dysfunction in Mice by Inducing Microglial Activation Through the VLA-4 Receptor.

Postoperative cognitive dysfunction (POCD) is a severe postsurgical complication, but its underlying mechanisms remain unclear. Neuroinflammation mediated by microglial activation plays a major role in POCD pathophysiology. Upregulation of vascular cell adhesion molecule 1 (VCAM1) on brain endothelial cells is closely correlated with microglial activation in the mouse hippocampus. However, the role of VCAM1 upregulation in microglial activation remains unknown. Soluble VCAM1 (sVCAM1) activates the very late antigen-4 (VLA-4) receptor under inflammatory conditions. Therefore, we hypothesized that sVCAM1 which is shed from VCAM1 contributes to POCD by triggering hippocampal microglial activation through the VLA-4 receptor. We found that VCAM1 and sVCAM1 expression in the mouse hippocampus was upregulated after surgery, and the upregulation was accompanied by hippocampal microglial activation. sVCAM1 levels in mouse and human serum were increased after surgery. Anti-VCAM1 treatment inhibited microglial activation, proinflammatory cytokine production, VLA-4 expression and P38 mitogen-associated protein kinase (MAPK) pathway activation and attenuated hippocampal-dependent cognitive dysfunction. In vitro, recombinant sVCAM1 promoted M1 polarization in BV2 cells, increased VLA-4 expression and activated the P38 MAPK pathway. These effects were reversed by VLA-4 receptor blockade. Anti-VLA-4 treatment ameliorated hippocampal-dependent cognitive dysfunction after surgery by inhibiting microglial activation, proinflammatory cytokine production and P38 pathway activation. In conclusion, increased sVCAM1 in the hippocampus is involved in microglial activation and cognitive dysfunction induced by surgery. Inhibiting the sVCAM1-VLA-4 interaction in microglia may be a therapeutic strategy for POCD.

Changes in the Transcriptome and Chromatin Landscape in BRAFi-Resistant Melanoma Cells.

Metastatic and drug-resistant melanoma are leading causes of skin cancer–associated death. Mitogen-associated protein kinase (MAPK) pathway inhibitors and immunotherapies have provided substantial benefits to patients with melanoma. However, long-term therapeutic efficacy has been limited due to emergence of treatment resistance. Despite the identification of several molecular mechanisms underlying the development of resistant phenotypes, significant progress has still not been made toward the effective treatment of drug-resistant melanoma. Therefore, the identification of new targets and mechanisms driving drug resistance in melanoma represents an unmet medical need. In this study, we performed unbiased RNA-sequencing (RNA-seq) and assay for transposase-accessible chromatin with sequencing (ATAC-seq) to identify new targets and mechanisms that drive resistance to MAPK pathway inhibitors targeting BRAF and MAPK kinase (MEK) in BRAF-mutant melanoma cells. An integrative analysis of ATAC-seq combined with RNA-seq showed that global changes in chromatin accessibility affected the mRNA expression levels of several known and novel genes, which consequently modulated multiple oncogenic signaling pathways to promote resistance to MAPK pathway inhibitors in melanoma cells. Many of these genes were also associated with prognosis predictions in melanoma patients. This study resulted in the identification of new genes and signaling pathways that might be targeted to treat MEK or BRAF inhibitors resistant melanoma patients. The present study applied new and advanced approaches to identify unique changes in chromatin accessibility regions that modulate gene expression associated with pathways to promote the development of resistance to MAPK pathway inhibitors.

Small Extracellular Vesicles From Brown Adipose Tissue Mediate Exercise Cardioprotection.

Long-term exercise provides reliable cardioprotection via mechanisms still incompletely understood. Although traditionally considered a thermogenic tissue, brown adipose tissue (BAT) communicates with remote organs (eg, the heart) through its endocrine function. BAT expands in response to exercise, but its involvement in exercise cardioprotection remains undefined. This study investigated whether small extracellular vesicles (sEVs) secreted by BAT and their contained microRNAs (miRNAs) regulate cardiomyocyte survival and participate in exercise cardioprotection in the context of myocardial ischemia/reperfusion (MI/R) injury. Four weeks of exercise resulted in a significant BAT expansion in mice. Surgical BAT ablation before MI/R weakened the salutary effects of exercise. Adeno-associated virus 9 vectors carrying short hairpin RNA targeting Rab27a (a GTPase required for sEV secretion) or control viruses were injected in situ into the interscapular BAT. Exercise-mediated protection against MI/R injury was greatly attenuated in mice whose BAT sEV secretion was suppressed by Rab27a silencing. Intramyocardial injection of the BAT sEVs ameliorated MI/R injury, revealing the cardioprotective potential of BAT sEVs. Discovery-driven experiments identified miR-125b-5p, miR-128-3p, and miR-30d-5p (referred to as the BAT miRNAs) as essential BAT sEV components for mediating cardioprotection. BAT-specific inhibition of the BAT miRNAs prevented their upregulation in plasma sEVs and hearts of exercised mice and attenuated exercise cardioprotection. Mechanistically, the BAT miRNAs cooperatively suppressed the proapoptotic MAPK (mitogen-associated protein kinase) pathway by targeting a series of molecules (eg, Map3k5, Map2k7, and Map2k4) in the signaling cascade. Delivery of BAT sEVs into hearts or cardiomyocytes suppressed MI/R-related MAPK pathway activation, an effect that disappeared with the combined use of the BAT miRNA inhibitors. The sEVs secreted by BAT participate in exercise cardioprotection via delivering the cardioprotective miRNAs into the heart. These results provide novel insights into the mechanisms underlying the BAT-cardiomyocyte interaction and highlight BAT sEVs and their contained miRNAs as alternative candidates for exercise cardioprotection.

Targeting BRAF and RAS in Colorectal Cancer.

Simple SummaryIn colorectal cancer, mutations of the KRAS and BRAF genes are quite common and can contribute to the activation of cell signaling pathways that lead to cell proliferation and differentiation. These processes promote cancer growth, and in some cases, they may cause cells to develop resistance to certain types of treatment, notably EGFR inhibitors. We summarize recent knowledge regarding the effects of KRAS and BRAF mutations in the setting of colorectal cancer and discuss the new therapies under development.Colorectal cancer (CRC) is still one of the most frequent forms of cancer in the world in terms of incidence. Around 40% of CRC patients carry a mutation of the Kirsten rat sarcoma (KRAS) gene, while 10% have a mutation in the B-Raf proto-oncogene serine/threonine kinase (BRAF) gene. These mutations are responsible for dysregulation of the mitogen-associated protein kinase (MAPK) pathway, leading to the proliferation, differentiation, angiogenesis, and resistance to apoptosis of cells. Activation of the MAPK pathway results in adaptive therapeutic resistance, rendering EGFR inhibitors ineffective. This review aims to highlight the recent findings that have improved our understanding of KRAS and BRAF mutations in colorectal cancer and to describe new targeted therapies, used alone or in combination.

Smooth Muscle Cell Derived Microparticles Acts as Autocrine Activation of Smooth Muscle Cell Proliferation by Mitogen Associated Protein Kinase Upregulation.

Microparticles (MP); also know as microvesicles are extracellular vesicles (0.1 to 1.0 μm) released by cells in response to cell activation or apoptosis. The high level of circulating MP is one of the important indicators of altered vascular function. Vascular smooth muscle cell (VSMC) derived MP could mediate proliferation and migration of VSMC leading to vascular inflammation. Proliferation of VSMC is mediated by mitogen associated protein kinase (MAPK) and proliferation cell nuclei antigen (PCNA) signaling pathway whereas migration is mediated by metalloproteinase and cytoskeletal remodeling pathway. In this study, VSMC-MP were isolated from confluent VSMC stimulated with tumor necrosis factor (TNF)-α. VSMC-MP were treated to VSMC to investigate the VSMC proliferation marker using in vitro assay. In comparison to normal (VSMC-MP untreated group), VSMC-MP treatment results in proliferation of VSMC as revealed by MTT assay. VSMC-MP and TNF-α induce proliferation by 34% and 67% respectively. VSMC-MP also induce over expression of PCNA in both immuno-fluorescence and western blot experiment. VSMC-MP and TNF-α increase the PCNA expression by 1.86-fold and 1.95-fold respectively. Similarly, VSMC-MP treatment results in over expression of MAPK pathway protein expression in VSMC. As compared to normal, the MAPK protein (pERK1/2, pP38 and pJNK) were increased by 1.41-fold, 1.42-fold and 1.48-fold, respectively in VSMC-MP treated VSMC. Our results provide the evidence of VSMC-MP involvement in proliferation of VSMC. Thus, VSMC-MP could be considered as a potential marker of vascular inflammatory disorder such as atherosclerosis.

Effects of masitinib compared with tadalafil for the treatment of monocrotaline-induced pulmonary arterial hypertension in rats

Targeting vascular remodeling in pulmonary arterial hypertension (PAH) remains a challenge given the lack of potent anti-remodeling abilities of the therapeutic drugs. Although sildenafil has been shown to ameliorate cardiopulmonary remodeling, that of tadalafil is questionable. Masitinib, a tyrosine kinase inhibitor appears safer and more potent than imatinib for treatment of malignancies, but its efficacy on PAH is unknown. Therefore, we investigated the anti-remodeling properties of masitinib (5, 15, 50 mg/kg) and tadalafil (5, 10 mg/kg) using a monocrotaline-induced rat model of PAH. The 14-day treatment with masitinib (15, 50 mg/kg) resulted in significantly decreased right ventricular (RV) systolic pressure (RVSP) and hypertrophy (RVH), and pulmonary vascular remodeling, whereas tadalafil showed weaker anti-remodeling properties. Besides, masitinib significantly blocked the mitogen-associated protein kinase (MAPK) pathway, and reduced phosphodiesterase (PDE)-5 mRNA expression in the lungs. By contrast, tadalafil did not significantly inhibit the MAPK pathway. Further, the 28-day treatment extension revealed that masitinib-treated rats (15 mg/kg) had significantly lower RVSP, and higher heart rate and serum cyclic guanosine monophosphate (cGMP) level, whereas those treated with tadalafil (10 mg/kg) showed insignificantly lower RVSP and higher cGMP level. Moreover, the RVH indices, heart rates, body weight gains, and survival rates of rats in both groups were comparable. Collectively, these results suggest that the treatment with a low-dose masitinib was non-inferior than tadalafil. A lower dose of masitinib may represent a novel approach to target both the cardiopulmonary remodeling and the dysregulated vasoconstriction in PAH.

Inhibiting TrxR suppresses liver cancer by inducing apoptosis and eliciting potent antitumor immunity.

Liver cancer is one of the most common malignant tumors worldwide. Thioredoxin reductase (TrxR) is highly expressed in liver cancer cells. The present study aimed to investigate the effect of inhibiting TrxR on liver cancer and to better understand the underlying molecular and immunological mechanisms associated with inhibition. It was demonstrated that targeting TrxR inhibited the growth and induced apoptosis of liver cancer cells, which was accompanied by activation of the mitogen associated protein kinase pathway. This inhibition was dependent on the production of reactive oxygen species (ROS). Blockage of ROS production reversed TrxR inhibitor-induced antitumor effects. Blocking the Trx/TrxR system activated the mammalian target of rapamycin pathway and inhibited autophagy, which occurred in a ROS-independent manner. TrxR inhibition led to lesions in the mitochondrial membrane, indicated by alterations in membrane potential. Mouse xenograft experiments were highly consistent with in vitro studies. Most importantly, blocking the Trx/TrxR system improved the tumor immune microenvironment. Together, these data demonstrated that TrxR is a potential target for liver cancer therapy, which could inhibit hepatocarcinogenesis and progression, and improve the antitumor immune response.

Trametinib after disease reactivation under dabrafenib in Erdheim-Chester disease with both BRAF and KRAS mutations

AbstractMajor advances have been made in understanding the pathogenesis of Erdheim-Chester disease (ECD) leading to novel treatment strategies. Targeted therapies such as BRAF inhibition have shown a significant impact on disease management, emphasizing the importance of the activated mitogen-associated protein kinase pathway in this disease. However, incomplete responsiveness, potentially limiting adverse effects, and the occurrence of treatment resistance to BRAF inhibition observed in other BRAF-mutant malignancies imply the importance of therapeutic strategies beyond BRAF inhibition. We report a patient with ECD who carried the BRAFV600E mutation and developed treatment resistance under BRAF inhibition despite initial treatment response. Genetic analyses of a newly developing ECD lesion revealed a somatic KRASQ61H mutation without the presence of BRAFV600E. Accordingly, the addition of MEK-inhibiting trametinib to BRAF-inhibiting dabrafenib was able to overcome acquired partial treatment resistance. This is the first report of treatment resistance as a result of a secondary MAPK pathway–activating mutation during BRAF inhibition in ECD. This case contributes to the ongoing efforts of simultaneous BRAF/MEK inhibition as a promising strategy in ECD.

Induced Quiescence of Lgr5+ Stem Cells in Intestinal Organoids Enables Differentiation of Hormone-Producing Enteroendocrine Cells

Lgr5+ adult intestinal stem cells are highly proliferative throughout life. Single Lgr5+ stem cells can be cultured into three-dimensional organoids containing all intestinal epithelial cell types at near-normal ratios. Conditions to generate the main cell types (enterocyte, goblet cells, Paneth cells, and M cells) are well established, but signals to induce the spectrum of hormone-producing enteroendocrine cells (EECs) have remained elusive. Here, we induce Lgr5+ stem cell quiescence invitro by blocking epidermal growth factor receptor (EGFR) or mitogen-associated protein kinase (MAPK) signaling pathways in organoids and show that their quiescent state is readily reverted. Quiescent Lgr5+ stem cells acquire a distinct molecular signature biased toward EEC differentiation. Indeed, combined inhibition of Wnt, Notch, and MAPK pathways efficiently generates a diversity of EEC hormone-expressing subtypes invitro. Our observations uncouple Wnt-dependent stem cell maintenance from EGF-dependent proliferation and provide an approach for the study of the elusive EECs in a defined environment.

Rhesus rotavirus VP6 regulates ERK-dependent calcium influx in cholangiocytes

The Rhesus rotavirus (RRV) induced murine model of biliary atresia (BA) is a useful tool in studying the pathogenesis of this neonatal biliary obstructive disease. In this model, the mitogen associated protein kinase pathway is involved in RRV infection of biliary epithelial cells (cholangiocytes). We hypothesized that extracellular signal-related kinase (ERK) phosphorylation is integral to calcium influx, allowing for viral replication within the cholangiocyte. Utilizing ERK and calcium inhibitors in immortalized cholangiocytes and BALB/c pups, we determined that ERK inhibition resulted in reduced viral yield and subsequent decreased symptomatology in mice. In vitro, the RRV VP6 protein induced ERK phosphorylation, leading to cellular calcium influx. Pre-treatment with an ERK inhibitor or Verapamil resulted in lower viral yields. We conclude that the pathogenesis of RRV-induced murine BA is dependent on the VP6 protein causing ERK phosphorylation and triggering calcium influx allowing replication in cholangiocytes.

ConcomitantRAS,RET/PTC, orBRAFMutations in Advanced Stage of Papillary Thyroid Carcinoma

RET/PTC rearrangement, RAS, and BRAF mutations are considered to be mutually exclusive in papillary thyroid carcinoma (PTC). However, although concomitant mutations of RET/PTC, RAS, or BRAF have been reported recently, their significance for tumor progression and survival remains unclear. We sought to examine the prognostic value of concomitant mutations in PTC. We investigated 88 PTC for concomitant mutations. Mutation in BRAF exon 15, KRAS, NRAS, and HRAS were studied by polymerase chain reaction (PCR)-sequencing of tumor DNA; RET/PTC rearrangement was determined by reverse transcription (RT)-PCR-sequencing of tumor cDNA. BRAF(V600E) was detected in 39 of 82 classic PTC (CPTC) and in all three tall-cell variants (49%, 42/85). KRAS mutation (p.Q61R and p.S65N) was detected in two CPTC (2%, 2/88) and NRAS(Q61R) in one CPTC and two follicular variant PTC (FVPTC; 3%, 3/88). KRAS(S65N) was identified for the first time in thyroid cancer and could activate mitogen-associated protein kinase (MAPK). RET/PTC-1 was detected in nine CPTC, one tall-cell variant, and two FVPTC. Concomitant BRAF(V600E) and KRAS, or BRAF(V600E) and RET/PTC-1 mutations were found in two CPTC, and six CPTC and one tall-cell variant, respectively. In total, 11 concomitant mutations were found in 88 PTC samples (13%), and most of them were in the advanced stage of disease (8/11, 73%; p<0.01). Our data show that concomitant mutations are a frequent event in advanced PTC and are associated with poor prognosis. The concomitant mutations may represent intratumor heterogeneity and could exert a gene dosage effect to promote disease progression. KRAS(S65N) can constitutively activate the MAPK pathway.

Thyroid carcinoma-associated genetic mutations also occur in thyroid lymphomas

Molecular testing for mutations activating the mitogen-associated protein kinase signaling pathway is being used to help diagnose thyroid carcinomas. However, the prevalence of these mutations in thyroid lymphomas has not been reported. Therefore, we studied the prevalence of BRAF, NRAS, HRAS, and KRAS mutations in 33 thyroid lymphomas and correlated the mutational status with the clinical, pathological, cytogenetic, and immunophenotypic findings. Eleven cases were also tested for PAX8/PPARγ translocations. The lymphomas included 25 diffuse large B-cell lymphomas, 6 extranodal marginal-zone lymphomas of mucosa-associated lymphoid tissue type, and 2 follicular lymphomas. Seventeen diffuse large B-cell lymphomas were germinal center type, six non-germinal center type, and two unclassifiable (Hans algorithm). None of the cases had an associated thyroid carcinoma. Mutations of the BRAF gene were identified in six (24%) diffuse large B-cell lymphomas (D594G in three germinal center diffuse large B-cell lymphomas, K601N in two germinal center diffuse large B-cell lymphomas, and V600E in one non-germinal center diffuse large B-cell lymphoma) and of the NRAS gene in two (8%) non-germinal center diffuse large B-cell lymphomas (Q61K and Q61H). BRAF and NRAS mutations were not found in any extranodal marginal-zone lymphomas of mucosa-associated lymphoid tissue type or follicular lymphomas. HRAS and KRAS mutations were not identified in any of the cases, nor were PAX8/PPARγ translocations found. Thus, interpretation of finding a BRAF or NRAS mutation in the thyroid, particularly in preoperative thyroid aspirates, must take into account the differential diagnosis of a lymphoma. In addition to the diagnostic importance, our data also demonstrate that alteration in the mitogen-associated protein kinase pathway may have a role in the pathogenesis of some large B-cell lymphomas of the thyroid with potential therapeutic implications.

The molecular pathology of cutaneous melanoma

Cutaneous melanoma is a highly aggressive cancer with still limited, but increasingly efficacious, standard treatment options. Recent preclinical and clinical findings support the notion that cutaneous melanoma is not one malignant disorder but rather a family of distinct molecular diseases. Incorporation of genetic signatures into the conventional histopathological classification of melanoma already has great implications for the management of cutaneous melanoma. Herein, we review our rapidly growing understanding of the molecular biology of cutaneous melanoma, including the pathogenic roles of the mitogen-associated protein kinase (MAPK) pathway, the phosphatidylinositol 3 kinase [PI3K]/phosphatase and tensin homologue deleted on chromosome 10 [PTEN]/Akt/mammalian target of rapamycin [mTOR])PTEN (phosphatase and tensin homolog) pathway, MET (hepatocyte growth factor), Notch signaling, and other key molecules regulating cell cycle progression and apoptosis. The mutation Val600Glu in the BRAF oncogene (designated BRAF(V600E)) has been associated with clinical benefit from agents that inhibit BRAF(V600E) or MEK (a kinase in the MAPK pathway). Cutaneous melanomas arising from mucosal, acral, chronically sun-damaged surfaces sometimes have oncogenic mutations in KIT, against which several inhibitors have shown clinical efficacy. These findings suggest that prospective genotyping of patients with melanoma, combined with the growing availability of targeted agents, which can be used to rationally exploit these findings, should be used increasingly as we work to develop new and more effective treatments for this devastating disease.

Treatment implications of the emerging molecular classification system for melanoma

Melanoma is an aggressive disease with few standard treatment options. The conventional classification system for this disease is based on histological growth patterns, with division into four subtypes: superficial spreading, lentigo maligna, nodular, and acral lentiginous. Major limitations of this classification system are absence of prognostic importance and little correlation with treatment outcomes. Recent preclinical and clinical findings support the notion that melanoma is not one malignant disorder but rather a family of distinct molecular diseases. Incorporation of genetic signatures into the conventional histopathological classification of melanoma has great implications for development of new and effective treatments. Genes of the mitogen-associated protein kinase (MAPK) pathway harbour alterations sometimes identified in people with melanoma. The mutation Val600Glu in the BRAF oncogene (designated BRAF(V600E)) has been associated with sensitivity in vitro and in vivo to agents that inhibit BRAF(V600E) or MEK (a kinase in the MAPK pathway). Melanomas arising from mucosal, acral, chronically sun-damaged surfaces sometimes have oncogenic mutations in KIT, against which several inhibitors have shown clinical efficacy. Some uveal melanomas have activating mutations in GNAQ and GNA11, rendering them potentially susceptible to MEK inhibition. These findings suggest that prospective genotyping of patients with melanoma should be used increasingly as we work to develop new and effective treatments for this disease.

Mitogen‐associated protein kinase‐ and protein kinase A‐dependent regulation of rhodopsin promoter expression in zebrafish rod photoreceptor cells

Mitogen-associated protein kinase (MAPK)- and protein kinase A (PKA)-dependent signal transductions play important roles in the regulation of gene expression. Both MAPK and PKA pathways can be activated by light exposure. In this study, we investigated the effect of light on MAPK and PKA signal transduction and their roles in the regulation of rhodopsin promoter expression by using transgenic zebrafish [Tg(rhod::GFP)]. The Tg(rhod::GFP) fish express short half-life GFP that is under the transcriptional control of the zebrafish rhodopsin promoter and can therefore be used for in vivo studies of rhodopsin gene transcription in live cells. Blue light plays a role in the regulation of rhodopsin promoter expression via an MAPK-mediated signal transduction cascade. Blue light excites cryptochromes (CRY), which activate the downstream PKC-dependent MAPK signal pathway. White light, on the other hand, regulates rhodopsin promoter expression via a G-protein-coupled cAMP-dependent PKA pathway. White light promotes dopamine release in the retina, which activates dopamine receptors and the downstream PKA pathway. Blocking MAPK signaling diminishes the blue light-induced increases in rhodopsin promoter expression, but this treatment has no effect on white light-mediated rhodopsin promoter expression. Conversely, blocking the PKA pathway diminishes the white light-induced rhodopsin promoter expression but does not affect rhodopsin promoter expression regulated by blue light. Together, the data suggest that MAPK and PKA regulate rhodopsin transcription through parallel signal transduction pathways.

A Lipase-Free PLC-Like Protein

Phospholipase C (PLC) cleaves phosphatidylinositol 4,5-bisphosphate to form the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. Recently, PLC-L2, a PLC-like protein that lacks lipase activity, has been identified in skeletal muscle, as well as in B and T lymphocytes. Takenaka et al. generated PLC-2-deficient mice to investigate the possible role of PLC-2 signaling in B lymphocytes. PLC-L2-deficient mature B cells showed an enhanced proliferative response and increased expression of the activation marker CD69 after B cell receptor (BCR) stimulation in vitro, and mice displayed increased production of immunoglobulin M (IgM), IgG1, and IgG3 in response to antigen treatment in vivo. The authors used fura-2 imaging to show that Ca 2+ influx after BCR stimulation was enhanced in PLC-2-deficient cells; moreover, translocation of nuclear factor of activated T cells (which is regulated by calcineurin, which is itself calcium-dependent) was enhanced. Finally, PLC-2-deficient cells showed increased phosphorylation of extracellular signal-regulated kinase and activation of the transcription factor AP-1 after BCR stimulation, which indicated that signaling through the mitogen-associated protein kinase pathway was enhanced. Thus, B cells from PLC-2-deficient mice showed a hyperreactive response to BCR stimulation; the authors suggest that PLC-2 acts as an inhibitory regulator of BCR signaling and of the B cell immune response. K. Takenaka, K. Fukami, M. Otsuki, Y. Nakamura, Y. Kataoka, M. Wada, K. Tsuji, S.-I. Nishikawa, N. Yoshida, T. Takenawa, Role of phospholipase C-L2, a novel phospholipase-C-like protein that lacks lipase activity in B-cell receptor signaling. Mol. Cell. Biol. 23 , 7329-7338 (2003). [Abstract] [Full Text]

How H. pylori Leads Cells Astray

The gastric bacterium Helicobacter pylori is associated with gastritis, gastroduodenal ulcers, gastric adenocarcinoma, and gastric lymphoma. The cytotoxin-associated gene A antigen (CagA) is translocated into gastric epithelial cells, where it undergoes tyrosine phosphorylation and triggers cell scattering and morphological changes, and it has been implicated in H. pylori 's pathogenic effects. The mechanism by which CagA affects cell behavior and morphology, however, remainsunclear. Mimuro et al. determined that CagA interacted with the adaptor protein Grb2 (growth factor receptor-binding protein 2) in AGS gastric adenocarcinoma cells independently of having undergone tyrosine phosphorylation, which leads to activation of the mitogen-associated protein kinase pathway and to cell scattering and proliferation. Using CagA mutants expressed in H. pylori that lacked endogenous CagA, the authors determined that substituting phenylalanine for tyrosine in the phosphorylated region of CagA (PY region) did not interfere with CagA-evoked scattering in infected cells, whereas deletion of the PY region abolished cell scattering. Western analysis using fusion proteins containing Grb2 domains indicated that both phosphorylated and nonphosphorylated CagA bound to Grb2, as did the phenylalanine-substituted CagA mutant, but not the mutant lacking the PY region. A dominant-negative Grb2 mutant that interfered with Ras activation by Grb2 inhibited H. pylori -induced cell scattering. Consistent with Ras pathway implication in the response, a sustained late phase of mitogen-activated and extracellular signal-regulated kinase kinase (MEK) activation in response to H. pylori infection depended on CagA expression. Analysis of the effects of various CagA constructs stably transfected into MDCK cells implicated the PY region in CagA-induced MEK phosphorylation, as well as scattering and proliferation. H. Mimuro, T. Suzuki, J. Tanaka, M. Asahi, R. Haas, C. Sasakawa, Grb2 is a key mediator of Helicobacter pylori CagA protein activities. Mol. Cell 10 , 745-755 (2002). [Online Journal]

Activity-dependent neurotrophic factor-14 requires protein kinase C and mitogen-associated protein kinase kinase activation to protect the developing mouse brain against excitotoxicity.

Activity-dependent neurotrophic factor (ADNF) is a newly identified compound that prevents in vitro neuronal death when present in fentomolar concentrations. ADNF-14, a 14 amino acid peptide derived from ADNF, has the same effects on growth as the parent molecule. However, the transduction pathways and target cells for these highly potent trophic factors are still unknown. We previously described a mouse model of excitotoxic lesions of the developing neocortex mimicking several hypoxic or hypoxic-like brain lesions observed in human fetuses and neonates. In this model, cotreatment with the excitotoxin ibotenate and ADNF-14 prevented both neuronal death in pups injected on the day of birth and white matter cystic lesions in pups treated 5 d after birth. In the present study, coadministration of ibotenate, ADNF-14, and selective transduction pathway inhibitors showed that activation of protein kinase C (PKC) and mitogen-associated protein kinase kinase was critical for neuroprotection. Immunocytochemistry revealed that ADNF-14 activated PKC and mitogen-associated protein kinase in cortical neurons on the day of birth and in white matter astrocytes on the fifth postnatal day. Taken in concert, these data identify PKC and mitogen-associated protein kinase pathways as critical to ADNF-14-induced neuroprotection of the developing brain against excitotoxic damage.

Effect of Taxol and Taxotere on Gene Expression in Macrophages: Induction of the Prostaglandin H Synthase-2 Isoenzyme

Induction of genes encoding cytokines or other, unidentified proteins may contribute to the pharmacological effects of taxol. We hypothesized that prostaglandin H synthase-2 (PGHS-2) was one of the unidentified genes induced by taxol. Taxol alone or taxol plus IFN-gamma increased PGE2 formation, PGHS-2 protein expression, and PGHS-2 mRNA expression in RAW 264.7 murine macrophages. The kinetics for mRNA induction, protein expression, and catalysis were self-consistent. A selective inhibitor of PGHS-2 blocked PGE2 formation by cells incubated with taxol; a selective inhibitor of PGHS-1 had no effect. A glucocorticoid blocked the induction of mRNA, the expression of PGHS-2 protein, and the formation of PGE2. Neither taxol alone nor taxol plus IFN-gamma altered the expression of the PGHS-1 isoenzyme in RAW 264.7 cells. Taxotere, an analogue that stabilizes microtubules as potently as taxol, did not alter the expression of PGHS-2, implying that its induction in RAW 264.7 murine macrophages did not originate from microtubule stabilization. Taxol and taxotere each induced PGHS-2 expression in human monocytes suspended in 10% human serum. However, human monocytes suspended in 10% bovine serum responded only to LPS, not to taxol or taxotere, implying that they act independently of the LPS-mimetic process that is prominent in mice. Taxol induced PGHS-2 in human and murine monocytes via a p38 mitogen-associated protein kinase pathway. The inclusion of PGHS-2 among the early response genes induced in leukocytes may be relevant to the beneficial and adverse effects encountered during taxol administration.

Regulation of neuroprotective action of vasoactive intestinal peptide in the murine developing brain by protein kinase C and mitogen-activated protein kinase cascades: in vivo and in vitro studies.

Intracerebral administration of the excitotoxin ibotenate to newborn mice induces white matter lesions mimicking periventricular leukomalacia, the most frequent brain lesion occurring in premature human babies. In this model, coinjection of vasoactive intestinal peptide prevents white matter lesions. In the present study, coadministration of ibotenate, vasoactive intestinal peptide, and selective transduction inhibitors showed that protein kinase C and mitogen-associated protein kinase pathways were critical for neuroprotection. In vivo and in vitro immunocytochemistry revealed that vasoactive intestinal peptide activated protein kinase C in astrocytes and neurons, and mitogen-associated protein kinase in neurons. In vitro neuronal transduction activation was indirect and required medium conditioned by astrocytes in which protein kinase C had been activated by vasoactive intestinal peptide. Although vasoactive intestinal peptide did not prevent the initial in vivo appearance of white matter lesion, it promoted a secondary repair of this lesion with axonal regrowth. Through protein kinase C activation, vasoactive intestinal peptide also prevented ibotenate-induced white matter astrocyte death. These data support the following hypothetical model: Vasoactive intestinal peptide activates protein kinase C in astrocytes, which promotes astrocytic survival and release of soluble factors; these released factors activate neuronal mitogen-associated protein kinase and protein kinase C, which will permit axonal regrowth.