Downstream Signaling Components Research Articles

Metastasis-Associated Protein 1 Short Form Stimulates Wnt1 Pathway in Mammary Epithelial and Cancer Cells

Although Wnt1 downstream signaling components as well as cytoplasmic level of metastatic tumor antigen 1 short form (MTA1s) are elevated in human breast cancer, it remains unknown whether a regulatory cross-talk exists between these two pathways. Here, we provide evidence of a remarkable correlation between the levels of MTA1s and stimulation of the Wnt1 signaling components, leading to increased stabilization of beta-catenin and stimulation of Wnt1 target genes in the murine mammary epithelial and human breast cancer cells. We found that MTA1s influences Wnt1 pathway through extracellular signal-regulated kinase (ERK) signaling as selective silencing of the endogenous MTA1s or ERK, or its target glycogen synthase kinase 3beta resulted in a substantial decrease in beta-catenin expression, leading to the inhibition of Wnt1 target genes. Furthermore, downregulation of beta-catenin in cells with elevated MTA1s level was accompanied by a corresponding decrease in the expression of Wnt1 target genes, establishing a mechanistic role for the ERK/glycogen synthase kinase 3beta/beta-catenin pathway in the stimulation of the Wnt1 target genes by MTA1s in mammary epithelial cells. In addition, mammary glands from the virgin MTA1s transgenic mice mimicked the phenotypic changes found in the Wnt1 transgenic mice and exhibited an overall hyperactivation of the Wnt1 signaling pathway, leading to increased stabilization and nuclear accumulation of beta-catenin. Mammary glands from the virgin MTA1s-TG mice revealed ductal hyperplasia and ductal carcinoma in situ, and low incidence of palpable tumors. These findings reveal a previously unrecognized role for MTA1s as an important modifier of the Wnt1 signaling in mammary epithelial and cancer cells.

CPLA2 Regulates the Expression of Type I Interferons and Intracellular Immunity to Chlamydia trachomatis

Infection with the obligate bacterial intracellular pathogen Chlamydia trachomatis leads to the sustained activation of the small GTPase RAS and many of its downstream signaling components. In particular, the mitogen-activated protein kinase ERK and the calcium-dependent phospholipase cPLA(2) are activated and are important for the onset of inflammatory responses. In this study we tested if activation of ERK and cPLA(2) occurred as a result of RAS signaling during infection and determined the relative contribution of these signaling components to chlamydial replication and survival. We provide genetic and pharmacological evidence that during infection RAS, ERK, and, to a lesser extent, cPLA(2) activation are uncoupled, suggesting that Chlamydia activates individual components of this signaling pathway in a non-canonical manner. In human cell lines, inhibition of ERK or cPLA(2) signaling did not adversely impact C. trachomatis replication. In contrast, in murine cells, inhibition of ERK and cPLA(2) played a significant protective role against C. trachomatis. We determined that cPLA(2)-deficient murine cells are permissive for C. trachomatis replication because of their impaired expression of beta interferon and the induction of immunity-related GTPases (IRG) important for the containment of intracellular pathogens. Furthermore, the MAPK p38 was primarily responsible for cPLA(2) activation in Chlamydia-infected cells and IRG expression. Overall, these findings define a previously unrecognized role for cPLA(2) in the induction of cell autonomous cellular immunity to Chlamydia and highlight the many non-canonical signaling pathways engaged during infection.

A Septin Diffusion Barrier at the Base of the Primary Cilium Maintains Ciliary Membrane Protein Distribution

In animal cells, the primary cilium transduces extracellular signals through signaling receptors localized in the ciliary membrane, but how these ciliary membrane proteins are retained in the cilium is unknown. We found that ciliary membrane proteins were highly mobile, but their diffusion was impeded at the base of the cilium by a diffusion barrier. Septin 2 (SEPT2), a member of the septin family of guanosine triphosphatases that form a diffusion barrier in budding yeast, localized at the base of the ciliary membrane. SEPT2 depletion resulted in loss of ciliary membrane protein localization and Sonic hedgehog signal transduction, and inhibited ciliogenesis. Thus, SEPT2 is part of a diffusion barrier at the base of the ciliary membrane and is essential for retaining receptor-signaling pathways in the primary cilium.

Transcription factor mutations in myelodysplastic/myeloproliferative neoplasms

Aberrant activation of tyrosine kinases, caused by either mutation or gene fusion, is of major importance for the development of many hematologic malignancies, particularly myeloproliferative neoplasms. We hypothesized that hitherto unrecognized, cytogenetically cryptic tyrosine kinase fusions may be common in non-classical or atypical myeloproliferative neoplasms and related myelodysplastic/myeloproliferative neoplasms. To detect genomic copy number changes associated with such fusions, we performed a systematic search in 68 patients using custom designed, targeted, high-resolution array comparative genomic hybridization. Arrays contained 44,000 oligonucleotide probes that targeted 500 genes including all 90 tyrosine kinases plus downstream tyrosine kinase signaling components, other translocation targets, transcription factors, and other factors known to be important for myelopoiesis. No abnormalities involving tyrosine kinases were detected; however, nine cytogenetically cryptic copy number imbalances were detected in seven patients, including hemizygous deletions of RUNX1 or CEBPA in two cases with atypical chronic myeloid leukemia. Mutation analysis of the remaining alleles revealed non-mutated RUNX1 and a frameshift insertion within CEBPA. A further mutation screen of 187 patients with myelodysplastic/myeloproliferative neoplasms identified RUNX1 mutations in 27 (14%) and CEBPA mutations in seven (4%) patients. Analysis of other transcription factors known to be frequently mutated in acute myeloid leukemia revealed NPM1 mutations in six (3%) and WT1 mutations in two (1%) patients with myelodysplastic/myeloproliferative neoplasms. Univariate analysis indicated that patients with mutations had a shorter overall survival (28 versus 44 months, P=0.019) compared with patients without mutations, with the prognosis for cases with CEBPA, NPM1 or WT1 mutations being particularly poor. We conclude that mutations of transcription and other nuclear factors are frequent in myelodysplastic/myeloproliferative neoplasms and are generally mutually exclusive. CEBPA, NPM1 or WT1 mutations may be associated with a poor prognosis, an observation that will need to be confirmed by detailed prospective studies.

Abstract LB-135: The transcriptome of p300 loss in MCF-7 cells

Abstract Introduction: p300 is a transcriptional co-activator that promotes gene transcription by acetylating DNA-binding factors and bridging them to the basal transcription machinery. Recent studies have revealed decreased levels of p300 in breast cancer cells, suggesting that p300 may be involved in the control of breast cancer growth and progression. Preliminary data from our laboratory also indicates that decreased p300 function promotes MCF-7 breast cancer cell proliferation in vitro and tumorigenesis in vivo. Methodology: In order to identify the effects of p300 loss on gene expression in breast cancer cells, microarray analysis was performed on total RNA from MCF-7 cells, stably transduced with lentiviruses expressing one of 3 p300 shRNAs or a non-silencing sequence. Silencing of p300 was > 50% in response to each of the shRNAs. Endogenous RNA was converted to cDNA using random primers and then to biotinylated cRNA for hybridization to human exonic evidence-based oligonucleotide microarrays (Ocean Ridge Biosciences, Palm Beach Gardens, FL). 877 genes were found to be differentially expressed in p300 knockdown MCF-7 cells compared to non-silenced controls. Differentially regulated genes were ranked according to the significance of change (p value < 0.0005) and compared to previously reported data in the Oncomine database, for both directionality (up versus down-regulation) and positive correlation with decreased p300 levels. As an additional strategy we ranked the differentially expressed genes from our microarray with good level of significance (p<0.05) by fold change and repeated the comparative analysis with Oncomine as described above. Results: In our analysis, 877 genes were found to be differentially expressed out of which 27 genes were either significantly up-regulated or down-regulated. The rest of the gene expression levels found in the microarray analysis were not as highly significant as the top 27 genes. Among those 27 genes, four (SPAG7, OAS2, RARRES3, SERPINA6) showed highly significant (p<0.0005) increase in their expression and five genes (DHX9, EIF4G2, POLR2D, MCM6, CYP4F2) showed highly significant decrease. Among the genes with the largest fold changes, 10 (IFI27, IFI6, ATF-3, PIK3IP1, TNFAIP3, P8, RARRES3, FOS, IRF9, and RNF39) showed significantly increased expression and 8 were decreased (WNT3A, CAMK1, CENPH, DCTN1, CASC5, HSPA4, CDC25A, and CDKN2C). The second set of genes showed correlations with p300 levels as indicated in analyses of Oncomine data. Some of these genes may be contributing to epithelial to mesenchymal- transition (EMT) under p300 knock-down conditions. Conclusions: Our microarray analysis in MCF-7 cells with partial p300 loss has identified several key genes that are related to breast cancer growth. These genes may be important in mediating the function of p300 loss in tumorigenicity, and future studies will focus on the use of p300 or its downstream signaling components as therapeutic targets in breast cancer treatment. 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 LB-135.

Abstract 5686: Linoleic acid supplementation upregulates phosphatidylinositol-3-kinase signaling by increasing the association between Gab1 and EGFR

Abstract The omega-6 polyunsaturated fatty acid, linoleic acid (LA), is prevalent in Western diets and is known to enhance the tumorigenesis of mammary cancer models; with previous work demonstrating an upregulation of phosphatidylinositol-3-kinase (PI3K) signaling may play a key role. The Grb2-associated binding protein 1 (Gab1) is a scaffolding protein that can complex with the epidermal growth factor receptor (EGFR) to activate PI3K signaling. Recent studies in our laboratory reveal LA supplementation of the breast cancer cell lines MDA-MB-231 and BT-474 greatly increases the association between Gab1 and EGFR, while at the same time dramatically decreasing Gab1 protein levels. These changes are concomitant with increases in activated Akt (pAkt), a downstream signaling component in the PI3K signaling pathway. Moreover, inhibitors of EGFR and Gab1-specific siRNAs are capable of reversing LA-induced upregulation of pAkt, as well as observed increases in cell proliferation for these models. Finally, we were able to confirm these findings in the A549 lung cancer cell line, validating the potential relevance to tissues other than breast cancer cell lines. These data establish Gab1 as major target in LA-induced enhancement of tumorigenesis. 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 5686.

Abstract 1563: Role of EGFR during the transition of inflammation to dysplasia in a colitis-associated colon cancer model

Abstract Background: The underlying mechanisms in ulcerative colitis-associated dysplasia are poorly understood. Neurokinin-1 receptors (NK-1R) have been shown to be involved in chronic colonic inflammation, and we have demonstrated their over-expression in patients with dysplasia. The NK-1R has been found to crosstalk with the epidermal growth factor receptor (EGFR). EGFR is related to the development of some cancers including colorectal cancer. Erlotinib, a potent inhibitor of the EGFR tyrosine kinase, has been shown to inhibit the intracellular EGFR signaling pathway and to block non-small cell lung cancer and pancreatic tumor growth; however its role in the genesis of dysplasia is unknown. Aim: The aim of this study was to investigate the role of EGFR in a novel model of colitis-associated dysplasia using a small molecule inhibitor, Erlotinib. Methods: Sprague Dawley rats received trinitrobenzene sulfonic acid (TNBS; 30 mg in 50% ethanol, ic.), followed six weeks later by reactivation with TNBS (5 mg/kg, iv.) for three days. To induce colitis-associated dysplasia, the rats then received TNBS (iv.) twice a week for ten weeks. One group received the Erlotinib (10 mg/kg, ip.) for one week before the start of the reactivation of the colitis and two weeks after (21 days); the rest received the vehicle. After sacrifice, the colons were removed and analyzed for damage and expression of NK-1R, Cox-2, EGFR, and the downstream signaling components. Results: Animals receiving Erlotinib had no significant difference in macroscopic or microscopic damage from controls. Pathological analysis however revealed a decrease of the incidence of dysplasia in animals treated with Erlotinib when compared with the vehicle-treated animals. This decrease was most evident in both the mid and the distal colon (15% vs. 53% and 20% vs. 61%). Colonic tissues from Erlotinib-treated animals also had decreased expression of NK-1R, Cox-2, pErk ½, and Akt compared to the vehicle-treated group. Conclusion: Our results suggest that Erlotinib can delay the development of colitis associated dysplasia, suggesting a potential therapeutic use for Erlotinib in patients with long-standing colitis. Supported in part by 1U56 CA126379-01 and 1F31 GM078951. 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 1563.

Abstract 1058: E-cadherin mutations contribute to gastric carcinogenesis by modulating multiple EGFR-dependent downstream signaling pathways

Abstract Mutations in the gene encoding the cell adhesion molecule E-cadherin and overexpression of the epidermal growth factor receptor (EGFR) represent fundamental genetic alterations associated with diffuse-type gastric cancer. Mutations, e.g. in frame deletion of exon 8 (del 8), lead to loss of putative extracellular Ca2+-binding sites of E-cadherin, thereby impairing its functionality. Several studies have shown a bidirectional crosstalk between E-cadherin and EGFR. Functional wild-type (wt) E-cadherin inhibits ligand-induced EGFR activation, but mutations lead to a loss of its suppressive function, resulting in increased EGFR activation and recruitment of downstream signalling components. The phenotype of cells harboring such mutations is characterized by increased proliferation and elevated migratory and invasive potential. Our project aims at studying the impact of somatic mutations of E-cadherin on EGFR-mediated signalling pathways, in order to identify signalling molecules of these pathways as molecular targets for selective therapies in gastric cancer. Cell lines expressing wt or mutant E-cadherin (del 8) were EGF-stimulated and cellular lysates were analyzed by commercial proteome profiler antibody array (R&D Systems). The array allowed detection of the phosphorylation status of 48 signalling molecules and facilitated identification of differential activation. We found differential response to EGF stimulation of proteins involved in DNA damage and repair, such as p53 and CHK2, in both cell lines. As both proteins are tumorsuppressors, their dysfunction could contribute to tumor progression. Furthermore, Src-family kinases (SFKs) like Src, Yes, Fgr, Lck and Lyn exhibited different activation profiles in response to EGF-stimulation in both cell lines. As SFKs are key players in multiple cellular processes like cell growth, differentiation, cellular adhesion, migration and invasion, the impact of their differential activation on the cellular phenotype is subject of ongoing experiments. To determine the role of the identified targets in gastric carcinogenesis and the mechanisms of crosstalk between E-cadherin and EGFR a protein microarray platform was established, allowing analysis of multiple samples under the same experimental conditions. This approach will reveal new insights in EGFR-mediated signalling pathways contributing to gastric carcinogenesis and complete our data describing how tumor associated mutations of E-cadherin affect cellular signalling. 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 1058.

Specific deletion of TRAF3 in B lymphocytes leads to B lymphoma development in mice (138.6)

Abstract TRAF3 is a cytoplasmic adaptor protein utilized by the tumor necrosis factor receptor superfamily and Toll-like receptors for signaling. To explore the in vivo functions of TRAF3 in B cells, we recently generated B cell-specific TRAF3-deficient (B-TRAF3-/-) mice, and reported that TRAF3 is a critical regulator of peripheral B cell survival. In this study, we examined the hypothesis that the prolonged survival of TRAF3-/- B cells may predispose them to transformation. We found that approximately 50% of B-TRAF3-/- mice spontaneously developed B lymphomas by the age of 18 months. Our histopathological and immunophenotyping data revealed that B1 lymphomas, marginal zone B lymphomas, and immunoblastic B lymphomas developed in different individual B-TRAF3-/- mice. TRAF3-/- B lymphoma cells were malignant, as demonstrated by the identification of clonal B lymphoma cells in multiple organs in diseased mice, including spleen, ascites, bone marrow, cervical and mesenteric lymph nodes, and occasionally, kidney and lung. Corroborating our findings, recent evidence demonstrates that deletions and mutations of the TRAF3 gene frequently occur in human patients with B-cell lymphomas and multiple myeloma. Thus, our results, together with the evidence from human patients, indicate that TRAF3 is a tumor suppressor gene in B lymphocytes. Our findings suggest that TRAF3 downstream signaling components may serve as important therapeutic targets for the treatment of B lymphomas.

GIPC mediates the generation of reactive oxygen species and the regulation of cancer cell proliferation by insulin-like growth factor-1/IGF-1R signaling

Insulin-like growth factor-1 (IGF-1)/IGF-1 receptor signaling participates in a variety of cellular processes, including cell survival, growth, and proliferation. Increased expression of IGF-1R and activation of its downstream signaling components have been implicated in human cancers. Although a regulatory role for IGF-1R has been established, the relationship between IGF-1R and its binding partner, GAIP-interacting protein C-terminus (GIPC), in terms of promoting cell proliferation, remains unclear. We found that siRNA-mediated silencing of GIPC expression decreased IGF-1-mediated IGF-1R phosphorylation and cellular proliferation in breast cancer models. IGF-1-mediated cellular proliferation was also inhibited by N-acetylcysteine, which implicates reactive oxygen species generation. siRNA-mediated silencing of GIPC expression also decreased IGF-1-mediated reactive oxygen species generation. Taken together, these data suggest that GIPC contributes to IGF-1-induced cancer cell proliferation via the regulation of reactive oxygen species production.

SAMe prevents the up regulation of toll-like receptor signaling in Mallory–Denk body forming hepatocytes

Mallory–Denk body (MDB) formation is a component of alcoholic and non alcoholic hepatitis. In the present study, the role of the toll-like receptor (TLR) signaling pathway was investigated in the mechanism of MDB formation in the DDC-fed mouse model. Microarray analysis data mining, performed on the livers of drug-primed mice refed DDC, showed that TLR2/4 gene expression was significantly up regulated by DDC refeeding. SAMe supplementation prevented this up regulation and prevented the formation of MDBs. qRT-PCR analysis confirmed these results. TLR2/4 activates the adapter protein MyD88. The levels of MyD88 were increased by DDC refeeding. The increase of MyD88 was also prevented by SAMe supplementation. Results showed that MyD88-independent TLR3/4-TRIF-IRF3 pathway was not up regulated in the liver of DDC refed mice. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is the downstream protein recruited by the MyD88/IRAK protein complex, and is involved in the regulation of innate immune responses. Results showed a significant increase in the levels of TRAF-6. TRAF-6 activation leads to activation of NFkB and the mitogen-activated protein kinase (MAPK) cascade. The TRAF-6 increase was ameliorated by SAMe supplementation. These results suggest that DDC induces MDB formation through the TLR2/4 and MyD88-dependent signaling pathway. In conclusion, SAMe blocked the over-expression of TLR2/4, and their downstream signaling components MyD88 and TRAF-6. SAMe prevented the DDC-induced up regulation of the TLR signaling pathways, probably by preventing the up regulation of INF-γ receptors by DDC feeding. INFγ stimulates the up regulation of TLR2. The ability of SAMe feeding to prevent TLR signaling up regulation has not been previously described.

Nitric oxide modulates sensitivity to ABA

Nitric oxide (NO) is a gas with crucial signaling functions in plant defence and development. As demonstrated by generating a triple nia1nia2noa1-2 mutant with extremely low levels of NO (February 2010 issue of Plant Physiology), NO is synthesized in plants through mainly two different pathways involving nitrate reductase (NR/NIA) and NO Associated 1 (AtNOA1) proteins. Depletion of basal NO levels leads to a priming of ABA-triggered responses that causes hypersensitivity to this hormone and results in enhanced seed dormancy and decreased seed germination and seedling establishment in the triple mutant. NO produced under non-stressed conditions represses inhibition of seed developmental transitions by ABA. Moreover, NO plays a positive role in post-germinative vegetative development and also exerts a critical control of ABA-related functions on stomata closure. The triple nia1nia2noa1-2 mutant is hypersensitive to ABA in stomatal closure thus resulting in a extreme phenotype of resistance to drought. In the light of the recent discovery of PYR/PYL/RCAR as a family of potential ABA receptors, regulation of ABA sensitivity by NO may be exerted either directly on ABA receptors or on downstream signalling components; both two aspects that deserve our present and future attention.

Inhibitory mechanisms of dihydroginsenoside Rg3 in platelet aggregation: Critical roles of ERK2 and cAMP

Abstract Ginsenoside Rg3, a single ginseng saponin, is known to be a major anti-platelet component of protopanaxadiol that is isolated from Korean red ginseng. In this study, we investigated whether dihydroginsenoside Rg3, a stable chemical derivative of ginsenoside Rg3, also demonstrated anti-platelet activity. Dihydroginsenoside Rg3 inhibited thrombin-induced platelet aggregation in a concentration-dependent manner with an IC50 (concentration producing 50% inhibition) of 18.8 ± 0.4 μM. Ginsenoside Rg3 inhibited platelet aggregation which was induced by thrombin (0.1 U mL−1) with an IC50 of 40.2 ± 0.9 μM. We next determined whether dihydroginsenoside Rg3 affected different types of ligand-induced platelet aggregation. We found that dihydroginsenoside Rg3 inhibited collagen-induced platelet aggregation with an IC50 of 20.0 ± 0.9 μM. To elucidate the inhibitory mechanism of dihydroginsenoside Rg3 on aggregation, we analysed its downstream signalling pathway. It was interesting to note that dihydroginsenoside Rg3 elevated cyclic AMP production in resting platelets, but did not affect cyclic GMP production. In addition, we found that dihydroginsenoside Rg3 potently suppressed phosphorylation of extracellular signal-regulated kinase 2 (ERK2), which was stimulated by collagen (2.5 μg mL−1), but not of p38 mitogen-activated protein kinase. Taken together, our results indicate that dihydroginsenoside Rg3 potently inhibited platelet aggregation via the modulation of downstream signalling components such as cAMP and ERK2.

Ionotropic and metabotropic mechanisms in chemoreception: 'chance or design'?

Chemosensory receptors convert an enormous diversity of chemical signals from the external world into a common language of electrical activity in the brain. Mammals and insects use several families of transmembrane receptor proteins to recognize distinct classes of volatile and non-volatile chemicals that are produced by conspecifics or other environmental sources. A comparison of the signalling mechanisms of mammalian and insect receptors has revealed an unexpected functional distinction: mammals rely almost exclusively on metabotropic ligand-binding receptors, which use second messenger signalling cascades to indirectly activate ion channels, whereas insects use ionotropic receptors, which are gated directly by chemical stimuli, thereby leading to neuronal depolarization. In this review, we consider possible reasons for this dichotomy, taking into account biophysical, cell biological, ecological and evolutionary influences on how information is extracted from chemosensory cues by these animal classes.

Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals

The upper respiratory tract is continually assaulted with harmful dusts and xenobiotics carried on the incoming airstream. Detection of such irritants by the trigeminal nerve evokes protective reflexes, including sneezing, apnea, and local neurogenic inflammation of the mucosa. Although free intra-epithelial nerve endings can detect certain lipophilic irritants (e.g., mints, ammonia), the epithelium also houses a population of trigeminally innervated solitary chemosensory cells (SCCs) that express T2R bitter taste receptors along with their downstream signaling components. These SCCs have been postulated to enhance the chemoresponsive capabilities of the trigeminal irritant-detection system. Here we show that transduction by the intranasal solitary chemosensory cells is necessary to evoke trigeminally mediated reflex reactions to some irritants including acyl-homoserine lactone bacterial quorum-sensing molecules, which activate the downstream signaling effectors associated with bitter taste transduction. Isolated nasal chemosensory cells respond to the classic bitter ligand denatonium as well as to the bacterial signals by increasing intracellular Ca(2+). Furthermore, these same substances evoke changes in respiration indicative of trigeminal activation. Genetic ablation of either G alpha-gustducin or TrpM5, essential elements of the T2R transduction cascade, eliminates the trigeminal response. Because acyl-homoserine lactones serve as quorum-sensing molecules for gram-negative pathogenic bacteria, detection of these substances by airway chemoreceptors offers a means by which the airway epithelium may trigger an epithelial inflammatory response before the bacteria reach population densities capable of forming destructive biofilms.

Delineation of downstream signalling components during acrosome reaction mediated by heat solubilized human zona pellucida

BackgroundHuman egg is enveloped by a glycoproteinaceous matrix, zona pellucida (ZP), responsible for binding of the human spermatozoa to the egg and induction of acrosomal exocytosis in the spermatozoon bound to ZP. In the present manuscript, attempts have been made to delineate the downstream signalling components employed by human ZP to induce acrosome reaction.MethodsHeat-solubilized human ZP (SIZP) was used to study the induction of acrosome reaction in capacitated human spermatozoa using tetramethylrhodamine isothiocyanate conjugated Pisum sativum agglutinin (TRITC-PSA) in absence or presence of various pharmacological inhibitors. In addition, intracellular calcium ([Ca2+]i) levels in sperm using Fluo-3 acetoxymethyl ester as fluorescent probe were also estimated in response to SIZP.ResultsSIZP induces acrosomal exocytosis in capacitated human sperm in a dose dependent manner accompanied by an increase in [Ca2+]i. Human SIZP mediated induction of acrosome reaction depends on extracellular Ca2+ and involves activation of Gi protein-coupled receptor, tyrosine kinase, protein kinases A & C and phosphoinositide 3 (PI3)- kinase. In addition, T-type voltage operated calcium channels and GABA-A receptor associated chloride (Cl-) channels play an important role in SIZP mediated induction of acrosome reaction.ConclusionsResults described in the present study provide a comprehensive account of the various downstream signalling components associated with human ZP mediated acrosome reaction.

Regulatory roles of transforming growth factor beta family members in folliculogenesis

Bidirectional signaling between the oocyte and surrounding somatic cells is absolutely essential for successful germ cell development in mammals. Oocytes secrete proteins that are necessary for granulosa cells growth and differentiation, whilst granulosa cells regulate oocyte development and integrate ovarian function with the rest of the body by orchestrating gonadal steroidogenesis. The importance of communication between the oocyte and granulosa cells is highlighted by genetic deletion of members of the transforming growth factor beta (TGFβ) family and their downstream signaling components. Such knockout models have uncovered an interesting spectrum of reproductive phenotypes that have greatly advanced our knowledge of ovarian function and dysfunction. The current review focuses on some of the more recent transgenic mouse models that elucidate the intraovarian TGFβ signaling vital for oocyte and granulosa cell development.

Modulation of LPS-mediated Inflammation by Fenofibrate via the TRIF-dependent TLR4 Signaling Pathway in Vascular Smooth Muscle Cells

Lipopolysaccharide (LPS) induced-vascular inflammation plays a central role in vasculitis and atherosclerosis. The stimulation of toll-like receptor 4 (TLR4) by LPS elicits the release of major proinflammatory cytokines that aggravates cardiovascular disorders. Peroxisome proliferator- activated receptor Α (PPARΑ) agonists have been shown to reduce cardiovascular events by controlling lipid metabolism as well as inflammation. However, the role of PPARΑ agonist fenofibrate in modulating LPS-mediated inflammatory responses in vascular smooth muscle cells (VSMCs) remains elusive. The present study demonstrated that fenofibrate exerted a potent anti-inflammatory action through reducing interleckin-1(IL-18), tissue inhibitor of metalloproteinase-1(TIMP-1), TLR4 and enhancing PPARΑ in LPS-stimulated VSMCs. Additionally, treatment of VSMCs with the TLR4 inhibition or TLR4 small-interfering RNA illustrated that the modulatory effects of fenofibrate on LPS-mediated inflammatory responses in VSMCs were reliant on TLR4. Especially, the results suggested that beneficial effects of fenofibrate on LPS-stimulated inflammatory responses in VSMCs were mediated through interference of TLR4 and its downstream signaling components such as Toll-interleckin-1(IL-1) receptor domain- containing adaptor inducing interferon-Β (TRIF), interferon regulatory factor 3 (IRF3) and interferon-gamma inducible protein 10 (IP-10). In conclusion, PPARΑ agonist fenofibrate exerts anti-inflammatory property by antagonizing LPS-mediated inflammatory responses in VSMCs. More importantly, the modulation of the TRIF-dependent signaling pathway (TLR4/TRIF/IRF3/IP-10) might be a useful and novel anti-inflammatory strategy of fenofibrate.

The neurodevelopmental implications of PI3K signaling.

Our understanding of the mechanisms involved in the formation of the complex arrangement of neurons and their interconnections within the brain has made significant progress in recent years. Current research has uncovered a network of intracellular signaling events that provide precise coordination of a diverse array of cellular responses, including trafficking events, cytoskeletal remodeling, gene transcription, and protein ubiquitination and translation. This chapter considers the specific cellular responses controlled by the phosphatidylinositol 3-kinase (PI3K) signaling pathway, which is instructive with regard to a number of important steps involved in the development of the brain. These range from the mediation of extrinsic signals - such as growth factors, axon guidance cues, and extracellular matrix components - to intrinsic effectors, such as downstream signaling components that act, for example, at the translation level. PI3K signaling is, consequently, at the heart of controlling neuronal migration and neuronal morphogenesis, as well as dendrite and synapse development. Many neurobehavioral disorders arise as a consequence of subtle developmental abnormalities. Unsurprisingly, therefore, aberrant PI3K signaling has been indicated by many studies to be a contributing factor to the pathophysiology of disorders such as schizophrenia and autism. In this chapter, we will focus on the specific, yet divergent, cellular processes that are achieved through PI3K signaling in neurons and are key to brain development.

Dasatinib synergizes with JSI-124 to inhibit growth and migration and induce apoptosis of malignant human glioma cells

Background:Src family kinases (SFK) collectively regulate a variety of cellular functions in many cancer types, including proliferation, invasion, motility, survival, differentiation, and angiogenesis. Although Dasatinib (BMS-354825), an ATP-competitive, small molecule tyrosine kinase inhibitor, suppresses the activity of SFKs at nanomolar concentrations, IC50 values for antiproliferative effects in glioma cell lines were well above the clinically achievable range, suggesting the need to interfere with other components of receptor-induced downstream signaling in order to achieve an optimal therapeutic effect.Materials and Methods:The cytotoxic effects of combining Src and STAT3 inhibition on glioma cell lines were evaluated using assays to measure cell proliferation, apoptosis and migration. Western blotting and immunocytochemistry was used to monitor its effects on cell signaling and morphology.Results:Silencing Src and STAT3 expression each partially inhibited cell proliferation and migration. In addition, JSI-124 significantly enhanced the efficacy of dasatinib in vitro. Combination of dasatinib and JSI-124 achieved significant inhibition of migration in all cell lines, which correlated with the inhibition of Src and downstream mediators of adhesion (e.g. focal adhesion kinase). Cells exposed to dasatinib and JSI-124 exhibited morphological changes that were consistent with an upstream role for Src in regulating focal adhesion complexes.Conclusions:Targeting the Src and STAT pathways may contribute to the treatment of cancers that demonstrate increased levels of these signaling mediators, including malignant human glioma. Clinical studies in these tumor types are warranted.