Browsing by Subject "Phosphatidylinositol 3-Kinases"
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Item B-Cell Adapter for Phosphoinositide 3-Kinase Is a Signaling Adapter in the Toll-Like Receptor/Interleukin-1 Receptor Superfamily(2014-02-17) Troutman, Ty Dale; van Oers, Nicolai S. C.; Pasare, Chandrashekhar; Hooper, Lora V.; Chen, Zhijian J.; Krämer, HelmutToll-like receptor (TLR)/Interleukin-1 receptor (IL1R) superfamily members share signaling components and (with the exception of TLR3) depend on the adapter myeloid differentiation primary response gene 88 (MyD88) for engagement of downstream pathways. Signals from the receptor to the adapter are transmitted through homotypic interaction of TIR (Toll-Interleukin-1 receptor) homology domains found in all TLR/IL1R family members and their adapters. The present work defines a novel TLR/IL1R signaling adapter, termed BCAP (B-cell adapter for PI3K), which was identified based on the presence of a cryptic N-terminal TIR domain. I show here that BCAP (B-cell adapter for PI3K) contains a functional TIR domain enabling its participation in the TLR signaling pathway. Through its TIR domain, BCAP associates with the TLR/IL1R signaling adapter MyD88, as well as the TLR signaling adapter toll-interleukin 1 receptor domain containing adapter protein (TIRAP). Importantly, BCAP plays an obligate role in linking TLRs to activation of phosphoinositide 3-kinase (PI3K) through recruitment of PI3K to the signaling complex and relief of inhibitory influences on PI3K activity. Importantly, BCAP selectively mediates TLR signaling towards the PI3K branch without affecting signaling to NFκB nor MAP kinases. In this capacity, BCAP inhibits secretion of inflammatory cytokines and regulates susceptibility to inflammatory colitis. Because the TLR/IL1R family shares signaling components, BCAP may also function in IL1R family signaling. To test this hypothesis, T cells were chosen as a model cell type responding to IL1R family signals. T helper cells utilize IL18 and IL1 (which engage the IL18R or the IL1R respectively, both IL1R family members) cytokines provided by myeloid cells to achieve optimal Th1 and Th17 effector capacities. I show here that BCAP intrinsically regulates differentiation of naïve T cells towards Th1 and Th17 effector lineages by participation in the IL1R family signaling pathways. Further, BCAP intrinsically regulates both T cell proliferation and survival during priming. The significance of this work lies in the revelation of a TLR signaling adapter serving as a node connecting TLRs to PI3K. Further, the findings here will increase the understanding of key signaling pathways involved in disease and inflammation.Item MiR-10a Regulation of Drug Response and Cancer Stem Cell Populations in Non-Small Cell Lung Carcinomas(2013-09-26) DeSevo, Christopher Gerard; Castrillon, Diego H.; Shay, Jerry W.; Scaglioni, Pier Paolo; Pertsemlidis, Alexander; Minna, John D.Phosphatidylinositol 3-kinases (PI3Ks) are enzymes involved in diverse cellular functions including cell growth, proliferation, differentiation, motility, survival and apoptosis. Many of these functions relate to class I PI3Ks, heterodimers composed of regulatory and catalytic subunits that convert extracellular cues to intracellular responses upon activation. Overall, this signaling pathway is under tight regulation and even slight perturbations can lead to aberrant pathway activation. In NSCLC cell lines, we found that manipulation of miR-10a results in significant changes to both mRNA and protein levels of PI3K. In the context of cellular response to front-line chemotherapeutic agents used to treat NSCLC, I uncovered that miR-10a mimic decreases cell viability10-fold in the presence of paclitaxel relative to drug alone, while inhibiting miR-10a results in a 10-fold increase, suggesting that high levels of miR-10a may be predictive of response to such agents. To assess its prognostic value, we interrogated miR-10a expression in NSCLC tumors and found that high miR-10a levels correlate with longer overall patient survival. miRNAs can target hundreds of genes, meaning that miR-10a may regulate PIK3CA expression both directly and indirectly. We identified the transcription factor GATA6 as both a target of miR-10a with a predicted miR-10a target site in its 3’UTR and a regulator of PI3K expression, with several conserved binding sites in the promoter of PIK3CA. These findings demonstrate that miR-10a regulates the PI3K pathway at two distinct levels. Microarray expression profiling of NSCLC cells treated with miR-10a mimic had significant down regulation of ALDH1A3, a marker of cancer stem cells. This relationship was confirmed through functional validation of ALDH activity. Multiple miRNA target prediction algorithms showed that ALDH1A3 is not a direct target of miR-10a. To uncover the direct target of miR-10a we used a targeted siRNA screen containing genes implicated in stem cell maintenance to reveal that the WNT and Notch pathway are important for cell survival. Both pathways are down-regulated when cells are treated with a miR-10a mimic. Bioinformatic analysis identified DVL3 as a miR-10a target gene. Manipulation of miR-10a levels resulted in significant changes in both mRNA and protein levels of DVL3. Finally, loss of DVL3 expression significantly decreased ALDH1A3 protein levels and the population of ALDH+ cells. Collectively, my work has uncovered miR-10a as mediator of the potent PI3K oncogenic pathway through both direct and indirect mechanisms, a modulator of cellular response to paclitaxel and finally its identification in NSCLC stem cell maintenance through regulation of the WNT and NOTCH pathways.Item Proteomic Discovery of Functionally Important Pathways in Myocardial Ischemia-Reperfusion Injury(2014-02-04) Ahmed, Kamran; Luo, Yang; Taneja, Shikha; Keshishian, Hasmik; Carr, Steven; Rosenzweig, AnthonyBACKGROUND: Coronary heart disease, a source of myocardial ischemia-reperfusion injury (IRI), is the world's leading cause of death and disability. Insulin-like growth factor 1 (IGF1) transgenic (Tg) mouse hearts are protected from IRI, whereas Akt-Tg mouse hearts recover poorly from IRI. Surprisingly, Akt is a downstream component of IGF1 signaling. The Akt-Tg phenotype can be rescued by cardiac gene transfer of activated PI3-kinase (PI3K), another component of the IGF1 pathway, suggesting that PI3K-dependent but Akt-independent pathways are key determinants of IRI. To discern such pathways, we analyzed the proteomic and phosphoproteomic changes in wild-type (WT), IGF1-Tg, and Akt-Tg mouse hearts, identified 20 differentially regulated candidates as potential modifiers of IRI, and began testing their functional roles in an in vitro model. We hypothesize that the cardioprotection observed in IGF1 overexpression is a result of PI3K-dependent but Akt-independent signaling pathways. METHODS: WT hearts were collected at 4 time points of ex-vivo Langendorff IRI and analyzed with liquid chromatography-tandem mass spectrometry to determine protein expression and phosphorylation changes. IGF1-Tg and Akt-Tg hearts were analyzed at baseline. Protein network analysis was performed using Cytoscape software. The functional effects of candidates with expression or phosphorylation differences ≥2-fold were assessed in rat neonatal ventricular myocytes using in vitro redox-based viability assays and cell proliferation studies. RESULTS: In the WT IRI studies, 6403 proteins and 22833 phosphopeptides were quantified. During IRI, no proteins changed in expression, 45 phosphopeptides were upregulated, and 975 phosphopeptides were downregulated. In the IGF1-Tg and Akt-Tg hearts, 6700 proteins and 23000 phosphopeptides were quantified. In vitro knockdown of rho-associated protein kinase 2 (ROCK2) increased the viability signal by 17% in normoxia and 33% in simulated IRI (p<0.05) and increased EdU incorporation from 28.9% to 40.15% (p<0.00001). Network analysis of Akt-Tg hearts revealed significant downregulation of 23 out of 45 subunits of Complex I (p<0.05). CONCLUSIONS: Dephosphorylation of the cardiac phosphoproteome is the dominant pattern in IRI, which may reflect phosphatase activation or reduced ATP levels inhibiting kinase activity. ROCK2 knockdown increased the viability signal by stimulating proliferation in vitro. Whether ROCK2 is involved in cardiomyogenesis in the adult heart will be addressed in future studies. Akt-Tg hearts may be susceptible to IRI due to a reduced ATP reserve caused by Complex I downregulationItem Proteomic Discovery of Functionally Important Pathways in Myocardial Ischemia-Reperfusion Injury(2016-04-01) Ahmed, Kamran; Hill, Joseph A.; Rosenzweig, Anthony; Munshi, Nikhil; Sadek, Hesham A.BACKGROUND: Coronary heart disease, a source of myocardial ischemia-reperfusion injury (IRI), is the world's leading cause of death and disability. Insulin-like growth factor 1 (IGF1) transgenic (Tg) mouse hearts are protected from IRI, whereas Akt-Tg mouse hearts recover poorly from IRI. Surprisingly, Akt is a downstream component of IGF1 signaling. The Akt-Tg phenotype can be rescued by cardiac gene transfer of activated PI3-kinase (PI3K), another component of the IGF1 pathway, suggesting that PI3K-dependent but Akt-independent pathways are key determinants of IRI. To discern such pathways, we analyzed the proteomic and phosphoproteomic changes in wild-type (WT) mouse hearts subjected to IRI ex vivo, and IGF1-TG and Akt-Tg mouse hearts in order to identify 20 differentially regulated candidates as potential modifiers of IRI, and began testing their functional roles in an in vitro model. OBJECTIVE: We hypothesize that the cardioprotection observed in IGF1 overexpression is a result of PI3K-dependent but Akt-independent signaling pathways. METHODS: WT hearts were collected at 4 time points of ex-vivo Langendorff IRI and analyzed with liquid chromatography-tandem mass spectrometry to determine protein abundance and phosphorylation changes. IGF1-Tg and Akt-Tg hearts were analyzed at baseline. Protein network analysis was performed using Cytoscape software. The functional effects of candidates with abundance or phosphorylation differences ≥2-fold were assessed in rat neonatal ventricular myocytes using in vitro redox-based viability assays and cell proliferation studies. RESULTS: In the WT IRI studies, 6403 proteins and 22833 phosphopeptides were quantified. During IRI, no proteins changed in abundance, 10 phosphopeptides were upregulated, and 330 phosphopeptides were downregulated. In the IGF1-Tg and Akt-Tg hearts, 6700 proteins and 23000 phosphopeptides were quantified. Out of the significantly regulated proteins, in vitro knockdown of rho-associated protein kinase 2 (ROCK2) increased the viability signal by 17% in normoxia and 33% in simulated IRI (p<0.05) and increased EdU incorporation from 28.9% to 40.15% (p<0.00001). Network analysis of Akt-Tg hearts revealed significant downregulation of 24 out of 45 subunits of Complex I of the electron transport chain (p<0.05). CONCLUSION: Dephosphorylation of the cardiac phosphoproteome is the dominant pattern in IRI, which may reflect phosphatase activation or reduced ATP levels inhibiting kinase activity. ROCK2 knockdown increased the viability signal by stimulating proliferation in vitro. Whether ROCK2 is involved in cardiomyogenesis in the adult heart will be addressed in future studies. Akt-Tg hearts may be susceptible to IRI due to a reduced ATP reserve caused by Complex I downregulation.Item Study of the Mechanisms Underlying Hippocampal Neuron Synaptogenesis: The Roles of Neurotrophin Signaling and MicroRNAs(2010-11-02) Zhang, Wei; Parada, Luis F.Synapse formation requires contacts between dendrites and axons. Although this process is often viewed as axon mediated, dendritic filopodia may be actively involved in mediating synaptogenic contacts. Brain-derived neurotrophic factor (BDNF) increases the density of dendritic filopodia and the conditional deletion of tyrosine receptor kinase B (TrkB) reduces synapse density in vivo (Luikart et al., 2005). Here, we report that TrkB associates with dendritic growth cones and filopodia, mediates filopodial motility, and does so via the phosphoinositide 3 kinase (PI3K) pathway. We used genetic and pharmacological manipulations of mouse hippocampal neurons to assess signaling downstream of TrkB. Conditional knock-out of two downstream negative regulators of TrkB signaling, Pten (phosphatase with tensin homolog) and Nf1 (neurofibromatosis type 1), enhanced filopodial motility. This effect was PI3K-dependent and correlated with synapse density. Phosphatidylinositol 3,4,5- trisphosphate (PIP3) was preferentially localized in filopodia and this distribution was enhanced by BDNF application. Thus, intracellular control of filopodial dynamics converged on PI3K activation and PIP3 accumulation, a cellular paradigm conserved for chemotaxis in other cell types. Our results suggest that filopodial movement is not random, but responsive to synaptic guidance cues. In order to further elucidate the mechanisms of BDNF-TrkB-PI3K pathway downstream signaling involved in regulating dendritic filopodial motility, we used a pharmacological approach as well as a gene expression approach to show that Rac1 and RhoA may play a role in this pathway. Rac1 positively regulated dendritic filopodial motility while RhoA had a negative effect. Our data suggest that BDNF-TrkB signaling might function to regulate the balance between Rac1 and RhoA, thus controlling dendritic filopodial motility. The developing nervous system is shaped by highly orchestrated programs of gene expression. This tight regulation is regulated by various transcriptional and post-transcriptional events that control individual gene expression. The recent discovery of small, non-coding RNAs has greatly expanded our understanding of the mechanisms that regulate gene expression at the post-transcriptional level. Here, I characterized the expression pattern of one neuronal microRNA, miR-381, and used in vitro cultured hippocampal neurons to show that miR-381 regulates neurite growth, as overexpression of miR-381 promotes neuronal dendritic branching. The effect of miR-381 on neuronal dendritic branching might be through a net regulation of multiple target genes.Item T Cell Intrinsic BCAP Links IL1R to the PI3K-mTOR Pathway and Regulates Pathogenic Th17 Differentiation(2018-07-24) Deason, Krystin Leigh; Hooper, Lora V.; Pasare, Chandrashekhar; van Oers, Nicolai S. C.; Satterthwaite, Anne B.Toll-IL-1R homology (TIR) domains are found within adaptor proteins involved in the signaling of Toll like receptors (TLRs) and Interleukin 1 receptor (IL1R) families. Previous work by our lab identified a TIR domain in the protein B cell adaptor for phosphoinositide 3-kinase (BCAP) and determined a role for BCAP in the TLR signaling pathway in myeloid cells. Due to the shared use of TIR domains by TLR and IL1R signaling pathways, I hypothesized that BCAP would also be involved in signaling downstream of the IL1 family of receptors. The IL1 cytokine family has been shown to play a major role in T cell activation, survival, and differentiation; IL1b, in particular, plays a critical role in the differentiation of Th17 lineage cells. Here, I discovered that BCAP functions downstream of IL1R in CD4 T cells and thereby regulates Th17 lineage differentiation and function. IL1b-induced PI3K-Akt-mTOR signaling is compromised in BCAP deficient T cells which leads to decreased mTOR activation, decreased glycolysis, and defective Th17 lineage commitment. Transcriptional analysis of BCAP deficient CD4 T cells revealed that BCAP is critical for the expression of genes associated with pathogenic Th17 lineage cells. Mice specifically lacking BCAP in T cells have normal development of steady state Th17 cells in vivo yet have decreased development of pathogenic Th17 diseases, such as experimental autoimmune encephalomyelitis (EAE) and T cell transfer colitis. Further, the use of a potent inhibitor of mTOR, which is downstream of BCAP activation, mimics BCAP deficiency by preventing IL1b induced differentiation of pathogenic Th17 cells. This study establishes BCAP as a critical link between IL1R and the metabolic status of activated Th17 cells and further demonstrates that BCAP is critical for the generation of pathogenic Th17 cells in vitro and in vivo.Item [UT Southwestern Medical Center News](2010-07-07) Shear, Kristen Holland