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1.
Nucleic Acids Res ; 45(22): 12681-12699, 2017 Dec 15.
Article in English | MEDLINE | ID: mdl-29036586

ABSTRACT

Crosstalk between growth factors (GFs) and steroid hormones recurs in embryogenesis and is co-opted in pathology, but underlying mechanisms remain elusive. Our data from mammary cells imply that the crosstalk between the epidermal GF and glucocorticoids (GCs) involves transcription factors like p53 and NF-κB, along with reduced pausing and traveling of RNA polymerase II (RNAPII) at both promoters and bodies of GF-inducible genes. Essentially, GCs inhibit positive feedback loops activated by GFs and stimulate the reciprocal inhibitory loops. As expected, no alterations in DNA methylation accompany the transcriptional events instigated by either stimulus, but forced demethylation of regulatory regions broadened the repertoire of GF-inducible genes. We report that enhancers, like some promoters, are poised for activation by GFs and GCs. In addition, within the cooperative interface of the crosstalk, GFs enhance binding of the GC receptor to DNA and, in synergy with GCs, promote productive RNAPII elongation. Reciprocally, within the antagonistic interface GFs hyper-acetylate chromatin at unmethylated promoters and enhancers of genes involved in motility, but GCs hypoacetylate the corresponding regions. In conclusion, unmethylated genomic regions that encode feedback regulatory modules and differentially recruit RNAPII and acetylases/deacetylases underlie the crosstalk between GFs and a steroid hormone.


Subject(s)
Epidermal Growth Factor/pharmacology , Epigenesis, Genetic , Gene Expression Regulation/drug effects , Glucocorticoids/pharmacology , Promoter Regions, Genetic/genetics , Cell Line , Cell Movement/drug effects , Cell Movement/genetics , DNA Methylation , Dexamethasone/pharmacology , Humans , NF-kappa B/metabolism , Protein Processing, Post-Translational/drug effects , RNA Polymerase II/metabolism , Transcription Factors/metabolism , Tumor Suppressor Protein p53/metabolism
2.
Nucleic Acids Res ; 44(3): 1370-83, 2016 Feb 18.
Article in English | MEDLINE | ID: mdl-26657629

ABSTRACT

Circular RNAs (circRNAs) are widespread circles of non-coding RNAs with largely unknown function. Because stimulation of mammary cells with the epidermal growth factor (EGF) leads to dynamic changes in the abundance of coding and non-coding RNA molecules, and culminates in the acquisition of a robust migratory phenotype, this cellular model might disclose functions of circRNAs. Here we show that circRNAs of EGF-stimulated mammary cells are stably expressed, while mRNAs and microRNAs change within minutes. In general, the circRNAs we detected are relatively long-lived and weakly expressed. Interestingly, they are almost ubiquitously co-expressed with the corresponding linear transcripts, and the respective, shared promoter regions are more active compared to genes producing linear isoforms with no detectable circRNAs. These findings imply that altered abundance of circRNAs, unlike changes in the levels of other RNAs, might not play critical roles in signaling cascades and downstream transcriptional networks that rapidly commit cells to specific outcomes.


Subject(s)
Epidermal Growth Factor/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , RNA/genetics , Breast/cytology , Cell Line , Gene Expression/drug effects , Gene Expression Profiling/methods , Heparin-binding EGF-like Growth Factor/genetics , Humans , MicroRNAs/genetics , Oligonucleotide Array Sequence Analysis , RNA Stability/drug effects , RNA Stability/genetics , RNA, Circular , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transforming Growth Factor alpha/genetics
3.
Nat Commun ; 5: 5073, 2014 Oct 03.
Article in English | MEDLINE | ID: mdl-25278152

ABSTRACT

Signal transduction by receptor tyrosine kinases (RTKs) and nuclear receptors for steroid hormones is essential for body homeostasis, but the cross-talk between these receptor families is poorly understood. We observed that glucocorticoids inhibit signalling downstream of EGFR, an RTK. The underlying mechanism entails suppression of EGFR's positive feedback loops and simultaneous triggering of negative feedback loops that normally restrain EGFR. Our studies in mice reveal that the regulation of EGFR's feedback loops by glucocorticoids translates to circadian control of EGFR signalling: EGFR signals are suppressed by high glucocorticoids during the active phase (night-time in rodents), while EGFR signals are enhanced during the resting phase. Consistent with this pattern, treatment of animals bearing EGFR-driven tumours with a specific kinase inhibitor was more effective if administered during the resting phase of the day, when glucocorticoids are low. These findings support a circadian clock-based paradigm in cancer therapy.


Subject(s)
ErbB Receptors/metabolism , Glucocorticoids/metabolism , Neoplasms/pathology , Signal Transduction , Animals , Cell Line, Tumor , Cell Movement , Circadian Rhythm , Disease Progression , Female , Humans , Ligands , MAP Kinase Signaling System , Mice , Mice, Inbred C57BL , Mice, Knockout , Oscillometry , Receptors, Glucocorticoid/metabolism , Treatment Outcome
4.
FEBS Lett ; 588(15): 2407-14, 2014 Aug 01.
Article in English | MEDLINE | ID: mdl-24873881

ABSTRACT

Tumor progression can be understood as a collaborative effort of mutations and growth factors, which propels cell proliferation and matrix invasion, and also enables evasion of drug-induced apoptosis. Concentrating on EGFR, we discuss downstream signaling and the initiation of transcriptional events in response to growth factors. Specifically, we portray a wave-like program, which initiates by rapid disappearance of two-dozen microRNAs, followed by an abrupt rise of immediate early genes (IEGs), relatively short transcripts encoding transcriptional regulators. Concurrent with the fall of IEGs, some 30-60 min after stimulation, a larger group, the delayed early genes, is up-regulated and its own fall overlaps the rise of the final wave of late response genes. This late wave persists and determines long-term phenotype acquisition, such as invasiveness. Key regulatory steps in the orderly response to growth factors provide a trove of potential oncogenes and tumor suppressors.


Subject(s)
Epidermal Growth Factor/metabolism , Gene Expression Regulation, Neoplastic , Neoplasms/metabolism , Transcription, Genetic , Animals , Genes, Immediate-Early , Genes, erbB-1 , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Neoplasms/genetics
5.
Nature ; 485(7396): 55-61, 2012 Feb 22.
Article in English | MEDLINE | ID: mdl-22367541

ABSTRACT

The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth and cancer. However, the downstream translationally regulated nodes of gene expression that may direct cancer development are poorly characterized. Using ribosome profiling, we uncover specialized translation of the prostate cancer genome by oncogenic mTOR signalling, revealing a remarkably specific repertoire of genes involved in cell proliferation, metabolism and invasion. We extend these findings by functionally characterizing a class of translationally controlled pro-invasion messenger RNAs that we show direct prostate cancer invasion and metastasis downstream of oncogenic mTOR signalling. Furthermore, we develop a clinically relevant ATP site inhibitor of mTOR, INK128, which reprograms this gene expression signature with therapeutic benefit for prostate cancer metastasis, for which there is presently no cure. Together, these findings extend our understanding of how the 'cancerous' translation machinery steers specific cancer cell behaviours, including metastasis, and may be therapeutically targeted.


Subject(s)
Neoplasm Metastasis , Prostatic Neoplasms/pathology , Protein Biosynthesis , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Benzoxazoles/pharmacology , Cell Cycle Proteins , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factors/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Genome/genetics , Humans , Male , Mice , Mice, Inbred C57BL , Neoplasm Invasiveness/genetics , Neoplasm Metastasis/drug therapy , Neoplasm Metastasis/genetics , Phosphoproteins/metabolism , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/genetics , Pyrimidines/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Repressor Proteins/metabolism , TOR Serine-Threonine Kinases/antagonists & inhibitors
6.
Invest New Drugs ; 30(6): 2219-25, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22270257

ABSTRACT

ATP-competitive mammalian target of rapamycin (mTOR) inhibitors are in early phase clinical trials. These novel targeted agents, including PP242, are mechanistically distinct from the allosteric, partial mTOR inhibitor, rapamycin. The goal of this study was to evaluate how PP242 best combines with standard chemotherapies for colorectal cancer (CRC), and which subsets of patients are most likely to benefit. The combination index for PP242 plus 5-fluorouracil, oxaliplatin, or irinotecan was determined in CRC cell lines with different mutational backgrounds. In KRAS mutant CRC cell lines, sensitivity to PP242 increases with co-mutation of PIK3CA. Mutation of p53 predicts resistance to chemotherapy, but not PP242. Efficacy of PP242 was comparable to that of standard chemotherapies over the dose range tested. Sensitivity or resistance to PP242 dictates relative synergy or antagonism, respectively, when PP242 is combined with 5-fluorouracil. The same trend exists for PP242 + oxaliplatin, but with a narrower dynamic range. Conversely potency of PP242 and the combination index for PP242 + irinotecan were unrelated, but synergy exists across all dose levels in PP242 and irinotecan sensitive, p53 wild-type cell lines. Overall, our in vitro analysis predicts that mutational status can be used to rank sensitivity to PP242 and standard chemotherapies. Single agent potency can in turn be used to predict the combination index in a drug-specific manner. Our data suggest a clinical trial to determine whether ATP-competitive mTOR inhibitors provide benefit in combination with standard chemotherapies for patients with PIK3CA mutant metastatic CRC, stratified by the presence or absence of KRAS co-mutation.


Subject(s)
Antineoplastic Agents/administration & dosage , Camptothecin/analogs & derivatives , Fluorouracil/administration & dosage , Indoles/administration & dosage , Organoplatinum Compounds/administration & dosage , Purines/administration & dosage , TOR Serine-Threonine Kinases/antagonists & inhibitors , Adenosine Triphosphate , Camptothecin/administration & dosage , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Drug Combinations , Humans , Irinotecan , Mutation , Nuclear Proteins/genetics , Oxaliplatin , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , Transcription Factors/genetics , ras Proteins/genetics
7.
Genes Dev ; 24(23): 2627-39, 2010 Dec 01.
Article in English | MEDLINE | ID: mdl-21123650

ABSTRACT

All viruses require cellular ribosomes to translate their mRNAs. Viruses producing methyl-7 (m7) GTP-capped mRNAs, like Herpes Simplex Virus-1 (HSV-1), stimulate cap-dependent translation by activating mTORC1 to inhibit the translational repressor 4E-binding protein 1 (4E-BP1). Here, we establish that the HSV-1 kinase Us3 masquerades as Akt to activate mTORC1. Remarkably, Us3 displays no sequence homology with the cellular kinase Akt, yet directly phosphorylates tuberous sclerosis complex 2 (TSC2) on the same sites as Akt. TSC2 depletion rescued Us3-deficient virus replication, establishing that Us3 enhances replication by phosphorylating TSC2 to constitutively activate mTORC1, effectively bypassing S6K-mediated feedback inhibition. Moreover, Us3 stimulated Akt substrate phosphorylation in infected cells, including FOXO1 and GSK3. Thus, HSV-1 encodes an Akt surrogate with overlapping substrate specificity to activate mTORC1, stimulating translation and virus replication. This establishes Us3 as a unique viral kinase with promising drug development potential.


Subject(s)
Enzyme Activation/physiology , Gene Expression Regulation, Viral , Herpes Simplex/virology , Herpesvirus 1, Human/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Transcription Factors/metabolism , Virus Replication , Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins , Cell Line , Cell Line, Tumor , Eukaryotic Initiation Factor-4F/metabolism , HEK293 Cells , Herpes Simplex/physiopathology , Herpesvirus 1, Human/enzymology , Humans , Phosphoproteins/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/metabolism , RNA, Viral/metabolism , Signal Transduction , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor Proteins/metabolism , Viral Proteins/metabolism
8.
Curr Top Microbiol Immunol ; 347: 241-62, 2010.
Article in English | MEDLINE | ID: mdl-20549474

ABSTRACT

mTOR (mammalian Target of Rapamycin) is the hub of the phosphoinositide 3-Kinase (PI3-K)→Akt→mTOR pathway, which is one of the most commonly mutated pathways in cancer. PI3-Ks and mTOR are related kinases which share an evolutionarily related kinase domain, although the former is a lipid kinase and the latter is a protein kinase. As a result of their similar ATP sites, the prototypical PI3-K inhibitors LY294002 and wortmannin inhibit both kinases, although the compounds have been primarily thought of as inhibitors of PI3-Ks. The widespread use of these reagents to understand PI3-K signaling and the likelihood that many of their effects are confounded by dual inhibition of PI3-K and mTOR make it essential to develop selective mTOR inhibitors in part to understand the unique cellular effects of inhibition of this key downstream component in the growth factor pathway. Rapamycin has historically provided a means for selective mTOR inhibition, yet it is not a typical ATP competitive inhibitor, making its effects difficult to reconcile with LY294002 and wortmannin. Several groups have recently reported pharmacological agents which inhibit mTOR but not PI3-K, providing a new pharmacological approach to selective mTOR inhibition. The TOR kinase domain inhibitors of mTOR have been termed TORKinibs to distinguish their mode of action from rapamycin and its analogs (rapalogs). These inhibitors bind to the ATP binding site of the kinase domain of mTOR and as a result inhibit both mTOR complexes, TORC1 (rapamycin sensitive) and TORC2 (rapamycin resistant). These molecules have allowed a reinvestigation of mTOR and in particular a reinvestigation of the mechanistic basis for incomplete proliferative arrest of cells by Rapamycin. A consensus has quickly emerged from the study of various TORKinibs that Rapamycin is ineffective at blocking cell proliferation because it only partially inhibits the activity of mTORC1. The profound anti-proliferative effect of TORKinibs suggests that as the molecules enter the clinic they may be successful in the treatment of cancers where rapamycin has failed.


Subject(s)
Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , TOR Serine-Threonine Kinases/antagonists & inhibitors , Animals , Cell Proliferation/drug effects , Enzyme Activation/drug effects , Humans , Mechanistic Target of Rapamycin Complex 1 , Multiprotein Complexes , Neoplasms/drug therapy , Phosphatidylinositol 3-Kinases/physiology , Phosphorylation , Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/physiology , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/physiology
9.
Cancer Cell ; 17(3): 249-61, 2010 Mar 16.
Article in English | MEDLINE | ID: mdl-20227039

ABSTRACT

We genetically dissect the contribution of the most prominent downstream translational components of mTOR signaling toward Akt-driven lymphomagenesis. While phosphorylation of rpS6 is dispensable for cancer formation, 4EBP-eIF4E exerts significant control over cap-dependent translation, cell growth, cancer initiation, and progression. This effect is mediated at least in part through 4EBP-dependent control of Mcl-1 expression, a key antiapoptotic protein. By using an active site inhibitor of mTOR, PP242, we show a marked therapeutic response in rapamycin-resistant tumors. The therapeutic benefit of PP242 is mediated through inhibition of mTORC1-dependent 4EBP-eIF4E hyperactivation. Thus, the 4EBP-eIF4E axis downstream of mTOR is a druggable mediator of translational control and Akt-mediated tumorigenesis that has important implications for the treatment of human cancers.


Subject(s)
Carrier Proteins/metabolism , Eukaryotic Initiation Factor-4E/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Adaptor Proteins, Signal Transducing , Animals , Apoptosis/drug effects , Carrier Proteins/physiology , Cell Cycle Proteins , Eukaryotic Initiation Factor-4E/physiology , Eukaryotic Initiation Factors , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Lymphoma/metabolism , Mice , Mice, Transgenic , Models, Genetic , Phosphoproteins/physiology , Phosphorylation , Protein Biosynthesis , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/physiology , Ribosomal Protein S6/metabolism , T-Lymphocytes/physiology , TOR Serine-Threonine Kinases
10.
J Am Soc Nephrol ; 21(5): 811-8, 2010 May.
Article in English | MEDLINE | ID: mdl-20338997

ABSTRACT

The serum- and glucocorticoid-induced kinase 1 (SGK1) plays a central role in hormone regulation of epithelial sodium (Na+) channel (ENaC)-dependent Na+ transport in the distal nephron. Phosphorylation within a carboxy-terminal domain, designated the hydrophobic motif (HM), determines the activity of SGK1, but the identity of the HM kinase is unknown. Here, we show that the highly conserved serine-threonine kinase mammalian target of rapamycin (mTOR) is essential for the phosphorylation of the HM of SGK1 and the activation of ENaC. We observed that mTOR, in conjunction with rictor (mTORC2), phosphorylated SGK1 and stimulated ENaC. In contrast, when mTOR assembled with raptor in the rapamycin-inhibited complex (mTORC1), it did not phosphorylate SGK1 or stimulate ENaC. Inhibition of mTOR blocked both SGK1 phosphorylation and ENaC-mediated Na+ transport, whereas specific inhibition of mTORC1 had no effect. Similarly, small hairpin RNA-mediated knockdown of rictor inhibited SGK1 phosphorylation and Na+ current, whereas knockdown of raptor had no effect. Finally, in co-immunoprecipitation experiments, SGK1 interacted selectively with rictor but not with raptor, suggesting selective recruitment of SGK1 to mTORC2. We conclude that mTOR, specifically mTORC2, is the HM kinase for SGK1 and is required for ENaC-mediated Na+ transport, thereby extending our understanding of the molecular mechanisms underlying Na+ balance.


Subject(s)
Epithelial Sodium Channels/metabolism , Immediate-Early Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Kidney Tubules/metabolism , Protein Serine-Threonine Kinases/metabolism , Transcription Factors/metabolism , Cell Line , Epithelial Cells/metabolism , Gene Knockdown Techniques , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 1 , Multiprotein Complexes , Phosphorylation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proteins , Sodium/metabolism , TOR Serine-Threonine Kinases
11.
PLoS Biol ; 7(2): e38, 2009 Feb 10.
Article in English | MEDLINE | ID: mdl-19209957

ABSTRACT

The mammalian target of rapamycin (mTOR) regulates cell growth and survival by integrating nutrient and hormonal signals. These signaling functions are distributed between at least two distinct mTOR protein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to the selective inhibitor rapamycin and activated by growth factor stimulation via the canonical phosphoinositide 3-kinase (PI3K)-->Akt-->mTOR pathway. Activated mTORC1 kinase up-regulates protein synthesis by phosphorylating key regulators of mRNA translation. By contrast, mTORC2 is resistant to rapamycin. Genetic studies have suggested that mTORC2 may phosphorylate Akt at S473, one of two phosphorylation sites required for Akt activation; this has been controversial, in part because RNA interference and gene knockouts produce distinct Akt phospho-isoforms. The central role of mTOR in controlling key cellular growth and survival pathways has sparked interest in discovering mTOR inhibitors that bind to the ATP site and therefore target both mTORC2 and mTORC1. We investigated mTOR signaling in cells and animals with two novel and specific mTOR kinase domain inhibitors (TORKinibs). Unlike rapamycin, these TORKinibs (PP242 and PP30) inhibit mTORC2, and we use them to show that pharmacological inhibition of mTOR blocks the phosphorylation of Akt at S473 and prevents its full activation. Furthermore, we show that TORKinibs inhibit proliferation of primary cells more completely than rapamycin. Surprisingly, we find that mTORC2 is not the basis for this enhanced activity, and we show that the TORKinib PP242 is a more effective mTORC1 inhibitor than rapamycin. Importantly, at the molecular level, PP242 inhibits cap-dependent translation under conditions in which rapamycin has no effect. Our findings identify new functional features of mTORC1 that are resistant to rapamycin but are effectively targeted by TORKinibs. These potent new pharmacological agents complement rapamycin in the study of mTOR and its role in normal physiology and human disease.


Subject(s)
Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Sirolimus/pharmacology , 3T3 Cells , Actins , Animals , Catalytic Domain/drug effects , Cell Proliferation/drug effects , Fibroblasts , Gene Expression Regulation , Insulin/metabolism , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Knockout , Multiprotein Complexes , Phosphorylation/drug effects , Protein Kinases/genetics , Proteins , Pyrimidines/metabolism , TOR Serine-Threonine Kinases , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism
12.
Blood ; 113(13): 2945-54, 2009 Mar 26.
Article in English | MEDLINE | ID: mdl-19139077

ABSTRACT

Gram-negative bacterial infections, unlike viral infections, do not typically protect against subsequent viral infections. This is puzzling given that lipopolysaccharide (LPS) and double-stranded (ds) RNA both activate the TIR domain-containing adaptor-inducing interferon beta (TRIF) pathway and, thus, are both capable of eliciting an antiviral response by stimulating type I interferon (IFN) production. We demonstrate herein that SH2-containing inositol-5'-phosphatase (SHIP) protein levels are dramatically increased in murine macrophages via the MyD88-dependent pathway, by up-regulating autocrine-acting transforming growth factor-beta (TGFbeta). The increased SHIP then mediates, via inhibition of the phosphatidylinositol-3-kinase (PI3K) pathway, cytosine-phosphate-guanosine (CPG)- and LPS-induced tolerance and cross-tolerance and restrains IFN-beta production induced by a subsequent exposure to LPS or dsRNA. Intriguingly, we found, using isoform-specific PI3K inhibitors, that LPS- or cytosine-phosphate-guanosine-induced interleukin-6 (IL-6) is positively regulated by p110alpha, -gamma, and -delta but negatively regulated by p110beta. This may explain some of the controversy concerning the role of PI3K in Toll-like receptor-induced cytokine production. Consistent with our in vitro findings, SHIP(-/-) mice overproduce IFN-beta in response to LPS, and this leads to antiviral hypothermia. Thus, up-regulation of SHIP in response to Gram-negative bacterial infections probably explains the inability of such infections to protect against subsequent viral infections.


Subject(s)
Immunity, Innate/drug effects , Immunity, Innate/genetics , Lipopolysaccharides/pharmacology , Phosphoric Monoester Hydrolases/genetics , Viruses/immunology , Animals , Cells, Cultured , CpG Islands/immunology , CpG Islands/physiology , Gene Expression Regulation/drug effects , Gene Expression Regulation/immunology , Hypothermia/genetics , Hypothermia/immunology , Immune Tolerance/drug effects , Immune Tolerance/genetics , Inositol Polyphosphate 5-Phosphatases , Interferon-beta/metabolism , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/physiology , Phosphoric Monoester Hydrolases/metabolism , RNA, Double-Stranded/immunology , RNA, Double-Stranded/pharmacology , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/pharmacology
13.
PLoS One ; 3(6): e2526, 2008 Jun 25.
Article in English | MEDLINE | ID: mdl-18575589

ABSTRACT

BACKGROUND: Hollow smooth muscle organs such as the bladder undergo significant changes in wall tension associated with filling and distension, with attendant changes in muscle tone. Our previous study indicated that stretch induces Ca(2+) release occurs in the form of Ca(2+) sparks and Ca(2+) waves in urinary bladder myocytes. While, the mechanism underlying stretch-induced Ca2+ release in smooth muscle is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We examined the transduction mechanism linking cell stretch to Ca(2+) release. The probability and frequency of Ca(2+) sparks induced by stretch were closely related to the extent of cell extension and the time that the stretch was maintained. Experiments in tissues and single myocytes indicated that mechanical stretch significantly increases the production of nitric oxide (NO) and the amplitude and duration of muscle contraction. Stretch-induced Ca(2+) sparks and contractility increases were abrogated by the NO inhibitor L-NAME and were also absent in eNOS knockout mice. Furthermore, exposure of eNOS null mice to exogenously generated NO induced Ca(2+) sparks. The soluble guanylyl cyclase inhibitor ODQ did not inhibit SICR, but this process was effectively blocked by the PI3 kinase inhibitors LY494002 and wortmannin; the phosphorylation of Akt and eNOS were up-regulated by 204+/-28.6% and 258+/-36.8% by stretch, respectively. Moreover, stretch significantly increased the eNOS protein expression level. CONCLUSIONS/SIGNIFICANCE: Taking together, these results suggest that stretch-induced Ca2+ release is NO dependent, resulting from the activation of PI3K/Akt pathway in smooth muscle.


Subject(s)
Calcium/metabolism , Muscle, Smooth/metabolism , Nitric Oxide/physiology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Urothelium/metabolism , Androstadienes/pharmacology , Animals , Enzyme Activation , Enzyme Inhibitors/pharmacology , Mice , Mice, Knockout , Muscle, Smooth/enzymology , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide/antagonists & inhibitors , Nitric Oxide/biosynthesis , Nitric Oxide Synthase Type III/genetics , Nitric Oxide Synthase Type III/metabolism , Oxadiazoles/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Quinoxalines/pharmacology , Urothelium/enzymology , Wortmannin
14.
J Gen Physiol ; 127(3): 225-35, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16505145

ABSTRACT

Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) occurs in smooth muscle as spontaneous SR Ca(2+) release or Ca(2+) sparks and, in some spiking tissues, as Ca(2+) release that is triggered by the activation of sarcolemmal Ca(2+) channels. Both processes display spatial localization in that release occurs at a higher frequency at specific subcellular regions. We have used two-photon flash photolysis (TPFP) of caged Ca(2+) (DMNP-EDTA) in Fluo-4-loaded urinary bladder smooth muscle cells to determine the extent to which spatially localized increases in Ca(2+) activate SR release and to further understand the molecular and biophysical processes underlying CICR. TPFP resulted in localized Ca(2+) release in the form of Ca(2+) sparks and Ca(2+) waves that were distinguishable from increases in Ca(2+) associated with Ca(2+) uncaging, unequivocally demonstrating that Ca(2+) release occurs subsequent to a localized rise in [Ca(2+)](i). TPFP-triggered Ca(2+) release was not constrained to a few discharge regions but could be activated at all areas of the cell, with release usually occurring at or within several microns of the site of photolysis. As expected, the process of CICR was dominated by ryanodine receptor (RYR) activity, as ryanodine abolished individual Ca(2+) sparks and evoked release with different threshold and kinetics in FKBP12.6-null cells. However, TPFP CICR was not completely inhibited by ryanodine; Ca(2+) release with distinct kinetic features occurred with a higher TPFP threshold in the presence of ryanodine. This high threshold release was blocked by xestospongin C, and the pharmacological sensitivity and kinetics were consistent with CICR release at high local [Ca(2+)](i) through inositol trisphosphate (InsP(3)) receptors (InsP(3)Rs). We conclude that CICR activated by localized Ca(2+) release bears essential similarities to those observed by the activation of I(Ca) (i.e., major dependence on the type 2 RYR), that the release is not spatially constrained to a few specific subcellular regions, and that Ca(2+) release through InsP(3)R can occur at high local [Ca(2+)](i).


Subject(s)
Calcium Signaling/physiology , Calcium/metabolism , Ion Channel Gating/physiology , Membrane Potentials/physiology , Myocytes, Smooth Muscle/physiology , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic Reticulum/physiology , Animals , Humans , Rabbits
15.
ACS Chem Biol ; 1(12): 755-60, 2006 Dec 15.
Article in English | MEDLINE | ID: mdl-17240973

ABSTRACT

The homeodomain (HD)-DNA interface has been conserved over 500 million years of evolution. Despite this conservation, we have successfully re-engineered the engrailed HD to specifically recognize an unnatural nucleotide using a phage display selection. Here we report the synthesis of novel nucleosides and the selection of mutant HDs that bind these nucleotides using phage display. The high-resolution crystal structure of one mutant in complex with modified and unmodified DNA demonstrates that, even with the substantial perturbation to the interface, this selected mutant retains a canonical HD structure. Dissection of the contributions due to each of the selected mutations reveals that the majority of the modification-specific binding is accomplished by a single mutation (I47G) but that the remaining mutations retune the stability of the HD. These results afford a detailed look at a re-engineered protein-DNA interaction and provide insight into the opportunities for re-engineering highly conserved interfaces.


Subject(s)
DNA , Homeodomain Proteins , Protein Engineering , Recombinant Fusion Proteins/chemistry , Transcription Factors , Base Sequence , Binding Sites , Crystallography, X-Ray , DNA/chemistry , DNA/genetics , Homeodomain Proteins/chemistry , Homeodomain Proteins/genetics , Models, Molecular , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , Peptide Library , Protein Binding , Transcription Factors/chemistry , Transcription Factors/genetics
16.
Nucleic Acids Res ; 33(2): e14, 2005 Jan 19.
Article in English | MEDLINE | ID: mdl-15659575

ABSTRACT

Conditional inactivation of individual genes in mice using site-specific recombinases is an extremely powerful method for determining the complex roles of mammalian genes in developmental and tissue-specific contexts, a major goal of post-genomic research. However, the process of generating mice with recombinase recognition sequences placed at specific locations within a gene, while maintaining a functional allele, is time consuming, expensive and technically challenging. We describe a system that combines gene trap and site-specific DNA inversion to generate mouse embryonic stem (ES) cell clones for the rapid production of conditional knockout mice, and the use of this system in an initial gene trap screen. Gene trapping should allow the selection of thousands of ES cell clones with defined insertions that can be used to generate conditional knockout mice, thereby providing extensive parallelism that eliminates the time-consuming steps of targeting vector construction and homologous recombination for each gene.


Subject(s)
Gene Targeting/methods , Mice, Knockout/genetics , Recombination, Genetic , Animals , Cell Line , Embryo, Mammalian/cytology , Humans , Integrases/genetics , Mice , Mice, Knockout/embryology , Mice, Transgenic , Mutagenesis, Insertional , Recombinases/metabolism , Stem Cells/cytology , Transcription, Genetic
17.
J Allergy Clin Immunol ; 114(2 Suppl): S18-31, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15309016

ABSTRACT

The study of isolated airway myocytes has provided important information relative to specific processes that regulate contraction, proliferation, and synthetic properties of airway smooth muscle (ASM). To place this information in physiological context, however, improved methods to examine airway biology in vivo are needed. Advances in genetic, biochemical, and optical methods provide unprecedented opportunities to improve our understanding of in vivo physiology and pathophysiology. This article describes 4 important methodologic advances in the study of ASM: (1) the development of transgenic mice that could be used to investigate ASM proliferation and phenotype switching during the development of hypersensitivity, and to investigate excitation-contraction coupling; (2) the use of CD38-deficient mice to confirm the role of CD38-dependent, cyclic adenosine diphosphate-ribose-mediated calcium release in airway responsiveness; (3) investigation of the role of actin filament length and p38 mitogen-activated protein kinase activity in regulating the mechanical plasticity-elasticity balance in contracted ASM; and (d) the use of bronchial biopsies to study ASM structure and phenotype in respiratory science.


Subject(s)
Bronchi/cytology , Myocytes, Smooth Muscle/physiology , Trachea/cytology , ADP-ribosyl Cyclase/physiology , ADP-ribosyl Cyclase 1 , Actins/physiology , Animals , Antigens, CD/physiology , Asthma/etiology , Calcium Signaling , Cyclic ADP-Ribose/physiology , Elasticity , Humans , Membrane Glycoproteins , Mice , Mice, Transgenic , Mitogen-Activated Protein Kinases/physiology , Myosin Heavy Chains/physiology , p38 Mitogen-Activated Protein Kinases
18.
J Gen Physiol ; 123(4): 377-86, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15024040

ABSTRACT

Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca(2+) release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca(2+) release events (Ca(2+) sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca(2+) release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca(2+) sparks, Ca(2+)-induced Ca(2+) release, and Ca(2+) waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6(-/-), and RYR3(-/-) mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca(2+) sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca(2+) sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca(2+) -induced Ca(2+) release was similarly augmented in FKBP12.6(-/-), but not in RYR3 null cells relative to wild-type. Finally, Ca(2+) wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca(2+) wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca(2+) sparks in smooth muscle, and the finding of normal Ca(2+) sparks and CICR in RYR3 null mice, indicate that Ca(2+) release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca(2+) sparks, and associated STOCs, in smooth muscle.


Subject(s)
Calcium Signaling/physiology , Muscle, Smooth/physiology , Ryanodine Receptor Calcium Release Channel/metabolism , Tacrolimus Binding Proteins/metabolism , Urinary Bladder/physiology , Animals , Membrane Potentials/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Smooth/cytology , Myocytes, Smooth Muscle/physiology , Patch-Clamp Techniques , Ryanodine Receptor Calcium Release Channel/genetics , Tacrolimus Binding Proteins/genetics , Urinary Bladder/cytology
19.
J Biol Chem ; 279(20): 21461-8, 2004 May 14.
Article in English | MEDLINE | ID: mdl-14990564

ABSTRACT

Genetically encoded signaling proteins provide remarkable opportunities to design and target the expression of molecules that can be used to report critical cellular events in vivo, thereby markedly extending the scope and physiological relevance of studies of cell function. Here we report the development of a transgenic mouse expressing such a reporter and its use to examine postsynaptic signaling in smooth muscle. The circularly permutated, Ca2+-sensing molecule G-CaMP (Nakai, J., Ohkura, M., and Imoto, K. (2001) Nat. Biotechnol. 19, 137-141) was expressed in vascular and non-vascular smooth muscle and functioned as a lineage-specific intracellular Ca2+ reporter. Detrusor tissue from these mice was used to identify two separate types of postsynaptic Ca2+ signals, mediated by distinct neurotransmitters. Intrinsic nerve stimulation evoked rapid, whole-cell Ca2+ transients, or "Ca2+ flashes," and slowly propagating Ca2+ waves. We show that Ca2+ flashes occur through P2X receptor stimulation and ryanodine receptor-mediated Ca2+ release, whereas Ca2+ waves arise from muscarinic receptor stimulation and inositol trisphosphate-mediated Ca2+ release. The distinct ionotropic and metabotropic postsynaptic Ca2+ signals are related at the level of Ca2+ release. Importantly, individual myocytes are capable of both postsynaptic responses, and a transition between Ca2+ -induced Ca2+ release and inositol trisphosphate waves occurs at higher synaptic inputs. Ca2+ signaling mice should provide significant advantages in the study of processive biological signaling.


Subject(s)
Calcium Signaling/genetics , Calcium/physiology , Muscle, Smooth/physiology , Animals , Cloning, Molecular , Mice , Mice, Transgenic , Muscle Cells/physiology , Myosin Heavy Chains/genetics , Promoter Regions, Genetic , Receptors, Calcium-Sensing/genetics , Receptors, Calcium-Sensing/physiology , Recombinant Proteins/metabolism , Signal Transduction , Synapses/physiology
20.
Neuron ; 35(6): 1085-97, 2002 Sep 12.
Article in English | MEDLINE | ID: mdl-12354398

ABSTRACT

Perhaps synaptic vesicles can recycle so rapidly because they avoid complete exocytosis, and release transmitter through a fusion pore that opens transiently. This view emerges from imaging whole terminals where the fluorescent lipid FM1-43 seems unable to leave vesicles during transmitter release. Here we imaged single, FM1-43-stained synaptic vesicles by evanescent field fluorescence microscopy, and tracked the escape of dye from single vesicles by watching the increase in fluorescence after exocytosis. Dye left rapidly and completely during most or all exocytic events. We conclude that vesicles at this terminal allow lipid exchange soon after exocytosis, and lose their dye even if they connected with the plasma membrane only briefly. At the level of single vesicles, therefore, observations with FM1-43 provide no evidence that exocytosis of synaptic vesicles is incomplete.


Subject(s)
Exocytosis/physiology , Membrane Lipids/metabolism , Presynaptic Terminals/metabolism , Retina/metabolism , Synaptic Membranes/metabolism , Synaptic Transmission/physiology , Synaptic Vesicles/metabolism , Animals , Diffusion/drug effects , Fluorescent Dyes/metabolism , Fluorescent Dyes/pharmacology , Goldfish , Presynaptic Terminals/ultrastructure , Protein Transport/physiology , Pyridinium Compounds/metabolism , Pyridinium Compounds/pharmacology , Quaternary Ammonium Compounds/metabolism , Quaternary Ammonium Compounds/pharmacology , Reaction Time/physiology , Retina/ultrastructure , Synaptic Membranes/ultrastructure , Synaptic Vesicles/ultrastructure
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