lithium-chloride and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Lithium chloride (LiCl) is a widely used drug for the treatment of bipolar disorders, but as a side effect, 40% of the patients develop diabetes insipidus. LiCl affects the activity of the glycogen synthase kinase 3 (GSK3), and mice deficient for GSK3β showed a reduction in the urine concentration capability. The cellular and molecular mechanisms are not fully understood. We used primary cultured inner medullary collecting duct cells to analyze the underlying mechanisms. LiCl and the inhibitor of GSK3 (SB216763) induced a decrease in the aquaporin-2 (Aqp2) protein level. LiCl induced downregulation of Aqp2 mRNA expression while SB216763 had no effect and TWS119 led to increase in expression. The inhibition of the lysosomal activity with bafilomycin or chloroquine prevented both LiCl- and SB216763-mediated downregulation of Aqp2 protein expression. Bafilomycin and chloroquine induced the accumulation of Aqp2 in lysosomal structures, which was prevented in cells treated with dibutyryl cyclic adenosine monophosphate (dbcAMP), which led to phosphorylation and membrane localization of Aqp2. Downregulation of Aqp2 was also evident when LiCl was applied together with dbcAMP, and dbcAMP prevented the SB216763-induced downregulation. We showed that LiCl and SB216763 induce downregulation of Aqp2 via different mechanisms. While LiCl also affected the mRNA level, SB216763 induced lysosmal degradation. Specific GSK3β inhibition had an opposite effect, indicating a more complex regulatory mechanism.

Topics: Animals; Aquaporin 2; Aquaporin 3; Aquaporin 4; Cells, Cultured; Chloroquine; Cyclic AMP Response Element-Binding Protein; Down-Regulation; Female; Glycogen Synthase Kinase 3; Indoles; Kidney Tubules, Collecting; Leupeptins; Lithium Chloride; Macrolides; Maleimides; Protein Kinase Inhibitors; Proteolysis; Pyrimidines; Pyrroles; Rats, Wistar; RNA, Messenger

Molecular hydrogen (H2) is effective for many diseases. However, molecular bases of H2 have not been fully elucidated. Cumulative evidence indicates that H2 acts as a gaseous signal modulator. We found that H2 suppresses activated Wnt/β-catenin signaling by promoting phosphorylation and degradation οf β-catenin. Either complete inhibition of GSK3 or mutations at CK1- and GSK3-phosphorylation sites of β-catenin abolished the suppressive effect of H2. H2 did not increase GSK3-mediated phosphorylation of glycogen synthase, indicating that H2 has no direct effect on GSK3 itself. Knock-down of adenomatous polyposis coli (APC) or Axin1, which form the β-catenin degradation complex, minimized the suppressive effect of H2 on β-catenin accumulation. Accordingly, the effect of H2 requires CK1/GSK3-phosphorylation sites of β-catenin, as well as the β-catenin degradation complex comprised of CK1, GSK3, APC, and Axin1. We additionally found that H2 reduces the activation of Wnt/β-catenin signaling in human osteoarthritis chondrocytes. Oral intake of H2 water tended to ameliorate cartilage degradation in a surgery-induced rat osteoarthritis model through attenuating β-catenin accumulation. We first demonstrate that H2 suppresses abnormally activated Wnt/β-catenin signaling, which accounts for the protective roles of H2 in a fraction of diseases.

Topics: Adenomatous Polyposis Coli Protein; Animals; Axin Protein; beta Catenin; Casein Kinase I; Cell Line; Chondrocytes; Gases; Glycogen Synthase Kinase 3; HCT116 Cells; HeLa Cells; HT29 Cells; Humans; Hydrogen; Leupeptins; Lithium Chloride; Male; Microscopy, Fluorescence; Osteoarthritis; Phosphorylation; Rats; Rats, Sprague-Dawley; RNA Interference; RNA, Small Interfering; SOX9 Transcription Factor; Water; Wnt Signaling Pathway; Wnt3A Protein

Although the pathogenesis and epileptogenesis of mesial temporal lobe epilepsy (MTLE) have been studied for years, many questions remain. The ubiquitin-proteasome system (UPS) is one factor that might regulate ion channels, inflammation and neuron excitability. Nedd4-2 is an E3 ubiquitin ligase linked with ion channels and synaptic vesicle recycling. Here, we explore the role of the UPS and its E3 ligase Nedd4-2 in the pathogenesis of MTLE. Our western blot results revealed that ubiquitin and Nedd4-2 were expressed differentially in different stages of MTLE. Co-immunoprecipitation and double immunostaining results indicated that Nedd4-2 was the substrate protein of ubiquitin both in vivo and in vitro. Inhibition of the UPS aggravated the epileptogenesis of MTLE, causing early and frequent spontaneous seizures, more obvious neuron loss and aberrant mossy fiber sprouting. Inhibition of ubiquitin also enhanced the activation of Nedd4-2, and switched ion channel α-ENaC downstream. Our study is the first to report that the UPS participates in the pathogenesis of MTLE, inhibition of UPS could aggravate the epileptogenesis, and that Nedd4-2 is a critical E3 ligase involved in this process.

Topics: Adenosine Monophosphate; Analysis of Variance; Animals; Animals, Newborn; Antimanic Agents; Cells, Cultured; Cysteine Proteinase Inhibitors; Disease Models, Animal; Endosomal Sorting Complexes Required for Transport; Epilepsy, Temporal Lobe; Female; Gene Expression Regulation; Hippocampus; Immunoprecipitation; Leupeptins; Lithium Chloride; Male; Muscarinic Agonists; Nedd4 Ubiquitin Protein Ligases; Neurons; Pilocarpine; Rats, Sprague-Dawley; RNA, Small Interfering; Time Factors; Transfection; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitins

Mutations in ribosomal protein (RP) genes can result in the loss of erythrocyte progenitor cells and cause severe anemia. This is seen in patients with Diamond-Blackfan anemia (DBA), a pure red cell aplasia and bone marrow failure syndrome that is almost exclusively linked to RP gene haploinsufficiency. While the mechanisms underlying the cytopenia phenotype of patients with these mutations are not completely understood, it is believed that stabilization of the p53 tumor suppressor protein may induce apoptosis in the progenitor cells. In stark contrast, tumor cells from zebrafish with RP gene haploinsufficiency are unable to stabilize p53 even when exposed to acute DNA damage despite transcribing wild type p53 normally. In this work we demonstrate that p53 has a limited role in eliciting the anemia phenotype of zebrafish models of DBA. In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells. Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53. By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53. Our work indicates that the anemia phenotype of zebrafish models of DBA is dependent on factors other than p53, and may hold clinical significance for both DBA and the increasing number of cancers revealing spontaneous mutations in RP genes.

Topics: Anemia, Diamond-Blackfan; Animals; Animals, Genetically Modified; Apoptosis; Disease Models, Animal; DNA Damage; DNA Repair; Glycogen Synthase Kinase 3; Haploinsufficiency; Insulin; Leupeptins; Lithium Chloride; Morpholinos; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-akt; Ribosomal Proteins; RNA, Messenger; Signal Transduction; Transcription, Genetic; Tumor Suppressor Protein p53; Zebrafish; Zebrafish Proteins

Tumor cells exposed to sub-lethal photodynamic therapy (PDT) cause cellular rescue responses that lead to resistance to the therapy, including expression of angiogenic factors and survival molecules. However, the mechanisms contributing to the resistance are yet to be fully understood. Here, we show for the first time that Mcl-1, an anti-apoptotic protein, plays an important role in protecting cells from PDT-induced apoptosis. In contrast to the reduction in the anti-apoptotic proteins Bcl-2 and Bcl-xl, sub-lethal PDT induces an increase in Mcl-1 expression. Silencing Mcl-1 sensitizes tumor cells to PDT-induced apoptosis, and ectopic expression of Mcl-1 significantly delays Bax translocation to mitochondria and inhibits caspase-3 activity following PDT. Mcl-1 expression is associated closely with activated AKT signaling following PDT. AKT can regulate Mcl-1 expression through GSK-3β and NF-κB at the protein and transcriptional levels, respectively. Inhibition of AKT by Wortmannin or siRNA significantly reduces the levels of Mcl-1 mRNA and protein and enhances PDT-induced apoptosis. Treatment with Celecoxib, a non-steroidal anti-inflammatory drug (NSAID), is shown to downregulate Mcl-1 expression, and enhances PDT-induced apoptosis both in vitro and in vivo. This down-regulation is closely related to the inhibition effect of Celecoxib on the AKT/GSK-3β pathway, and was blocked upon addition of GSK-3β inhibitor LiCl or the proteasome inhibitor MG132. These results suggest that Mcl-1 is a potential target for improving the antitumor efficiency of PDT. A loss in Mcl-1 by inhibiting AKT promotes PDT-induced apoptosis through the mitochondrial pathway. This also provides a novel rationale for utilizing Celecoxib to improve the efficacy of PDT.

Topics: Androstadienes; Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Celecoxib; Cell Survival; Dihematoporphyrin Ether; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HeLa Cells; Humans; Leupeptins; Lithium Chloride; Mice; Mice, Inbred BALB C; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; NF-kappa B; Photochemotherapy; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Pyrazoles; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Sulfonamides; Wortmannin; Xenograft Model Antitumor Assays

Differentiation-inducing factor-1 (DIF-1), a morphogen for Dictyostelium discoideum, inhibits the proliferation of human cancer cell lines by suppressing the Wnt/β-catenin signaling pathway. In this study, we examined the effect of DIF-1 on c-Myc, a target gene product of the Wnt/β-catenin signaling pathway, mainly using HCT-116 colon cancer cells. DIF-1 strongly reduced the amount of c-Myc protein in time- and concentration-dependent manners and reduced c-Myc mRNA expression by inhibiting promoter activity through the TCF binding sites. The effect of DIF-1 on c-Myc was also confirmed using the human cervical cell line HeLa. Pretreatment with the proteasome inhibitor MG132 or glycogen synthase kinase-3β (GSK-3β) inhibitors (LiCl and SB216763) attenuated the effect of DIF-1, suggesting that DIF-1 induced c-Myc protein degradation through GSK-3β activation. Furthermore, we examined whether c-Myc was involved in the anti-proliferative effect of DIF-1 using c-Myc-overexpressing cells and found that c-Myc was associated with the anti-proliferative effect of this compound. These results suggest that DIF-1 inhibits c-Myc expression by inhibiting promoter activity and inducing protein degradation via GSK-3β activation, resulting in the inhibition of cell proliferation. Since c-Myc seems to be profoundly involved in accelerated proliferation of various malignant tumors, DIF-1 may have a potential to develop into a novel anti-cancer agent.

Topics: Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hexanones; Humans; Hydrocarbons, Chlorinated; Indoles; Leupeptins; Lithium Chloride; Maleimides; Proteasome Inhibitors; Proto-Oncogene Proteins c-myc; Signal Transduction

Artemisinin and its derivatives (ARTs) are effective antimalarial drugs and also possess profound anticancer activity. However, the mechanism accounted for its distinctive activity in tumor cells remains unelucidated. We computed Pair wise Pearson correlation coefficients to identify genes that show significant correlation with ARTs activity in NCI-55 cell lines using data obtained from studies with HG-U133A Affymetrix chip. We found c-myc is one of the genes that showed the highest positive correlation coefficients among the probe sets analyzed (r=0.585, P<0.001). Dihydroartemisinin (DHA), the main active metabolite of ARTs, induced significant apoptosis in HL-60 and HCT116 cells that express high levels of c-MYC. Stable knockdown of c-myc abrogated DHA-induced apoptosis in HCT116 cells. Conversely, forced expression of c-myc in NIH3T3 cells sensitized these cells to DHA-induced apoptosis. Interestingly, DHA irreversibly down-regulated the protein level of c-MYC in DHA-sensitive HCT116 cells, which is consistent to persistent G1 phase arrest induced by DHA. Further studies demonstrated that DHA accelerated the degradation of c-MYC protein and this process was blocked by pretreatment with the proteasome inhibitor MG-132 or GSK 3beta inhibitor LiCl in HCT116 cells. Taken together, ARTs might be useful in the treatment of c-MYC-overexpressing tumors. We also suggest that c-MYC may potentially be a biomarker candidate for prediction of the antitumor efficacies of ARTs.

Topics: Animals; Antimalarials; Apoptosis; Artemisinins; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Down-Regulation; G1 Phase; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HCT116 Cells; HL-60 Cells; Humans; Leupeptins; Lithium Chloride; Mice; NIH 3T3 Cells; Proto-Oncogene Proteins c-myc

By studying primary isogenic murine embryonic fibroblasts (MEFs), we have shown that PLK3 null MEFs contain a reduced level of phosphatase and tensin homolog (PTEN) and increased Akt1 activation coupled with decreased GSK3β activation under normoxia and hypoxia. Purified recombinant Plk3, but not a kinase-defective mutant, efficiently phosphorylates PTEN in vitro. Mass spectrometry identifies threonine 366 and serine 370 as two putative residues that are phosphorylated by Plk3. Immunoblotting using a phosphospecific antibody confirms these sites as Plk3 phosphorylation sites. Moreover, treatment of MEFs with LiCl, an inhibitor of GSK3β and CK2, only partially suppresses the phosphorylation, suggesting Plk3 as an additional kinase that phosphorylates these sites in vivo. Plk3-targeting mutants of PTEN are expressed at a reduced level in comparison with the wild-type counterpart, which is associated with an enhanced activity of PDK1, an upstream activator of Akt1. Furthermore, the reduced level of PTEN in PLK3 null MEFs is stabilized by treatment with MG132, a proteosome inhibitor. Combined, our study identifies Plk3 as a new player in the regulation of the PI3K/PDK1/Akt signaling axis by phosphorylation and stabilization of PTEN.

Topics: Adjuvants, Immunologic; Animals; Cysteine Proteinase Inhibitors; Embryo, Mammalian; Enzyme Stability; Fibroblasts; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HEK293 Cells; Humans; Leupeptins; Lithium Chloride; Mice; Mice, Knockout; Mutation; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Tumor Suppressor Proteins

The expression level of cyclin D1 plays a vital role in the control of proliferation. This protein is reported to be degraded following phosphorylation by glycogen synthase kinase 3 (GSK3) on Thr-286. We recently showed that phosphorylation of Thr-286 is responsible for a decline in cyclin D1 levels during S phase, an event required for efficient DNA synthesis. These studies were undertaken to test the possibility that phosphorylation by GSK3 is responsible for the S phase specific decline in cyclin D1 levels, and that this event is regulated by the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway which controls GSK3.. We found, however, that neither PI3K, AKT, GSK3, nor proliferative signaling activity in general is responsible for the S phase decline in cyclin D1 levels. In fact, the activity of these signaling kinases does not vary through the cell cycle of proliferating cells. Moreover, we found that GSK3 activity has little influence over cyclin D1 expression levels during any cell cycle phase. Inhibition of GSK3 activity by siRNA, LiCl, or other chemical inhibitors failed to influence cyclin D1 phosphorylation on Thr-286, even though LiCl efficiently blocked phosphorylation of beta-catenin, a known substrate of GSK3. Likewise, the expression of a constitutively active GSK3 mutant protein failed to influence cyclin D1 phosphorylation or total protein expression level.. Because we were unable to identify any proliferative signaling molecule or pathway which is regulated through the cell cycle, or which is able to influence cyclin D1 levels, we conclude that the suppression of cyclin D1 levels during S phase is regulated by cell cycle position rather than signaling activity. We propose that this mechanism guarantees the decline in cyclin D1 levels during each S phase; and that in so doing it reduces the likelihood that simple over expression of cyclin D1 can lead to uncontrolled cell growth.

Topics: Animals; beta Catenin; Cell Cycle; Cell Division; Cell Line; Chromones; Cyclin D; Cyclins; Fibroblasts; Genes, bcl-1; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Leupeptins; Lithium Chloride; Mice; Morpholines; NIH 3T3 Cells; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Phosphothreonine; Protein Biosynthesis; Protein Kinases; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Recombinant Fusion Proteins; S Phase; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Degradation of IkappaB is an essential step in nuclear factor (NF)-kappaB activation. However, the determinants regulating this process have not been defined in vascular smooth muscle cells (VSMCs). We hypothesized that the E3-ligase, beta-transducin repeat-containing protein 1 (beta-TrCP1), was a rate-determining mediator that regulates the ubiquitin-mediated degradation of IkappaBalpha (in VSMC).. Upregulation of beta-TrCP1 accelerated the rate of IkappaBalpha degradation, leading to increased NF-kappaB activity. In contrast, VSMCs harboring a dominant-negative beta-TrCP1 transgene lacking the F-box domain exhibited a reduction in serum-stimulated NF-kB activity but no alteration in response to tumor necrosis factor (TNF). These findings suggest that beta-TrCP1 increases the rate of NF-kappaB activation but is not rate-limiting in response to TNF in VSMCs. Endogenous beta-TrCP1 expression was regulated through the conserved Wnt cascade. Upregulation of Wnt1 resulted in beta-catenin-mediated activation of Tcf-4, leading to increased beta-TrCP1 expression and NF-kappaB activity. Furthermore, VSMCs harboring a Tcf-4 mutant lacking a beta-catenin binding domain exhibited a significant reduction in beta-TrCP1 expression along with abolishment of NF-kappaB activity.. We provide the first evidence of crosstalk between the Wnt cascade and NF-kappaB signaling in VSMCs. This crosstalk is mediated through the E3-ligase, beta-TrCP1.

Topics: Animals; Aorta; beta Catenin; beta-Transducin Repeat-Containing Proteins; Cells, Cultured; Cytoskeletal Proteins; Gene Expression Regulation; Humans; I-kappa B Proteins; Leupeptins; Lithium Chloride; Muscle, Smooth, Vascular; NF-kappa B; NF-KappaB Inhibitor alpha; Protein Transport; Proto-Oncogene Proteins; Rats; Signal Transduction; Trans-Activators; Transcription, Genetic; Transfection; Tumor Necrosis Factor-alpha; Wnt Proteins; Wnt1 Protein; Zebrafish Proteins