Inhibition of MUC1-C Increases ROS and Cell Death in Mouse Embryonic Stem Cells

Background and Objectives@#Embryonic stem (ES) cells have the capacity to self-renew and generate all types of cells.MUC1-C, a cytoplasmic subunit of MUC1, is overexpressed in various carcinomas and mediates signaling pathways to regulate intracellular metabolic processes and gene expression involved in the maintenance of cancer cells. However, the functional role of MUC1-C in ES cells is not well understood. In this study, we investigated the role of MUC1-C on growth, survival,: and differentiation of mouse ES (mES) cells. @*Methods@#and Results: Undifferentiated mES cells expressed the MUC1-C protein and the expression level was decreased during differentiation. Inhibition of MUC1-C, by the specific inhibitor GO201, reduced proliferation of mES cells.However, there was no prominent effect on pluripotent markers such as Oct4 expression and STAT3 signaling, and MUC1-C inhibition did not induce differentiation. Inhibition of MUC1-C increased the G1 phase population, decreased the S phase population, and increased cell death. Furthermore, inhibition of MUC1-C induced disruption of the ROS balance in mES cells. @*Conclusions@#These results suggest that MUC1-C is involved in the growth and survival of mES cells.

Inhibition of MUC1-C Increases ROS and Cell Death in Mouse Embryonic Stem Cells

Background and Objectives@#Embryonic stem (ES) cells have the capacity to self-renew and generate all types of cells.MUC1-C, a cytoplasmic subunit of MUC1, is overexpressed in various carcinomas and mediates signaling pathways to regulate intracellular metabolic processes and gene expression involved in the maintenance of cancer cells. However, the functional role of MUC1-C in ES cells is not well understood. In this study, we investigated the role of MUC1-C on growth, survival,: and differentiation of mouse ES (mES) cells. @*Methods@#and Results: Undifferentiated mES cells expressed the MUC1-C protein and the expression level was decreased during differentiation. Inhibition of MUC1-C, by the specific inhibitor GO201, reduced proliferation of mES cells.However, there was no prominent effect on pluripotent markers such as Oct4 expression and STAT3 signaling, and MUC1-C inhibition did not induce differentiation. Inhibition of MUC1-C increased the G1 phase population, decreased the S phase population, and increased cell death. Furthermore, inhibition of MUC1-C induced disruption of the ROS balance in mES cells. @*Conclusions@#These results suggest that MUC1-C is involved in the growth and survival of mES cells.

Splitomicin, a SIRT1 Inhibitor, Enhances Hematopoietic Differentiation of Mouse Embryonic Stem Cells

BACKGROUND AND OBJECTIVES: Embryonic stem (ES) cells have pluripotent ability to differentiate into multiple tissue lineages. SIRT1 is a class III histone deacetylase which modulates chromatin remodeling, gene silencing, cell survival, metabolism, and development. In this study, we examined the effects of SIRT1 inhibitors on the hematopoietic differentiation of mouse ES cells. METHODS AND RESULTS: Treatment with the SIRT1 inhibitors, nicotinamide and splitomicin, during the hematopoietic differentiation of ES cells enhanced the production of hematopoietic progenitors and slightly up-regulated erythroid and myeloid specific gene expression. Furthermore, treatment with splitomicin increased the percentage of erythroid and myeloid lineage cells. CONCLUSIONS: Application of the SIRT1 inhibitor splitomicin during ES cell differentiation to hematopoietic cells enhanced the yield of specific hematopoietic lineage cells from ES cells. This result suggests that SIRT1 is involved in the regulation of hematopoietic differentiation of specific lineages and that the modulation of the SIRT1 activity can be a strategy to enhance the efficiency of hematopoietic differentiation.

HSP60 is required for stemness and proper differentiation of mouse embryonic stem cells

Embryonic stem cells (ESCs) are metabolically distinct from their differentiated counterparts. ESC mitochondria are less complex and fewer in number than their differentiated progeny. However, few studies have examined the proteins responsible for differences in mitochondrial structure and function between ESCs and somatic cells. Therefore, in this study, we aimed to investigate the differences between mitochondrial proteins in these two cell types. We demonstrate that HSP60 is more abundant in mouse ESC mitochondria than in mouse embryonic fibroblasts. Depletion of HSP60 inhibited mouse ESC proliferation and self-renewal, characterized by decreased OCT4 expression. HSP60 depletion also enhanced apoptosis during mouse ESC differentiation into embryoid bodies. Our results suggest that HSP60 expression has an essential role in ESC self-renewal and survival of differentiated cells from ESCs.

Suppression of the ERK–SRF axis facilitates somatic cell reprogramming

The molecular mechanism underlying the initiation of somatic cell reprogramming into induced pluripotent stem cells (iPSCs) has not been well described. Thus, we generated single-cell-derived clones by using a combination of drug-inducible vectors encoding transcription factors (Oct4, Sox2, Klf4 and Myc) and a single-cell expansion strategy. This system achieved a high reprogramming efficiency after metabolic and epigenetic remodeling. Functional analyses of the cloned cells revealed that extracellular signal-regulated kinase (ERK) signaling was downregulated at an early stage of reprogramming and that its inhibition was a driving force for iPSC formation. Among the reprogramming factors, Myc predominantly induced ERK suppression. ERK inhibition upregulated the conversion of somatic cells into iPSCs through concomitant suppression of serum response factor (SRF). Conversely, SRF activation suppressed the reprogramming induced by ERK inhibition and negatively regulated embryonic pluripotency by inducing differentiation via upregulation of immediate early genes, such as c-Jun, c-Fos and EGR1. These data reveal that suppression of the ERK-SRF axis is an initial molecular event that facilitates iPSC formation and may be a useful surrogate marker for cellular reprogramming.

Protein tyrosine phosphatase 1B is a mediator of cyclic ADP ribose-induced Ca²⁺ signaling in ventricular myocytes

Cyclic ADP-ribose (cADPR) releases Ca²⁺ from ryanodine receptor (RyR)-sensitive calcium pools in various cell types. In cardiac myocytes, the physiological levels of cADPR transiently increase the amplitude and frequency of Ca²⁺ (that is, a rapid increase and decrease of calcium within one second) during the cardiac action potential. In this study, we demonstrated that cADPR levels higher than physiological levels induce a slow and gradual increase in the resting intracellular Ca²⁺ ([Ca²⁺](i)) level over 10 min by inhibiting the sarcoendoplasmic reticulum Ca²⁺ ATPase (SERCA). Higher cADPR levels mediate the tyrosine-dephosphorylation of α-actin by protein tyrosine phosphatase 1B (PTP1B) present in the endoplasmic reticulum. The tyrosine dephosphorylation of α-actin dissociates phospholamban, the key regulator of SERCA, from α-actin and results in SERCA inhibition. The disruption of the integrity of α-actin by cytochalasin B and the inhibition of α-actin tyrosine dephosphorylation by a PTP1B inhibitor block cADPR-mediated Ca²⁺ increase. Our results suggest that levels of cADPR that are relatively higher than normal physiological levels modify calcium homeostasis through the dephosphorylation of α-actin by PTB1B and the subsequent inhibition of SERCA in cardiac myocytes.

Dendroaspis natriuretic peptide regulates the cardiac L-type Ca2+ channel activity by the phosphorylation of alpha1c proteins

Dendroaspis natriuretic peptide (DNP), a new member of the natriuretic peptide family, is structurally similar to atrial, brain, and C-type natriuretic peptides. However, the effects of DNP on the cardiac function are poorly defined. In the present study, we examined the effect of DNP on the cardiac L-type Ca2+ channels in rabbit ventricular myocytes. DNP inhibited the L-type Ca2+ current (ICa,L) in a concentration dependent manner with a IC50 of 25.5 nM, which was blocked by an inhibitor of protein kinase G (PKG), KT5823 (1 microM). DNP did not affect the voltage dependence of activation and inactivation of ICa,L. The alpha1c subunit of cardiac L-type Ca2+ channel proteins was phosphorylated by the treatment of DNP (1 microM), which was completely blocked by KT5823 (1 microM). Finally, DNP also caused the shortening of action potential duration in rabbit ventricular tissue by 22.3 +/- 4.2% of the control (n = 6), which was completely blocked by KT5823 (1 microM). These results clearly indicate that DNP inhibits the L-type Ca2+ channel activity by phosphorylating the Ca2+ channel protein via PKG activation.

SIRT1 Inhibits p53 but not NF-kappaB Transcriptional Activity during Differentiation of Mouse Embryonic Stem Cells into Embryoid Bodies

BACKGROUND AND OBJECTIVES: SIRT1, a histone diacetylase, modify transactivation function of various transcription factor including p53 and NF-kappaB. p53 and NF-kappaB is involved in in vitro differentiation of mouse embryonic stem cells (mESC) into mouse embryoid body (mEB). These suggest that SIRT1 might affect in vitro differentiation of mESC into mEB by regulation of p53 and NF-kappaB. METHODS AND RESULTS: In this study we analyzed the effect of SIRT1 in in vitro differentiation of mESC into mEB using wild and SIRT1 knockout mESC. To examine SIRT1-specific gene in mESC, this study conducted microarray-based differential gene expression analysis between wild and SIRT1 knockout mESC. Comparing their gene expression patterns, this study determined a list of genes regulated by SIRT1. cDNA microarray data-set analysis revealed that genes associated with transcription and signal transduction are significantly modified in SIRT1 knockout mESC. cDNA microarray data-set analysis between mESC and EB in wild and SIRT1 showed that SIRT1 inhibits p53 signaling pathway but not affect NF-kappaB signaling pathway. CONCLUSIONS: This study suggests that SIRT1 modify mESC differentiation by regulation of p53 transcriptional activity.

TNF-alpha upregulates PTEN via NF-kappaB signaling pathways in human leukemic cells

TNF-alpha plays a variety of biological functions such as apoptosis, inflammation and immunity. PTEN also has various cellular function including cell growth, proliferation, migration and differentiation. Thus, possible relationships between the two molecules are suggested. TNF-alpha has been known to downregulate PTEN via NF-kappaB pathway in the human colon cell line, HT-29. However, here we show the opposite finding that TNF-alpha upregulates PTEN via activation of NF-kappaB in human leukemic cells. TNF-alpha increased PTEN expression at HL-60 cells in a time- and dose-dependent manner, but the response was abolished by disruption of NF-kappaB with p65 anisense phosphorothioate oligonucleotide or pyrrolidine dithiocarbamate. We found that TNF-alpha activated the NF-kappaB pathways, evidenced by the translocation of p65 to the nucleus in TNF-alpha-treated cells. We conclude that TNF-alpha induces upregulation of PTEN expression through NF-kappaB activation in human leukemic cells.

Low-density lipoprotein protects Vibrio vulnificus-induced lethality through blocking lipopolysaccharide action

Lipoprotein plays a role in the host defense against bacterial infection, and its serum level has been demonstrated to be an important prognosis factor of survival. We have previously demonstrated that LDL directly inactivates the hemolytic activity of Vibrio vulnificus cytolysin (VVC) in vitro. The object of this study was therefore to examine whether the LDL-mediated inactivation of VVC leads to protection against lethal infection of V. vulnificus in vivo, using wild and VVC-deficient V. vulnificus strains. Unexpectedly, we found that LDL protects mouse lethality induced by VVC-deficient as well as wild V. vulnificus strain. We also demonstrated that LDL blocks V. vulnificus LPS-induced lethality in mice. These results suggest that LDL preferentially act on endotoxin rather than exotoxin in the protection against V. vulnificus-induced mice lethality.

Enzymatic characterization of Mycobacterium smegmatis ADP-ribosyltransferase

ADP-ribosyltransferase (ADPRT) catalyzes the reaction in which the ADP-ribose moiety of beta-NAD+ is transferred to specific amino acid residues in target proteins. The ADPRT of Mycobacterium smegmatis has been known to inactivate rifampin through ADP-ribosylation. However, the enzymatic characteristics and functions of the enzyme have not been elucidated yet. In this study, the ADPRT-glutathione S-transferase (GST) fusion protein was expressed in Escherichia coli and enzymatic characteristics of the fusion protein were investigated. ADPRT-GST fusion protein was an ADPribosyltransferase that had no NAD glycohydrolase activity. ADPRT-GST fusion protein showed no self-inactivation phenomenon that is a universal nature for all NAD glycohydrolases and is important in regulating its activity. ADPRT activity of the enzyme was decreased by novobiocin and isonicotinic acid hydrazide. These results suggest that Mycobacterium smegmatis ADPRT could be regulated by a different way from other NADases and involved in bacterial physiological process through a post-translational modification of cytosolic proteins.

Epigallocatechin gallate, a constituent of green tea, suppresses cytokine-induced pancreatic beta-cell damage

Cytokines produced by immune cells infiltrating pancreatic islets have been implicated as one of the important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. In this study, the protective effects of epigallocatechin gallate (EGCG) on cytokine-induced beta-cell destruction were investigated. EGCG effectively protected IL-1beta and IFN-g-mediated cytotoxicity in insulinoma cell line (RINm5F). EGCG induced a significant reduction in IL-1b and IFN-gamma-induced nitric oxide (NO) production and reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein levels on RINm5F cells. The molecular mechanism by which EGCG inhibited iNOS gene expression appeared to involve the inhibition of NF-kB activation. These findings revealed EGCG as a possible therapeutic agent for the prevention of diabetes mellitus progression.