Pancreatic ß-Cell-Specific Deletion of VPS41 Causes Diabetes Due to Defects in Insulin Secretion.

Insulin secretory granules (SGs) mediate the regulated secretion of insulin, which is essential for glucose homeostasis. The basic machinery responsible for this regulated exocytosis consists of specific proteins present both at the plasma membrane and on insulin SGs. The protein composition of insulin SGs thus dictates their release properties, yet the mechanisms controlling insulin SG formation, which determine this molecular composition, remain poorly understood. VPS41, a component of the endolysosomal tethering homotypic fusion and vacuole protein sorting (HOPS) complex, was recently identified as a cytosolic factor involved in the formation of neuroendocrine and neuronal granules. We now find that VPS41 is required for insulin SG biogenesis and regulated insulin secretion. Loss of VPS41 in pancreatic ß-cells leads to a reduction in insulin SG number, changes in their transmembrane protein composition, and defects in granule-regulated exocytosis. Exploring a human point mutation, identified in patients with neurological but no endocrine defects, we show that the effect on SG formation is independent of HOPS complex formation. Finally, we report that mice with a deletion of VPS41 specifically in ß-cells develop diabetes due to severe depletion of insulin SG content and a defect in insulin secretion. In sum, our data demonstrate that VPS41 contributes to glucose homeostasis and metabolism.

TGF-ß1 accelerates the DNA damage response in epithelial cells via Smad signaling.

The evidence suggests that transforming growth factor-beta (TGF-ß) regulates the DNA-damage response (DDR) upon irradiation, and we previously reported that TGF-ß1 induced DNA ligase IV (Lig4) expression and enhanced the nonhomologous end-joining repair pathway in irradiated cells. In the present study, we investigated the effects of TGF-ß1 on the irradiation-induced DDRs of A431 and HaCaT cells. Cells were pretreated with or without TGF-ß1 and irradiated. At 30 min post-irradiation, DDRs were detected by immunoblotting of phospho-ATM, phospho-Chk2, and the presence of histone foci (γH2AX). The levels of all three factors were similar right after irradiation regardless of TGF-ß1 pretreatment. However, they soon thereafter exhibited downregulation in TGF-ß1-pretreated cells, indicating the acceleration of the DDR. Treatment with a TGF-ß type I receptor inhibitor (SB431542) or transfections with siRNAs against Smad2/3 or DNA ligase IV (Lig4) reversed this acceleration of the DDR. Furthermore, the frequency of irradiation-induced apoptosis was decreased by TGF-ß1 pretreatment in vivo, but this effect was abrogated by SB431542. These results collectively suggest that TGF-ß1 could enhance cell survival by accelerating the DDR via Smad signaling and Lig4 expression.

Heat Shock Protein 90 Regulates Subcellular Localization of Smads in Mv1Lu Cells.

Heat shock protein 90 (HSP90) regulates the stability of various proteins and plays an essential role in cellular homeostasis. Many client proteins of HSP90 are involved in cell growth, survival, and migration; processes that are generally accepted as participants in tumorigenesis. HSP90 is also up-regulated in certain tumors. Indeed, the inhibition of HSP90 is known to be effective in cancer treatment. Recently, studies showed that HSP90 regulates transforming growth factor ß1 (TGF-ß1)-induced transcription by increasing the stability of the TGF-ß receptor. TGF-ß signaling also has been implicated in cancer, suggesting the possibility that TGF-ß1 and HSP90 function cooperatively during the cancer cell progression. Here in this paper, we investigated the role of HSP90 in TGF-ß1-stimulated Mv1Lu cells. Treatment of Mv1Lu cells with the HSP90 inhibitor, 17-allylamino-demethoxy-geldanamycin (17AAG), or transfection with truncated HSP90 (ΔHSP90) significantly reduced TGF-ß1-induced cell migration. Pretreatment with 17AAG or transfection with ΔHSP90 also reduced the levels of phosphorylated Smad2 and Smad3. In addition, the HSP90 inhibition interfered the nuclear localization of Smads induced by constitutively active Smad2 (S2EE) or Smad3 (S3EE). We also found that the HSP90 inhibition decreased the protein level of importin-ß1 which is known to regulate R-Smad nuclear translocation. These data clearly demonstrate a novel function of HSP90; HSP90 modulates TGF-ß signaling by regulating Smads localization. Overall, our data could provide a detailed mechanism linking HSP90 and TGF-ß signaling. The extension of our understanding of HSP90 would offer a better strategy for treating cancer.

TGFß1 protects cells from γ-IR by enhancing the activity of the NHEJ repair pathway.

UNLABELLED: Several groups have reported that TGFß1 regulates cellular responses to γ-irradiation; however, the exact mechanism has not been fully elucidated. In the current study, the role of TGFß1 in cellular responses to γ-irradiation was investigated in detail. The data indicate that TGFß1 pretreatment decreased the aftermath of ionizing radiation (IR)-induced DNA damage in a SMAD-dependent manner. To determine the underlying mechanism for these effects, the extent of IR-induced DNA repair activity in the presence or absence of TGFß1 was examined. Studies reveal that TGFß1 upregulated DNA ligase IV (Lig4), augmented IR-induced nuclear retention of the DNA ligase, and enhanced nonhomologous end-joining (NHEJ) repair activity. In addition, knockdown of Lig4 reduced the TGFß1-induced protection against IR. Overall, these data indicate that TGFß1 facilitates the NHEJ repair process upon γ-irradiation and thereby enhances long-term survival. IMPLICATIONS: These findings provide new insight and a possible approach to controlling genotoxic stress by the TGFß signaling pathway.

TGF-ß signaling plays an important role in resisting γ-irradiation.

Transforming growth factor-ß1 (TGF-ß1) regulates various biological processes, including differentiation, bone remodeling and angiogenesis, and is particularly important as a regulator of homeostasis and cell growth in normal tissue. Interestingly, some studies have reported that TGF-ß1 induces apoptosis through induction of specific genes, whereas others suggest that TGF-ß1 inhibits apoptosis and facilitates cell survival. Resolving these discrepancies, which may reflect differences in cellular context, is an important research priority. Here, using the parental mink lung epithelial cell line, Mv1Lu, and its derivatives, R1B and DR26, lacking TGF-ß receptors, we investigated the involvement of TGF-ß signaling in the effects of γ-irradiation. We found that canonical TGF-ß signaling played an important role in protecting cells from γ-irradiation. Introduction of functional TGF-ß receptors or constitutively active Smads into R1B and DR26 cell lines reduced DNA fragmentation, Caspase-3 cleavage and γ-H2AX foci formation in γ-irradiated cells. Notably, we also found that de novo protein synthesis was required for the radio-resistant effects of TGF-ß1. Our data thus indicate that TGF-ß1 protected against γ-irradiation, decreasing DNA damage and reducing apoptosis, and thereby enhanced cell survival.

Substance P stimulates the recovery of bone marrow after the irradiation.

The therapeutic use of ionizing radiation (e.g., X-rays and γ-rays) needs to inflict minimal damage on non-target tissue. Recent studies have shown that substance P (SP) mediates multiple activities in various cell types, including cell proliferation, anti-apoptotic responses, and inflammatory processes. The present study investigated the effects of SP on γ-irradiated bone marrow stem cells (BMSCs). In mouse bone marrow extracts, SP prolonged activation of Erk1/2 and enhanced Bcl-2 expression, but attenuated the activation of apoptotic molecules (e.g., p38 and cleaved caspase-3) and down-regulated Bax. We also observed that SP-decreased apoptotic cell death and stimulated cell proliferation in γ-irradiated mouse bone marrow tissues through TUNEL assay and PCNA analysis. To determine how SP affects bone marrow stem cell populations, mouse bone marrow cells were isolated and colony-forming unit (CFU) of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) was estimated. SP-pretreated ones showed higher CFUs of MSC and HSC than untreated ones. Furthermore, when SP was pretreated in cultured human MSC, it significantly decreased apoptotic cells at 48 and 72 h after γ-irradiation. Compared with untreated cells, SP-treated human MSCs showed reduced cleavage of apoptotic molecules such as caspase-8, -9, -3, and poly ADP-ribose polymerase (PARP). Thus, our results suggest that SP alleviates γ-radiation-induced damage to mouse BMSCs and human MSCs via regulation of the apoptotic pathway.

Monodisperse polystyrene-silica core-shell particles and silica hollow spheres prepared by the Stöber method.

Monodisperse polystyrene (PS) bead particles (diameter approximately 750 nm) have been synthesized with an initiator, 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AIBA), using a surfactant-free emulsion polymerization. The PS particles charged positively by the cationic initiator exhibit uniform size with a narrow size distribution. The Stöber method was adopted to coat silica on the surface of the PS particles. The silica coatings were performed on different conditions; pH (10.0-12.5), tetraethylorthosilicate (TEOS) concentration (10-40 mM), and reaction time (1-5 h). The reaction rates are too low at low pH (10.0) so that no or little silica is coated on the PS bead particles. At high pH condition (approximately 12.5) relatively rough silica shell and heterogeneous nucleation of silica colloids were observed due to high reaction rate of TEOS hydrolysis and condensation reaction. Smooth surface with uniform thickness of silica shells were obtained at a pH value around 11. Also, the thickness of silica coated on the PS spheres increases with increase of TEOS concentration and reaction time. Silica hollow spheres were also obtained by calcination of the PS-silica core-shell particles.

A study on the synthesis of polystyrene-silica nanocomposite particles by soap-free emulsion polymerization using cationic initiator in company with colloidal silica sol solution.

Nano-sized polystyrene (PS)-silica nanocomposite particles have been prepared by soap-free emulsion polymerization using a cationic initiator, 2,2'-azobis(isobutyramidine) hydrochloride (AIBA) with a colloidal silica (Ludox SM30, 7 nm diameter). The cationic initiator leads to the formation of the PS-silica nanocomposite particles by electrostatic interaction with negatively charged silica particles. Morphology, particle size distribution, reactivity and silica content of the particles were monitored on different reaction conditions such as pH, the addition time of silica sol and the amount of the silica sol. It is found that the nucleation of styrene monomer depends on the pH of water medium, the addition time of silica, the presence of silica in polymerization system. The reaction whose styrene monomer didn't react in water medium with pH 10 was progressed in the presence of silica sol to give nanocomposite particles. In the condition of constant pH 10 in the polymerization system, the increase of the amount of silica gave little influence to the changes in the particle size and particle size distribution of nanocomposite particles. The changes in the pH of medium gave much influence on the particle size and particle size distribution due to the changes in ionic interaction of silica and initiator. The silica content absorbed on the nanocomposite particles decreases with decreasing the pH values in the polymerization media.

Micronucleus-centromere assay and DNA repair gene polymorphism in lymphocytes of industrial radiographers.

The micronucleus-centromere assay using a pan-centromeric probe was used to assess chromosomal damage in lymphocytes of 47 industrial radiographers occupationally exposed to low dose ionizing radiation and 47 controls. The influence of genotype of DNA repair genes (XRCC1(399), XRCC3(241) and XPD(751)) on micronuclei (MN) frequency was also investigated. Centromere negative micronuclei (MNC-) frequency was significantly higher in radiographers than in controls, whereas similar centromere positive micronuclei (MNC+) frequency was observed in both groups. Poisson regression analyses revealed that the MNC- frequency was significantly associated with radiation occupational exposure and with cumulative-radiation doses in radiographers, after adjusting for confounding variables such as age, smoking, alcohol intake and genotypes. Compared to homozygous wild-type subjects, MNC- frequency in radiographers with variant XRCC3 genotype was significantly higher using univariate analysis. There were no differences in MNC- or MNC+ frequencies by genotype in controls. In conclusion, scoring of MNC- is a useful cytogenetic biomonitoring method for radiographers. Polymorphisms in XRCC3 might contribute to the increased genetic damage in individuals occupationally exposed to chronic ionizing radiation.