Role for Btg1 and Btg2 in growth arrest of WEHI-231 cells through arginine methylation following membrane immunoglobulin engagement

Engagement of membrane Ig (mIg) on WEHI-231 murine B lymphoma cells, a cell line model representative of primary immature B cells, results in growth arrest and subsequent apoptosis. Of the several dozen genes upregulated greater than two-fold by anti-IgM treatment through DNA microarray analysis, we focused on B cell translocation gene 1 (Btg1) and Btg2, member of Btg/Tob family of proteins. WEHI-231 cells were infected with the Btg1/EGFP or Btg2/EGFP retroviral vectors, and those expressing either Btg1 or Btg2 accumulated in G1 phase at significantly higher proportions than that seen for cells expressing control vector. Btg1 or Btg2 bound to protein arginine methyltransferase (PRMT) 1 via the box C region, an interaction required for anti-IgM-induced growth inhibition. The arginine methyltransferase inhibitor AdOx partially abrogated growth inhibition induced by Btg1, Btg2, or anti-IgM. The Btg1- or Btg2-induced growth inhibition was also abrogated in PRMT1-deficient cells via introduction of small interference RNA. In addition, we observed anti-IgM-induced arginine methylation of two proteins, a 28-kDa and a 36-kDa protein. Methylation, detected by a monoclonal antibody specific for asymmetric, but not symmetric methyl residues, was observed as early as 1 h-2 h after stimulation and was sustained for up to 24 h. The anti-IgM-induced p36 arginine methylation was abrogated in the PRMT1-deficient cells, suggesting that PRMT1 induces p36 methylation. Together, these results suggest that anti-IgM-induced growth inhibition is mediated via upregulation of Btg1 and Btg2, resulting in the activation of arginine methyltransferase activity and culminating in growth inhibition of WEHI-231 cells.     

alpha-Tomatine, the major saponin in tomato, induces programmed cell death mediated by reactive oxygen species in the fungal pathogen Fusarium oxysporum

The tomato saponin alpha-tomatine has been proposed to kill sensitive cells by binding to cell membranes followed by leakage of cell components. However, details of the modes of action of the compound on fungal cells are poorly understood. In the present study, mechanisms involved in alpha-tomatine-induced cell death of fungi were examined using a filamentous pathogenic fungus Fusarium oxysporum. alpha-Tomatine-induced cell death of F. oxysporum (TICDF) occurred only under aerobic conditions and was blocked by the mitochondrial F(0)F(1)-ATPase inhibitor oligomycin, the caspase inhibitor D-VAD-fmk, and protein synthesis inhibitor cycloheximide. Fungal cells exposed to alpha-tomatine showed TUNEL-positive nuclei, depolarization of transmembrane potential of mitochondria, and reactive oxygen species (ROS) accumulation. These results suggest that TICDF occurs through a programmed cell death process in which mitochondria play a pivotal role. Pharmacological studies using inhibitors suggest that alpha-tomatine activates phosphotyrosine kinase and monomeric G-protein signaling pathways leading to Ca(2+) elevation and ROS burst in F. oxysporum cells.     

Repulsive guidance molecule RGMa alters utilization of bone morphogenetic protein (BMP) type II receptors by BMP2 and BMP4

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily of multifunctional ligands that transduce their signals through type I and II serine/threonine kinase receptors and intracellular Smad proteins. Recently, we identified the glycosylphosphatidylinositol-anchored repulsive guidance molecules RGMa, DRAGON (RGMb), and hemojuvelin (RGMc) as coreceptors for BMP signaling (Babbit, J. L., Huang, F. W., Wrighting, D. W., Xia, Y., Sidis, Y., Samad, T. A., Campagna, J. A., Chung, R., Schneyer, A., Woolf, C. J., Andrews, N. C., and Lin, H. Y. (2006) Nat. Genet. 38, 531-539; Babbit, J. L., Zhang, Y., Samad, T. A., Xia, Y., Tang, J., Schneyer, A., Woolf, C. J., and Lin, H. Y. (2005) J. Biol. Chem. 280, 29820-29827; Samad, T. A., Rebbapragada, A., Bell, E., Zhang, Y., Sidis, Y., Jeong, S. J., Campagna, J. A., Perusini, S., Fabrizio, D. A., Schneyer, A. L., Lin, H. Y., Brivanlou, A. H., Attisano, L., and Woolf, C. J. (2005) J. Biol. Chem. 280, 14122-14129). However, the mechanism by which RGM family members enhance BMP signaling remains unknown. Here, we report that RGMa bound to radiolabeled BMP2 and BMP4 with Kd values of 2.4+/-0.2 and 1.4+/-0.1 nm, respectively. In KGN human ovarian granulosa cells and mouse pulmonary artery smooth muscle cells, BMP2 and BMP4 signaling required BMP receptor type II (BMPRII), but not activin receptor type IIA (ActRIIA) or ActRIIB, based on changes in BMP signaling by small interfering RNA inhibition of receptor expression. In contrast, cells transfected with RGMa utilized both BMPRII and ActRIIA for BMP2 or BMP4 signaling. Furthermore, in BmpRII-null pulmonary artery smooth muscle cells, BMP2 and BMP4 signaling was reduced by inhibition of endogenous RGMa expression, and RGMa-mediated BMP signaling required ActRIIA expression. These findings suggest that RGMa facilitates the use of ActRIIA by endogenous BMP2 and BMP4 ligands that otherwise prefer signaling via BMPRII and that increased utilization of ActRIIA leads to generation of an enhanced BMP signal.     

AMPK mediates autophagy during myocardial ischemia in vivo

We have recently shown that autophagy is induced by ischemia and reperfusion in the mouse heart in vivo. Ischemia stimulates autophagy through an AMP activated protein kinase (AMPK)-dependent mechanism, whereas reperfusion after ischemia stimulates autophagy through a Beclin 1-dependent, but AMPK-independent, mechanism. Autophagy plays distinct roles during ischemia and reperfusion: autophagy may be protective during ischemia, whereas it may be detrimental during reperfusion. We will discuss the role of AMPK in mediating autophagy during myocardial ischemia in vivo.     

Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage

Mitochondrial damage is a well known cause of mitochondria-related diseases. A major mechanism underlying the development of mitochondria-related diseases is thought to be an increase in intracellular oxidative stress produced by impairment of the mitochondrial electron transport chain (ETC). However, clear evidence of intracellular free radical generation has not been clearly provided for mitochondrial DNA (mtDNA)-damaged cells. In this study, using the novel fluorescence dye, 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF), which was designed to detect hydroxyl radicals (*OH), intracellular free radical formation was examined in 143B cells (parental cells), 143B-rho(0) cells (mtDNA-lacking cells), 87 wt (cybrid), and cybrids of 4977-bp mtDNA deletion (common deletion) cells containing the deletion with 0%, 5%, 50% and >99% frequency (HeLacot, BH5, BH50 and BH3.12, respectively), using a laser confocal microscope detection method. ETC inhibitors (rotenone, 3-nitropropionic acid, thenoyltrifluoroacetone, antimycin A and sodium cyanide) were also tested to determine whether inhibitor treatment increased intracellular reactive oxygen species (ROS) generation. A significant increase in ROS for 143B-rho(0) cells was observed compared with 143B cells. However, for the 87 wt cybrid, no increase was observed. An increase was also observed in the mtDNA-deleted cells BH50 and BH3.12. The ETC inhibitors increased intracellular ROS in both 143B and 143B-rho(0) cells. Furthermore, in every fluorescence image, the fluorescence dye appeared localized around the nuclei. To clarify the localization, we double-stained cells with the dye and MitoTracker Red. The resulting fluorescence was consistently located in mitochondria. Furthermore, manganese superoxide dismutase (MnSOD) cDNA-transfected cells had decreased ROS. These results suggest that more ROS are generated from mitochondria in ETC-inhibited and mtDNA-damaged cells, which have impaired ETC.     

Circumnutational movement in rice coleoptiles involves the gravitropic response: analysis of an agravitropic mutant and space‐grown seedlings

Plants exhibit helical growth movements known as circumnutation in growing organs. Some studies indicate that circumnutation involves the gravitropic response, but this notion is a matter of debate. Here, using the agravitropic rice mutant lazy1 and space‐grown rice seedlings, we found that circumnutation was reduced or lost during agravitropic growth in coleoptiles. Coleoptiles of wild‐type rice exhibited circumnutation in the dark, with vigorous oscillatory movements during their growth. The gravitropic responses in lazy1 coleoptiles differed depending on the growth stage, with gravitropic responses detected during early growth and agravitropism during later growth. The nutation‐like movements observed in lazy1 coleoptiles at the early stage of growth were no longer detected with the disappearance of the gravitropic response. To verify the relationship between circumnutation and gravitropic responses in rice coleoptiles, we conducted spaceflight experiments in plants under microgravity conditions on the International Space Station. Wild‐type rice seeds were germinated, and the resulting seedlings were grown under microgravity or a centrifuge‐generated 1 g environment in space. We began filming the seedlings 2 days after seed imbibition and obtained images of seedling growth every 15 min. The seed germination rate in space was 92–100% under both microgravity and 1 g conditions. LED‐synchronized flashlight photography induced an attenuation of coleoptile growth and circumnutational movement due to cumulative light exposure. Nevertheless, wild‐type rice coleoptiles still showed circumnutational oscillations under 1 g but not microgravity conditions. These results support the idea that the gravitropic response is involved in plant circumnutation.     

The cis-Regulatory Atlas of the Mouse Immune System

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Co-culture of fibroblasts and hepatic parenchymal cells induces metabolic changes and formation of a three-dimensional structure

Co-culturing of tendon fibroblasts and liver parenchymal cells in Williams' medium E supplemented with fetal bovine serum, hormones, and L-ascorbic acid 2-phosphate, a long acting vitamin C derivative, resulted in formation of three-dimensional structure. Both growth of fibroblasts and their production of collagen were inhibited, however production of albumin by the hepatocytes was much better preserved than when individual cells were cultured separately, indicating epithelial-mesenchymal interactions stimulate reorganization of the liver-like tissue from isolated cells.     

Downregulation of the voltage-dependent calcium channel (VDCC) beta-subunit mRNAs in pancreatic islets of type 2 diabetic rats

The present study was undertaken to determine whether altered expression of the VDCC beta-subunits in pancreatic beta-cells could play a role in the changes in beta-cell sensitivity to glucose that occur with diabetes. Application of competitive RT-PCR procedure revealed that in normal Wistar rats, LETO and prediabetic OLETF rats, the beta(2)-subunit mRNA levels were 60-200-fold greater than the levels for the beta(3)-subunit. These findings suggest that the beta(2)-subunit as well as the beta-cell type VDCC1 alpha(1)-subunit may be the predominant form of the VDCC expressed in pancreatic beta-cells. The levels of mRNA encoding the beta-subunits and the beta-cell type alpha(1)-subunit as well as insulin were significantly reduced in diabetic rats. Perfusion experiments revealed that diabetic rats showed the higher basal insulin secretion and profoundly impaired insulin secretory responses to glucose compared with non-diabetic rats. Alternatively, impaired insulin secretory responses to glucose in high dose glucose-infused rats were recovered partly with the elevation of mRNA levels of the VDCC beta(2)- and beta(3)-subunits as well as the alpha(1)-subunit by the treatment with diazoxide. Thus, considering the possibility that the most striking effect of the VDCC alpha(1) beta-subunit coexpression in pancreatic beta-cells might occur on activation kinetics like the skeletal muscle, the impairment of further activation of the VDCCs to acute glucose challenge caused by the reduced expressions of the alpha(1) beta-subunits mRNAs in type 2 diabetic animals might be at least partly associated with the alterations in beta-cell sensitivity to glucose.