Essential Role of the Zinc Transporter ZIP9/SLC39A9 in Regulating the Activations of Akt and Erk in B-Cell Receptor Signaling Pathway in DT40 Cells

The essential trace element zinc is important for all living organisms. Zinc functions not only as a nutritional factor, but also as a second messenger. However, the effects of intracellular zinc on the B cell-receptor (BCR) signaling pathway remain poorly understood. Here, we present data indicating that the increase in intracellular zinc level induced by ZIP9/SLC39A9 (a ZIP Zrt-/Irt-like protein) plays an important role in the activation of Akt and Erk in response to BCR activation. In DT40 cells, the enhancement of Akt and Erk phosphorylation following BCR activation requires intracellular zinc. To clarify this event, we used chicken ZnT5/6/7-gene-triple-knockout DT40 (TKO) cells and chicken Zip9-knockout DT40 (cZip9KO) cells. The levels of Akt and ERK phosphorylation significantly decreased in cZip9KO cells. In addition, the enzymatic activity of protein tyrosine phosphatase (PTPase) increased in cZip9KO cells. These biochemical events were restored by overexpressing the human Zip9 (hZip9) gene. Moreover, we found that the increase in intracellular zinc level depends on the expression of ZIP9. This observation is in agreement with the increased levels of Akt and Erk phosphorylation and the inhibition of total PTPase activity. We concluded that ZIP9 regulates cytosolic zinc level, resulting in the enhancement of Akt and Erk phosphorylation. Our observations provide new mechanistic insights into the BCR signaling pathway underlying the regulation of intracellular zinc level by ZIP9 in response to the BCR activation.     

TNF-alpha-dependent activation of NF-kappa B in human osteoblastic HOS-TE85 cells is repressed in vector-averaged gravity using clinostat rotation

Effects of vector-averaged gravity on tumor necrosis factor (TNF)-alpha-dependent activation of nuclear factor kappa B (NF-kappa B) in human osteoblastic HOS-TE85 cells were investigated by culturing the cells using clinostat rotation (clinorotation). Cell cultures were rotated for 72 h at 40 rpm in a clinostat. At the end of clinorotation, the cells were treated with TNF-alpha for 30 min under stationary conditions. Electrophoretic mobility shift assays revealed that TNF-alpha-dependent activation of NF-kappa B was markedly reduced in the clinorotated cells when compared with the cells in control stationary cultures or after horizontal rotation (motional controls). The NF-kappa B-dependent transactivation was also impaired in the clinorotated cells, as evidenced by a transient transfection assay with a reporter plasmid containing multimerized NF-kappa B sites. Consistent with these findings, the TNF-alpha-dependent induction of endogenous NF-kappa B-responsive genes p105, I kappa B-alpha, and IL-8, was significantly attenuate in clinorotated cells. These results demonstrate that vector-averaged gravity inhibits the responsiveness of osteoblasts to TNF-alpha by repressing NF-kappa B activation.     

1-Butyryl-glycerol: a novel angiogenesis factor secreted by differentiating adipocytes.

Differentiation of adipocytes is accompanied by secretion of molecules stimulating angiogenesis in vivo and endothelial cell growth and motility in vitro. We demonstrate that the angiogenic and motility-stimulating activities secreted by adipocytes are separable from the endothelial cell mitogenic activity by fractionation of adipocyte-conditioned medium. The major differentiation-dependent angiogenic molecule was purified and identified by GCMS as 1-butyryl-glycerol (monobutyrin). Monobutyrin levels increase at least 200-fold during adipocyte differentiation and represent a major fraction of the total angiogenic activity. Synthetic monobutyrin shows the same spectrum of biological activities as the adipocyte-derived factor: stimulation of angiogenesis in vivo and microvascular endothelial cell motility in vitro, with no effect on endothelial cell proliferation. Angiogenesis is stimulated at doses as low as 20 pg when tested in the chick chorioallantoic membrane assay. These results strongly suggest that monobutyrin is a key regulatory molecule in an angiogenic process linked to normal cellular and tissue development.     

Identification of 6-octadecynoic acid from a methanol extract of Marrubium vulgare L. as a peroxisome proliferator-activated receptor γ agonist

6-Octadecynoic acid (6-ODA), a fatty acid with a triple bond, was identified in the methanol extract of Marrubium vulgare L. as an agonist of peroxisome proliferator-activated receptor γ (PPARγ). Fibrogenesis caused by hepatic stellate cells is inhibited by PPARγ whose ligands are clinically used for the treatment of diabetes. Plant extracts of Marrubium vulgare L., were screened for activity to inhibit fibrosis in the hepatic stellate cell line HSC-T6 using Oil Red-O staining, which detects lipids that typically accumulate in quiescent hepatic stellate cells. A methanol extract with activity to stimulate accumulation of lipids was obtained. This extract was found to have PPARγ agonist activity using a luciferase reporter assay. After purification using several chromatographic methods, 6-ODA, a fatty acid with a triple bond, was identified as a candidate of PPARγ agonist. Synthesized 6-ODA and its derivative 9-octadecynoic acid (9-ODA), which both have a triple bond but in different positions, activated PPARγ in a luciferase reporter assay and increased lipid accumulation in 3T3-L1 adipocytes in a PPARγ-dependent manner. There is little information about the biological activity of fatty acids with a triple bond, and to our knowledge, this is the first report that 6-ODA and 9-ODA function as PPARγ agonists.     

Pituitary Adenylate Cyclase-activating Polypeptide Type 1 Receptor (PAC1) Gene Is Suppressed by Transglutaminase 2 Activation

Pituitary adenylate cyclase-activating polypeptide (PACAP) functions as a neuroprotective factor through the PACAP type 1 receptor, PAC1. In a previous work, we demonstrated that nerve growth factor augmented PAC1 gene expression through the activation of Sp1 via the Ras/MAPK pathway. We also observed that PAC1 expression in Neuro2a cells was transiently suppressed during in vitro ischemic conditions, oxygen-glucose deprivation (OGD). Because endoplasmic reticulum (ER) stress is induced by ischemia, we attempted to clarify how ER stress affects the expression of PAC1. Tunicamycin, which induces ER stress, significantly suppressed PAC1 gene expression, and salubrinal, a selective inhibitor of the protein kinase RNA-like endoplasmic reticulum kinase signaling pathway of ER stress, blocked the suppression. In luciferase reporter assay, we found that two Sp1 sites were involved in suppression of PAC1 gene expression due to tunicamycin or OGD. Immunocytochemical staining demonstrated that OGD-induced transglutaminase 2 (TG2) expression was suppressed by salubrinal or cystamine, a TG activity inhibitor. Further, the OGD-induced accumulation of cross-linked Sp1 in nuclei was suppressed by cystamine or salubrinal. Together with cystamine, R283, TG2-specific inhibitor, and siRNA specific for TG2 also ameliorated OGD-induced attenuation of PAC1 gene expression. These results suggest that Sp1 cross-linking might be crucial in negative regulation of PAC1 gene expression due to TG2 in OGD-induced ER stress.     

Amino Acid Sequences of Glycopeptides from Cucumisin

Oxidation of short-chain iso-alkanes (isobutane, isopentane, 2-methylpentane, and 3-methylpentane) was studied with propane-grown resting mycelia of Scedosporium sp. A-4. Isobutane was oxidized to terf-butanol, but both isobutane and tert-butanol were not used for growth. Isopentane was oxidized to 3-methyl-1-butanol, 2-methyl-2-butanol, and 3-methyl-2-butanol but not to 2-methyl-1-butanol. 2-Methylpentane was oxidized to 4-methyl-1-pentanol, 2-methyl-2-pentanol, and 4-methyl-2-pentanol but not to 2-methyl-1-pentanol or 2-methyl-3-pentanol. 3-Methylpentane was not oxidized. Oxidation of branched alcohols was also studied.     

Tex261 modulates the excitotoxic cell death induced by N-methyl-D-aspartate (NMDA) receptor activation

N-methyl-D-aspartate (NMDA) receptor is a calcium-permeable ionotropic glutamate receptor and plays a role in many neurologic disorders such as brain ischemia through its involvement in excitotoxicity. We have performed differential display PCR to identify changes in gene expression that occur in the hippocampus of the mouse brain after intraperitoneal injection of NMDA and identified a gene, Tex261 as an inducible gene by NMDA stimulation in vivo. Tex261 mRNA was gradually induced in response to NMDA and reached about 4.5-fold at 24 h. When HEK 293 cells are transfected with NMDA receptors, the cells die in a manner that mimics excitotoxicity in neurons. HEK 293 cells transfected with the combination of Tex261 and the NMDA receptors NR1/NR2A produced the greater cell death compared with the cells transfected with the NMDA receptors alone. These findings suggest that Tex261 modulates the excitotoxic cell death induced by NMDA receptor activation.     

Heparin potentiation of 3T3-adipocyte stimulated angiogenesis: mechanisms of action on endothelial cells

We have examined the cellular mechanisms by which heparin potentiates the ability of 3T3-adipocytes to stimulate the formation of new blood vessels. Both anticoagulant and non-anticoagulant heparin species enhanced the angiogenic activity of adipocyte-secreted products in the chick chorioallantoic membrane assay, indicating that the angiotropic effect of this glycosaminoglycan is independent of its effect on the coagulation cascade. Heparin alone was unable to produce a neovascular response. The ability of heparin to modulate three endothelial functions in vitro thought to be related to angiogenesis were examined: protease activity, motility, and mitogenesis. Heparin caused a 100% increase in the adipocyte-induced stimulation of endothelial cell plasminogen activator activity and motility, but had no effect on proliferation. The enhancement of plasminogen activator and chemoattractant activities had a similar ED50 (1-2 micrograms/ml) and optimum dose (10-30 micrograms/ml). When we examined the direct effect of heparin on the activity of two distinct plasminogen activator enzymes--urokinase and tissue-type--a dual action of heparin was observed: tissue-type enzyme activity was stimulated 100% by heparin at 10 micrograms/ml, whereas urokinase activity was inhibited by 77% at this dose. These data suggest that heparin potentiates angiogenesis in vivo by stimulating endothelial cell plasminogen activator, motility, or both. Our results further suggest that for adipocyte-induced blood vessel formation, in contrast to other angiogenesis systems, heparin does not appear to affect the mitogenic activity.     

Cloning of a complementary deoxyribonucleic acid coding for human thyroxine-binding globulin (TBG): existence of two TBG messenger ribonucleic acid species possessing different 3'-untranslated regions

An adult human liver cDNA library constructed in expression vector, bacteriophage lambda gt11, was screened with polyclonal antibody directed against human T4-binding globulin (TBG). TBG cDNA cloned in the present study was 944 nucleotides in length. It contained approximately 70% of the coding region and complete 3'-untranslated region. When the sequence was compared with that of TBG cDNA recently cloned by I. L. Flint, T. J. Bailey, T. A., Gustafson, B. E. Markham, and E. Morkin, the 3'-untranslated region of our cDNA was 231 nucleotides shorter than their cDNA. These results indicated that two TBG mRNAs with different length of 3'-untranslated regions may exist in human liver. Indeed, Northern blot analysis revealed that two TBG mRNAs differing in the length approximately 200 base pairs were present in normal human liver as well as in human hepatoma cell line (HepG2). It was demonstrated that this size difference was due to the length of 3'-untranslated region by hybridization with a probe specific to the longer 3'-end. Together with the sequence data, it was suggested that these two TBG mRNA species may be produced by alternative processing and polyadenylation at two different sites.