BBA - Molecular Cell Research (v.1540, #2)

Hyperosmotic stress activates the insulin receptor in CHO cells by D.Margriet Ouwens; Daniel S. Gomes de Mesquita; Jeroen Dekker; J.Antonie Maassen (97-106).
Stress factors, such as osmotic stress and genotoxic agents, activate stress kinases, whereas growth factors preferentially stimulate the structurally homologous mitogen-activated protein kinases, ERK1/2. Hyperosmolarity also has insulin-mimicking action as reflected by ERK1/2 activation and by the stimulation of glucose uptake in adipocytes. We examined to what extent hyperosmolarity activates components of the insulin receptor (IR) signalling pathway. CHO cells expressing the human IR were treated with 500 mM NaCl or 700 mM sorbitol and the activation of insulin signalling intermediates was studied. Hyperosmolarity induced tyrosine phosphorylation of the IR β-subunit, and the adaptor proteins p52-Shc, p66-Shc, and IRS1. Furthermore, the stress kinases JNK and p38 were activated. When CHO cells were transfected with a kinase-dead IR (K1030R) mutant, hyperosmolarity did not induce tyrosine phosphorylation of the IR, indicating that hyperosmolarity induced IR autophosphorylation directly, rather than inducing phosphorylation by an exogenous tyrosine kinase. A partially purified and detergent-solubilized IR was not phosphorylated in response to hyperosmolarity, suggesting that hyperosmolarity activates the receptor only when present in the plasma membrane. In cells stably expressing the kinase-dead IR, IRS1 and Shc Tyr phosphorylation was abrogated, indicating that the hyperosmolarity signalling was dependent on an active IR tyrosine kinase. In contrast, the stress kinases p38 and JNK were normally activated by hyperosmolarity in the IR-K1030R mutant. We conclude that, at least in CHO cells, hyperosmolarity signals partially through IR autophosphorylation and subsequent activation of the IR downstream targets. This may be responsible for some of the insulin-mimicking effects of hyperosmolarity. The activation of stress kinases by hyperosmolarity occurs independent of the IR.
Keywords: Hyperosmolarity; Insulin receptor; IRS-1; SAPK/JNK; p38;

The structures and mechanisms of activation of non-selective cation channels (NSCCs) are not well understood although NSCCs play important roles in the regulation of metabolism, ion transport, cell volume and cell shape. It has been proposed that TRP (transient receptor potential) proteins are the molecular correlates of some NSCCs. Using fura-2 and patch-clamp recording, it was shown that the maitotoxin-activated cation channels in the H4-IIE rat liver cell line admit Ca2+, Mn2+ and Na+, have a high selectivity for Na+ compared with Ca2+, and are inhibited by Gd3+ (half-maximal inhibition at 1 μM). Activation of the channels by maitotoxin was inhibited by increasing the extracellular Ca2+ concentration or by inclusion of 10 mM EGTA in the patch pipette. mRNA encoding TRP proteins 1, 2 and 3 at levels comparable with those in brain was detected using reverse transcriptase–polymerase chain reaction in poly(A)+ RNA prepared from H4-IIE cells and freshly-isolated rat hepatocytes. In H4-IIE cells transiently transfected with cDNA encoding hTRPC-1, the expressed hTRPC-1 protein was chiefly located at intracellular sites and at the plasma membrane. Cells expressing hTRPC-1 exhibited a substantial enhancement of maitotoxin-initiated Ca2+ inflow and a modest enhancement of thapsigargin-initiated Ca2+ inflow (measured using fura-2) and no enhancement of the highly Ca2+-selective store-operated Ca2+ current (measured using patch-clamp recording). In cells expressing hTRPC-1, maitotoxin activated channels which were not found in untransfected cells, have an approximately equal selectivity for Na+ and Ca2+, and are inhibited by Gd3+ (half-maximal inhibition at 3 μM). It is concluded that in liver cells (i) maitotoxin initiates the activation of endogenous NSCCs with a high selectivity for Na+ compared with Ca2+; (ii) TRP proteins 1, 2 and 3 are expressed; (iii) maitotoxin is an effective initiator of activation of heterologously expressed hTRPC-1 channels; and (iv) the endogenous TRP-1 protein is unlikely to be the molecular counterpart of the maitotoxin-activated NSCCs nor the highly Ca2+-selective store-operated Ca2+ channels.
Keywords: H4-IIE cell; Patch-clamp recording; Reverse transcriptase–polymerase chain reaction; Ca2+, Mn2+ and Na+ inflow; Heterologous expression; Gd3+;

The effect of chitosan on stiffness and glycolytic activity of human bladder cells by M Lekka; P Laidler; J Ignacak; M Łabędź; J Lekki; H Struszczyk; Z Stachura; A.Z Hrynkiewicz (127-136).
The cell’s cytoskeleton together with the cell membrane and numerous accessory proteins determines the mechanical properties of cell. Any factors influencing cell organization and structure can cause alterations in mechanical properties of cell (its ability for deformation and adhesion). The determination of the local elastic properties of cells in their culture conditions has opened the possibility for the measurement of the influence of different factors on the mechanical properties of the living cells. The effect of the chitosan on the stiffness of the non-malignant transitional epithelial cells of ureter (HCV 29) and the transitional cell cancer of urine bladder (T24) was determined using scanning force microscopy. The investigations were performed in the culture medium (RPMI 1640) containing 10% fetal calf serum in the presence of the microcrystalline chitosan of the three different deacetylation degrees. In parallel, the effect of chitosan on production of lactate and ATP level was determined. The results showed the strong correlation between the decrease of the energy production and the increase in Young’s modulus values obtained for the cancer cells treated with chitosan.
Keywords: Scanning force microscopy; Cell stiffness; Glycolytic activity; Cytoskeleton; Microcrystalline chitosan;

Necrotic cell death by hydrogen peroxide in immortal DF-1 chicken embryo fibroblast cells expressing deregulated MnSOD and catalase by Hyunggee Kim; Seungkwon You; Byung-Whi Kong; Linda K. Foster; James Farris; Douglas N. Foster (137-146).
The reactive oxygen species are known as endogenous toxic oxidant damaging factors in a variety of cell types, and in response, the antioxidant genes have been implicated in cell proliferation, senescence, immortalization, and tumorigenesis. The expression of manganese superoxide dismutase mRNA was shown to increase in most of the immortal chicken embryo fibroblast (CEF) cells tested, while expression of catalase mRNA appeared to be dramatically decreased in all immortal CEF cells compared to their primary counterparts. The expression of copper–zinc superoxide dismutase mRNA was shown to increase slightly in some immortal CEF cells. The glutathione peroxidase expressed relatively similar levels in both primary and immortal CEF cells. As primary and immortal DF-1 CEF cells were treated with 10–100 μM of hydrogen peroxide (concentrations known to be sublethal in human diploid fibroblasts), immortal DF-1 CEF cells were shown to be more sensitive to hydrogen peroxide, and total cell numbers were dramatically reduced when compared with primary cell counterparts. This increased sensitivity to hydrogen peroxide in immortal DF-1 cells occurred without evident changes in either antioxidant gene expression, mitochondrial membrane potential, cell cycle distribution or chromatin condensation. However, the total number of dead cells without chromatin condensation was dramatically elevated in immortal DF-1 CEFs treated with hydrogen peroxide, indicating that the inhibition of immortal DF-1 cell growth by low concentrations of hydrogen peroxide is due to increased necrotic cell death, but not apoptosis. Taken together, our observation suggests that the balanced antioxidant function might be important for cell proliferation in response to toxic oxidative damage by hydrogen peroxide.
Keywords: Manganese superoxide dismutase; Catalase; Hydrogen peroxide; Chicken embryo fibroblast; DF-1;

Focal adhesion kinase (FAK) is known to be located at the intersection between extracellular matrix and growth factor signaling pathways to regulate cell motility. We have shown previously that an activated form (BCR-FLTm1) of Flt-1 kinase, a receptor for vascular endothelial growth factor, had a tubulogenic activity not only in endothelial cells but also in fibroblastic cells. Here we show that tubulogenesis by BCR-FLTm1 depends on FAK and that FAK tyrosine phosphorylation and association with an activated Flt-1 receptor complex is increased after vascular endothelial growth factor stimulation of NIH3T3 cells overexpressing Flt-1.
Keywords: Tubulogenesis; Focal adhesion kinase; Flt-1; Vascular endothelial growth factor;

Homocysteine inhibits tumor necrosis factor-induced activation of endothelium via modulation of nuclear factor-κb activity by Johannes Roth; Matthias Goebeler; Stephan Ludwig; Liliane Wagner; Karin Kilian; Clemens Sorg; Erik Harms; Klaus Schulze-Osthoff; Hans-Georg Koch (154-165).
Homocystinuria is a metabolic disorder associated with an increased incidence of vascular disease. Here, we analyzed the effects of homocysteine on endothelial cell activation that is a prerequisite for the recruitment of leukocytes to sites of evolving atherosclerotic plaques. Exposure of human umbilical vein endothelial cells to homocysteine alone did not modulate expression of the adhesion molecules E-selectin, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and the chemokines monocyte chemotactic protein-1 and interleukin-8. In contrast, tumor necrosis factor (TNF)-induced upregulation of these molecules was almost completely inhibited by homocysteine, but not by related thiol amino acids. Using electrophoretic mobility shift and reporter gene assays, the inhibitory effect of homocysteine could be attributed to inhibition of DNA binding and transcriptional activity of NF-κB. TNF-induced phosphorylation and degradation of IκB-α, however, were not affected. Neither was NF-κB-independent activation of endothelial cells by interferon-γ influenced by homocysteine. In summary, our data indicate that homocysteine alters the response to injury of endothelial cells which may have fundamental impacts on mechanisms of leukocyte recruitment to sites of inflammation. Our findings might refer to a novel pathway by which homocysteine is involved in vascular disorders associated with homocystinuria.
Keywords: Homocystinuria; Endothelial activation; Nuclear factor-κB; Atherosclerosis;

Dibenzyl trisulphide (DTS), a main lipophilic compound in Petiveria alliacea L. (Phytolaccaceae), was identified as one of the active immunomodulatory compounds in extracts of the plant. To learn more about its biological activities and molecular mechanisms, we conducted one-dimensional NMR interaction studies with bovine serum albumin (BSA) and tested DTS and related compounds in two well-established neuronal cell-and-tissue culture systems. We found that DTS preferentially binds to an aromatic region of BSA which is rich in tyrosyl residues. In SH-SY5Y neuroblastoma cells, DTS attenuates the dephosphorylation of tyrosyl residues of MAP kinase (erk1/erk2). In the same neuroblastoma cell line and in Wistar 38 human lung fibroblasts, DTS causes a reversible disassembly of microtubules, but it did not affect actin dynamics. Probably due to the disruption of the microtubule dynamics, DTS also inhibits neuroblastoma cell proliferation and neurite outgrowth from spinal cord explants. Related dibenzyl compounds with none, one, or two sulphur atoms were found to be significantly less effective. These data confirmed that the natural compound DTS has a diverse spectrum of biological properties, including cytostatic and neurotoxic actions in addition to immunomodulatory activities.
Keywords: Dibenzyl trisulphide; Neurite outgrowth; Cell proliferation; Microtubules; Tyrosine phosphorylation;