BBA - Molecular Cell Research (v.1498, #1)
The role of NF-AT transcription factors in T cell activation and differentiation 1 1 We dedicate this review to Prof. Dr. Rigomar Rieger (Gatersleben), a great scientist and man, on the occasion of his 70th birthday. One of us (E.S.) had the pleasure of working in his department. by Edgar Serfling; Friederike Berberich-Siebelt; Sergei Chuvpilo; Eriks Jankevics; Stefan Klein-Hessling; Thomas Twardzik; Andris Avots (1-18).
The family of genuine NF-AT transcription factors consists of four members (NF-AT1 [or NF-ATp], NF-AT2 [or NF-ATc], NF-AT3 and NF-AT4 [or NF-ATx]) which are characterized by a highly conserved DNA binding domain (is designated as Rel similarity domain) and a calcineurin binding domain. The binding of the Ca2+-dependent phosphatase calcineurin to this region controls the nuclear import and exit of NF-ATs. This review deals (1) with the structure of NF-AT proteins, (2) the DNA binding of NF-AT factors and their interaction with AP-1, (3) NF-AT target genes, (4) signalling pathways leading to NF-AT activation: the role of protein kinases and calcineurin, (5) the nuclear entry and exit of NF-AT factors, (6) transcriptional transactivation by NF-AT factors, (7) the structure and expression of the chromosomal NF-AT2 gene, and (8) NF-AT factors in Th cell differentiation. The experimental data presented and discussed in the review show that NF-AT factors are major players in the control of T cell activation and differentiation and, in all likelihood, also of the cell cycle and apoptosis of T lymphocytes.
Keywords: AP-1; Cyclosporin A; Interleukin; NF-AT transcription factor; T cell activation; T cell differentiation;
Phorbol ester synergistically increases interferon regulatory factor-1 and inducible nitric oxide synthase induction in interferon-γ-treated RAW 264.7 cells by Isamu Momose; Masaharu Terashima; Yukie Nakashima; Masako Sakamoto; Hiroshi Ishino; Toumei Nabika; Yu Hosokawa; Yoshinori Tanigawa (19-31).
The roles of PKC in iNOS induction by IFN-γ have been shown in some cell types. The effect of a PKC activator, phorbol ester, in iNOS induction is thought to be due to multiple mechanisms, and it is necessary to examine the involvement of phorbol ester on IFN-γ-induced iNOS in detail. In the present study, we investigated the mechanisms of phorbol ester on IFN-γ-induced iNOS in RAW 264.7 cells. PMA synergistically increased iNOS activity, protein and mRNA levels in IFN-γ-treated RAW 264.7 cells. PMA together with IFN-γ increased iNOS mRNA without affecting the iNOS mRNA degradation, suggesting that the synergistic effect of PMA on IFN-γ-induced iNOS mRNA production may depend on the elevation of the transcription rate rather than a prolongation of mRNA stability. The DNA binding proteins that are involved in the regulation of iNOS expression are mainly NF-κB and IRF-1. IRF-1 transcriptionally regulates many IFN-inducible genes such as iNOS whose promoter contains an IRF-1 binding site. PMA might modulate iNOS induction as a cosignal with IFN-γ in RAW 264.7 cells because the synergistic effect of PMA was mediated through IRF-1, rather than NF-κB. Ro 31-8220, a PKC inhibitor, decreased iNOS activity, protein, mRNA levels and IRF-1 activity, indicating that the effect of PMA on iNOS induction might occur via the PKC pathway. It is evidence that PKC plays an important role in IRF-1 activation and that phorbol ester has a synergistic effect on iNOS induction through IRF-1 activation in IFN-γ-treated RAW 264.7 cells. The synergistic effect of PMA on IFN-γ-induced IRF-1 binding activity was observed in macrophage cell line J774 cells as well as RAW 264.7 cells, but not in thioglycollate-elicited peritoneal macrophages.
Keywords: Phorbol ester; Interferon-γ; Inducible nitric oxide synthase; Interferon regulatory factor 1; Macrophage;
Evidence for the presence of cGMP-dependent protein kinase-II in human distal colon and in T84, the colonic cell line by Nataraja G. Selvaraj; Roli Prasad; Jay L. Goldstein; Mrinalini C. Rao (32-43).
Heat-stable enterotoxin (STa) stimulates intestinal Cl− secretion by activating guanylate cyclase C (GCC) to increase intracellular cyclic GMP (cGMP). In the colon, cGMP action could involve protein kinase (PK) G-II or PKA pathways, depending on the segment and species. In the human colon, both PKG and PKA pathways have been implicated, and, therefore, the present study examined the mechanism of cGMP-mediated Cl− transport in primary cultures of human distal colonocytes and in T84, the colonic cell line. Both cell preparations express mRNA for CFTR, Na+-K+-2Cl− cotransporter (NKCC1), GCC and PKG-II as detected by RT-PCR. The effects of STa and the PKG-specific cGMP analogues, 8Br-cGMP and 8pCPT-cGMP, on Cl− transport were measured using a halide-sensitive probe. In primary human colonocytes and T84 cells, STa, the cGMP analogues and the cAMP-dependent secretagogue, prostaglandin E1 (PGE1), enhanced Cl− transport. The effects of 8Br-cGMP and 8pCPT-cGMP suggested the involvement of PKG, and this was explored further in T84 cells. The effects of 8pCPT-cGMP were dose-dependent and sensitive to the PKG inhibitor, H8 (70 μM), but H8 had no effect on PGE1-induced Cl− secretion. In contrast, a PKA inhibitor, H7 (50 μM), blocked PGE1-mediated but not 8pCPT-cGMP-induced Cl− transport. 8pCPT-cGMP enhanced phosphorylation of the PKG-specific substrate, 2A3, by T84 membranes in vitro. This phosphorylation was inhibited by H8. These results strongly suggest that cGMP activates Cl− transport through a PKG-II pathway in primary cells and in the T84 cell line of the human colon.
Keywords: Human colon; T84; Protein kinase G-II; Chloride transport;
CHO cell enlargement oscillates with a temperature-compensated period of 24 min by Rhea Pogue; Dorothy M. Morré; D.James Morré (44-51).
The rate of increase in cell area of CHO cells when measured at intervals of 1 min using a light microscope equipped with a video measurement system, oscillated with a minimum period of about 24 min. The pattern of oscillations paralleled those of the 24 min period observed with the oxidation of NADH by an external cell surface or plasma membrane NADH oxidase. The increase in cell area was non-linear. Intervals of rapid increase in area alternated with intervals of rapid decrease in area. The length of the 24 min period was temperature-compensated (approximately the same when measured at 14°C, 24°C or 34°C) while the rate of cell enlargement increased with temperature over this same range of temperatures.
Keywords: NADH oxidase; Cell enlargement; Growth; Temperature compensation; Ubiquinone; Chinese hamster ovary cell; Coenzyme Q;
Periodic NADH oxidase activity associated with an endoplasmic reticulum fraction from pig liver. Response to micromolar concentrations of retinol by Peichuan Sun; D.James Morré; Dorothy M. Morré (52-63).
An endoplasmic reticulum fraction from pig liver enriched in transitional endoplasmic reticulum vesicles capable of forming 50–60 nm buds in the presence of ATP and retinol was assayed for retinol-responsive oxidation of NADH and cleavage of a dithiodipyridine (DTDP) protein disulfide–thiol interchange substrate. Maxima for the two activities alternated giving rise to a 24 min period. The NADH oxidase activity was inhibited by micromolar and submicromolar concentrations of retinol. Retinol at 0.1 mM stimulated the activity. The inhibition was confined to two activity maxima separated in time by about 5 min. In contrast, with the DTDP substrate, the activity was stimulated by retinol and the stimulations were in the part of the oscillatory pattern where retinol inhibition of NADH oxidation was observed. The findings support an earlier proposed mechanism whereby retinol exerted opposing effects on NADH oxidation and protein disulfide reductions.
Keywords: NADH oxidase; Transitional endoplasmic reticulum; Protein disulfide–thiol interchange; Retinol;
Subcellular localization of cyclic ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities in porcine airway smooth muscle by Thomas A. White; Sonja Johnson; Timothy F. Walseth; Hon Cheung Lee; Richard M. Graeff; Cyrus B. Munshi; Y.S. Prakash; Gary C. Sieck; Mathur S. Kannan (64-71).
Recent studies have provided evidence for a role of cyclic ADP-ribose (cADPR) in the regulation of intracellular calcium in smooth muscles of the intestine, blood vessels and airways. We investigated the presence and subcellular localization of ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of β-NAD+ to cADPR, and cADPR hydrolase, the enzyme that degrades cADPR to ADPR, in tracheal smooth muscle (TSM). Sucrose density fractionation of TSM crude membranes provided evidence that ADP-ribosyl cyclase and cADPR hydrolase activities were associated with a fraction enriched in 5′-nucleotidase activity, a plasma membrane marker enzyme, but not in a fraction enriched in either sarcoplasmic endoplasmic reticulum calcium ATPase or ryanodine receptor channels, both sarcoplasmic reticulum markers. The ADP-ribosyl cyclase and cADPR hydrolase activities comigrated at a molecular weight of approximately 40 kDa on SDS–PAGE. This comigration was confirmed by gel filtration chromatography. Investigation of kinetics yielded K m values of 30.4±1.5 and 695.3±171.2 μM and V max values of 330.4±90 and 102.8±17.1 nmol/mg/h for ADP-ribosyl cyclase and cADPR hydrolase, respectively. These results suggest a possible role for cADPR as an endogenous modulator of [Ca2+]i in porcine TSM cells.
Keywords: Airway smooth muscle; Cyclic ADP-ribose; ADP-ribosyl cyclase; Cyclic ADP-ribose hydrolase;
Nitric oxide induces oxidative stress and apoptosis in neuronal cells by Taotao Wei; Chang Chen; Jingwu Hou; Wenjuan Xin; Akitane Mori (72-79).
Within the central nervous system and under normal conditions, nitric oxide (NO) is an important physiological signaling molecule. When produced in large excess, NO also displays neurotoxicity. In our previous report, we have demonstrated that the exposure of neuronal cells to NO donors induced apoptotic cell death, while pretreatment with free radical scavengers L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt (EPC-K1) or superoxide dismutase attenuated apoptosis effectively, suggesting that reactive oxygen species (ROS) may be involved in the cascade of events leading to apoptosis. In the present investigation, we directly studied the kinetic generation of ROS in NO-treated neuronal cells by flow cytometry using 2′,7′-dichloro-fluorescein diacetate and dihydrorhodamine 123 as redox-sensitive fluorescence probes. The results indicated that exposure of cerebellar granule cells to the NO donor S-nitroso-N-acetylpenicillamine (SNAP) induced oxidative stress, which was characterized by the accumulation of cytosolic and mitochondrial ROS, the increase in the extracellular hydrogen peroxide level, and the formation of lipid peroxidation products. SNAP treatment also induced apoptotic cell death as confirmed by the formation of cytosolic mono- and oligonucleosomes. Pretreating cells with the novel antioxidant EPC-K1 effectively prevented oxidative stress induced by SNAP, and attenuated cells from apoptosis.
Keywords: Nitric oxide; Reactive oxygen species; Oxidative stress; Cerebellar granule cell; Apoptosis; EPC-K1;