Reactive Oxygen Species-dependent Manner Research Articles

Rac1, but Not Rac1B, Stimulates RelB-mediated Gene Transcription in Colorectal Cancer Cells

Increased NF-kappaB-mediated transcription has been extensively linked to tumorigenesis and can be stimulated by deregulated Rac1 signaling. For example, the overexpression of Rac1b, a highly activated splicing variant of Rac1 with increased expression in colorectal tumors, stimulates NF-kappaB-mediated G1/S progression and cell survival, and was shown to promote cell transformation and epithelial-mesenchymal transition. Here we show evidence of further complexity between Rac1b and Rac1 signaling toward NF-kappaB in colorectal cells. Consistent with data from other cell types we demonstrate that both Rac1 and Rac1b stimulate transcriptional activation from reporter genes driven by NF-kappaB motifs or the cyclin D1 promoter in an IkappaBalpha- and reactive oxygen species-dependent manner. However, we found that in colorectal cells Rac1, but not Rac1b, induces nuclear translocation of RelB and p52, activates transcription from a RelB-specific reporter, and can be isolated in a complex with endogenous RelB and its inhibitor NF-kappaB2/p100. In addition, Rac1 colocalizes at the plasma membrane with RelB, p100, and cullin-1, a core subunit of the E3 ubiquitin ligase that marks p100 for proteolytic processing to p52. Interestingly, this Rac1-specific pathway is not mediated via the production of reactive oxygen species. These data provide evidence that both Rac1 and Rac1b activate the canonical RelA-IkappaBalpha pathway, whereas Rac1 further stimulates NF-kappaB by inducing the RelB-NF-kappaB2/p100 pathway. The RelB pathway was reported to down-regulate canonical NF-kappaB activation during the inflammatory response, suggesting that increased levels of Rac1b in colorectal tumors may promote tumorigenesis by stimulating canonical NF-kappaB signaling while circumventing a negative feedback from the RelB pathway.

Cytochrome c Is Released in a Reactive Oxygen Species-Dependent Manner and Is Degraded via Caspase-Like Proteases in Tobacco Bright-Yellow 2 Cells en Route to Heat Shock-Induced Cell Death

To gain some insight into the mechanism of plant programmed cell death, certain features of cytochrome c (cyt c) release were investigated in heat-shocked tobacco (Nicotiana tabacum) Bright-Yellow 2 cells in the 2- to 6-h time range. We found that 2 h after heat shock, cyt c is released from intact mitochondria into the cytoplasm as a functionally active protein. Such a release did not occur in the presence of superoxide anion dismutase and catalase, thus showing that it depends on reactive oxygen species (ROS). Interestingly, ROS production due to xanthine plus xanthine oxidase results in cyt c release in sister control cultures. Maximal cyt c release was found 2 h after heat shock; later, activation of caspase-3-like protease was found to increase with time. Activation of this protease did not occur in the presence of ROS scavenger enzymes. The released cyt c was found to be progressively degraded in a manner prevented by either the broad-range caspase inhibitor (zVAD-fmk) or the specific inhibitor of caspase-3 (AC-DEVD-CHO), which have no effect on cyt c release. In the presence of these inhibitors, a significant increase in survival of the cells undergoing programmed cell death was found. We conclude that ROS can trigger release of cyt c, but do not cause cell death, which requires caspase-like activation.

Interleukin-1β induces death in chondrocyte-like ATDC5 cells through mitochondrial dysfunction and energy depletion in a reactive nitrogen and oxygen species-dependent manner

IL-1 (interleukin-1) acts as a key mediator of the degeneration of articular cartilage in RA (rheumatoid arthritis) and OA (osteoarthritis),where chondrocyte death is observed. It is still controversial, however, whether IL-1 induces chondrocyte death. In the present study, the viability of mouse chondrocyte-like ATDC5 cells was reduced by the treatment with IL-1beta for 48 h or longer. IL-1beta augmented the expression of the catalytic gp91 subunit of NADPH oxidase, gp91phox, as well as inducible NO synthase in ATDC5 cells. Generation of nitrated guanosine and tyrosine suggested the formation of reactive nitrogen species including ONOO- (peroxynitrite), a reaction product of NO and O2-, in ATDC5 cells and rat primary chondrocytes treated with IL-1beta. Death of ATDC5 cells after IL-1beta treatment was prevented by an NADPH-oxidase inhibitor, AEBSF[4-(2-aminoethyl)benzene-sulphonyl fluoride], an NO synthase inhibitor, L-NAME (NG-nitro-L-arginine methyl ester), and a ONOO- scavenger, uric acid. The viability of ATDC5 cells was reduced by the ONOO(-)-generator 3-(4-morpholinyl)sydnonimine hydrochloride, but not by either the NO-donor 1-hydroxy-2-oxo-3-(N-methyl-2-aminopropyl)-3-methyl-1-triazene or S-nitrosoglutathione. Disruption of mitochondrial membrane potential and ATP deprivation were observed in IL-1beta-treated ATDC5 cells, both of which were restored by L-NAME, AEBSF or uric acid. On the other hand, no morphological or biochemical signs indicating apoptosis were observed in these cells. These results suggest that the death of chondrocyte-like ATDC5 cells was mediated at least in part by mitochondrial dysfunction and energy depletion through ONOO- formation after IL-1beta treatment.

Activation of the JAK/STAT pathway in vascular smooth muscle by serotonin

Serotonin (5-hydroxytryptamine, 5-HT) is a vasoconstrictor and mitogen whose levels are elevated in diabetes. Previous studies have shown the presence of 5-HT2A, 5-HT2B, and 5-HT1B receptors in vascular smooth muscle cells (VSMCs). There are currently no data regarding 5-HT2B and 5-HT1B receptor activation of the JAK/STAT pathway in VSMCs and resultant potential alterations in 5-HT signaling in diabetes. Therefore, we tested the hypothesis that 5-HT differentially activates the JAK/STAT pathway in VSMCs under conditions of normal (5 mM) and high (25 mM) glucose. Treatment of rat VSMCs with 5-HT (10(-6) M) resulted in time-dependent activation ( approximately 2-fold) of JAK2, JAK1, and STAT1, but not STAT3 (maximal at 5 min, returned to baseline by 30 min). The 5-HT2B receptor agonist BW723C86 and the 5-HT1B receptor agonist CGS12066A (10(-9)-10(-5) M, 5-min stimulation) did not activate the JAK/STAT pathway. Treatment with the 5-HT2A receptor antagonist ketanserin (10 nM) inhibited JAK2 activation by 5-HT. Treatment of streptozotocin-induced diabetic rats with ketanserin (5 mg.kg-1.day-1) reduced activation of JAK2 and STAT1 but not STAT3 in endothelium-denuded thoracic aorta in vivo. 5-HT (10(-6) M) treatment resulted in increased cell proliferation and increased DNA synthesis, which were inhibited by the JAK2 inhibitor AG490. Further studies with apocynin, diphenyleneiodonium chloride, catalase, and virally transfected superoxide dismutase had no effect at either glucose concentration on activation of the JAK/STAT pathway by 5-HT. Therefore, we conclude that 5-HT activates JAK2, JAK1, and STAT1 via the 5-HT2A receptors in a reactive oxygen species-independent manner under both normal and high glucose conditions.

Glucuronic acid is a novel inducer of heat shock response.

The elevated expression of 70 kDa heat shock protein (Hsp70) induces resistance to stress-induced apoptosis. We have screened a variety of natural products for their ability to enhance Hsp70 expression as anti-apoptotic agent. We found that glucuronic acid (GA) induced the synthesis of Hsp70 and that cells pretreated with GA were significantly tolerant to stress including heat shock and hydrogen peroxide. We also found that GA induces the production of reactive oxygen species (ROS), a process inhibited by NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI) and antioxidant N-acetylcysteine (NAC). GA-induced ROS production was also inhibited in RacN17 cell line overexpressing a dominant negative mutant of Rac1. Furthermore, GA treatment induces MAPKs activation (SAPK/JNK and p38) and Hsp70 expression in ROS dependent manner, suggesting that GA turns on the signaling pathway by generation of ROS through Rac1. We analyzed the profiles of newly synthesized proteins by GA with 2-dimensional gel electrophoresis and MALDI-TOF MS and found that two families of proteins were expressed by GA. One was similar to the protein family synthesized by heat shock (Hsp70, Hsp73, Hsp65, Hsp90, vimentin, tubulin, Ras homolog); and the other was a family of protein specific to GA (calreticulin, annexin III, thioredoxin peroxidase). These results suggest that GA-induced stress responses are mediated by ROS generation and are similar, in part, to heat shock-induced responses and GA can be possibly adopted for the protecting agent from cell death.