Hydrogen sulfide (H2S) is produced inside the intestine and is known as a poison that inhibits cellular respiration at the level of cytochrome oxidase. However, sulfide is used as an energetic substrate by many photo- and chemoautotrophic bacteria and by animals such as the lugworm Arenicola marina. The concentrations of sulfide present in their habitats are comparable with those present in the human colon. Using permeabilized colonic cells to which sulfide was added by an infusion pump we show that the maximal respiratory rate of colonocyte mitochondria in presence of sulfide compares with that obtained with succinate or L-alpha-glycerophosphate. This oxidation is accompanied by mitochondrial energization. In contrast, other cell types not naturally exposed to high concentration of sulfide showed much lower oxidation rates. Mitochondria showed a very high affinity for sulfide that permits its use as an energetic substrate at low micromolar concentrations, hence, below the toxic level. However, if the supply of sulfide exceeds the oxidation rate, poisoning renders mitochondria inefficient and our data suggest that an anaerobic mechanism involving partial reversion of Krebs cycle already known in invertebrates takes place. In conclusion, this work provides additional and compelling evidence that sulfide is not only a toxic compound. According to our study, sulfide appears to be the first inorganic substrate for mammalian cells characterized thus far.

High expression of EpCAM and the tetraspanin CO-029 has been associated with colorectal cancer progression. However, opposing results have been reported on CD44 variant isoform v6 (CD44v6) expression. We recently noted in rat gastrointestinal tumors that EpCAM, claudin-7, CO-029, and CD44v6 were frequently coexpressed and could form a complex. This finding suggested the possibly that the complex, rather than the individual molecules, could support tumor progression. The expression of EpCAM, claudin-7, CO-029, and CD44v6 expression was evaluated in colorectal cancer (n = 104), liver metastasis (n = 66), and tumor-free colon and liver tissue. Coexpression and complex formation of the molecules was correlated with clinical variables and apoptosis resistance. EpCAM, claudin-7, CO-029, and CD44v6 expression was up-regulated in colon cancer and liver metastasis. Expression of the four molecules did not correlate with tumor staging and grading. However, coexpression inversely correlated with disease-free survival. Coexpression was accompanied by complex formation and recruitment into tetraspanin-enriched membrane microdomains (TEM). Claudin-7 contributes to complex formation inasmuch as in the absence of claudin-7, EpCAM hardly associates with CO-029 and CD44v6 and is not recruited into TEMs. Notably, colorectal cancer lines that expressed the EpCAM/claudin-7/CO-029/CD44v6 complex displayed a higher degree of apoptosis resistance than lines devoid of any one of the four molecules. Expression of EpCAM, claudin-7, CO-029, and CD44v6 by themselves cannot be considered as prognostic markers in colorectal cancer. However, claudin-7 -associated EpCAM is recruited into TEM and forms a complex with CO-029 and CD44v6 that facilitates metastasis formation. (Mol Cancer Res 2007;5(6):553 -67)

The mitochondrion is a major organelle contributing to energy metabolism but also a main site of ROS (reactive oxygen species) production. LPS (lipopolysaccharide)-induced ROS signalling is a critical event in macrophage activation. In the present paper we report that part of LPS-mediated ROS signalling comes from mitochondria inside a signal amplification loop that enhances MAPK (mitogen-activated protein kinase) activation. More precisely, we have identified the inner mitochondrial membrane UCP2 (uncoupling protein 2) as a physiological brake on ROS signalling. Stimulation of murine bone marrow-derived macrophages by LPS quickly down-regulated UCP2 through the JNK (c-Jun N-terminal kinase) and p38 pathways. UCP2 down-regulation was shown to be necessary to increase mitochondrial ROS production in order to potentiate MAPK activation. Consistent with this, UCP2-deficient macrophages exhibit an enhanced inflammatory state characterized by increased nitric oxide production and elevated migration ability. Additionally, we found that the absence of UCP2 renders macrophages more resistant to nitric oxide-induced apoptosis.

EpCAM has been described as a therapeutically relevant tumor marker. We noted an interaction between EpCAM and the tight junction protein claudin-7 and here explored the nature of this interaction and its effect on EpCAM-mediated functions. The interaction between EpCAM and claudin-7 was defined in HEK293 cells transfected with rat claudin-7 and EpCAM cDNA. Deletions of the epidermal growth factor -like and the thyroglobin repeat domains of EpCAM or the cytoplasmic domain of EpCAM or claudin-7 did not prevent the EpCAM-claudin-7 association. A chimeric EpCAM molecule with an exchange of the cytoplasmic and transmembrane domains and an EpCAM molecule with point mutations in an AxxxG motif in the transmembrane region do not associate with claudin-7. HEK cells and the rat pancreatic tumor line BSp73AS, transfected with (mutated) EpCAM and claudin-7 cDNA, revealed that the association of both molecules severely alters the functional activity of EpCAM. Claudin-7 -associated

a b s t r a c tUncoupling protein 2 (UCP2) belongs to the family of mitochondrial carriers. Here, we highlight recent findings regarding UCP2 function in the immune system. UCP2 controls immune cell activation by modulating MAPK pathways and the production of mitochondrial reactive oxygen species. In several models of infection, inflammation and autoimmunity, a regulatory impact of UCP2 was demonstrated by its direct implication in the production of cytokines and nitric oxide and in cell migration. In addition, UCP2 is reported as a key protein for oxidation of fatty acids, glutamine and glucose. Therefore we present a model of how the regulation of nutrient oxidation by UCP2 promotes immune cell activation.

Infiltration of inflammatory cells into pancreatic islets of Langerhans and selective destruction of insulin-secreting ␤-cells are characteristics of type 1 diabetes. Uncoupling protein 2 (UCP2) is a mitochondrial protein expressed in immune cells. UCP2 controls macrophage activation by modulating the production of mitochondrial reactive oxygen species (ROS) and MAPK signaling. We investigated the role of UCP2 on immune cell activity in type 1 diabetes in Ucp2-deficient mice. Using the model of multiple low-dose streptozotocin (STZ)-induced diabetes, we found that autoimmune diabetes was strongly accelerated in Ucp2-KO mice, compared with Ucp2-WT mice with increased intraislet lymphocytic infiltration. Macrophages from STZ-treated Ucp2-KO mice had increased IL-1␤ and nitric oxide (NO) production, compared with WT macrophages. Moreover, more macrophages were recruited in islets of STZ-treated Ucp2-KO mice, compared with Ucp2-WT mice. This finding also was accompanied by increased NO/ROS-induced damage. Altogether, our data show that inflammation is stronger in Ucp2-KO mice and islets, leading to the exacerbated disease in these mice. Our results highlight the mitochondrial protein UCP2 as a new player in autoimmune diabetes.inflammation ͉ nitric oxide M itochondria are important organelles for cellular functions, including ATP synthesis. Oxidative glucose metabolism in pancreatic ␤-cells increases the ATP/ADP ratio, leading to insulin release. Uncoupling protein 2 (UCP2) is a mitochondrial carrier protein expressed in pancreatic ␤-cells (1, 2) and immune cells (3,4).In pancreatic ␤-cells, UCP2 was reported to alter the yield of ATP synthesis from glucose, and it has been proposed as a negative regulator of glucose-stimulated insulin secretion (2). In other respects, there is much evidence for the influence of UCP2 on immune responses. First, Ucp2-KO mice exhibit increased resistance to a Toxoplasma gondii or Listeria monocytogenes infection (4, 5). Second, in an LDL receptor-null background, Ucp2-KO mice develop greater and more unstable atherosclerotic plaques than WT mice (6). Third, in experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis, Ucp2-KO mice develop higher disease scores than Ucp2-WT mice (7). We showed that UCP2 regulates LPS-induced reactive oxygen species (ROS) signaling in macrophages (8). MAPK activation in Ucp2-KO macrophages is quicker and stronger than in WT macrophages (8), leading to increased nitric oxide (NO), cytokine production, and migration ability (8, 9). Consistent with these findings, overexpression of UCP2 in macrophages is associated with diminished NO production (10) and decreased migration capacity (11).Given the evidence for a role of UCP2 in the immune system, in macrophages, and in ␤-cell function, we investigated whether UCP2 affects the development of autoimmune diabetes by using the model of multiple low-dose of streptozotocin (STZ). In this report, we show that the absence of UCP2 renders animals more sensitive to the onset of diabetes in mice....

E-selectin mediated cell-cell adhesion plays an important role in inflammatory processes and extravasation of tumor cells. Tumor necrosis factor-alpha (TNF-alpha) induces E-selectin gene and protein expression in primary human endothelial cells (HUVEC) and in an endothelial cell line (EA.hy-926). As shown by ELISA and FACS analyses, HMG-CoA reductase inhibitors (e.g., lovastatin) impair the TNF-alpha stimulated increase in E-selectin protein expression. Similar results were obtained for E-selectin mRNA expression and promoter activity, indicating that the effect of lovastatin is based on inhibition of gene expression. The effective inhibitory concentration of lovastatin was in a physiologically relevant range (IC50<0.1 microM). Lovastatin-mediated block of TNF-alpha induced E-selectin expression is due to inhibition of protein geranylgeranylation rather than farnesylation. Down-regulation of Rho signaling by coexpression of dominant-negative Rho mutants (i.e RhoA, RhoB and Rac) impaired TNF-alpha driven E-selectin gene expression, indicating Rho signaling to be essential for transcriptional activation of the E-selectin gene. Inhibition of E-selectin expression by lovastatin gives rise to a significant reduction in TNF-alpha stimulated adhesion of colon carcinoma cells to HUVEC. Furthermore, low concentration of lovastatin (i.e., < or =1 microM) attenuated TNF-alpha induced tumor cell invasion in vitro. The data support the view that statins might be clinically useful in protection against E-selectin mediated metastasis.

Purpose: 3-hydroxy-3-methylglutaryl CoA reductase inhibitors (statins) are frequently used lipid-lowering drugs. Moreover, they are reported to exert pleiotropic effects on cellular stress responses, proliferation, and apoptosis. Whether statins affect the sensitivity of primary human cells to ionizing radiation (IR) is still unknown. The present study aims at answering this question. Experimental Design: The effect of lovastatin on IR-provoked cytotoxicity was analyzed in primary human umbilical vein endothelial cells (HUVEC). To this end, cell viability, proliferation, and apoptosis as well as DNA damage^related stress responses were investigated. Results: The data show that lovastatin protects HUVEC from IR-induced cell death. Lovastatin did not confer radioresistance to human fibroblasts. The radioprotective, antiapoptotic effect of lovastatin was observed at low, physiologically relevant dose level (1 Amol/L). Lovastatin affected various IR-induced stress responses in HUVEC: It attenuated the increase in p53/p21protein level and impaired the activation of nuclear factor-nB, Chk-1, and Akt kinase but did not inhibit extracellular signal-regulated kinase activation. Exposure of HUVEC to IR did not change the level of Bax and Bcl-2 and did not cause activation of caspase-3, indicating that radioprotection by lovastatin does not depend on the modulation of the mitochondrial death pathway. Also, IR-induced DNA double-strand break formation and repair were not influenced by lovastatin. Conclusions:The data show that lovastatin has multiple inhibitory effects on IR-stimulated DNA damage^dependent stress responses in HUVEC. Because lovastatin causes radioresistance, it might be useful in the clinic for attenuating side effects of radiation therapy that are related to endothelial cell damage.Cellular sensitivity to ionizing radiation (IR) is determined by numerous factors. Most important are DNA repair (1) and radiation-induced signaling mechanisms that cause changes in gene expression, cell cycle progression, and apoptosis (2). DNA damage induced by IR (e.g., DNA strand breaks) causes activation of the DNA damage -specific kinases ATM/ATR and DNA-PK cs (2 -4). Subsequently, downstream functions, such as p53 and checkpoint kinases, become activated, resulting in changes in repair and cell cycle progression and, possibly, induction of cell death (5). Apart from DNA damage -triggered functions, IR also causes activation of cell surface receptors that eventually lead to the activation of mitogen-activated protein kinases and transcription factors, e.g., activator protein-1 (AP-1) and nuclear factor-nB . Similar to DNA damage -triggered stress responses, signal mechanisms originating from activated cell receptors also affect the cellular susceptibility to radiation (9, 10).A pharmacologic approach for intervening with radiationinduced stress responses is based on the fact that Ras and Rho GTPases, which are required for genotoxic stress-stimulated activation of mitogen-activated protein kinases (8) and , are su...