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The transcription factor AP-1 subunit JUNB has been shown to play a pivotal role in angiogenesis. It positively controls angiogenesis by regulating Vegfa as well as the transcriptional regulator Cbfb and its target Mmp13. In line with these findings, it has been demonstrated that tumor cell-derived JUNB promotes tumor growth and angiogenesis. In contrast to JUNB's function in tumor cells, the role of host-derived stromal JUNB has not been elucidated so far. Here, we show that ablation of Junb in stromal cells including endothelial cells (ECs), vascular smooth muscle cells (SMCs) and fibroblasts does not affect tumor growth in two different syngeneic mouse models, the B16-F1 melanoma and the Lewis lung carcinoma model. In-depth analyses of the tumors revealed that tumor angiogenesis remains unaffected as assessed by measurements of the microvascular density and relative blood volume in the tumor. Furthermore, we could show that the maturation status of the tumor vasculature, analyzed by the SMC marker expression, α-smooth muscle actin and Desmin, as well as the attachment of pericytes to the endothelium, is not changed upon ablation of Junb. Taken together, these results indicate that the pro-angiogenic functions of stromal JUNB are well compensated with regard to tumor angiogenesis and tumor growth., (© 2013 UICC.)

The metabolically competent hepatocyte-like human HepaRG cells represent a suitable alternative in vitro cell model to human primary hepatocytes. Here, we describe the culture procedure required to expand progenitor HepaRG cells and to differentiate them into hepatocyte-like cells. Transient transfection of gene and siRNA into cultured cells, using nonviral strategies, is an invaluable technique to decipher gene functions. In this chapter, we detail transfection protocols for efficient transfer of plasmid DNA or siRNAs into proliferating progenitor or quiescent differentiated HepaRG cells as well as into primary hepatocytes.

Cellular contractility and, thus, the ability to alter cell shape are prerequisites for a number of important biological processes such as cytokinesis, movement, differentiation, and substrate adherence. The contractile capacity of vascular smooth muscle cells (VSMCs) is pivotal for the regulation of vascular tone and thus blood pressure and flow. Here, we report that conditional ablation of the transcriptional regulator Junb results in impaired arterial contractility in vivo and in vitro. This was exemplified by resistance of Junb-deficient mice to DOCA-salt-induced volume-dependent hypertension as well as by a decreased contractile capacity of isolated arteries. Detailed analyses of Junb-deficient VSMCs, mouse embryonic fibroblasts, and endothelial cells revealed a general failure in stress fiber formation and impaired cellular motility. Concomitantly, we identified myosin regulatory light chain 9 (Myl9), which is critically involved in actomyosin contractility and stress fiber assembly, as a Junb target. Consistent with these findings, reexpression of either Junb or Myl9 in Junb-deficient cells restored stress fiber formation, cellular motility, and contractile capacity. Our data establish a molecular link between the activator protein-1 transcription factor subunit Junb and actomyosin-based cellular motility as well as cellular and vascular contractility by governing Myl9 transcription.

Uncoupling 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., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

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 EpCAM is recruited into tetraspanin-enriched membrane microdomains (TEM). The TEM-located claudin-7-EpCAM complex supports proliferation accompanied by sustained extracellular signal-regulated kinase-1/2 phosphorylation, up-regulation of antiapoptotic proteins, and drug resistance, but not EpCAM-mediated cell-cell adhesion. Enhanced motility may be supported by colocalization of claudin-7 with actin bundles, which is only seen in EpCAM-claudin-7-expressing cells. The EpCAM-claudin-7 complex strongly promotes tumorigenicity, accelerates tumor growth, and supports ascites production and thymic metastasis formation. High expression of the tumor marker EpCAM is frequently associated with poor prognosis, which could well rely on the EpCAM-claudin-7 association that prohibits EpCAM-mediated cell-cell adhesion but promotes migration, proliferation, apoptosis resistance, and tumorigenicity.

Uncoupling protein 2 (UCP2) belongs to a family of transporters of the mitochondrial inner membrane and is reported to uncouple respiration from ATP synthesis. Our observation that the amino acid glutamine specifically induces UCP2 protein expression prompted us to investigate metabolic consequences of a UCP2 knockdown (Ucp2-KO) when glutamine is offered as a substrate. We found that Ucp2-KO macrophages incubated in the presence of glutamine exhibit a lower ammonium release, a decreased respiratory rate, and an intracellular accumulation of aspartate. Therefore, we conclude that UCP2 expression is required for efficient oxidation of glutamine in macrophages. This role of UCP2 in glutamine metabolism appears independent from the uncoupling activity of UCP2.

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.

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.

Uncoupling proteins (UCPs) are specialized members of the mitochondrial transporter family. They allow passive proton transport through the mitochondrial inner membrane. This activity leads to uncoupling of mitochondrial respiration and to energy waste, which is well documented with UCP1 in brown adipose tissue. The uncoupling activity of the new UCPs (discovered after 1997), such as UCP2 and UCP3 in mammals or avUCP in birds, is more difficult to characterize. However, extensive data support the idea that the new UCPs are involved in the control of reactive oxygen species (ROS) generation. This fits with the hypothesis that mild uncoupling caused by the UCPs prevents ROS production. Activators and inhibitors regulate the proton transport activity of the UCPs. In the absence of activators of proton transport, the UCP allows the permeation of other ions. We suggest that this activity has physiological significance and, for example, UCP3 expressed in glycolytic muscle fibres may be a passive pyruvate transporter ensuring equilibrium between glycolysis and oxidative phosphorylation. Induction of UCP2 expression by glutamine strengthens the proposal that new UCPs could act to determine the choice of mitochondrial substrate. This would obviously have an impact on mitochondrial bioenergetics and ROS production.

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 micromol/L). Lovastatin affected various IR-induced stress responses in HUVEC: It attenuated the increase in p53/p21 protein level and impaired the activation of nuclear factor-kappaB, 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.

HMG-CoA reductase inhibitors (statins) exert pleiotropic physiological effects. Among others they attenuate cellular responses to genotoxic and inflammatory stress. We investigated the effect of lovastatin on the expression level of TNF receptors (TNFR) in primary human endothelial cells (HUVEC). ELISA, FACS and immunocytochemical analyses show that lovastatin selectively increases the cell surface expression of TNFR2 without affecting the expression level of TNFR1. This effect of lovastatin is independent from inhibition of cell-cycle progression since cells both in G1- and G2-phase showed elevated levels of TNFR2 after lovastatin treatment. To analyze the physiological relevance of lovastatin-mediated upregulation of TNFR2, we investigated the expression of the cell adhesion molecule E-selectin, which is inducible by TNFalpha. While lovastatin on its own did not change the number of HUVEC expressing E-selectin protein, it promoted the TNFalpha-stimulated increase in the percentage of E-selectin expressing endothelial cells in a dose-dependent manner. This indicates that lovastatin sensitizes HUVEC towards TNFalpha-induced signaling by upregulation of TNFR2 expression. Based on the data, we suggest that statins have impact on endothelial responses to inflammatory stress by modulation of the expression of cytokine receptors.

E-selectin mediated tumor cell adhesion plays an important role in metastasis. Here we show that ionizing radiation (IR) induces E-selectin gene and protein expression in human endothelial cells at therapeutically relevant dose level. E-selectin expression is accompanied by an increase in the adhesion of human colon carcinoma cells to primary human umbilical vein endothelial cells (HUVEC). The HMG-CoA reductase inhibitor lovastatin impairs IR-stimulated E-selectin expression as analyzed at the level of the protein, mRNA and promoter. Inactivation of Rho GTPases either by use of Clostridium difficile toxin A or by co-expression of dominant-negative Rho blocked IR-induced E-selectin gene induction, indicating Rho GTPases to be essential. Radiation-induced expression of E-selectin was also blocked by all-trans retinoic acid (at-RA), whereas 9-cis retinoic acid was ineffective. Abrogation of IR-stimulated E-selectin expression by lovastatin and at-RA reduced tumor cell adhesion in a dose-dependent manner. Combined treatment with lovastatin and at-RA exerted additive inhibitory effects on radiation-induced E-selectin expression and tumor cell adhesion. Therefore, application of statins and at-RA might have clinical impact in protecting against E-selectin-promoted metastasis, which might arise as an unwanted side effect from radiation treatment.

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.

The sulfide-dependent reduction of exogenous ubiquinone by membranes of the hyperthermophilic chemotrophic bacterium Aquifex aeolicus (VF5), the sulfide-dependent consumption of oxygen and the reduction of cytochromes by sulfide in membranes were studied. Sulfide reduced decyl-ubiquinone with a maximal rate of up to 3.5 micromol (mg protein)(-1) min(-1) at 20 degrees C. Rates of 220 nmol (mg protein)(-1) min(-1)] for the sulfide-dependent consumption of oxygen and 480 nmol (mg protein)(-1) min(-1) for the oxidation of sulfide at 20 C were estimated. The reactions were sensitive towards 2-n-nonyl-4-hydroxyquinoline-N-oxide, but insensitive towards cyanide. Both reduction of decyl-ubiquinone and consumption of oxygen by sulfide rapidly increased with increasing temperature. For the sulfide-dependent respiratory activity, a sulfide-to-oxygen ratio of 2.3+/-0.2 was measured. This indicates that sulfide was oxidized to the level of zero-valent sulfur. Reduction of cytochromes by sulfide was monitored with an LED-array spectrophotometer. Reduction of cytochrome b was stimulated by 2-n-nonyl-4-hydroxyquinoline-N-oxide in the presence of excess sulfide under oxic conditions. This "oxidant-induced reduction" of cytochrome b suggests that electron transport from sulfide to oxygen in A. aeolicus employs the cytochrome bc complex via the quinone pool. Comparison of the amino acid sequence with the sequence of the sulfide:quinone oxidoreductase from Rhodobacter capsulatus and of the flavocytochrome c from Allochromatium vinosum revealed that the sulfide:quinone oxidoreductase from A. aeolicus belongs to the glutathione reductase family of flavoproteins.