Heme oxygenase-1 inhibits apoptosis in Caco-2 cells via activation of Akt pathway

Heme oxygenase-1 can play a protective role against cellular stress. In colon cancer cells, these effects would be relevant to oncogenesis and resistance to chemotherapy. The aim of the study was to examine the effects of heme oxygenase-1 induction on cell survival in a human colon cancer cell line, Caco-2. Serum deprivation induced apoptosis, reduced Akt and p38 phosphorylation, and increased p21(Cip/WAF1) levels. Heme oxygenase-1 induction by treatment with cobalt protoporphyrin IX resulted in resistance to apoptosis, activation of Akt, reduction in p21(Cip/WAF1) levels and modification of bcl2/bax ratio towards survival. Indomethacin reduced apoptosis but in contrast to heme oxygenase-1, arrested cells in G0/G1. Apoptosis was also inhibited by the heme oxygenase metabolites bilirubin and biliverdin but the CO donor tricarbonyldichlororuthenium(II) dimer did not exert significant effects. Protection against apoptosis in cells treated with cobalt protoporphyrin IX was reverted by incubation with heme oxygenase-1 small interfering RNA. This study shows an antiapoptotic effect of heme oxygenase-1 in colon cancer cells which could be mediated by the formation of bilirubin and biliverdin. Our results support an antiapoptotic role for HO-1 in these cells and provide a mechanism by which overexpression of HO-1 may promote tumor resistance to stress in conditions of limited nutrient supply. We have extended these observations by demonstrating that these effects are independent of p38 but are mediated via Akt pathway.     

GDI-1 phosphorylation switch at serine 96 induces RhoA activation and increased endothelial permeability

We identified the GDI-1-regulated mechanism of RhoA activation from the Rho-GDI-1 complex and its role in mediating increased endothelial permeability. Thrombin stimulation failed to induce RhoA activation and actin stress fiber formation in human pulmonary arterial endothelial cells transduced with full-length GDI-1. Expression of a GDI-1 mutant form (C-GDI) containing the C terminus (aa 69 to 204) also prevented RhoA activation, whereas further deletions failed to alter RhoA activation. We observed that protein kinase Calpha-mediated phosphorylation of the C terminus of GDI-1 at Ser96 reduced the affinity of GDI-1 for RhoA and thereby enabled RhoA activation. Rendering GDI-1 phosphodefective with a Ser96 --> Ala substitution rescued the inhibitory activity of GDI-1 toward RhoA but did not alter the thrombin-induced activation of other Rho GTPases, i.e., Rac1 and Cdc42. Phosphodefective mutant GDI-1 also suppressed myosin light chain phosphorylation, actin stress fiber formation, and the increased endothelial permeability induced by thrombin. In contrast, expressing the phospho-mimicking mutant S96D-GDI-1 protein induced RhoA activity and increased endothelial permeability independently of thrombin stimulation. These results demonstrate the crucial role of the phosphorylation of the C terminus of GDI-1 at S96 in selectively activating RhoA. Inhibiting GDI-1 phosphorylation at S96 is a potential therapeutic target for modulating RhoA activity and thus preventing the increase in endothelial permeability associated with vascular inflammation.     

Monochloramine inhibits the expression of E-selectin and intercellular adhesion molecule-1 induced by TNF-alpha through the suppression of NF-kappaB activation in human endothelial cells

Reactive oxygen species have various effects on the expression of cell adhesion molecules induced by proinflammatory cytokines, such as tumor necrosis factor a (T-NF-alpha). We studied the effects of monochloramine (NH2Cl), a physiological oxidant derived from activated neutrophils, on the TNF-alpha-induced expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVEC). HUVEC were pretreated with or without NH2Cl (20-90 microM for 20 min), then stimulated with TNF-alpha (10 ng/ml), and the expression of E-selectin and ICAM-1 was measured. Without NH2Cl, TNF-alpha induced marked expression of e-selectin and ICAM-1. Pretreatment with NH2Cl resulted in a significant, but transient inhibition of the expression of adhesion molecules. Higher dose of NH2Cl showed more pronounced inhibition, and the inhibitory effect lasted for 8h when 70 microM of NH2Cl was added. TNF-alpha stimulation also induced marked activation of nuclear factor KB (NF-kappaB). Notably, NH2Cl also inhibited this NF-kappaB activation in a dose- and time-dependent manner, which was similar to the inhibition of E-selectin and ICAM-1 expression. In addition, IkappaB-alpha phosphorylation and degradation were also inhibited by NH2Cl pretreatment. These observations indicated that NH2Cl inhibited TNF-alpha-induced expression of E-selectin and ICAM-1 through the inhibition of NF-kappaB activation. We speculate that neutrophil-derived chloramines may have a regulatory role in the recruitment of leukocytes.     

Silymarin inhibits TNF-alpha-induced expression of adhesion molecules in human umbilical vein endothelial cells

Silymarin is known to have an anti-atherosclerotic activity, but the mechanism responsible for it remains unclear. Here, we demonstrate a possible mechanism involved in the anti-atherosclerotic activity of silymarin. Silymarin inhibited THP-1 cell adhesion to human umbilical vein endothelial cells (HUVECs). Silymarin also suppressed the TNF-alpha-induced protein and mRNA expression of adhesion molecules, such as VCAM-1, ICAM-1 and E-selectin, in HUVECs. Moreover, silymarin suppressed the TNF-alpha-induced DNA binding of NF-kappaB/Rel in HUVECs. Taken together, these results demonstrate that silymarin exerts an anti-atherosclerotic activity, at least in part, by inhibiting the expression of adhesion molecules.     

T cell receptor-stimulated generation of hydrogen peroxide inhibits MEK-ERK activation and lck serine phosphorylation

Previous studies indicated that antigen receptor (TcR) stimulation of mature T cells induced rapid generation of reactive oxygen species (ROS). The goal of the current study was to examine the role(s) of ROS in TcR signal transduction, with a focus upon the redox-sensitive MAPK family. TcR cross-linking of primary human T blasts and Jurkat human T cells rapidly activated the ERK, JNK, p38 and Akt kinases within minutes, and was temporally associated with TcR-stimulated production of hydrogen peroxide (H(2)O(2)). TcR-induced activation of ERK was selectively augmented and sustained in the presence of pharmacologic antioxidants that can quench or inhibit H(2)O(2) production (NAC, MnTBAP and Ebselen, but not DPI), while activation of JNK and Akt were largely unaffected. This was paralleled by concurrent changes in MEK1/2 phosphorylation, suggesting that ROS acted upstream of MEK-ERK activation. Molecular targeting of H(2)O(2) by overexpression of peroxiredoxin II, a thioredoxin dependent peroxidase, also increased and sustained ERK and MEK activation upon TcR cross-linking. Enhancement of ERK phosphorylation by antioxidants correlated with increased and sustained serine phosphorylation of the src-family kinase lck, a known ERK substrate. Thus, the data suggest that TcR-stimulated production of hydrogen peroxide negatively feeds back to dampen antigen-stimulated ERK activation and this redox-dependent regulation may serve to modulate key steps in TcR signaling.     

Ox-PAPC activation of PMET system increases expression of heme oxygenase-1 in human aortic endothelial cell

Oxidized-1-palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine (Ox-PAPC) has been demonstrated to accumulate in atherosclerotic lesions and regulates expression of more than 1,000 genes in human aortic endothelial cell (HAEC). Among the most highly induced is heme oxygenase-1 (HO-1), a cell-protective antioxidant enzyme, which is sensitively induced by oxidative stress. To identify the pathway by which Ox-PAPC induces HO-1, we focused on the plasma membrane electron transport (PMET) complex, which contains ecto-NADH oxidase 1 (eNOX1) and NADPH:quinone oxidoreductase 1 (NQO1) and affects cellular redox status by regulating levels of NAD(P)H. We demonstrated that Ox-PAPC and its active components stimulated electron transfer through the PMET complex in HAECs from inside to outside [as determined by extracellular 2-(4-iodophenyl)-3-(44-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) reduction] and from outside to inside of the cell (as determined by intracellular NBT reduction). Chemical inhibitors of PMET system and siRNAs to PMET components (NQO1 and eNOX1) significantly decreased HO-1 induction by Ox-PAPC. We present evidence that Ox-PAPC activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in HAEC plays an important role in the induction of HO-1 and PMET inhibitors blocked Nrf2 activation by Ox-PAPC. We hypothesized that PMET activation by Ox-PAPC causes intracellular NAD(P)H depletion, which leads to the increased oxidative stress and HO-1 induction. Supporting this hypothesis, cotreatment of cells with exogenous NAD(P)H and Ox-PAPC significantly decreased oxidative stress and HO-1 induction by Ox-PAPC. Taken together, we demonstrated that the PMET system in HAEC plays an important role in the regulation of cellular redox status and HO-1 expression by Ox-PAPC.     

Pharmacological inhibition of MALT1 protease activity suppresses endothelial activation via enhancing MCPIP1 expression

Mucosa associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is not only an intracellular signaling scaffold protein but also a paracaspase that plays a key role in the signal transduction and cellular activation of lymphocytes and macrophages. However, its role in endothelial cells remains unknown. Here we report that pharmacological inhibition of MALT1 protease activity strongly suppresses endothelial activation via enhancing MCPIP1 expression. Treatment with MALT1 protease inhibitors selectively inhibited TNFα-induced VCAM-1 expression in HUVECs and LPS-induced VCAM-1 expression in mice. In addition, Inhibition of MALT1 protease activity also significantly inhibited TNFα-induced adhesion of THP-1 monocytic cells to HUVECs. To explore the mechanisms, MALT1 inhibitors does not affect the activation of NF-κB signaling pathway in HUVEC. However, they can stabilize MCPIP1 protein and significantly enhance MCPIP1 protein level in endothelial cells. These results suggest that MALT1 paracaspase also targets MCPIP1 and degrade MCPIP1 protein in endothelial cells similar as it does in immune cells. Taken together, the study suggest inhibition of MALT1 protease activity may represent a new strategy for prevention/therapy of vascular inflammatory diseases such as atherosclerosis.