Role of Nrf2 signaling in regulation of antioxidants and phase 2 enzymes in cardiac fibroblasts: protection against reactive oxygen and nitrogen species-induced cell injury

Understanding the molecular pathway(s) of antioxidant gene regulation is of crucial importance for developing antioxidant-inducing agents for the intervention of oxidative cardiac disorders. Accordingly, this study was undertaken to determine the role of Nrf2 signaling in the basal expression as well as the chemical inducibility of endogenous antioxidants and phase 2 enzymes in cardiac fibroblasts. The basal expression of a scope of key cellular antioxidants and phase 2 enzymes was significantly lower in cardiac fibroblasts derived from Nrf2-/- mice than those from wild type control. These include catalase, reduced glutathione (GSH), glutathione reductase (GR), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase-1 (NQO1). Incubation of Nrf2+/+ cardiac fibroblasts with 3H-1,2-dithiole-3-thione (D3T) led to a significant induction of superoxide dismutase (SOD), catalase, GSH, GR, glutathione peroxidase (GPx), GST, and NQO1. The inducibility of SOD, catalase, GSH, GR, GST, and NQO1, but not GPx by D3T was completely abolished in Nrf2-/- cells. The Nrf2-/- cardiac fibroblasts were much more sensitive to reactive oxygen and nitrogen species-mediated cytotoxicity. Upregulation of antioxidants and phase 2 enzymes by D3T in Nrf2+/+ cardiac fibroblasts resulted in a dramatically increased resistance to the above species-induced cytotoxicity. In contrast, D3T-treatment of the Nrf2-/- cells only provided a slight cytoprotection. Taken together, this study demonstrates for the first time that Nrf2 is critically involved in the regulation of the basal expression and chemical induction of a number of antioxidants and phase 2 enzymes in cardiac fibroblasts, and is an important factor in controlling cardiac cellular susceptibility to reactive oxygen and nitrogen species-induced cytotoxicity.     

Antioxidants and phase 2 enzymes in macrophages: regulation by Nrf2 signaling and protection against oxidative and electrophilic stress

Macrophages play important roles in immunity and other physiological processes. They are also target cells of various toxic agents, including oxidants and electrophiles. However, little is known regarding the molecular regulation and chemical inducibility of a spectrum of endogenous antioxidants and phase 2 enzymes in normal macrophages. Understanding the molecular pathway(s) controlling the coordinated expression of various macrophage antioxidants and phase 2 defenses is of importance for developing strategies to protect against macrophage injury induced by oxidants and electrophiles. Accordingly, this study was undertaken to determine the role of the nuclear factor E2-related factor 2 (Nrf2) in regulating both constitutive and chemoprotectant-inducible expression of various antioxidants and phase 2 enzymes in mouse macrophages. The constitutive expression of a series of antioxidants and phase 2 enzymes was significantly lower in macrophages derived from Nrf2-null (Nrf2(-/-)) mice than those from wild-type (Nrf2(+/+)) littermates. Incubation of wild-type macrophages with 3H-1,2-dithiole-3-thione (D3T) led to significant induction of various antioxidants and phase 2 enzymes, including catalase, glutathione, glutathione peroxidase (GPx), glutathione reductase, glutathione S-transferase, and NAD(P)H:quinone oxidoreductase 1. The inducibility of the above cellular defenses except for GPx by D3T was completely abolished in Nrf2(-/-) macrophages. As compared with wild-type cells, Nrf2(- /-) macrophages were much more susceptible to cell injury induced by reactive oxygen/nitrogen species, as well as two known macrophage toxins, acrolein and cadmium. Up-regulation of the antioxidants and phase 2 enzymes by D3T in wild-type macrophages resulted in increased resistance to the above oxidant-and electrophile-induced cell injury, whereas D3T treatment of Nrf2(- /-) macrophages provided only marginal or no cytoprotec-tion. This study demonstrates that Nrf2 is an indispensable factor in controlling both constitutive and inducible expression of a wide spectrum of antioxidants and phase 2 enzymes in macrophages as well as the susceptibility of these cells to oxidative and electrophilic stress.     

Biology of marrow stromal cell lines derived from long-term bone marrow cultures of Trp53-deficient mice.

To investigate the effect of Trp53 (formerly known as p53) on stromal cells of the hematopoietic microenvironment, long-term bone marrow cultures were established from mice in which the Trp53 gene had been inactivated by homologous recombination (Trp53(-/-)) or their wild-type littermates (Trp53(+/+)). Long-term bone marrow cultures from Trp53(-/-) mice continued to produce nonadherent cells for 22 weeks, while Trp53(+/+) cultures ceased production after 15 weeks. There was a significant increase in the number of nonadherent cells produced in Trp53(-/-) long-term bone marrow cultures beginning at week 9 and continuing to week 22 (P < 0.02). The Trp53(-/-) cultures also showed significantly increased cobblestone island formation indicative of early hematopoietic stem cell-containing colonies beginning at week 10 (P < 0.01). Cobblestone islands persisted until weeks 15 and 22 in Trp53(+/+) and Trp53(-/-) cultures, respectively. Co-cultivation experiments in which Trp53(+/+) Sca1(+)lin- enriched hematopoietic stem cells were plated on Trp53(-/-) stromal cells showed increased cobblestone island formation compared to Trp53(-/-) Scal+lin- cells plated on Trp53(+/+) or Trp53(-/-) stromal cells. Radiation survival curves for clonal bone marrow stromal cells revealed a similar D0 for the Trp53(+/+) and Trp53(-/-) cell lines (1.62 +/- 0.16 and 1.49 +/- 0. 08 Gy, respectively; P = 0.408), and similar n (8.60 +/- 3.23 and 10.71 +/- 0.78, respectively) (P = 0.491). Cell cycle analysis demonstrated a G2/M-phase arrest that occurred 6 h after irradiation for both Trp53(+/+) and Trp53(-/-) stromal cell lines. After 10 Gy irradiation, there was no significant increase in the frequency of apoptosis detected in Trp53(+/+) compared to Trp53(-/-) marrow stromal cell lines. In the stromal cell lines, ICAM-1 was constitutively expressed on Trp53(+/+) but not Trp53(-/-) cells; however, a 24-h exposure to TNF-alpha induced detectable ICAM-1 on Trp53(-/-) cells and increased expression on Trp53(+/+) cells. To test the effect of Trp53 on the radiation biology of hematopoietic progenitor cells, the 32D cl 3 cell line was compared with a subclone in which expression of an E6 inserted transgene accelerates ubiquitin-dependent degradation of Trp53, thus preventing accumulation of Trp53 after genotoxic stress. The radiation survival curves were similar with no significant difference in the D0 or n, or in the percentage of cells undergoing apoptosis after 10 Gy irradiation between the two cell lines. Cells of the 32D-E6 cell line displayed a G2/M-phase arrest 6 h after 10 Gy, while cells of the parent line exhibited both a G2/M-phase arrest and a G1-phase arrest at 24 and 48 h. The results suggest a complex mechanism of action of Trp53 on the interactions between stromal and hematopoietic cells in long-term bone marrow cultures.     

Antioxidants and phase 2 enzymes in cardiomyocytes: Chemical inducibility and chemoprotection against oxidant and simulated ischemia-reperfusion injury

The increasing recognition of the role for oxidative stress in cardiac disorders has led to extensive investigation on the protection by exogenous antioxidants against oxidative cardiac injury. On the other hand, another strategy for protecting against oxidative cardiac injury may be through upregulation of the endogenous antioxidants and phase 2 enzymes in the myocardium by chemical inducers. However, our current understanding of the chemical inducibility of cardiac cellular antioxidants and phase 2 enzymes is very limited. In this study, using rat cardiac H9c2 cells we have characterized the concentration- and time-dependent induction of cellular antioxidants and phase 2 enzymes by 3H-1,2-dithiole-3-thione (D3T), and the resultant chemoprotective effects on oxidative cardiac cell injury. Incubation of H9c2 cells with D3T resulted in a marked concentration- and time-dependent induction of a number of cellular antioxidants and phase 2 enzymes, including catalase, reduced glutathione (GSH), GSH peroxidase, glutathione reductase (GR), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase-1 (NQO1). D3T treatment of H9c2 cells also caused an increase in mRNA expression of catalase, gamma-glutamylcysteine ligase catalytic subunit, GR, GSTA1, M1 and P1, and NQO1. Moreover, both mRNA and protein expression of Nrf2 were induced in D3T-treated cells. D3T pretreatment led to a marked protection against H9c2 cell injury elicited by various oxidants and simulated ischemia-reperfusion. D3T pretreatment also resulted in decreased intracellular accumulation of reactive oxygen in H9c2 cells after exposure to the oxidants as well as simulated ischemia-reperfusion. This study demonstrates that a series of endogenous antioxidants and phase 2 enzymes in H9c2 cells can be induced by D3T in a concentration- and time-dependent fashion, and that the D3T-upregulated cellular defenses are accompanied by a markedly increased resistance to oxidative cardiac cell injury.     

Increased radioresistance, g(2)/m checkpoint inhibition, and impaired migration of bone marrow stromal cell lines derived from Smad3(-/-) mice

Smad3 protein is a prominent member of the Tgfb receptor signaling pathway. Smad3(-/-) mice display decreased radiation-induced skin fibrosis, suggesting a defect in both Tgfb-mediated fibroblast proliferation and migration. We established bone marrow stromal cell lines from Smad3(-/-) mice and homozygous littermate(+/+) mice. Smad3(-/-) cells displayed a significant increase in radiation resistance with a D(0)=2.25+/- 0.14 Gy compared to Smad3(+/+) cells with a D(0)=1.75+/- 0.03 (P=0.023). Radioresistance was abrogated by reinsertion of the human SMAD3 transgene, resulting in a D(0)=1.49 0.10 (P=0.028) for Smad3(-/-)(3) cells. More Smad3(-/-) cells than Smad3(+/+) cells were in the G(2)/M phase; Smad3(-/-)(3) cells were similar to Smad3(+/+) cells. Smad3(+/+) cells exhibited increased apoptosis 24 h after 5 Gy (15%) or 8 Gy (43%) compared to less than 1% in Smad3(-/-) cells exposed to either dose. The movement of Smad3(-/-) cells, measured in an automated cell tracking system, was slower than that of Smad3(+/+) cells. Smad3(-/-)(3) cells resembled Smad3(+/+) cells. These studies establish concordance of a defective Tgfb signal transduction pathway, an increased proportion of G(2)/M cells, and radioresistance. The decreased migratory capacity of Smad3(-/-) cells in vitro correlates with decreased radiation fibrosis in vivo in mice deficient in Tgfb signaling.     

Generation of osteoclasts from hemopoietic cells and a multipotential cell line in vitro

Osteoclasts are the cells that resorb bone. It is generally presumed, on the basis of indirect experiments, that they are derived from the hemopoietic stem cell. However, this origin has never been established. We have developed an assay for osteoclastic differentiation in which bone marrow cells are incubated in liquid culture on slices of cortical bone. The bone slices are inspected in the scanning electron microscope after incubation for the presence of excavations, which are characteristic of osteoclastic activity. We have now incubated bone marrow cells at low density, or a factor-dependent mouse hemopoietic cell line (FDCP-mix A4) with 1,25 dihydroxyvitamin D3 (a hormone which we have previously found induces osteoclastic differentiation) with and without murine bone marrow stromal cells, or with and without 3T3 cells, on bone slices. Neither the bone marrow cells nor the bone marrow stromal cells alone developed osteoclastic function even in the presence of 1,25 dihydroxyvitamin D3. However, extensive excavation of the bone surface was observed, only in the presence of 1,25 dihydroxyvitamin D3, on bone slices on which bone marrow stromal cells were cocultured with low-density bone marrow cells or the hemopoietic cell line. Similar results were obtained when the bone marrow stromal cells were killed by glutaraldehyde fixation; 3T3 cells were unable to substitute for stromal cells. These results are strong evidence that osteoclasts derive from the hemopoietic stem cell and suggest that although mature osteoclasts possess neither receptors for nor responsiveness to 1,25 dihydroxyvitamin D3, the hormone induces osteoclastic function through a direct effect on hemopoietic cells rather than through some accessory cell in the bone marrow stroma. The failure of 3T3 cells, which enable differentiation of other hemopoietic progeny from this cell line, to induce osteoclastic differentiation suggests that bone marrow stroma possesses additional characteristics distinct from those that induce differentiation of other hemopoietic cells that are specifically required for osteoclastic differentiation.     

Extracellular inorganic phosphate regulates gibbon ape leukemia virus receptor-2/phosphate transporter mRNA expression in rat bone marrow stromal cells

In mammalian cells, several observations indicate not only that phosphate transport probably regulates local inorganic phosphate (Pi) concentration, but also that Pi affects normal cellular metabolism, which in turn regulates apoptosis and the process of mineralization. To elucidate how extracellular Pi regulates cellular functions of pre-osteoblastic cells, we investigated the expression of type III sodium (Na)-dependent Pi transporters in rat bone marrow stromal cells and ROB-C26 pre-osteoblastic cells. The mRNA expression level of gibbon ape leukemia virus receptor (Glvr)-2 was increased by the addition of Pi in rat bone marrow stromal cells, but not in ROB-C26 or normal rat kidney (NRK) cells. In contrast, the level of Glvr-1 mRNA was not altered by the addition of extracellular Pi in these cells. The induction of Glvr-2 mRNA by Pi was inhibited in the presence of cycloheximide (CHX). Moreover, mitogen-activated protein kinase (MEK) /extracellular-signal-regulated kinase (ERK) pathway inhibitors; U0126 (1.4-diamino-2, 3-dicyano-1, 4-bis [2-amino-phenylthio] butadiene) and PD98059 (2'-Amino-3'-methoxyflavone) inhibited inducible Glvr-2 mRNA expression, but p38 MEK inhibitor SB203580 [4-(4'-fluorophenyl)-2-(4'-methyl-sulfinylphenyl)-5-(4'pyridyl) imidazole] did not inhibit the induction of Glvr-2 mRNA expression, suggesting that extracellular Pi regulates de novo protein synthesis and MEK/ERK activity in rat bone marrow stromal cells, and through these, induction of Glvr-2 mRNA. Although Pi also induced osteopontin mRNA expression in rat bone marrow stromal cells but not in ROB-C26 and NRK cells, changes in cell viability with the addition of Pi were similar in both cell types. These data indicate that extracellular Pi regulates Glvr-2 mRNA expression, provide insights into possible mechanisms whereby Pi may regulate protein phosphorylation, and suggest a potential role for the Pi transporter in rat bone marrow stromal cells.     

Depletion of CD4 and CD8 T lymphocytes in mice in vivo enhances 1,25-dihydroxyvitamin D3-stimulated osteoclast-like cell formation in vitro by a mechanism that is dependent on prostaglandin synthesis

To investigate the role of T lymphocytes in osteoclastogenesis, we performed in vivo depletion of CD4 and/or CD8 T lymphocyte subsets and evaluated in vitro osteoclast-like cell (OCL) formation. T lymphocyte depletion (TLD) with mAbs was confirmed 24 h later by flow cytometry. OCL formation was stimulated with 1, 25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) in bone marrow and with recombinant mouse (rm) receptor activator of NF-kappaB ligand (RANK-L) and rmM-CSF in bone marrow and spleen cell cultures. OCL formation was up to 2-fold greater in 1,25-(OH)(2)D(3)-stimulated bone marrow cultures from TLD mice than in those from intact mice. In contrast, TLD did not alter OCL formation in bone marrow or spleen cell cultures that were stimulated with rmRANK-L and rmM-CSF. The effects of TLD seemed to be mediated by enhanced PG synthesis, because the PGE(2) concentration in the medium of 1, 25-(OH)(2)D(3)-stimulated bone marrow cultures from TLD mice was 5-fold higher than that in cultures from intact mice, and indomethacin treatment abolished the stimulatory effect of TLD on OCL formation. There was a 2-fold increase in RANK-L expression and an almost complete suppression of osteoprotegerin expression in 1, 25-(OH)(2)D(3)-stimulated bone marrow cultures from TLD mice compared with those from intact mice. Although there was a small (20%) increase in IL-1alpha expression in 1, 25-(OH)(2)D(3)-stimulated bone marrow cultures from TLD mice, TLD in mice lacking type I IL-1R and wild-type mice produced similar effects on OCL formation. Our data demonstrate that TLD up-regulates OCL formation in vitro by increasing PG production, which, in turn, produces reciprocal changes in RANK-L and osteoprotegerin expression. These results suggest that T lymphocytes influence osteoclastogenesis by altering bone marrow stromal cell function.     

Coordinated upregulation of a series of endogenous antioxidants and phase 2 enzymes as a novel strategy for protecting renal tubular cells from oxidative and electrophilic stress

In view of the crucial involvement of oxidative and electrophilic stress in various kidney disorders, this study was undertaken to test the hypothesis that pharmacologically-mediated coordinated upregulation of endogenous renal antioxidants and phase 2 enzymes is an effective strategy for renal protection. Notably, studies on the pharmacological inducibility of a series of antioxidants and phase 2 enzymes in renal tubular cells are lacking. Here we reported that incubation of normal rat kidney (NRK-52E) proximal tubular cells with low micromolar concentrations (10-50 microM) of the cruciferous nutraceutical, 1,2-dithiole-3-thione (D3T), led to a significant concentration-dependent induction of a wide spectrum of antioxidants and phase 2 enzymes, including catalase (CAT), reduced form of glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase 1 (NQO1), and heme oxygenase (HO). We further observed that D3T treatment also increased the protein and mRNA expression for CAT, gamma-glutamylcysteine ligase, GR, GST-A, GST-M, NQO1, and HO-1. Incubation of the renal tubular cells with H(2)O(2), SIN-1-derived peroxynitrite, or 4-hydroxy-2-nonenal led to concentration-dependent decreases in cell viability. Pretreatment of the renal tubular cells with 10-50 microM D3T afforded remarkable protection against the nephrocytotoxicity elicited by the above oxidative and electrophilic species. The D3T-mediated cytoprotection showed a concentration-dependent relationship. Taken together, this study for the first time comprehensively characterized the inducibility by a unique nutraceutical of a wide spectrum of antioxidative and phase 2 defenses in renal tubular cells at the levels of enzyme activity as well as protein and mRNA expression, and demonstrated that such a coordinated upregulation of cellular defenses led to remarkable protection of renal tubular cell from oxidative and electrophilic stress. Because of the crucial role of oxidative and electrophilic stress in inflammatory injury, D3T-mediated coordinated induction of endogenous antioxidative and phase 2 defenses may also serve as an important anti-inflammatory mechanism in kidneys.     

The chemical inducibility of mouse cardiac antioxidants and phase 2 enzymes in vivo

The recognition of the critical involvement of oxidative and electrophilic stress in cardiac disorders has led to extensive investigation of the protective effects of exogenous antioxidants on cardiac injury. On the other hand, another strategy for protecting against oxidative/electrophilic cardiac injury may be through induction of the endogenous antioxidants and phase 2 enzymes in myocardium by chemical inducers. However, our understanding of the chemical inducibility of cardiac antioxidants/phase 2 enzymes in vivo is very limited. In addition, careful studies on the basal levels of a scope of endogenous antioxidants/phase 2 enzymes in myocardium as compared with other tissues, such as liver, are lacking. Accordingly, this study was undertaken to determine the basal levels of endogenous antioxidants/phase 2 enzymes, including superoxide dismutase (SOD), catalase, reduced glutathione (GSH), GSH peroxidase (GPx), glutathione reductase (GR), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase 1 (NQO1), and investigate the inducibility of the above antioxidants/phase 2 enzymes by the chemoprotectant, 1,2-dithiole-3-thione (D3T), in cardiac as well as hepatic tissues in C57BL/6 mice. Our results demonstrated that in C57BL/6 mice, the levels of catalase, GSH, GPx, GR, and GST were significantly lower in cardiac tissue than in hepatic tissue. The level of total SOD did not differ significantly between mouse heart and liver. Notably, heart contained a much higher NQO1 activity than liver. Immunoblotting and RT-PCR analyses further demonstrated the high expression of NQO1 protein and mRNA in myocardium. Oral administration of D3T at 0.25 and 0.5 mmol/kg body weight for 3 consecutive days resulted in a significant induction of cardiac SOD, catalase, GR, GST, and NQO1. No significant induction of cardiac GSH and GPx was observed with the above D3T treatment. Only GR, GST, and NQO1 in mouse liver were induced by the D3T treatment. Unexpectedly, we observed a significant D3T dose-dependent decrease in hepatic GPx activity. Taken together, this study demonstrates for the first time that: (1) the expression of NQO1 is remarkably high in mouse myocardium though other cardiac antioxidants/phase 2 enzymes are relatively lower as compared with liver; (2) a number of endogenous antioxidants/phase 2 enzymes in mouse cardiac tissue can be significantly induced by D3T following oral administration; and (3) the inducibility of endogenous antioxidants/phase 2 enzymes by D3T differs between mouse cardiac and hepatic tissues. This study provides a basis for future investigation of the cardioprotection of chemically induced endogenous antioxidants and phase 2 enzymes in myocardium in animal models of oxidative/electrophilic cardiac disorders.     

Up-regulation of rat adipose tissue adiponectin gene expression by long-term but not by short-term food restriction

3H-1,2-Dithiole-3-thione (D3T), a potent member of dithiolethiones, induces phase 2 enzymes by activating an Nrf2/Keap1-dependent signaling pathway. It was proposed that interaction between D3T and two adjacent sulfhydryl groups of Keap1 might cause dissociation of Keap1 from Nrf2, leading to Nrf2 activation. This study was undertaken to investigate the reactions between D3T and thiols, including the dithiol compound, dithiothreitol (DTT), and the monothiol, glutathione (GSH). We reported here that under physiologically relevant conditions incubation of D3T with DTT caused remarkable oxygen consumption, indicating a redox reaction between D3T and the dithiol molecule. Incubation of D3T with GSH also led to oxygen consumption, but to a less extent. Electron paramagnetic resonance (EPR) studies showed that the redox reaction between D3T and DTT generated superoxide. Superoxide was also formed from the redox reaction of D3T with GSH. These findings demonstrate that D3T reacts with thiols, particularly a dithiol, generating superoxide, which may provide a mechanistic explanation for induction of Nrf2-dependent phase 2 enzymes by D3T.