Regina M. Day

Ras-dependent and -independent regulation of reactive oxygen species by mitogenic growth factors and TGF-β1

Mitogenic growth factors and transforming growth factor β1 (TGF‐β1) induce the generation of reactive oxygen species (ROS) in non‐phagocytic cells, but their enzymatic source(s) and regulatory mechanisms are largely unknown. We previously reported on the ability of TGF‐β1 to activate a cell surface‐associated NADH:flavin: O2 oxidoreductase (NADH oxidase) that generates extracellular H2O2. In this study, we compared the ROS‐generating enzymatic systems activated by mitogenic growth factors and TGF‐β1 with respect to the primary reactive species produced (O2 vs. H2O2), the site of generation (intracellular vs. extracellular) and regulation by Ras. We find that the mitogenic growth factors PDGF‐BB, FGF‐2, and TGF‐α (an EGF receptor ligand) are able to rapidly (within 5 min) induce the generation of intracellular O2 without detectable NADH oxidase activity or extracellular H2O2 release. In contrast, TGF‐β1 does not stimulate intracellular O2 production and the delayed induction of extracellular H2O2 release is not associated with O2 production. Expression of dominant‐negative Ras (N17Ras) protein by herpes simplex virus‐mediated gene transfer blocks mitogen‐stimulated intracellular O2 generation but has no effect on TGF‐β1‐induced NADH oxidase activation/H2O2 production. These results demonstrate that there are at least two distinctly different ROS‐generating enzymatic systems in lung fibroblasts regulated by mitogenic growth factors and TGF‐β1 via Ras‐dependent and ‐independent mechanisms, respectively. In addition, these findings suggest that endogenous production of ROS by growth factors/ cytokines may have different biological effects depending on the primary reactive species generated and site of production.—Thannickal, V. J., Day, R. M., Klinz, S. G., Bastien, M. C., Larios, J. M., Fanburg, B. L. Ras‐dependent and ‐independent regulation of reactive oxygen species by mitogenic growth factors and TGF‐β1. FASEB J. 14, 1741–1748 (2000)

RhoA Modulates Smad Signaling during Transforming Growth Factor-β-induced Smooth Muscle Differentiation

We recently reported that transforming growth factor (TGF)-β induced the neural crest stem cell line Monc-1 to differentiate into a spindle-like contractile smooth muscle cell (SMC) phenotype and that Smad signaling played an important role in this phenomenon. In addition to Smad signaling, other pathways such as mitogen-activated protein kinase (MAPK), phosphoinositol-3 kinase, and RhoA have also been shown to mediate TGF-β actions. The objectives of this study were to examine whether these signaling pathways contribute to TGF-β-induced SMC development and to test whether Smad signaling cross-talks with other pathway(s) during SMC differentiation induced by TGF-β. We demonstrate here that RhoA signaling is critical to TGF-β-induced SMC differentiation. RhoA kinase (ROCK) inhibitor Y27632 significantly blocks the expression of multiple SMC markers such as smooth muscle α-actin, SM22α, and calponin in TGF-β-treated Monc-1 cells. In addition, Y27632 reversed the cell morphology and abolished the contractility of TGF-β-treated cells. RhoA signaling was activated as early as 5 min following TGF-β addition. Dominant negative RhoA blocked nuclear translocation of Smad2 and Smad3 because of the inhibition of phosphorylation of both Smads and inhibited Smad-dependent SBE promoter activity, whereas constitutively active RhoA significantly enhanced SBE promoter activity. Consistent with these results, C3 exotoxin, an inhibitor of RhoA activation, significantly attenuated SBE promoter activity and inhibited Smad nuclear translocation. Taken together, these data point to a new role for RhoA as a modulator of Smad activation while regulating TGF-β-induced SMC differentiation.

Hepatocyte growth factor protects cardiac myocytes against oxidative stress-induced apoptosis.

Hepatocyte growth factor (HGF) has been proposed as an endogenous cardioprotective agent against oxidative stress. The mechanism of HGF action in the heart, however, has not yet been elucidated. The present study demonstrates that HGF protects adult cardiac myocytes against oxidative stress-induced apoptosis. HGF, at the concentrations which can be detected in the plasma of humans subsequent to myocardial infarction, effectively attenuated death of isolated adult rat cardiac myocytes and cultured HL-1 cardiac muscle cells induced by apoptosis-inducing oxidative stress stimuli such as daunorubicin, serum deprivation, and hydrogen peroxide. We identified expression of c-Met HGF receptor in adult cardiac myocytes, which can be rapidly tyrosine phosphorylated in response to HGF treatment. HGF also activated MEK, p44/42 MAPK, and p90RSK. To determine if MEK-MAPK pathway may be involved in the mechanism of HGF-mediated cardiac myocyte protection, effects of a specific MEK inhibitor, PD98059, were studied. Pretreatment of cells with PD98059 partially blocked HGF signaling for protection against hydrogen peroxide-induced cell death. Thus, HGF protects cardiac myocytes against oxidative stress, in part, via activating MEK-MAPK pathway.

Differential signaling by alternative HGF isoforms through c-Met: activation of both MAP kinase and PI 3-kinase pathways is insufficient for mitogenesis

HGF/NK2, a naturally occurring truncated HGF isoform, antagonizes the mitogenic and morphogenic activities of full length HGF, but stimulates cell scatter, or the motogenic response to HGF. We studied postreceptor signaling by these HGF isoforms in the human breast epithelial cell line 184B5, and in murine myeloid progenitor 32D cells transfected with c-Met, the human HGF receptor (32D/c-Met). HGF stimulated DNA synthesis in 184B5 and 32D/c-Met cells, while HGF/NK2 was mitogenically inactive, despite the ability of HGF/NK2 to stimulate c-Met autophosphorylation, mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) in both cell systems. In 184B5 cells, HGF stimulated sustained MAPK activation, while activation by HGF/NK2 declined rapidly. In contrast, both isoforms activated MAPK with rapidly attenuated kinetics in 32D/c-Met cells. In both cell systems the increased motility observed in response to either HGF or HGF/NK2 treatment was more potently blocked by the PI3 kinase inhibitor wortmannin, than by PD98059, an inhibitor of MAPK kinase (MEK1). These data suggest that (1) alternative HGF isoforms signaling through c-Met generate both common and distinct biological responses, (2) the extent and duration of ligand-stimulated c-Met and MAPK activities are dependent on the cellular context and are not predictive of mitogenic signaling, and (3) in at least some HGF target cells, the activation of both MAPK and PI3K signaling pathways is insufficient for mitogenesis elicited through c-Met.

Cell proliferation, reactive oxygen and cellular glutathione.

A variety of cellular activities, including metabolism, growth, and death, are regulated and modulated by the redox status of the environment. A biphasic effect has been demonstrated on cellular proliferation with reactive oxygen species (ROS)—especially hydrogen peroxide and superoxide—in which low levels (usually submicromolar concentrations) induce growth but higher concentrations (usually >10–30 micromolar) induce apoptosis or necrosis. This phenomenon has been demonstrated for primary, immortalized and transformed cell types. However, the mechanism of the proliferative response to low levels of ROS is not well understood. Much of the work examining the signal transduction by ROS, including H2O2, has been performed using doses in the lethal range. Although use of higher ROS doses have allowed the identification of important signal transduction pathways, these pathways may be activated by cells only in association with ROS-induced apoptosis and necrosis, and may not utilize the same pathways activated by lower doses of ROS associated with increased cell growth. Recent data has shown that low levels of exogenous H2O2 up-regulate intracellular glutathione and activate the DNA binding activity toward antioxidant response element. The modulation of the cellular redox environment, through the regulation of cellular glutathione levels, may be a part of the hormetic effect shown by ROS on cell growth.

Genistein induces radioprotection by hematopoietic stem cell quiescence

Purpose: In this study we addressed whether genistein-induced radioprotection in mice is associated with alterations of the cell cycle of hematopoietic stem and progenitor cells. Materials and methods: C57BL/6J female mice received a single subcutaneous injection of genistein (200 mg/kg) 24 h prior to a lethal dose (7.75 Gy, 60Co) of total body irradiation. Proliferation-associated Ki-67 protein/7-aminoactinomycin-D (Ki67/7AAD) cell cycle staining was used to differentiate between G0, G1, and S/G2/M in bone marrow cell populations negative for expression of mature hematopoietic lineage marker cells but positive for expression of stem cell antigen-1 and tyrosine kinase receptor for stem cell factor (Lin−Sca-1+cKit+, LSK+). Quantitative real-time polymerase chain reaction (qRT-PCR) microarrays were utilized to examine cell cycle specific genes. Results: At 24 h following radiation exposure, a greater percentage of LSK+ in genistein-treated mice accumulated in the G0 phase of the cell cycle, whereas a large percentage of LSK+ bone marrow cells from untreated and vehicle (PEG-400)-treated mice progressed into the G1 and S/G2/M phases. Moreover, the absolute number of marrow total LSK+, long-term LSK+, and short-term LSK+ increased 2.8, 12.1, and 4.2-fold, respectively, at 7 days post-irradiation in genistein-treated vs. untreated irradiated mice. Lin− cells from genistein-treated mice expressed fewer DNA damage responsive and cell cycle checkpoint genes than LSK+ from untreated or vehicle-treated mice. Conclusion: Pretreatment with genistein provides in vivo protection from acute myelotoxicity through extended quiescence followed by reduced senescence of marrow repopulating LSK+.

Angiotensin-II-induced apoptosis requires regulation of nucleolin and Bcl-xL by SHP-2 in primary lung endothelial cells

Angiotensin II (Ang II) is a key proapoptotic factor in fibrotic tissue diseases. However, the mechanism of Ang-II-induced cell death in endothelial cells has not been previously elucidated. Using the neutral comet assay and specific receptor antagonists and agonists, we found that Ang-II-mediated apoptosis in primary pulmonary endothelial cells required the AT2 receptor. Ang II caused cytochrome c release from the mitochondria concurrent with caspase-3 activation and DNA fragmentation, and apoptosis was suppressed by an inhibitor of Bax-protein channel formation, implicating mitochondrial-mediated apoptosis. There was no evidence that the extrinsic apoptotic pathway was involved, because caspase-9, but not caspase-8, was activated by Ang-II treatment. Apoptosis required phosphoprotein phosphatase activation, and inhibition of the SHP-2 phosphatase (encoded by Ptpn11) blocked cell death. Reduced levels of anti-apoptotic Bcl-2-family members can initiate intrinsic apoptosis, and we found that Ang-II treatment lowered cytosolic Bcl-xL protein levels. Because the protein nucleolin has been demonstrated to bind Bcl-xL mRNA and prevent its degradation, we investigated the role of nucleolin in Ang-II-induced loss of Bcl-xL. RNA-immunoprecipitation experiments revealed that Ang II reduced the binding of nucleolin to Bcl-xL mRNA in an AU-rich region implicated in instability of Bcl-xL mRNA. Inhibition of SHP-2 prevented Ang-II-induced degradation of Bcl-xL mRNA. Taken together, our findings suggest that nucleolin is a primary target of Ang-II signaling, and that Ang-II-activated SHP-2 inhibits nucleolin binding to Bcl-xL mRNA, thus affecting the equilibrium between pro- and anti-apoptotic members of the Bcl-2 family.

X-irradiation induces ER stress, apoptosis, and senescence in pulmonary artery endothelial cells

Abstract Purpose: The use of clinical radiation for cancer treatment is limited by damage to underlying normal tissue including to the vascular endothelium. We investigated the mechanisms of X-ray-induced cell damage to endothelial cells. Methods: We evaluated necrosis, apoptosis, cellular senescence, and the contribution of endoplasmic reticulum (ER) stress in pulmonary artery endothelial cells (PAEC) irradiated with X-rays (2–50 Gray [Gy]). Results: Clonogenic assays showed that 10 Gy induced ∼99.9% loss of cell viability. No necrosis was detected using lactate dehydrogenase assays, but a low population underwent extrinsic and intrinsic apoptosis, as indicated by the activation of caspases 3, 8, and 9 as well as by neutral comet assay. A majority of PAEC underwent accelerated senescence, as indicated by morphological changes, increased 21 kD cyclin-dependent kinase inhibitor (p21/waf1), decreased sirtuin 1 (SIRT1), and elevated senescence-associated β-galactosidase (SA-β-gal). ER stress was detected by assays for glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), and growth arrest and DNA damage-inducible protein 34 (GADD34) mRNA, and transient phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). The ER stress inhibitor salubrinal blocked ∼50% of apoptosis with no effect on senescence. Conclusions: X-rays primarily induced cellular senescence with limited levels of apoptosis in endothelial cells. ER stress contributed to apoptosis but not to senescence.

New Approaches to Radiation Protection

Radioprotectors are compounds that protect against radiation injury when given prior to radiation exposure. Mitigators can protect against radiation injury when given after exposure but before symptoms appear. Radioprotectors and mitigators can potentially improve the outcomes of radiotherapy for cancer treatment by allowing higher doses of radiation and/or reduced damage to normal tissues. Such compounds can also potentially counteract the effects of accidental exposure to radiation or deliberate exposure (e.g., nuclear reactor meltdown, dirty bomb, or nuclear bomb explosion); hence they are called radiation countermeasures. Here, we will review the general principles of radiation injury and protection and describe selected examples of radioprotectors/mitigators ranging from small-molecules to proteins to cell-based treatments. We will emphasize agents that are in more advanced stages of development.

Bleomycin induces the extrinsic apoptotic pathway in pulmonary endothelial cells

Bleomycin, a chemotherapeutic agent, can cause pulmonary fibrosis in humans and is commonly used to induce experimental pulmonary fibrosis in rodents. In cell culture, bleomycin causes single- and double-stranded DNA breaks and produces reactive oxidative species, both of which require iron (Fe(2+)) and O(2). The mechanism of bleomycin-induced apoptosis is controversial due to its complexity. We investigated bleomycin apoptotic signaling events in primary pulmonary endothelial cells. Time course experiments revealed that bleomycin induced apoptosis within 4 h. Caspase-8, the initiator caspase for the extrinsic pathway, was activated within 2 h and preceded activation of the effector caspases-3 and -6 (4 h). Caspase-9, the initiator of the intrinsic pathway and release of cytochrome c from the mitochondria were not detected at these time points. Bleomycin induced the expression of Bcl-2 and Bcl-x(L), Bcl-2 family member proteins that protect cells from the mitochondria-dependent intrinsic apoptosis. Real-time quantitative RT-PCR results demonstrated that, at 4-8 h, bleomycin induced expression of TNF and TNF receptor family genes known to induce the extrinsic apoptotic pathway. Silencing of the death receptor adaptor protein Fas-associated death domain by short interfering RNA significantly reduced bleomycin-induced apoptosis. Apoptosis was also abrogated by caspase-8 inhibition, but only slightly reduced by caspase-3 inhibition. Together, these data suggest that bleomycin initiates apoptosis via the extrinsic pathway.