Crosstalk of moderate ROS and PARP-1 contributes to sustainable proliferation of conditionally reprogrammed keratinocytes

Conditionally reprogrammed cell (CRC) technique is a promising model for biomedical and toxicological research. In the present study, our data first demonstrated an increased level of PARP-1 in conditionally reprogrammed human foreskin keratinocytes (CR-HFKs). We then found that PARP inhibitor ABT-888 (ABT), reactive oxygen species (ROS) scavenger N-acetyl-l -cysteine (NAC), or combination (ABT + NAC) were able to inhibit cell proliferation, ROS, PARP-1, and ROS related protein, NRF2, and NOX1. Interestingly, knockdown of endogenous PARP-1 significantly inhibited cell proliferation, indicating that the increased PARP-1 expression was critical for CR. Importantly, we found that a moderate level of ROS contributed the cell proliferation and increased PARP-1 since knockdown of PARP-1 also inhibited the ROS. The similar inhibition of cell proliferation, ROS, and expression of PARP-1 and NRF2 proteins was observed when CR-HFKs were treated with hydroquinone (HQ), a key component from skin-lightening products. Moreover, the treatment of HQ plus treatment of ABT, NAC, or combination can further inhibit cell proliferation, ROS, expression of PARP-1, and NRF2 proteins. PARP-1 knockdown inhibited the population doubling (PDL) and treatment of HQ inhibited the PDL further, as well as the change of ROS. Finally, we discovered that pathways including cyclin D1, NRF2, Rb and pRb, CHK2, and p53, were involved in cell proliferation inhibition with HQ. Taken together, our findings demonstrated that crosstalk between ROS and PARP-1 involves in the cell proliferation in CR-HFKs, and that inhibition of CR-HFK proliferation with HQ is through modulating G1 cell cycle arrest.     

Role of reactive oxygen species in bradykinin-induced proliferation of vascular smooth muscle cells

In addition to the induction of cell proliferation and migration, bradykinin (BK) can increase c-fos mRNA expression, activate ERK 1/2 and generate reactive oxygen species (ROS) in vascular smooth muscle cells (VSMC). It is not known, however, whether BK can induce cellular proliferation and extracellular matrix production via redox-sensitive signaling pathways. We investigated the role(s) of ROS in proliferation, migration and collagen synthesis induced by BK in VSMC derived from Sprague Dawley rat aorta. BK (10 nM) increased VSMC proliferation by 30% (n=5); this proliferation was inhibited by the antioxidants N-acetylcysteine (20 mM) and alpha-lipoic acid (LA, 250 mM). In addition, BK induced an increase in cell migration and in collagen levels that were blocked by LA. ROS production induced by BK (n=10) was significantly inhibited by bisindolylmaleimide (4microM) and by PD98059 (40microM). These results suggest that: 1) ROS participate in the mechanism(s) used by bradykinin to induce cellular proliferation; 2) bradykinin induces ROS generation through a pathway that involves the kinases PKC and MEK; and 3) ROS participate in the pathways mediating cell migration and the production of collagen as a response to treatment with bradykinin. To our knowledge, this is the first report describing mechanisms to explain the participation of ROS in the cellular proliferation and extracellular matrix pathway regulated by BK.     

High glucose induces renal mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway, which is inhibited by resveratrol

Renal hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy. Hyperglycemia-induced oxidative stress is implicated in the etiology of diabetic nephropathy. Resveratrol has potent antioxidative and protective effects on diabetic nephropathy. We aimed to examine whether high glucose (HG)-induced NADPH oxidase activation and reactive oxygen species (ROS) production contribute to glomerular mesangial cell proliferation and fibronectin expression and the effect of resveratrol on HG action in mesangial cells. By using rat mesangial cell line and primary mesangial cells, we found that NADPH oxidase inhibitor (apocynin) and ROS inhibitor (N-acetyl cysteine) both inhibited HG-induced mesangial cell proliferation and fibronectin expression. HG-induced elevation of NADPH oxidase activity and production of ROS in mesangial cells was inhibited by apocynin. These results suggest that HG induces mesangial cell proliferation and fibronectin expression through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunits p22(phox) and p47(phox) expression through JNK/NF-κB pathway, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced mesangial cell proliferation and fibronectin expression through inhibiting HG-induced JNK and NF-κB activation, NADPH oxidase activity elevation and ROS production. These results demonstrate that HG enhances mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide novel therapeutic targets for diabetic nephropathy.     

The ROS derived mitochondrial respirstion not from NADPH oxidase plays key role in Celastrol against angiotensin II-mediated HepG2 cell proliferation

Angiotensin II (AngII) is an important factor that promotes the proliferation of cancer cells, whereas celastrol exhibits a significant antitumor activity in various cancer models. Whether celastrol can effectively suppress AngII mediated cell proliferation remains unknown. In this study, we studied the effect of celastrol on AngII-induced HepG2 cell proliferation and evaluated its underlying mechanism. The results revealed that AngII was able to significantly promote HepG2 cell proliferation via up-regulating AngII type 1 (AT₁) receptor expression, improving mitochondrial respiratory function, enhancing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, increasing the levels of reactive oxygen species (ROS) and pro-inflammatory cytokines. The excess ROS from mitochondrial dysfunction is able to cause the apoptosis of tumor cells via activating caspase3 signal pathway. In addition, the reaction between NO and ROS results in the formation of peroxynitrite (ONOO^?), and then promoting cell damage. celastrol dramatically enhanced ROS generation, thereby causing cell apoptosis through inhibiting mitochodrial respiratory function and boosting the expression levels of AngII type 2 (AT₂) receptor without influencing NADPH oxidase activity. PD123319 as a special inhibitor of AT₂R was able to effectively decreased the levels of inflammatory cytokines and endothelial nitric oxide synthase (eNOS) activity, but only partially attenuate the effect of celastrol on AnII mediated HepG2 cell proliferation. Thus, celastrol has the potential for use in liver cancer therapy. ROS derived from mitochondrial is an important factor for celastrol to suppress HepG2 cell proliferation.     

Signaling mechanism of protease activated receptor 1-induced proliferation of astrocytes: Stabilization of hypoxia inducible factor-1α triggers glucose metabolism and accumulation of cyclin D1

Thrombin was found to stimulate astrocytes proliferation. In this study, we want to clarify whether thrombin-activated protease-activated receptor will affect the glucose metabolism signaling pathways to accelerate the proliferation of astrocytes. In addition, we study if thrombin has effects on cell cycle transition to promote astrocytes proliferation. We firstly observed that thrombin activated protease-activated receptor 1 (PAR-1) inducing the increases of intracellular Ca²⁺ and ROS production, which contribute to the astrocytes' proliferation. We further confirmed that ROS stabilized HIF-1α, the latter subsequently accelerated glucose uptake in astrocytes. On the other hand, we demonstrated that thrombin triggered PI3K/Akt/cyclin D1 signal transduction, which may promote the cell cycle transition to enhance astrocytes proliferation. As a result, we discovered three signaling pathways mainly accounting for cell proliferation induced by thrombin: (1) thrombin-stimulated ERK, JNK/ROS/HIF-1α and (2) PI3K/Akt/ROS/HIF-1α pathways to increase expression of hexokinase 2 which mediated glucose metabolism in astrocytes, and (3) thrombin stimulates PAR-1/PI3K/Akt/cyclin D1 to promote the cell cycle transition and finally to increase cell proliferation.     

Mitochondrial reactive oxygen species originating from Romo1 exert an important role in normal cell cycle progression by regulating p27Kip1 expression

Reactive oxygen species (ROS) steady-state levels are required for entry into the S phase of the cell cycle in normal cells, as well as in tumour cells. However, the contribution of mitochondrial ROS to normal cell proliferation has not been well investigated thus far. A previous report showed that Romo1 was responsible for the high ROS levels in tumour cells. Here, we show that endogenous ROS generated by Romo1 are indispensable for cell cycle transition from G1 to S phase in normal WI-38 human lung fibroblasts. The ROS level in these cells was down-regulated by Romo1 knockdown, resulting in cell cycle arrest in the G1 phase. This arrest was associated with an increase in the level of p27^Kip1. These results demonstrate that mitochondrial ROS generated by Romo1 expression is required for normal cell proliferation and it is suggested that Romo1 plays an important role in redox signalling during normal cell proliferation.     

Proliferation and wound healing of vascular cells trigger the generation of extracellular reactive oxygen species and LDL oxidation

Cell proliferation of vascular cells is a key feature in vascular biology, wound healing, and pathophysiological processes such as atherosclerosis and restenosis. In atherosclerotic intima, cell proliferation colocalizes with oxidized LDL that indicate a local oxidative stress. This study aims to investigate whether cell proliferation is causally related with extracellular ROS generation and subsequent LDL oxidation. Sparse proliferating endothelial and smooth muscle cells generate higher levels of extracellular ROS (O₂^•− and H₂O₂) and LDL oxidation than confluent contact-inhibited cells. During wound healing of confluent cell layer, cell proliferation associated with healing also induced enhanced extracellular ROS generation and LDL oxidation. Proliferation-associated extracellular ROS generation is mediated through mitogenic signaling pathways, involving ERK1/2 and PKC, but is independent of de novo DNA synthesis, gene expression and protein synthesis. Data obtained with inhibitors of oxidases suggest that proliferation-associated extracellular ROS are not generated by a single ROS-generating system and are not essential for cell proliferation. In conclusion, our data show that proliferating vascular cells (in sparse culture or during wound healing) generate high levels of extracellular ROS and LDL oxidation through regulation of ROS-generating systems by mitogenic signaling. This constitutes a link between proliferative events and oxidative stress/LDL oxidation in atherosclerotic lesions and restenosis.     

A critical role for Romo1-derived ROS in cell proliferation

Low levels of endogenous reactive oxygen species (ROS) originating from NADPH oxidase have been implicated in various signaling pathways induced by growth factors and mediated by cytokines. However, the main source of ROS is known to be the mitochondria, and increased levels of ROS from the mitochondria have been observed in many cancer cells. Thus far, the mechanism of ROS production in cancer cell proliferation in the mitochondria is not well-understood. We recently identified a novel protein, ROS modulator 1 (Romo1), and reported that increased expression of Romo1-triggered ROS production in the mitochondria. The experiments conducted in the present study showed that Romo1-derived ROS were indispensable for the proliferation of both normal and cancer cells. Furthermore, whilst cell growth was inhibited by blocking the ERK pathway in cells transfected with siRNA directed against Romo1, the cell growth was recovered by addition of exogenous hydrogen peroxide. The results of this study suggest that Romo1-induced ROS may play an important role in redox signaling in cancer cells.     

Focal adhesion kinase regulation by oxidative stress in different cell types

Focal adhesion kinase (FAK) is a tyrosine kinase ubiquitously expressed in cells. It was initially shown to be the initiator of focal adhesion formation in adherent cells, after its binding to integrins which induce its autophosphorylation. However, it can be also activated by a great variety of other stimuli able to act on different intracellular signaling. Reactive oxygen species (ROS), which have been shown to act as external or internal cell stimuli, induce tyrosine phosphorylation of FAK. Its autophosphorylation is followed by a submembranous localization which is crucial for many of the biological roles of FAK, including cell spreading, cell migration, cell proliferation, and prevention of apoptosis. It plays an important role in development of tumor cells, its regulation could be thus a way of impairing cell proliferation in cancer. We describe in this review the structure, activity, and functions of FAK in different cells and how ROS are able, like other stimuli, to induce its phosphorylation and modification of cell morphology and structure. The link between ROS and FAK activation could explain the role of ROS in mediating cell proliferation, cell migration, or apoptosis.     

Regulation of oxidative stress responses by ataxia-telangiectasia mutated is required for T cell proliferation

Mutations in the gene encoding ataxia-telangiectasia (A-T) mutated (Atm) cause the disease A-T, characterized by immunodeficiency, the molecular basis of which is not known. Following stimulation through the TCR, Atm-deficient T cells and normal T cells in which Atm is inhibited undergo apoptosis rather than proliferation. Apoptosis is prevented by scavenging reactive oxygen species (ROS) during activation. Atm therefore plays a critical role in T cell proliferation by regulating responses to ROS generated following T cell activation. The inability of Atm-deficient T cells to control responses to ROS is therefore the molecular basis of immunodeficiency associated with A-T.     

Reactive oxygen species and vascular cell apoptosis in response to angiotensin II and pro-atherosclerotic factors

Reactive oxygen species (ROS) are known to induce apoptotic cell death in various cell types. In the vessel wall, ROS can be formed by macrophages within the atherosclerotic plaque or can act on the endothelium after adhesion of monocytes or leucocytes. Moreover, ROS are endogenously synthesized by endothelial and vascular smooth muscle cells by NAD(P)H oxidase. Enhanced ROS production is a very early hallmark in the atherogenic process, suggesting a link between ROS and apoptosis. In endothelial cells, the endogenous generation of ROS is induced by different pro-inflammatory and pro-atherosclerotic factors such as Ang II, oxLDL or TNFalpha, which all promote the execution of programmed cell death. ROS synthesis is thereby causally involved in apoptosis induction, because antioxidants prevent endothelial cell death. The pro-apoptotic effects of endogenous ROS in endothelial cells mechanistically seems to involve the disturbance of mitochondrial membrane permeability followed by cytochrome c release, which finally activates the executioner caspases. In contrast to the pro-apoptotic capacity of ROS in endothelial cells, in vascular smooth muscle cells emerging evidence suggests that endogenous ROS synthesis promotes cell proliferation and hypertrophy and does not affect cell survival. However, high concentrations of exogenous ROS can also stimulate smooth muscle cell apoptosis as shown for other cell types probably via activation of p53. Taken together, the double-edged effects of endogenously derived ROS in endothelial cells versus VSMC may provide a mechanistic clue to the anti-atherosclerotic effects of antioxidants shown in experimental studies, given that the promotion of endothelial survival in combination with inhibition of VSMC proliferation blocks two very important steps in the pathogenesis of atherosclerosis.     

Urotensin-II-Mediated Reactive Oxygen Species Generation via NADPH Oxidase Pathway Contributes to Hepatic Oval Cell Proliferation

Urotensin II (UII), a somatostatin-like cyclic peptide, is involved in tumor progression due to its mitogenic effect. Our previous study demonstrated that UII and its receptor UT were up-regulated in human hepatocellular carcinoma (HCC), and exogenous UII promoted proliferation of human hepatoma cell line BEL-7402. Hepatic progenitor cell (HPCs) are considered to be one of the origins of liver cancer cells, but their relationship with UII remains unclear. In this work, we aimed to investigate the effect of UII on ROS generation in HPCs and the mechanisms of UII-induced ROS in promoting cell proliferation. Human HCC samples were used to examine ROS level and expression of NADPH oxidase. Hepatic oval cell line WB-F344 was utilized to investigate the underlying mechanisms. ROS level was detected by dihydroethidium (DHE) or 2’, 7’-dichlorofluorescein diacetate (DCF-DA) fluorescent probe. For HCC samples, ROS level and expression of NADPH oxidase were significantly up-regulated. In vitro, UII also increased ROS generation and expression of NADPH oxidase in WB-F344 cells. NADPH oxidase inhibitor apocynin pretreatment partially abolished UII-increased phosphorylation of PI3K/Akt and ERK, expression of cyclin E/cyclin-dependent kinase 2. Cell cycle was then analyzed by flow cytometry and UII-elevated S phase proportion was inhibited by apocynin pretreatment. Finally, bromodeoxyuridine (Brdu) incorporation assay showed that apocynin partially abolished UII induced cell proliferation. In conclusion, this study indicates that UII-increased ROS production via the NADPH oxidase pathway is partially associated with activation of the PI3K/Akt and ERK cascades, accelerates G1/S transition, and contributes to cell proliferation. These results showed that UII plays an important role in growth of HPCs, which provides novel evidence for the involvement of HPCs in the formation and pathogenesis of HCC.     

Reactive oxygen species stimulated human hepatoma cell proliferation via cross-talk between PI3-K/PKB and JNK signaling pathways

Reactive oxygen species (ROS) are important for intracellular signaling mechanisms regulating many cellular processes. Manganese superoxide dismutase (MnSOD) may regulate cell growth by changing the level of intracellular ROS. In our study, we investigated the effect of ROS on 7721 human hepatoma cell proliferation. Treatment with H2O2 (1-10 microM) or transfection with antisense MnSOD cDNA constructs significantly increased the cell proliferation. Recently, the mitogen-activated protein kinases (MAPK) and the protein kinase B (PKB) were proposed to be involved in cell growth. Accordingly, we assessed the ability of ROS to activate MAPK and PKB. PKB and extracellular signal-regulated kinase (ERK) were both rapidly and transiently activated by 10 microM H2O2, but the activities of p38 MAPK and JNK were not changed. ROS-induced PKB activation was abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002, suggesting that PI3-K is an upstream mediator of PKB activation in 7721 cells. Transfection with sense PKB cDNA promoted c-fos and c-jun expression in 7721 cells, suggesting that ROS may regulate c-fos and c-jun expression via the PKB pathway. Furthermore we found that exogenous H2O2 could stimulate the proliferation of PKB-AS7721 cells transfected with antisense PKB cDNA, which was partly dependent on JNK activation, suggesting that H2O2 stimulated hepatoma cell proliferation via cross-talk between the PI3-K/PKB and the JNK signaling pathways. However, insulin could stimulate 7721 cell proliferation, which is independent of cross-talk between PI3-K/PKB and JNK pathways. In addition, H2O2 did not induce the cross-talk between the PI3-K/PKB and the JNK pathways in normal liver cells. Taken together, we found that ROS regulate hepatoma cell growth via specific signaling pathways (cross-talk between PI3-K/PKB and JNK pathway) which may provide a novel clue to elucidate the mechanism of hepatoma carcinogenesis.     

Reactive oxygen species regulate M-CSF-induced monocyte/macrophage proliferation through SHP1 oxidation

Macrophage colony-stimulating factor (M-CSF) stimulation results in the production of reactive oxygen species (ROS) that participate in the proliferation of monocyte/macrophage. However, the molecular mechanisms whereby ROS modulate the signaling processes of M-CSF remain poorly defined. We report here that the redox-sensitive Src homology region 2 domain-containing phosphatase 1 (SHP1) is a critical regulator of M-CSF-mediated signaling in bone marrow monocyte/macrophage lineage cells (BMMs). Application of diphenylene iodonium (DPI) inhibited the responses of BMMs to M-CSF, including ROS production, cell proliferation, and phosphorylation of c-Fms as well as Akt kinase, but not of MAP kinases such as ERK, p38, and JNK. Dysregulation of SHP1 by overexpression or RNA interference in BMMs showed that SHP1 specifically regulates PI3 kinase (PI3K)/Akt signaling, but not MAP kinases in a redox-dependent manner, thereby regulating proliferation of BMMs through cyclins D1 and D2. These findings demonstrate that M-CSF-mediated ROS generation leads to SHP1 oxidation, which promotes cell proliferation through the PI3K/Akt-dependent signaling pathway.     

Important role for Rac1 in regulating reactive oxygen species generation and pulmonary arterial smooth muscle cell growth

Vascular NADPH oxidases have been shown to be a major source of reactive oxygen species (ROS). Recent studies have also implicated ROS in the proliferation of vascular smooth muscle cells. However, the components required for activation of the NADPH oxidase complex have not been clearly elucidated. Here we demonstrate that ROS generation in ovine pulmonary arterial smooth muscle cells (PASMCs) requires the activation of Rac1, implicating this protein as an important subunit of the NADPH oxidase complex. Our results, using a geranylgeranyl transferase inhibitor (GGTI-287), demonstrated a dose-dependent inhibition of Rac1 activity and ROS production. This was associated with an inhibition of PASMC proliferation with an arrest at G(2)/M. The inhibition of Rac1 by GGTI-287 led us to more specifically target Rac1 to investigate its role in the generation of ROS and cellular proliferation. To accomplish this, we utilized a dominant negative Rac1 (N17Rac1) and a constitutively active Rac1 (V12Rac1). These two forms of Rac1 were transiently expressed in PASMCs using adenovirus-mediated gene transfer. N17Rac1 expression resulted in decreased cellular Rac1 activity, whereas V12Rac1 infection showed increased activity. Compared with controls, the V12Rac1-expressing cells had higher levels of ROS production and increased proliferation, whereas the N17Rac1-expressing cells had decreased ROS generation and proliferation and cell cycle arrest at G(2)/M. However, the inhibition of cell growth produced by N17Rac1 overexpression could be overcome if cells were co-incubated with the Cu,Zn superoxide dismutase inhibitor DETC. These results indicate the importance of Rac1 in ROS generation and proliferation of vascular smooth muscle cells.     

Manganese superoxide dismutase activity regulates transitions between quiescent and proliferative growth

In recent years, the intracellular reactive oxygen species (ROS) levels have gained increasing attention as a critical regulator of cellular proliferation. We investigated the hypothesis that manganese superoxide dismutase (MnSOD) activity regulates proliferative and quiescent growth by modulating cellular ROS levels. Decreasing MnSOD activity favored proliferation in mouse embryonic fibroblasts (MEF), while increasing MnSOD activity facilitated proliferating cells' transitions into quiescence. MnSOD +/- and -/- MEFs demonstrated increased superoxide steady-state levels; these fibroblasts failed to exit from the proliferative cycle, and showed increasing cyclin D1 and cyclin B1 protein levels. MnSOD +/- MEFs exhibited an increase in the percentage of G(2) cells compared to MnSOD +/+ MEFs. Overexpression of MnSOD in MnSOD +/- MEFs suppressed superoxide levels and G(2) accumulation, decreased cyclin B1 protein levels, and facilitated cells' transit into quiescence. While ROS are known to regulate differentiation and cell death pathways, both of which are irreversible processes, our results show MnSOD activity and, therefore, mitochondria-derived ROS levels regulate cellular proliferation and quiescence, which are reversible processes essential to prevent aberrant proliferation and subsequent exhaustion of normal cell proliferative capacity. These results support the hypothesis that MnSOD activity regulates a mitochondrial 'ROS-switch' favoring a superoxide-signaling regulating proliferation and a hydrogen peroxide-signaling supporting quiescence.     

Caveolae are an essential component of the pathway for endothelial cell signaling associated with abrupt reduction of shear stress

Abrupt cessation of flow representing the acute loss of shear stress (simulated ischemia) to flow-adapted pulmonary microvascular endothelial cells (PMVEC) leads to reactive oxygen species (ROS) generation that signals for EC proliferation. We evaluated the role of caveolin-1 on this cellular response with mouse PMVEC that were preconditioned for 72 h to laminar flow at 5 dyn/cm(2) followed by stop of flow ("ischemia"). Preconditioning resulted in a 2.7-fold increase in cellular expression of K(ATP) (K(IR) 6.2) channels but no change in expression level of caveolin-1, gp91(phox), or MAP kinases. The initial response to ischemia in wild type cells was cell membrane depolarization that was abolished by gene targeting of K(IR) 6.2. The subsequent response was increased ROS production associated with activation of NADPH oxidase (NOX2) and then phosphorylation of MAP kinases (Erk, JNK). After 24 h of ischemia in wild type cells, the cell proliferation index increased 2.5 fold and the % of cells in S+G(2)/M phases increased 6-fold. This signaling cascade (cell membrane depolarization, ROS production, MAP kinase activation and cell proliferation) was abrogated in caveolin-1 null PMVEC or by treatment of wild type cells with filipin. These studies indicate that caveolin-1 functions as a shear sensor in flow-adapted EC resulting in ROS-mediated cell signaling and endothelial cell proliferation following the abrupt reduction in flow.