Redox control of cell fate by MAP kinase: physiological roles of ASK1-MAP kinase pathway in stress signaling

The intracellular redox state is a key determinant of cell fate, such as cell survival, proliferation, differentiation, and apoptosis. Redox imbalance is closely linked to a variety of human diseases, so that the intracellular redox condition should be tightly regulated. The redox state of the cell is a consequence of the precise balance between the levels of oxidizing and reducing equivalents, such as reactive oxygen species (ROS) and endogenous antioxidants. ROS are not only toxicants to the cell, but also second messengers in intracellular signal transduction, and control the action of several signaling pathways, including mitogen-activated protein (MAP) kinases. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase of the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways, which is preferentially activated in response to various types of stress such as oxidative stress and plays pivotal roles in a wide variety of cellular responses. Recent studies have revealed that ASK1 is also required for innate immune response through ROS production. In this review, we focus on redox control of cell function by MAP kinase signaling, and provide the advanced mechanism of redox-regulated ASK1 activation and physiological roles of the ASK1-MAP kinase pathway in stress signaling.     

The Adenosine A3 Receptor Agonist Cl-IB-MECA Induces Cell Death Through Ca2+/ROS-Dependent Down Regulation of ERK and Akt in A172 Human Glioma Cells

Adenosine A₃ receptor (A3AR) is coupled to G proteins that are involved in a variety of intracellular signaling pathways and physiological functions. 2-Chloro-N ⁶-(3-iodobenzyl) adenosine-5??-N-methylcarboxamide (Cl-IB-MECA), an agonist of A3AR, has been reported to induce cell death in various cancer cells. However, the effect of CI-IB-MECA on glioma cell growth is not clear. This study was undertaken to examine the effect of CI-IB-MECA on glioma cell viability and to determine its molecular mechanism. CI-IB-MECA inhibited cell proliferation and induced cell death in a dose- and time-dependent manner. Treatment of CI-IB-MECA resulted in an increase in intracellular Ca²⁺ followed by enhanced reactive oxygen species (ROS) generation. EGTA and N-acetylcysteine (NAC) blocked the cell death induced by CI-IB-MECA, suggesting that Ca²⁺ and ROS are involved in the Cl-IB-MECA-induced cell death. Western blot analysis showed that CI-IB-MECA induced the down-regulation of extracellular signal-regulated kinases (ERK) and Akt, which was prevented by EGTA, NAC, and the A3AR antagonist MRS1191. Transfection of constitutively active forms of MEK, the upstream kinase of ERK, and Akt prevented the cell death. CI-IB-MECA induced caspase-3 activation and the CI-IB-MECA-induced cell death was blocked by the caspase inhibitors DEVD-CHO and z-VAD-FMK. In addition, expression of XIAP and Survivin were decreased in cells treated with Cl-IB-MECA. Collectively, these findings demonstrate that CI-IB-MECA induce a caspase-dependent cell death through suppression of ERK and Akt mediated by an increase in intracellular Ca²⁺ and ROS generation in human glioma cells. These suggest that A3AR agonists may be a potential therapeutic agent for induction of apoptosis in human glioma cells.     

Superoxide Mediates Direct Current Electric Field-Induced Directional Migration of Glioma Cells through the Activation of AKT and ERK

Direct current electric fields (DCEFs) can induce directional migration for many cell types through activation of intracellular signaling pathways. However, the mechanisms that bridge extracellular electrical stimulation with intracellular signaling remain largely unknown. In the current study, we found that a DCEF can induce the directional migration of U87, C6 and U251 glioma cells to the cathode and stimulate the production of hydrogen peroxide and superoxide. Subsequent studies demonstrated that the electrotaxis of glioma cells were abolished by the superoxide inhibitor N-acetyl-l-cysteine (NAC) or overexpression of mitochondrial superoxide dismutase (MnSOD), but was not affected by inhibition of hydrogen peroxide through the overexpression of catalase. Furthermore, we found that the presence of NAC, as well as the overexpression of MnSOD, could almost completely abolish the activation of Akt, extracellular-signal-regulated kinase (Erk)1/2, c-Jun N-terminal kinase (JNK), and p38, although only JNK and p38 were affected by overexpression of catalase. The presenting of specific inhibitors can decrease the activation of Erk1/2 or Akt as well as the directional migration of glioma cells. Collectively, our data demonstrate that superoxide may play a critical role in DCEF-induced directional migration of glioma cells through the regulation of Akt and Erk1/2 activation. This study provides novel evidence that the superoxide is at least one of the “bridges” coupling the extracellular electric stimulation to the intracellular signals during DCEF-mediated cell directional migration.     

H2O2致WB-F344细胞内活性氧的产生及机理

以双氢罗丹明123（DHR123）作为荧光探针,采用激光共聚焦扫描显微镜研究小剂量（800nmol/L)H2O2诱导大鼠肝卵细胞株WB－F344细胞内活性氧产生的动态变化过程及其机理。结果发现：（1）小剂量H2O2的一次作用可以引起胞内活性氧的产生;（2）胞内活性氧清除剂N－乙酰－L－半胱氨酸（NAC）处理2h时后,再加入小剂量H2O2,发现胞内活性氧的产生明显减少;（3）用广谱的蛋白激酶抑制剂2－氨基嘌呤（2－AP）、Ca^2 依赖性蛋白激酶（PKC）抑制剂Bisindolylmaleimide Ⅰ、酷氨酸蛋白激酶（TPK）抑制剂Tyrphostin25分别预处理15min后,H2O2诱导的胞内活性氧的产生现象均消失;（4）细胞在无外钙环境下,小剂量H2O2诱导的胞内活性氧的产生明显减少;（5）细胞在无外钙环境下用NAC预处理后,H2O2诱导的胞内活性氧的产生现象消失。结果表明,H2O2可以通过胞内信号转导系统诱使WB细胞胞内活性氧产生,这可能与小剂量H2O2调控细胞生物学功能（如增殖、转化）相关。     

Stretch-induced phosphorylation of focal adhesion kinase in endothelial cells: role of mitochondrial oxidants

Mechanical stretch activates a number of signaling pathways in endothelial cells, and it elicits a variety of functional responses including increases in the phosphorylation of focal adhesion kinase (FAK), a nonreceptor tyrosine kinase involved in integrin-mediated signal transduction. Stretch also triggers an increase in the generation of reactive oxygen species (ROS), which may function as second messengers in the signal transduction cascades that activate cellular responses to strain. Mitochondria represent an important source of ROS in the cell, and these organelles may release ROS in response to strain by virtue of their attachment to cytoskeletal proteins. We therefore tested whether cyclic stretch increases FAK phosphorylation at Tyr397 through a mitochondrial ROS signaling pathway in bovine pulmonary artery endothelial cells (BPAEC). Oxidant signaling, measured using 2'7'-dichlorofluorescin (DCFH), increased 152 +/- 16% during 1.5 h of cyclic strain relative to unstrained controls. The mitochondrial inhibitors diphenylene iodonium (5 microM) or rotenone (2 microM) attenuated this increase, whereas L-nitroarginine (100 microM), allopurinol (100 microM), or apocynin (30 microM) had no effect. The antioxidants ebselen (5 microM) and dithiodidiethyldithiocarbamate (1 mM) inhibited the strain-induced increase in oxidant signaling, but Hb (5 microM) had no effect. These results indicate that strain induces oxidant release from mitochondria. Treatment with cytochalasin D (5 microM) abrogated strain-induced DCFH oxidation in BPAEC, indicating that actin filaments were required for stretch-induced mitochondrial ROS generation. Cyclic strain increased FAK phosphorylation at Tyr397, but this was abolished by mitochondrial inhibitors as well as by antioxidants. Strain-induced FAK phosphorylation was abrogated by inhibition of protein kinase C (PKC) with Ro-31-8220 or G?-6976. These findings indicate that mitochondrial oxidants generated in response to endothelial strain trigger FAK phosphorylation through a signaling pathway that involves PKC.     

Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling

Reactive oxygen species (ROS) are generated during mitochondrial oxidative metabolism as well as in cellular response to xenobiotics, cytokines, and bacterial invasion. Oxidative stress refers to the imbalance due to excess ROS or oxidants over the capability of the cell to mount an effective antioxidant response. Oxidative stress results in macromolecular damage and is implicated in various disease states such as atherosclerosis, diabetes, cancer, neurodegeneration, and aging. Paradoxically, accumulating evidence indicates that ROS also serve as critical signaling molecules in cell proliferation and survival. While there is a large body of research demonstrating the general effect of oxidative stress on signaling pathways, less is known about the initial and direct regulation of signaling molecules by ROS, or what we term the "oxidative interface." Cellular ROS sensing and metabolism are tightly regulated by a variety of proteins involved in the redox (reduction/oxidation) mechanism. This review focuses on the molecular mechanisms through which ROS directly interact with critical signaling molecules to initiate signaling in a broad variety of cellular processes, such as proliferation and survival (MAP kinases, PI3 kinase, PTEN, and protein tyrosine phosphatases), ROS homeostasis and antioxidant gene regulation (thioredoxin, peroxiredoxin, Ref-1, and Nrf-2), mitochondrial oxidative stress, apoptosis, and aging (p66Shc), iron homeostasis through iron-sulfur cluster proteins (IRE-IRP), and ATM-regulated DNA damage response.     

Interactions of multiple signaling pathways in neuropeptide Y-mediated bimodal vascular smooth muscle cell growth

Neuropeptide Y (NPY), a sympathetic cotransmitter, acts via G protein-coupled receptors to stimulate constriction and vascular smooth muscle cell (VSMC) proliferation through interactions with its Y1 receptors. However, VSMC proliferation appears bimodal, with high- and low-affinity peaks differentially blocked by antagonists of both Y1 and Y5 receptors. Here, we sought to determine the signaling mechanisms of NPY-mediated bimodal mitogenesis. In rat aortic VSMCs, NPY's mitogenic effect at all concentrations was blocked by pertussis toxin and was associated with decreased forskolin-stimulated cAMP levels. NPY also increased intracellular calcium levels; in contrast to mitogenesis, this effect was dose dependent. The rise in intracellular Ca2+ depended on extracellular Ca2+ and was mediated via activation of Y1 receptors, but not Y5 receptors. Despite differences in calcium, the signaling pathways activated at low and high NPY concentrations were similar. The mitogenic effect of the peptide at all doses was completely blocked by inhibitors of calcium/calmodulin-dependent kinase II (CaMKII), protein kinase C (PKC), and mitogen-activated protein kinase kinase, MEK1/2. Thus, in VSMCs, NPY-mediated mitogenesis signals primarily via Y1 receptors activating 2 Ca2+-dependent, growth-promoting pathways -- PKC and CaMKII. At the high-affinity peak, these 2 pathways are amplified by Y5 receptor-mediated, calcium-independent inhibition of the adenylyl cyclase - protein kinase A (PKA) pathway. All 3 mechanisms converge to the extracellular signal-regulated kinases (ERK1/2) signaling cascade and lead to VSMC proliferation.     

Protein kinase C mediates induced secretion of vascular endothelial growth factor by human glioma cells

To understand how vascular endothelial growth factor (VEGF) production is activated in malignant glioma cells, we employed protein tyrosine kinase (PTK) and protein kinase C (PKC) inhibitors to evaluate the extent to which these protein kinases were involved in signal transduction leading to VEGF production. PTK inhibitors blocked glioma proliferation and epidermal growth factor (EGF)-induced VEGF secretion, while H-7, a PKC inhibitor, inhibited both EGF-induced and baseline VEGF secretion. Phorbol 12-myristate 13-acetate (PMA), a non-specific activator of PKC, induced VEGF secretion by glioma cells, which was enhanced by calcium ionophore A23187, but completely blocked after prolonged treatment of cells with 1 microM PMA, by presumably depleting PKC. All inhibitors (genistein, AG18, AG213, H-7, prolonged PMA treatment) which inhibited EGF-induced VEGF secretion in glioma cells also inhibited cell proliferation at similar concentrations. However, PKC inhibition only blocked 50% of the VEGF secretion induced by growth factors (EGF, platelet-derived growth factor-BB, or basic fibroblast growth factor). This reserve capacity could be ascribed to a PKC-independent effect, or to PKC isoenzymes not down-regulated by PMA. These findings extend our previous assertion that VEGF secretion is tightly coupled with proliferation by suggesting that activation of convergent growth factor signaling pathways will lead to increased glioma VEGF secretion. Understanding of signal transduction of growth factor-induced VEGF secretion should provide a rational basis for the development of novel strategies for therapy.     

A critical role of redox state in determining HL-60 cell granulocytic differentiation and apoptosis via involvement of PKC and NF-κB

The modifications of intracellular redox balance leads to important cellular changes in many cell types. Here, a causal relationship among redox state, granulocytic differentiation induced by all-trans retinoic acid (RA) or dibutyryl cAMP (dbcAMP) and apoptosis have been studied in the human acute promyelocytic leukaemia HL-60 cells. The modulation of intracellular reactive oxygen species levels by d, l-buthionine-(S, R) sulfoximide (BSO), and n-acetyl-l-cysteine (NAC) caused inducer- and time-dependent or stage-specific effects on HL-60 cell growth inhibition, differentiation and subsequent apoptosis. The presence of BSO during the commitment stage suppressed RA—but not dbcAMP-mediated differentiation, while NAC inhibited both. BSO alone and in combination with RA or dbcAMP-induced apoptosis, which was prevented by NAC in dbcAMP—but not in RA-treated cells. Using protein kinase C inhibitor, calphostin C, cross-talk effects between the intracellular redox state and PKC signalling was identified by demonstrating inducer-dependent changes in cell differentiation or apoptosis, which were associated with the changes in DNA-NF-κB binding activity. These observations suggest a critical role of redox state in determining HL-60 cell behaviour and provide new insights into the complex effects of redox perturbations on the intracellular signalling network via the involvement of PKC and NF-κB.     

G protein-linked cell signaling and cardiovascular functions in diabetes/hyperglycemia

Vascular complications, including impaired contractility and increased cell proliferation, are the most common complications with diabetes. Chronic hyperglycemia seems to be an important contributing factor in this process. Various signaling pathways are implicated in diabetes/hyperglycemia-induced impaired vascular functions. Nonenzymatic glycation, enhanced production of diacylglycerol, increased activity of membranous protein kinase C (PKC), and increased oxidative stress have been proposed to explain the adverse effects of hyperglycemia on vascular smooth muscle cells. Hyperglycemia-induced stimulation of L-type Ca²⁺ channel via G protein-coupled adenylyl cyclase/cAMP and phospholipase C/PKC pathways also has been shown. In addition, hyperglycemia has been reported to decrease the availability of nitric oxide in humans, which may contribute to all the hemodynamic and physiological changes occuring in diabetes. G protein-adenylyl cyclase signaling that plays an important role in the regulation of cardiovascular functions also has been reported to be impaired in diabetes and under hyperglycemic conditions. In this review article, various G protein-linked cell signaling and functions in diabetes and hyperglycemia are discussed.     

Phorbol ester potentiates the growth inhibitory effects of troglitazone via up-regulation of PPARgamma in A549 cells

The activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to induce growth arrest and differentiation of various cancer cells. In the current study, we investigated the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of PPARgamma and proliferation of A549 cells. TPA elicited a dose- and time-dependent increase in PPARgamma mRNA and protein levels. PPARgamma expression in response to TPA was attenuated by pretreatment with bisindolylmaleimide I, N-acetyl-L-cysteine (NAC) and PD98059. TPA-induced protein kinase C (PKC) activation was linked to the generation of reactive oxygen species (ROS), both of which were indispensable for PPARgamma expression in A549 cells. Pretreatment with bisindolylmaleimide I or NAC blocked TPA-induced phosphorylation of extracellular signal-regulated kinase (ERK), suggesting that ERK-mediated signaling is also involved in the induction of PPARgamma. Furthermore, the growth inhibitory effect of troglitazone was significantly potentiated by prolonged incubation with TPA and was attenuated in the presence of GW9662, a specific inhibitor of PPARgamma. These effects were associated with an induction of cell cycle arrest at G0/G1 phase, which was accompanied by the induction of p21Waf1/Cip1 expression and decreased cyclin D1 expression. Taken together, these observations indicate that TPA synergizes with PPARgamma ligand to inhibit cell growth through up-regulation of PPARgamma expression.     

Cancer-derived immunoglobulin G promotes tumor cell growth and proliferation through inducing production of reactive oxygen species

Cancer cells have been found to express immunoglobulin G (IgG), but the exact functions and underlying mechanisms of cancer-derived IgG remain elusive. In this study, we first confirmed that downregulation of IgG restrained the growth and proliferation of cancer cells in vitro and in vivo. To elucidate its mechanism, we carried out a co-immunoprecipitation assay in HeLa cells and identified 27 potential IgG-interacting proteins. Among them, receptor of activated protein kinase C 1 (RACK1), ras-related nuclear protein (RAN) and peroxiredoxin 1 (PRDX1) are closely related to cell growth and oxidative stress, which prompted us to investigate the mechanism of action of IgG in the above phenomena. Upon confirmation of the interactions between IgG and the three proteins, further experiments revealed that downregulation of cancer-derived IgG lowered levels of intracellular reactive oxygen species (ROS) by enhancing cellular total antioxidant capacity. In addition, a few ROS scavengers, including catalase (CAT), dimethylsulfoxide (DMSO), n-acetylcysteine (NAC) and superoxide dismutase (SOD), further inhibited the growth of IgG-deficient cancer cells through suppressing mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK) signaling pathway induced by a low level of intracellular ROS, whereas exogenous hydrogen peroxide (H₂O₂) at low concentration promoted their survival via increasing intracellular ROS levels. Similar results were obtained in an animal model and human tissues. Taken together, our results demonstrate that cancer-derived IgG can enhance the growth and proliferation of cancer cells via inducing the production of ROS at low level. These findings provide new clues for understanding tumor proliferation and designing cancer therapy.     

PKCδ phosphorylation is an upstream event of GSK3 inactivation-mediated ROS generation in TGF-β1-induced senescence

Abstract

Transforming growth factor β1 (TGF-β1) induces Mv1Lu cell senescence through inactivating glycogen synthase kinase 3 (GSK3), thereby inactivating complex IV and increasing intracellular ROS. In the present study, we identified protein kinase C delta (PKCδ) as an upstream regulator of GSK3 inactivation in this mechanism of TGF-β1-induced senescence. When Mv1Lu cells were exposed to TGF-β1, PKCδ phosphorylation simultaneously increased with GSK3 phosphorylation, and then AKT and ERK were phosphorylated. AKT phosphorylation and Smad signaling were independent of GSK3 phosphorylation, but ERK phosphorylation was downstream of GSK3 inactivation. TGF-β1-triggered GSK3 phosphorylation was blocked by inhibition of PKCδ, using its pharmacological inhibitor, Rottlerin, or overexpression of a dominant negative PKCδ mutant, but GSK3 inhibition with SB415286 did not alter PKCδ phosphorylation. Activation of PKCδ by PMA delayed cell growth and increased intracellular ROS level, but did not induce senescent phenotypes. In addition, overexpression of wild type or a constitutively active PKCδ mutant was enough to delay cell growth and decrease the mitochondrial oxygen consumption rate and complex IV activity, but weakly induce senescence. However, PMA treatment on Mv1Lu cells, which overexpress wild type and constitutively active PKCδ mutants, effectively induced senescence. These results indicate that PKCδ plays a key role in TGF-β1-induced senescence of Mv1Lu cells through the phosphorylation of GSK3, thereby triggering mitochondrial complex IV dysfunction and intracellular ROS generation.     

Reactive oxygen species,cellular redox systems,and apoptosis

Reactive oxygen species (ROS) are products of normal metabolism and xenobiotic exposure, and depending on their concentration, ROS can be beneficial or harmful to cells and tissues. At physiological low levels, ROS function as “redox messengers” in intracellular signaling and regulation, whereas excess ROS induce oxidative modification of cellular macromolecules, inhibit protein function, and promote cell death. Additionally, various redox systems, such as the glutathione, thioredoxin, and pyridine nucleotide redox couples, participate in cell signaling and modulation of cell function, including apoptotic cell death. Cell apoptosis is initiated by extracellular and intracellular signals via two main pathways, the death receptor- and the mitochondria-mediated pathways. Various pathologies can result from oxidative stress-induced apoptotic signaling that is consequent to ROS increases and/or antioxidant decreases, disruption of intracellular redox homeostasis, and irreversible oxidative modifications of lipid, protein, or DNA. In this review, we focus on several key aspects of ROS and redox mechanisms in apoptotic signaling and highlight the gaps in knowledge and potential avenues for further investigation. A full understanding of the redox control of apoptotic initiation and execution could underpin the development of therapeutic interventions targeted at oxidative stress-associated disorders.     

Differential recruitment of PKC isoforms in HeLa cells during redox stress

The protein kinase C (PKC) family is a major transducer of several intracellular pathways. In confirmation of this important role, PKCs exhibit high molecular heterogeneity, because they occur in at least 10 different isoforms differing in biochemical properties and sensitivity to activators. In this report we focused on the ability of different redox agents to induce modification of intracellular distribution of specific PKC isoforms in HeLa cells. To this end we utilized a panel of green fluorescent protein (GFP) chimeras and a high-speed digital imaging system. We observed a remarkable complexity of PKC signalling patterns occurring during redox stress with marked differences among PKC isoforms also belonging to the same subgroup. Moreover our results suggest that modifications of the intracellular redox state can modulate the responsiveness of specific PKC isoforms and, in turn, change the sensitivity of the different isoforms to cell stimulation.     

Involvement of p21(Waf1/Cip1) in protein kinase C alpha-induced cell cycle progression

Protein kinase C (PKC) plays an important role in the regulation of glioma growth; however, the identity of the specific isoform and mechanism by which PKC fulfills this function remain unknown. In this study, we demonstrate that PKC activation in glioma cells increased their progression through the cell cycle. Of the six PKC isoforms that were present in glioma cells, PKC alpha was both necessary and sufficient to promote cell cycle progression when stimulated with phorbol 12-myristate 13-acetate. Also, decreased PKC alpha expression resulted in a marked decrease in cell proliferation. The only cell cycle-regulatory molecule whose expression was rapidly altered and increased by PKC alpha activity was the cyclin-cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1). Coimmunoprecipitation studies revealed that p21(Waf1/Cip1) upregulation was accompanied by an incorporation of p21(Waf1/Cip1) into various cyclin-CDK complexes and that the kinase activity of these complexes was increased, thus resulting in cell cycle progression. Furthermore, depletion of p21(Waf1/Cip1) by antisense strategy attenuated the PKC-induced cell cycle progression. These results suggest that PKC alpha activity controls glioma cell cycle progression through the upregulation of p21(Waf1/Cip1), which facilitates active cyclin-CDK complex formation.     

In vitro identification of mitochondrial oxidative stress production by time-resolved fluorescence imaging of glioma cells

Oxidative phosphorylation and glycolysis are important features, by which cells could bypass oxidative stress. The level of oxidative stress, and the ability of cells to promote oxidative phosphorylation or glycolysis, significantly determined proliferation or cell demise. In the present work, we have employed selective mitochondrial probe MitoTracker? Orange CMTM/Ros (MTO) to estimate the level of oxidative stress in cancer cells at different stressed conditions. MTO is partially sensitive to decrease of mitochondrial membrane potential and to reactive oxygen species (ROS) generated in mitochondria. We have demonstrated, that fluorescence lifetime of MTO is much more sensitive to oxidative stress than intensity-based approaches. This method was validated in different cancer cell lines. Our approach revealed, at relatively low ROS levels, that Gö 6976, a protein kinase C (PKC) α inhibitor, and rottlerin, an indirect PKCδ inhibitor, increased mitochondrial ROS level in glioma cell. Their involvement in oxidative phosphorylation and apoptosis was investigated with oxygen consumption rate estimation, western blot and flow-cytometric analysis. Our study brings new insight to identify feeble differences in ROS production in living cells.     

Leukotriene D4 mediates survival and proliferation via separate but parallel pathways in the human intestinal epithelial cell line Int 407

We demonstrated previously that leukotriene D4 (LTD4) regulates proliferation of intestinal epithelial cells through a CysLT receptor by protein kinase C (PKC)epsilon-dependent stimulation of the mitogen-activated protein kinase ERK1/2. Our current study provides the first evidence that LTD4 can activate 90-kDa ribosomal S6 kinase (p90RSK) and cAMP-responsive element-binding protein (CREB) via pertussis-toxin-sensitive Gi protein pathways. Transfection and inhibitor experiments revealed that activation of p90RSK, but not CREB, is a PKCepsilon/Raf-1/ERK1/2-dependent process. LTD4-mediated CREB activation was not affected by expression of kinase-dead p90RSK but was abolished by transfection with the regulatory domain of PKCalpha (a specific dominant-inhibitor of PKCalpha). Kinase-negative mutants of p90RSK and CREB (K-p90RSK and K-CREB) blocked the LTD4-induced increase in cell number and DNA synthesis (thymidine incorporation). Compatible with these results, flow cytometry showed that LTD4 caused transition from the G0/G1 to the S+G2/M cell cycle phase, indicating increased proliferation. Similar treatment of cells transfected with K-p90RSK resulted in cell cycle arrest in the G0/G1 phase, consistent with a role of p90RSK in LTD4-induced proliferation. On the other hand, expression of K-CREB caused a substantial buildup in the sub-G0/G1 phase, suggesting a role for CREB in mediating LTD4-mediated survival in intestinal epithelial cells. Our results show that LTD4 regulates proliferation and survival via distinct intracellular signaling pathways in intestinal epithelial cells.     

Novel evidence that testosterone promotes cell proliferation and differentiation via G protein-coupled receptors in the rat L6 skeletal muscle myoblast cell line

Androgens are known to modulate the skeletal muscle proliferation and differentiation processes. Recent in vitro studies have shown that dihydrotestosterone and anabolic steroids have functions in promoting the proliferation and differentiation of the mouse skeletal muscle myoblast C2C12 cell line through the classical androgen receptor (AR) signaling pathway. But there are contradictory reports that androgen plays its roles through the membrane signaling pathways. In the present study, we show that there is no expression of the classical AR in L6 cells both at gene and protein levels. We then investigated the effects of testosterone (T) on L6 cell proliferation and differentiation. The results show that T promotes L6 cell proliferation after a 24 h treatment, which followed by enhancing L6 cell differentiation, but these effects are not inhibited by flutamide (F), an antagonist of intracellular AR. Further, we tested the effect of testosterone covalently bounding to albumin (T-BSA), which does not cross the plasma membrane. The results demonstrate that T-BSA and free T have similar effects on L6 cell proliferation and differentiation, and that these effects involve G protein-coupled receptors and different downstream pathways. The L6 cell proliferation induced by T involves PKC and ERK1/2 signaling pathways and cell differentiation happens via the PKA signaling pathway. These results suggest that T promotes cell proliferation and differentiation via G protein-coupled receptors and different downstream pathways in the L6 cell line, although the related molecular mechanisms need to be elucidated in future studies.     

Mitochondrial respiratory chain and NAD(P)H oxidase are targets for the antiproliferative effect of carbon monoxide in human airway smooth muscle

Carbon monoxide (CO), one of the end products of heme oxygenase activity, inhibits smooth muscle proliferation by decreasing ERK1/2 phosphorylation and cyclin D1 expression, a signaling pathway that is known to be modulated by reactive oxygen species (ROS) in airway smooth muscle cells (ASMCs). Two important sources of ROS involved in cell signaling are the membrane NAD(P)H oxidase and the mitochondrial respiratory chain. Thus, that CO could modulate redox signaling in ASMCs by interacting with the heme moiety of NAD(P)H oxidase and/or the respiratory chain is a plausible hypothesis. Here we show that a recently identified carbon monoxide-releasing molecule, [Ru(CO)3Cl2]2 (or CORM-2) 1) inhibits NAD(P)H oxidase cytochrome b558 activity, 2) increases oxidant production by the mitochondria, and 3) inhibits ASMC proliferation and phosphorylation of the ERK1/2 mitogen-activated protein kinase and expression of cyclin D1, two critical pathways involved in muscle proliferation. No such effects were observed with the negative control (Ru(Me2SO)4Cl2), which does not contain CO groups. Because both diphenylene iodinium or apocynin (inhibitors of NAD(P)H oxidase) and rotenone (a molecule that increases mitochondrial ROS production by blocking the respiratory chain) mimicked the effect of CORM-2 on cyclin D1 expression and ASMC proliferation, the antiproliferative effect of CORM-2 is probably related to inhibition of cytochromes on both NAD(P)H oxidase and the respiratory chain. The involvement of increased mitochondria-derived oxidants is substantiated by the findings showing that the antioxidant N-acetylcysteine partially inhibited the effects of CORM-2. This study provides a new mechanism to explain redox signaling by CO.