Cigarette smoke extract induces HO‐1 expression in mouse cerebral vascular endothelial cells: Involvement of c‐Src/NADPH oxidase/PDGFR/JAK2/STAT3 pathway

Several chemicals present in cigarette smoke (CS) have been reported to induce heme oxygenase‐1 (HO‐1) expression, which represents a prime defense mechanism in protecting the cells from stress‐dependent adverse effects on peripheral vascular system. However, the effects of cigarette smoke extract (CSE) on HO‐1 induction and the mechanisms underlying CSE‐induced HO‐1 expression in brain vessels are not completely understood. Here, we used a mouse brain endothelial cell culture (bEnd.3) to investigate the effect of CSE on HO‐1 induction and the mechanisms underlying CSE‐induced HO‐1 expression in cerebral vessels. We demonstrated that sublethal concentrations of CSE (30 µg/ml) induced submaximal HO‐1 expression in bEnd.3 cells. NADPH oxidase‐dependent ROS generation played a key role in CSE‐induced HO‐1 expression. CSE‐induced HO‐1 expression was mediated through PDGFR/JAK2/STAT3 cascade, which was observed by pretreatment with the respective pharmacological inhibitors or transfection with PDGFR shRNA. CSE activated NADPH oxidase through c‐Src in bEnd.3 cells. Taken together, these results suggested that, in bEnd.3 cells, CSE‐induced HO‐1 expression was mediated through PDGFR/JAK2/STAT3 cascade, which was regulated by c‐Src or c‐Src activated‐NADPH oxidase/ROS. J. Cell. Physiol. 225: 741–750, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of low dose rate (0.003-0.025 Gy/h) chronic X-irradiation on radioresistant and radiosensitive L5178Y mouse lymphoma cells

Cultures of radioresistant (LY-R) and radiosensitive (LY-S) strains of L5178Y mouse lymphoma cells were exposed continuously to X-rays delivered at dose rates ranging from 0.003 to 0.025 Gy/h for up to 35 days. Populations of both strains proliferated actively during the exposure, but the growth rates were reduced in a dose rate-dependent manner. The reduction of growth rate occurred for strain LY-S earlier during the exposure and at lower dose rates than for strain LY-R. The survival (as measured by colony forming ability) of strain LY-R was affected only slightly at all dose rates applied. For strain LY-S, a decrease in the surviving fraction was observed in the initial part of the exposure. This decrease was followed by a plateau and eventually by an increase, in some cases to values close to the control level. The increase in the surviving fraction indicated that the radioresistance of the exposed LY-S cells had increased. This pattern was particularly clear for dose rates greater than 0.014 Gy/h. The pre-irradiated cells exhibited radioresistance when exposed to acute X-radiation after termination of the chronic exposure. The increase in radiation resistance was stable for at least 70 days after termination of the protracted exposure. These results show that mutagenic and/or selective phenomena leading to an increase in radiation resistance of mammalian cells can be caused by protracted exposures to X-rays at dose rates permitting active proliferation.     

Impact of lek mating on the sterile insect technique: A modeling study

Modelling studies are presented which describe the effect of lek mating on the control of a wild population by sterile male release. The mixed leks are assumed to follow a Poisson-binomial distribution and the system includes three parts: territory defense, matings inside a lek and matings outside a lek. The effects of parameters on the hatchability are discussed. Among the parameters, sterile type effect (W_s), female choice (f_s) and mating competitiveness (C_m) are the most important. The application to determining the effects of sterile male release and on the proportion of sterile males required for eradication are also discussed.     

The LuxR family protein SpnR functions as a negative regulator of N-acylhomoserine lactone-dependent quorum sensing in Serratia marcescens

Serratia marcescens SS-1 produces at least four N-acylhomoserine lactones (AHLs) which were identified using high-resolution mass spectrometry and chemical synthesis, as N-(3-oxohexanoyl) homo-serine lactone (3-oxo-C6-HSL), N-hexanoyl- (C6-HSL), N-heptanoyl (C7-HSL) and N-octanoyl- (C8-HSL) homoserine lactone. These AHLs are synthesized via the LuxI homologue SpnI, and regulate via the LuxR homologue SpnR, the production of the red pigment, prodigiosin, the nuclease, NucA, and a biosurfactant which facilitates surface translocation. spnR overexpression and spnR gene deletion show that SpnR, in contrast to most LuxR homologues, acts as a negative regulator. spnI overexpression, the provision of exogenous AHLs and spnI gene deletion suggest that SpnR is de-repressed by 3-oxo-C6-HSL. In addition, long chain AHLs antagonize the biosurfactant-mediated surface translocation of S. marcescens SS-1. Upstream of spnI there is a gene which we have termed spnT. spnI and spnT form an operon and although database searches failed to reveal any spnT homologues, overexpression of this novel gene negatively affected both sliding motility and prodigiosin production.     

ROS sets the stage for macrophage differentiation

While M1 macrophages are highly pro-inflammatory and microbicidal, M2 macrophages and the related tumor associated macrophages (TAMs) regulate tissue remodeling and angiogenesis and can display immunomodulatory activity. In July issue of Cell Research, Zhang et al. show that ROS production, critical for the activation and functions of M1 macrophages, is necessary for the differentiation of M2 macrophages and TAMs, and that antioxidant therapy blocks TAM differentiation and tumorigenesis in mouse models of cancer.Macrophages are key orchestrators in both the initiation and resolution stages of inflammation, and function as sentinel cells that maintain homeostasis and protect against infection. They are activated by many stimuli including pathogen-associated molecular patterns (PAMPs), endogenous danger-associated molecular patterns (DAMPs), and cytokines found in the tissue microenvironment¹. During their activation, macrophages can polarize to pro-inflammatory or anti-inflammatory states with distinct phenotypes and physiological responses — the classical pro-inflammatory M1 state induced by LPS and interferon-γ (IFN-γ) and the “alternative” M2 state triggered by IL-4 and IL-13². The M1 state is characterized by increased expression of pro-inflammatory cytokines as well as microbicidal activity, while M2 macrophages upregulate the anti-inflammatory cytokine IL-10 and participate in tissue remodeling, wound repair, and host defense against large parasites.M2-like macrophage polarization is of particular pathophysiological consequence in the setting of cancer. Early in tumor development, monocytes are recruited by tumor and stromal cell-derived chemokines to take up residence at the tumor site, where they differentiate into macrophages in response to MCSF produced by tumor cells. Such tumor-associated macrophages (TAMs) facilitate multiple steps in tumorigenesis, including promotion of tumor cell proliferation and resistance to apoptosis as well as secretion of pro-angiogenic factors and proteolytic enzymes that aid tumor cell metastasis. TAMs also display some immunosuppressive features, such as IL-10 and TGF-β production and poor antigen presentation, which conspire to prevent tumor cell killing by infiltrating T cells. Thus, the characteristics most critical for the tumor-promoting profile of TAMs bear semblance to the M2 phenotype. Although the details of such M2 polarization are not well characterized, IL-4 produced by T-cells in the tumor, as well as other tumor-derived factors, may be critical³.In July issue of Cell Research, a study by Zhang et al.⁴ provides new insights into control of macrophage differentiation and activation. In particular, the authors show that ROS production is important in M2 but not M1 macrophage differentiation. Their experimental protocol is to treat monocytes for 6 days with M-CSF or GM-CSF to induce differentiation to macrophages, followed by polarization with IL-4 (M2 state) or LPS and IFN-γ (M1 state). Interestingly, pre-treating monocytes with the antioxidant butylated hydroxyanisole (BHA) prior to differentiation inhibits M2 but not M1 polarization, as indicated by analysis of macrophage differentiation markers and M1/M2 polarization markers. The authors attribute this to the effects of BHA, i.e., block of ROS production, in inhibiting ERK activation during macrophage differentiation, consistent with previous reports implicating a role for ROS as well as MAP kinases in macrophage differentiation⁵. Furthermore, LPS and IFN-γ but not IL-4 stimulation can “rescue” ERK activation, perhaps in a manner dependent on ROS production, thus explaining why M2 but not M1 polarization is impaired by antioxidant treatment (Figure 1).Open in a separate windowFigure 1M1 macrophages are highly pro-inflammatory and microbicidal and are polarized by treatment with LPS+IFNγ, while M2 macrophages mediate tissue repair, angiogenesis and immunomodulation. Tumor associated macrophages (TAMs), which are M2-like, are associated with worsened clinical prognosis in many cancers and are thought to be skewed by a combination of tumor-derived factors and other cytokines present in the tumor microenvironment. ROS production increases during M-CSF- or GM-CSF-induced macrophage differentiation from monocytes, and the antioxidant BHA specifically inhibits M2 and TAM polarization. LPS+IFNγ treatment is able to overcome the effects of BHA to induce normal M1 polarization, revealing a specific role for ROS in macrophage polarization.As the M2-like properties of TAMs are thought to promote tumorigenesis, Zhang et al. go on to investigate the consequences of BHA administration in mouse models of cancer. They demonstrate that in vivo treatment of BHA can attenuate cancer initiation, progression, and metastasis in multiple models. As ROS can promote tumor cell proliferation, survival, and DNA damage, BHA could be acting directly on the tumor cells to prevent growth and metastasis⁶. However, BHA had no effects on the proliferation of three tumor cell lines in vitro. The authors propose that TAM differentiation may be a critical target, as BHA administration reduced TAM numbers as well as levels of TAM markers. Moreover, in at least one of the models, BHA administration was ineffective when macrophages were depleted by clodronate injection.Collectively, the findings of Zhang et al. are intriguing for several reasons. First, ROS production is usually associated with the activation and functions of M1 rather than M2 macrophages. ROS production downstream of LPS signaling mediates production of pro-inflammatory cytokines (in part through MAP kinase activation). ROS and nitric oxide (NO) production by NADPH oxidase and iNOS, respectively, as well as mROS upregulation are key to the antimicrobial activity of M1 macrophages⁷. Indeed NO production can inhibit oxidative metabolism, pivotal to the survival and function of M2 macrophages⁸. Thus ROS production may be important in M1 activation and function while the requirement for ROS in M2 differentiation may be most critical during MCSF-mediated differentiation rather than IL-4-triggered polarization. Future studies to better understand the role of ROS production in macrophage differentiation and activation may be informative. Second, it would be interesting to further probe the effects of BHA in inhibiting tumorigenesis. The authors'' in vitro studies suggest inhibition of TAM differentiation as one underlying mechanism, but one can envision additional possibilities. At least in some cancers, tumor cells and other immune cells in the microenvironment produce ROS that promote inflammation⁹, thus contributing to tumorigenesis. mROS has been linked to activation of HIF1α, which can facilitate angiogenesis and metastasis. Indeed, it is worth pointing out that ROS can regulate many cellular processes, some of which have already been alluded to, including signal transduction (e.g., downstream of growth factor receptors and innate immune signaling pathways as well as MAP kinase activation), redox signaling, autophagy, and respiratory burst and other antimicrobial activities¹⁰. Thus it is likely that other cellular processes perturbed by antioxidant treatment contribute to the effects of BHA in reducing tumorigenesis.Finally, the study by Zhang et al. suggests that treatment with BHA or perhaps other antioxidants could be considered in therapeutic control of cancer. Indeed, there is tremendous interest in the clinical use of antioxidants for treating many diseases. Given the pleiotropic activities of ROS mentioned above, it would be important to better understand the molecular pathways by which antioxidants exert their effects.     

Effects of sphondin,isolated from Heracleum laciniatum,on IL-1beta-induced cyclooxygenase-2 expression in human pulmonary epithelial cells

Recently, under large-scale screening experiments, we found that sphondin, a furanocoumarin derivative isolated from Heracleum laciniatum, possessed an inhibitory effect on IL-1beta-induced increase in the level of COX-2 protein and PGE(2) release in A549 cells. Accordingly, we examined in the present study the action mechanism of sphondin on the inhibition of IL-1beta-induced COX-2 protein expression and PGE(2) release in a human pulmonary epithelial cell line (A549). Pretreatment of cells with sphondin (10-50 microM) concentration-dependently attenuated IL-1beta-induced COX-2 protein expression and PGE(2) release. The IL-1beta-induced increase in COX-2 mRNA expression was also attenuated by sphondin (50 microM). The selective COX-2 inhibitor, NS-398 (0.01-1 microM), inhibited the activity of the COX-2 enzyme in a concentration-dependent manner, while sphondin (10-50 microM) had no effect. Sphondin (50 microM) did not affect the IL-1beta-induced activations of p44/42 MAPK, p38 MAPK, and JNK. Treatment of cells with sphondin (50 microM) or the NF-kappaB inhibitor, PDTC (50 microM) partially inhibited IL-1beta-induced degradation of IkappaB-alpha in the cytosol and translocation of p65 NF-kappaB from the cytosol to the nucleus. Furthermore, IL-1beta-induced NF-kappaB-specific DNA-protein complex formation in the nucleus was partially inhibited by sphondin (50 microM) or PDTC (50 microM). Taken together, we demonstrate that sphondin inhibits IL-1beta-induced PGE(2) release in A549 cells; this inhibition is mediated by suppressing of COX-2 expression, rather than by inhibiting COX-2 enzyme activity. The inhibitory mechanism of sphondin on IL-1beta-induced COX-2 expression may be, at least in part, through suppression of NF-kappaB activity. We conclude that sphondin may have the therapeutic potential as an anti-inflammatory drug on airway inflammation.     

Ammonia excretion by the skin of zebrafish (Danio rerio) larvae

The mechanism of ammonia excretion in freshwater teleosts is not well understood. In this study, scanning ion-selective electrode technique was applied to measure H(+) and NH(4)(+) fluxes in specific cells on the skin of zebrafish larvae. NH(4)(+) extrusion was relatively high in H(+) pump-rich cells, which were identified as the H(+)-secreting ionocyte in zebrafish. Minor NH(4)(+) extrusion was also detected in keratinocytes and other types of ionocytes in larval skin. NH(4)(+) extrusion from the skin was tightly linked to acid secretion. Increases in the external pH and buffer concentration (5 mM MOPS) diminished H(+) and NH(4)(+) gradients at the larval surface. Moreover, coupled decreases in NH(4)(+) and H(+) extrusion were found in larvae treated with an H(+)-pump inhibitor (bafilomycin A1) or H(+)-pump gene (atp6v1a) knockdown. Knockdown of Rhcg1 with morpholino-oligonucleotides also decreased NH(4)(+) excretion. This study demonstrates ammonia excretion in epithelial cells of larval skin through an acid-trapping mechanism, and it provides direct evidence for the involvement of the H(+) pump and an Rh glycoprotein (Rhcg1) in ammonia excretion.