Effects of E2 on the FC-induced IκBα phosphorylation and nuclear translocation of NFκB in the CEC.

<p>(a) The FC-induced increases of the levels of p-IκBα in the CEC were abolished by E2 (100 nM) treatment. Membrane was probed with anti-G3PDH antibody to verify equivalent loading. (b) The FC-induced an increase of p65 nuclear translocation in the CEC was abolished by E2 (100 nM) treatment. PARP and G3PDH were used as a nuclear and cytosolic protein marker, respectively, to confirm the purities of isolation and to verify equivalent loading. (c) E2 and FC induced nuclear translocation of ER and p65, respectively. After treatment with FC for 4 h, the CEC was fixed and then labeled with an anti-p65 antibody followed by a Rhodamine-conjugated secondary antibody and an anti-ER antibody followed by a FITC-conjugated secondary antibody. The nuclei were visualized with Hoechst (1 µg/mL) staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084320#s2" target="_blank">Materials and Methods</a>. Bar  = 25 µm. Co, control; E2, estradiol; FC, ferrous citrate.</p

Involvement of the ER subtype on the E2-mediated inhibition of the FC-induced NOS2 up-regulation.

<p>(a) Pre-transfection of CEC with ERα siRNA did not significantly affect the E2-mediated inhibition on the FC-induced increases of the levels of NOS2 protein. (b) Pre-transfection of CEC with ERβ siRNA abolished the E2-mediated inhibition on the FC-induced increases of the levels of NOS2 protein. Top panel: representative results of NOS2 and G3PDH protein levels determined by Western blot analysis. Membrane was probed with anti-G3PDH antibody to verify equivalent loading. Bottom panel: quantitative results of NOS2 protein levels, which were adjusted with G3PDH protein level and expressed as fold-induction of its own control. Values represent the means±s.e.mean. (n = 3). ^*<i>P</i><0.05, different from cells treated with FC. (c) Pre-transfection of CEC with ERα siRNA did not significantly affect the E2-mediated inhibition on the FC-induced increases of the levels of NOS2 mRNA. (d) Pre-transfection of CEC with ERβ siRNA abolished the E2-mediated inhibition on the FC-induced increases of the levels of NOS2 mRNA. The levels of mRNA were detected by RT-PCR (top panel) or real-time quantitative PCR (bottom panel). ER, estrogen receptor; N-T Si, non-target siRNA; Si, small interfering RNA.</p

Effects of E2 on the FC-induced increases of NFκB binding onto the NOS2 promoter and NOS2 luciferase promoter activity in the CEC.

<p>(a) The FC-induced increases of p65 binding onto the NOS2 promoter DNA in the CEC was abolished by E2 (100 nM) treatment. (b) The prevention effect of E2 (100 nM) on the FC-induced increases of p65 binding onto the NOS2 promoter DNA in the CEC was blocked by pre-treatment of the cell with an ER antagonist, ICI 182,780 (5 µM). (c) E2 increased the formation of the p65-ER complex in the nucleus of the CEC. This effect was blocked by pre-treatment of the cell with ICI (5 µM). P65 was immunoprecipitated by anti-p65 antibody, and ER-p65 association was detected by anti-ER antibody. PARP and G3PDH were used as a nuclear and cytosolic protein marker, respectively, to confirm the purities of isolation and to verify equivalent loading. (d) The CEC was transiently transfected with the mouse NOS2 promoter DNA for 24 h, and then treated with vehicle, FC (100 µM), E2 (100 nM), ICI (5 µM), or in combination for 16 h. Subsequently, the cell was processed for the luciferase activity assay. Quantitative results of the NOS2 promoter activity were shown and expressed as fold induction of the CEC treated with vehicle (control). Values represent the means±s.e.mean. (n = 4). ^*<i>P</i><0.05, different from cells treated with FC. CHIP, chromatin immunoprecipitation; IB, immunoblotting; IP, immunoprecipitation; E2, estradiol; ER, estrogen receptor; FC, ferrous citrate; ICI, ICI 182,780.</p

Effects of E2 on the FC-induced increases of intracellular ROS levels in the CEC.

<p>The FC-induced increases of ROS in the CEC were reduced by E2 (100 nM) treatment. This effect was blocked by pre-treatment of the cell with an ER antagonist, ICI (5 µM). Top panel: representative photographs of ROS generation. ROS levels were assayed using 5 µM DCF as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084320#s2" target="_blank">Materials and Methods</a>. DCF fluorescence images and DIC images were taken using Leica TCS SP5 fluorescent microscope imaging system (Wetzlar, Germany). Bottom panel: quantitative results of ROS levels. Values represent the means±s.e.mean. (n = 4). ^*<i>P</i><0.05 different from FC-treated group. DCF, dichlorodihydrofluorescein diacetate [CM-H2DCF-DA]; DIC, differential interference contrast; E2, estradiol; FC, ferrous citrate; ICI, ICI 182,780.</p

Effects of E2 on the FC-induced increases of the levels of NOS2 mRNA and protein in the CEC.

<p>The FC-induced increases of the levels of NOS2 protein (a) at 24 h after FC treatment and mRNA (b) at 16 h after FC (100 µM) treatment in the CEC were abolished by E2 (100 nM) treatment. This effect was blocked by pre-treatment of the cell with an ER antagonist, ICI (5 µM). Quantitative results of NOS2 protein levels, which were adjusted with G3PDH protein level and expressed as fold-induction of its own control. Values represent the means±s.e.mean. (n = 4). ^*<i>P</i><0.05 different from control group. ^#<i>P</i><0.05 different from FC-treated group. ^§<i>P</i><0.05 different from combined treatment with FC and E2 group. E2, estradiol; FC, ferrous citrate; ICI, ICI 182,780.</p

Proposed signaling pathway associated with E2-caused reduction of the FC-induced NOS2 up-regulation in the CEC.

<p>E2 suppressed the NFκB-induced increases of NOS2 through (1) maintenance of steady level of inhibitor IκBα in the cytoplasm; (2) prevention of cytosolic NFκB from translocation into the nucleus; and (3) interference of NFκB binding onto the <i>NOS2</i> promoter DNA by physical association of ERβ with NFκB.</p

Effects of FC on NOS2 expression in the CEC.

<p>(A) The levels of NOS2 mRNA in the CEC were significantly increased at 8 h after FC treatment. A peak of NOS2 mRNA level was observed at 8–16 h after FC treatment, and then began to decline. The levels of NOS2 mRNA were determined using quantitative real time-PCR, adjusted with G3PDH mRNA, and expressed as ratio over control. (B) FC (100 µM) treatment increased the levels of NOS2 protein in the CEC. FC (100 µM) time-dependently increased the level of NOS2 protein. Top panel: representative results of NOS2 and G3PDH protein levels determined by Western blot analysis. Bottom panel: quantitative results of NOS2 protein levels, which were adjusted with G3PDH protein level and expressed as fold-induction of its own control. Values represent the means±s.e.mean. (n = 3). ^*<i>P</i> < 0.05 different from corresponding control. (C) The FC-induced increases of the level of NOS2 protein were in a concentration-dependent manner. Top panel: representative results of the levels of NOS2 and G3PDH protein determined by Western blot analysis. Bottom panel: quantitative results of NOS2 protein levels, which were adjusted with G3PDH protein level and expressed as fold of control. Values represent the means±s.e.mean. (n = 3). ^*<i>P</i> < 0.05 different from corresponding control. (D) FC-induced NOS2 expression is mainly located in the CEC. Micrographs show NOS2 (green) and vWF (red) immunoreactivity detected by dual immunofluorescent staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046239#s2" target="_blank">Materials and Methods</a>. NOS2, inducible nitric oxide synthase 2; G3PDH, glyceraldehyde3-phosphate dehydrogenase, vWF, von Willebrand Factor.</p

FC increases ROS generation in the CEC.

<p>(A) FC (1–100 µM) concentration-dependently increased ROS generation in the CEC. (B) The ROS generation was observed at 5 min after FC (100 µM) treatment. (C) FC (100 µM)-induced increases of ROS generation in CEC were prevented by pretreatment of the cell with a ROS scavenger, NAC (5 mM). ROS levels were assayed using 5 µM DCF as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046239#s2" target="_blank">Materials and Methods</a>. DCF fluorescence images and DIC images were taken using Leica TCS SP5 fluorescent microscope imaging system (Wetzlar, Germany) and the levels of ROS were quantified by flow cytometric analysis. Values represent the means±s.e.mean. (n = 4). ^*<i>P</i> < 0.05 different from control. ^#<i>P</i> < 0.05 different from FC-treated group. DIC, differential interference contrast; DCF, dichlorodihydrofluorescein diacetate [CM-H2DCF-DA].</p

FC induces NFκB activation through increases of the levels of ROS and phosphorylated IκBα.

<p>(A) Pretreatment of the CEC with a ROS scavenger, NAC (5 mM), for 1 h prevented the FC-induced increases of the levels of NOS2 protein. Top panel: representative results of NOS2 and G3PDH protein levels determined by Western blot analysis. Bottom panel: quantitative results of NOS2 protein levels, which were adjusted with G3PDH protein level and expressed as fold of control. Values represent the means±s.e.mean. (n = 3). ^*<i>P</i> < 0.05 different from corresponding control; ^#<i>P</i> < 0.05 different from the FC-treated CEC. (B) The FC (100 µM)-induced an increase of the NFκB (p65) DNA binding onto the NOS2 promoter was completely abolished by pretreatment of the cell with NAC (5 mM). The NOS2 DNA binding activity was assessed by using ChIP assay and detected by semiquantitative PCR (top panel) and quantitative real-time PCR (bottom panel). Values represent the means±s.e.mean. (n = 3). ^*<i>P</i> < 0.05 different from corresponding control; ^#<i>P</i> < 0.05 different from the FC-treated CEC. (C) FC-induced an increase of the NOS2 promoter activity was abolished by pretreatment of the cell with NAC. Values represent the means±s.e.mean. (n = 3). ^*<i>P</i> < 0.05, different from control. (D) Pretreatment of the CEC with NAC (5 mM) for 1 h prevented the FC-induced increases of the levels of phosphorylated IκBα (p-IκBα) protein. Top panel: representative results of the levels of p-IκBα and total IκBα (t-IκBα) protein determined by Western blot analysis. Bottom panel: quantitative results of p-IκBα protein levels, which were adjusted with t-IκBα protein level and expressed as fold of control. Values represent the means±s.e.mean. (n = 3). ^*<i>P</i> < 0.05 different from corresponding control; ^#<i>P</i> <0.05 different from the FC-treated CEC. (E) FC increased phosphorylation of the protein IκBα and NFκB binding on the NOS2 promoter, and these effects were blocked by a ROS scavenger (NAC), an IKK inhibitor (Bay 11-7082), or NFκB translocation inhibitors (PTDC). The levels of NOS2 protein were quantified by Western blot analysis. The NFκB DNA binding activity was assessed by using ChIP assay and quantitative real-time PCR (bottom panel). Values represent the means±s.e.mean. (n = 3). ^*<i>P</i> < 0.05, different from corresponding control; ^#<i>P</i> < 0.05, different from FC-treated without ROS scavenger (NAC).</p

Involvement of NFκB (p65) in regulating the NOS2 promoter activity in the FC-treated CEC.

<p>(A) FC (100 µM) induced NFκB translocation from the cytosolic fraction into the nuclear fraction in the CEC. After treatment with FC for 4 h, the CEC was fixed and then labeled with an anti-NFκB antibody, followed by an FITC-conjugated secondary antibody. The nuclei were visualized with propidium iodide (50 µg/mL) staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046239#s2" target="_blank">Materials and Methods</a>. (B) FC (100 µM) increased nuclear translocation of NFκB in the CEC. Top panel: representative results of NFκB, PARP and G3PDH protein levels determined by Western blot analysis. Bottom panel: quantitative results of NFκB protein levels, which were adjusted with PARP protein level and expressed as fold of control. Values represent the means±s.e.mean. (n = 4). ^*<i>P</i> < 0.05 different from corresponding control. (C) FC (100 µM) induced an increase of NFκB binding onto the NOS2 promoter. The levels of NFκB binding onto the NOS2 promoter were assessed by using ChIP assay (top panel) and quantitative real-time PCR (bottom panel). Values represent the means±s.e.mean. (n = 4). ^*<i>P</i> < 0.05 different from control. P.I., propidium iodide.</p