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First published online 28 February 2006
doi: 10.1242/jcs.02798

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Regulation of cardiotrophin-1 expression in mouse embryonic stem cells by HIF-1 and intracellular reactive oxygen species

¹ Department of Physiology, University of Giessen, 35392 Giessen, Germany
² GKSS Research Institute, Department of Cell Biology, 14513 Teltow, Germany
³ Institute of Veterinary Physiology, Vetsuisse Faculty, University of Zurich, and Zurich Center for Integrative Human Physiology (ZIHP), 8057 Zurich, Switzerland

View larger version (17K): [in a new window]   Fig. 1. Protein expression of CT-1 (A) and gp130 (B) during of ES cell differentiation. Determination was performed within the three-dimensional tissue of embryoid bodies. Note that cardiac cell differentiation occurs during day 6 and 8 of embryoid body cell culture (Hescheler et al., 2002). *P<0.05, significantly different to levels at day 1 and day 2 of cell culture in A and B, respectively.

View larger version (32K): [in a new window]   Fig. 2. Regulation of CT-1 and HIF-1 expression in embryoid bodies by endogenous ROS. Embryoid bodies were incubated from day 2 to day 8 of cell culture with 20 µM vitamin E. On day 8 protein expression of CT-1 and HIF-1 was assessed by semiquantitative immunohistochemistry in whole-mount embryoid bodies. The images show representative embryoid bodies labeled with antibodies either against CT-1 or HIF-1 which remained untreated (a,c) or were treated with vitamin E (b,d). *P<0.05, significantly different to levels in the untreated control.

View larger version (44K): [in a new window]   Fig. 3. Upregulation of CT-1 and HIF-1 protein and mRNA upon treatment with prooxidants and hypoxia. (A,B) Embryoid bodies were treated on day 4 with menadione (20 µM) and H2O2 (10 µM). Protein (A) and mRNA (B) levels were examined 24 hours later. The images above A show representative embryoid bodies labeled with antibodies against CT-1 (left panel) and HIF-1 (right panel) which remained either untreated (a,d) or were treated with menadione (b,e) or H2O2 (c,f). (C,D) Embryoid bodies were treated on day 4 of cell culture with either physiological (1% O2) or chemical (CoCl2) hypoxia and protein (C) and mRNA (D) levels were examined 24 hours later. The images above C show representative embryoid bodies labeled with antibodies against CT-1 (left panel) and HIF-1 (right panel) which remained either untreated (a,d) or were treated with 1% O2 (b,e) or CoCl2 (50 µM) (c,f). *P<0.05, levels are significantly different to those in the untreated controls. Bars, 200 µm.

View larger version (12K): [in a new window]   Fig. 4. Time course of HIF-1 and CT-1 upregulation upon treatment with either menadione (A) or CoCl2 (B). Protein expression was assessed 2, 4, 8 and 24 hours after treatment. *,#P<0.05, significantly different when compared with levels in untreated samples.

View larger version (22K): [in a new window]   Fig. 5. Generation of ROS following exposure to menadione or chemical hypoxia (CoCl2 treatment). (A) Embryoid bodies were treated for 24 hours with 20 µM menadione. Subsequently, the cell culture medium was exchanged with medium devoid of menadione, and ROS were monitored at different times after removal of menadione. (B) ROS generation in the presence of either menadione (20 µM) or CoCl2 (50 µM). Determination was performed immediately after exposure. The upregulation of CT-1 by menadione (C) and CoCl2 (D) was inhibited by either the free radical scavenger vitamin E (20 µM) or NMPG (20 µM). Embryoid bodies were treated at day 4 of cell culture with either menadione (20 µM) or CoCl2 (50 µM) in the presence of free radical scavengers. After 24 hours CT-1 protein expression was determined by semiquantitative immunohistochemistry. *P<0.05, significantly different to levels in the relative controls. #P<0.05, significantly different to levels in the CoCl2 or menadione-treated samples before addition of scavengers.

View larger version (43K): [in a new window]   Fig. 6. Upregulation of NADPH oxidase subunits upon incubation of embryoid bodies with menadione and CoCl2. (A) Protein expression of the NADPH oxidase subunits p22-phox, p47-phox, p69-phox, Nox-1 and Nox-4 which was determined 24 hours after treatment of 4-day-old embryoid bodies by semiquantitative immunohistochemistry. (B) mRNA expression of Nox1 and Nox4 upon either menadione (20 µM) or CoCl2 (50 µM) treatment. (C) The upregulation of CT-1 by menadione and CoCl2 was inhibited by preincubation with the NADPH oxidase inhibitors DPI (10 µM) or apocynin (10 µM). Embryoid bodies were treated at day 4 of cell culture with either menadione (20 µM) or CoCl2 (50 µM) in the presence of either DPI or apocynin. After 24 hours, CT-1 protein expression was determined by semiquantitative immunohistochemistry. The micrographs are representative embryoid bodies either untreated (a), or treated with menadione (b), CoCl2 (c), DPI (d), DPI + menadione (e), DPI + CoCl2 (f), apocynin (g), apocynin + menadione (h), apocynin + CoCl2 (i). *P<0.05, significantly different from levels in the control. #P<0.05, significantly different to levels in menadione- and CoCl2-treated samples before addition of inhibitors. Bar, 400 µm.

View larger version (35K): [in a new window]   Fig. 7. Upregulation of gp130 leads to a complex signalling cascade upon treatment of embryoid bodies with prooxidants or chemical hypoxia. (A) Embryoid bodies were treated at day 4 of cell culture with either H2O2 (10 µM), menadione (20 µM) or CoCl2 (50 µM). After 24 hours, gp130 protein expression and phosphorylation was assessed by quantitative immunohistochemistry using an antibody directed against unphosphorylated gp130 or the phosphorylated form of gp130. (B) Activation of ERK1,2, JNK p38 and PI3-kinase upon incubation of embryoid bodies with either menadione (20 µM) or CoCl2 (50 µM). Activation was assessed by semiquantitative immunohistochemistry using phospho-specific antibodies. Maximum activation of ERK1,2 was achieved after 15 minutes; maximum activation of JNK was at 15 minutes for menadione treatment and 30 minutes for CoCl2 treatment; maximum activation of p38 was at 30 minutes for menadione treatment and 60 minutes for CoCl2 treatment; maximum activation of PI3-kinase was at 60 minutes. (C) Inhibition of menadione- and CoCl2-mediated upregulation of CT-1 by the ERK1,2 inhibitor UO126 (10 µM), the JNK inhibitor SP600125 (10 µM), the p38 inhibitor SKF86002 (10 µM), and the PI3-kinase inhibitor LY294002 (20 µM). Embryoid bodies were treated at day 4 of cell culture with either menadione (20 µM) or CoCl2 in the presence of the respective inhibitor. CT-1 protein expression was assessed after 24 hours by semiquantitative immunohistochemistry. *P<0.05, significantly different from levels in the untreated control; #P<0.05, significantly different to levels before addition of inhibitors as indicated.

View larger version (36K): [in a new window]   Fig. 8. Upregulation of CT-1 mediated by menadione and CoCl2 is inhibited by the HIF-1 inhibitor 2-ME (3 µM). Embryoid bodies were treated at day 4 of cell culture with either menadione (20 µM) or CoCl2 in the presence of 2-ME. CT-1 protein expression was assessed after 24 hours by semiquantitative immunohistochemistry. *P<0.05, significantly different from levels in the untreated control; #P<0.05, significantly different to levels before addition of 2-ME.

View larger version (21K): [in a new window]   Fig. 9. (A) Cardiac cell differentiation in wild-type (wt) and HIF-1-/- ES cells. Expression of CT-1 during differentiation (B) and upon incubation with menadione (20 µM) and CoCl2 (50 µM) (C,D). In HIF-1-/- ES cells cardiomyogenesis was completely absent as evaluated by counting the number of spontaneously contracting embryoid bodies (A). Furthermore, no upregulation of CT-1 mRNA was observed in HIF-1-/- compared with the wild type (B). Menadione as well as CoCl2 failed to upregulate CT-1 mRNA (C) and protein (D) in HIF-1-/- cells. *P<0.05, significantly different to levels in wild-type embryoid bodies.

View larger version (39K): [in a new window]   Fig. 10. Scheme of the proposed signal transduction cascade activated by ROS and hypoxia. Either hypoxia (initiating elevated ROS generation) or exogenous addition of ROS induces phosphorylation of gp130 and initiates a feed-forward cycle of NADPH oxidase activity and expression. ROS generated by NADPH oxidase act as signalling molecules within the CT-1/gp130 signalling cascade which finally results in HIF-1 upregulation and stimulation of CT-1 expression.