Opposing effects of oestradiol and progesterone on intracellular pathways and activation processes in the oxidative stress induced activation of cultured rat hepatic stellate cells - PubMed (original) (raw)

Opposing effects of oestradiol and progesterone on intracellular pathways and activation processes in the oxidative stress induced activation of cultured rat hepatic stellate cells

T Itagaki et al. Gut. 2005 Dec.

Erratum in

Abstract

Background: Oxidative stress, including the generation of reactive oxygen species (ROS), is involved in hepatofibrogenesis. The authors' previous studies have shown that oestradiol suppresses hepatic fibrosis in animal models and attenuates the activation of cultured rat hepatic stellate cells (HSCs), which possess oestrogen receptor subtype beta and are also activated by ROS.

Aims: To define the mechanisms by which female sex hormones play an antifibrogenic role in activated HSCs, the effects of oestradiol and progesterone on ROS generation processes and intracellular pathways, leading to the activation of HSCs undergoing oxidative stress, was examined.

Methods: HSCs, isolated from rats, were cultured for 7 days with oestradiol or progesterone for 24 hours as pretreatment, and oxidative stress was then induced by exposure to low doses of hydrogen peroxide for another 24 hours.

Results: Oestradiol inhibited ROS generation and antioxidant enzyme loss via the suppression of NADH/NADPH oxidase activity, and attenuated hydrogen peroxide induced transforming growth factor-beta1 (TGF-beta1) expression, HSC proliferation and transformation, and the activation of mitogen activated protein kinase (MAPK) pathways and transcription factors. Progesterone exerted a stimulatory effect through the progesterone receptor on the induction of ROS generation processes and intracellular pathways, resulting in TGF-beta1 expression and HSC activation, and fibrogenic effects were inhibited by oestradiol.

Conclusion: These findings show for the first time that oestradiol inhibits the activation of transcription factors by suppressing ROS generation processes and the MAPK pathways, and inactivates the downstream transcription processes involved in TGF-beta1 expression and HSC activation, whereas progesterone acts in opposition to the favourable effects of oestradiol and its effects are blocked by oestradiol.

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Figures

Figure 1

Figure 1

Western blot, immunohistochemical, and RT-PCR analyses of progesterone receptor in cultured HSCs from male and female rats. (A) On culture day seven, HSCs, preincubated in serum-free DMEM for 24 hours, were then incubated with (oxidative stress) and without (none) 10−5 mol/l hydrogen peroxide for another 24 hours. Cell lysates were subjected to 12% SDS-polyacrylamide gel electrophoresis and transferred onto nylon membranes, and progesterone receptor was detected immunologically. The graphs represent typical results of five independent experiments. (B) Reaction of cultured HSCs from males with an antibody against progesterone receptor (C-20). Original magnification ×200. (C) RT-PCR showed 320 bp transcripts for progesterone receptor in cultured HSCs from males as well as the uterus as a positive control. One of four similar studies is shown. PR, progesterone receptor.

Figure 2

Figure 2

Activation of MAPK pathways and transcription factors of AP-1 and NF-κB after exposure to hydrogen peroxide in cultured rat HSCs. On culture day seven, HSCs, preincubated in serum-free DMEM for 24 hours, were then exposed to 10−5 mol/l hydrogen peroxide for another 24 hours. Following exposure to hydrogen peroxide, MAPK pathways of ERK, p38, and JNK (A) and transcription factors of AP-1 and NF-κB (B) were activated and IκB-α was degraded (C) in a time dependent manner. The micrographs represent typical results of three independent experiments.

Figure 3

Figure 3

Stimulation of proliferation and αSMA expression and intracellular levels of TGF-β1 mRNA and protein after exposure to hydrogen peroxide in cultured rat HSCs. On culture day seven, HSCs, preincubated in serum-free DMEM for 24 hours, were then exposed to 10−5 mol/l hydrogen peroxide for another 24 hours. Following exposure to hydrogen peroxide, αSMA expression (A), DNA synthesis (B), and intracellular levels of TGF-β1 mRNA and protein (C) were increased after hydrogen peroxide exposure. DNA synthesis in the presence of PDGF-BB (5 ng/ml) for 1 hour, as a positive control, was 142 (SD 10)%. Results of densitometric analysis are presented as the mean percentages of β-actin signal intensity of for αSMA expression (B). The levels of TGF-β1 gene expression were quantitatively analysed by real-time PCR, and the results are expressed in arbitrary units (C). Values are means (SD) for six dishes. *p<0.05 compared with cultures before hydrogen peroxide exposure.

Figure 4

Figure 4

Effects of oestradiol and progesterone on the activation of MAPK pathways and transcription factors of AP-1 and NF-κB in cultured rat HSCs with and without hydrogen peroxide exposure. On culture day seven, oxidative stress was induced in HSCs by exposure to 10−5 mol/l hydrogen peroxide (oxidative stress) for 24 hours in pretreatment in serum-free DMEM with and without oestradiol (10−9–10−7 mol/l) or progesterone (10−7–10−6 mol/l) in the presence and absence of 10−6 mol/l ICI 182,780 (ICI) or 10−6 mol/l RU486 (RU) for 24 hours. After exposure to hydrogen peroxide, activation of the MAPK pathways of ERK, p38, and JNK (A) was evaluated at 30 minutes, activation of transcription factors of AP-1 and NF-κB (B) with the degradation of IκB-α (C) at 1 hour. The micrographs represent typical results of three independent experiments.

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