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Expression of PUMA in Follicular Granulosa Cells Regulated by FoxO1 Activation During Oxidative Stress.

ABSTRACT: Many studies have demonstrated that oxidative stress-induced apoptosis is a main cause of follicular atresia. Reactive oxygen species (ROS)-induced granulosa cell (GC) apoptosis is regulated by a variety of signaling pathways involving numerous genes and transcription factors. In this study, we found expression of the p53-upregulated modulator of apoptosis (PUMA), a BH3-only Bcl-2 subfamily protein, in ovarian GCs during oxidative stress. By overexpression and knockdown of Forkhead box O1 (FoxO1), we found that FoxO1 regulates PUMA at the protein level. Moreover, as c-Jun N-terminal kinase (JNK) has been shown to activate FoxO1 by promoting its nuclear import, we used a JNK inhibitor to reduce FoxO1 activation and detected decreased PUMA messenger RNA expression and protein levels during oxidative stress. In addition, in vivo oxidative stress-induced upregulation of PUMA was found following injection of 3 nitropropionic acid in mice. In conclusion, oxidative stress increases PUMA expression regulated by FoxO1 in follicular GCs.

SUBMITTER: Liu ZQ

PROVIDER: S-EPMC4502799 | biostudies-other | 2015 Jun

REPOSITORIES: biostudies-other

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Expression of PUMA in Follicular Granulosa Cells Regulated by FoxO1 Activation During Oxidative Stress.

Liu Ze-Qun ZQ Shen Ming M Wu Wang-Jun WJ Li Bo-Jiang BJ Weng Qian-Nan QN Li Mei M Liu Hong-Lin HL

Reproductive sciences (Thousand Oaks, Calif.) 20141125 6

Many studies have demonstrated that oxidative stress-induced apoptosis is a main cause of follicular atresia. Reactive oxygen species (ROS)-induced granulosa cell (GC) apoptosis is regulated by a variety of signaling pathways involving numerous genes and transcription factors. In this study, we found expression of the p53-upregulated modulator of apoptosis (PUMA), a BH3-only Bcl-2 subfamily protein, in ovarian GCs during oxidative stress. By overexpression and knockdown of Forkhead box O1 (FoxO1 ...[more]

PMID: 25425107

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Project description:Oxidative stress is the major culprits responsible for ovarian dysfunction by damaging granulosa cells (GCs). Ferritin heavy chain (FHC) may participate in the regulation of ovarian function by mediating GCs apoptosis. However, the specific regulatory function of FHC in follicular GCs remains unclear. Here, 3-nitropropionic acid (3-NPA) was utilized to establish an oxidative stress model of follicular GCs of Sichuan white geese. To explore the regulatory effects of FHC on oxidative stress and apoptosis of primary GCs in geese by interfering or overexpressing FHC gene. After transfection of siRNA-FHC to GCs for 60 h, the expressions of FHC gene and protein decreased significantly (P < 0.05). After FHC overexpression for 72 h, the expressions of FHC mRNA and protein upregulated considerably (P < 0.05). The activity of GCs was impaired after interfering with FHC and 3-NPA coincubated (P < 0.05). When overexpression of FHC combined with 3-NPA treatment, the activity of GCs was remarkably enhanced (P < 0.05). After interference FHC and 3-NPA treatment, NF-κB and NRF2 gene expression decreased (P < 0.05), the intracellular reactive oxygen species (ROS) level increased greatly (P < 0.05), BCL-2 expression reduced, BAX/BCL-2 ratio intensified (P < 0.05), the mitochondrial membrane potential decreased notably (P < 0.05), and the apoptosis rate of GCs aggravated (P < 0.05). While overexpression of FHC combined with 3-NPA treatment could promote BCL-2 protein expression and reduce BAX/BCL-2 ratio, indicating that FHC regulated the mitochondrial membrane potential and apoptosis of GCs by mediating the expression of BCL-2. Taken together, our research manifested that FHC alleviated the inhibitory effect of 3-NPA on the activity of GCs. FHC knockdown could suppress the expression of NRF2 and NF-κB genes, reduce BCL-2 expression and augment BAX/BCL-2 ratio, contributing to the accumulation of ROS and jeopardizing mitochondrial membrane potential, as well as exacerbating GCs apoptosis.

Project description:Polycystic ovarian ovary syndrome (PCOS) is the main cause of ovulatory infertility and a common reproductive endocrine disease of women in reproductive age. In addition, nearly half of PCOS patients are associated with obesity, and their total free fatty acids tend to increase. Arachidonic acid (AA) is a polyunsaturated fatty acid. Oxidation products of AA reacting with various enzymes[cyclooxygenases (COX), lipoxygenases (LOX), cytochrome P450s (CYP)] can change cellular mitochondrial distribution and calcium ion concentration, and increase reactive oxygen species (ROS) production. In this study, we analyzed the follicular fluid fatty acids and found higher levels of C20:4n6 (AA) in PCOS patients than in normal control subjects. Also, to determine whether AA induces oxidative stress (OS) in the human ovarian granulosa tumor cell line (KGN) and affects its function, we treated KGN cells with or without reduced glutathione (GSH) and then stimulated them with AA. The results showed that AA significantly reduced the total antioxidant capacity (TAC) and activity of antioxidant enzymes and increased the malondialdehyde (MDA), ROS and superoxide anion(O2-)levels in KGN cells. In addition, AA was also found to impair the secretory and mitochondrial functions of KGN cells and induce their apoptosis. We further investigated the downstream genes affected by AA in KGN cells and its mechanism of action. We found that AA upregulated the expression of growth differentiation factor 15 (GDF15), which had a protective effect on inflammation and tissue damage. Therefore, we investigated whether AA-induced OS in KGN cells upregulates GDF15 expression as an OS response.Through silencing of GDF15 and supplementation with recombinant GDF15 (rGDF15), we found that GDF15, expressed as an OS response, protected KGN cells against AA-induced OS effects, such as impairment of secretory and mitochondrial functions and apoptosis. Therefore, this study suggested that AA might induce OS in KGN cells and upregulate the expression of GDF15 as a response to OS.

Dataset Information

Expression of PUMA in Follicular Granulosa Cells Regulated by FoxO1 Activation During Oxidative Stress.

Publications

Expression of PUMA in Follicular Granulosa Cells Regulated by FoxO1 Activation During Oxidative Stress.

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