PIM2-mediated phosphorylation contributes to granulosa cell survival via resisting apoptosis during folliculogenesis.
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ABSTRACT:
SUBMITTER: Wang L
PROVIDER: S-EPMC7943893 | biostudies-literature | 2021 Mar
REPOSITORIES: biostudies-literature
Publications
PIM2-mediated phosphorylation contributes to granulosa cell survival via resisting apoptosis during folliculogenesis.
Wang Lei L Chen Yaru Y Wu Shang S Wang Ling L Tan Feng F Li Fenge F
Clinical and translational medicine 20210301 3
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Project description:Women with diminished ovarian reserve (DOR) have reduced fertility, but the underlying regulation of ovarian function remains unknown. Although differential microRNA (miRNA) expression has been described in several ovarian disorders, little is known about the role of miRNAs in the pathogenesis of DOR. In this study, we investigated the expression levels of miR-484 in granulosa cells (GCs) derived from human follicular fluid, and explored their correlation with female ovarian reserve function as well as clinical outcomes of assisted reproduction technology (ART). Additionally, we investigated the effects of miR-484 on the biological functions of GC cell lines in vitro. We found that miR-484 was highly expressed in GCs from DOR patients and was correlated with decreasing AMH levels and AFC, as well as increasing FSH levels, but not with LH, progesterone, or estradiol. Additionally, miR-484 was negatively related to the number of retrieved oocytes and the ratio of high-quality embryos. Moreover, we found that miR-484 repressed the proliferation of GCs and induced apoptosis, which can in part be attributed to mitochondrial dysfunction. Conversely, silencing miR-484 had the opposite effect. Multiple approaches, including bioinformatic analysis, RNA-seq, qPCR, immunofluorescence, western blotting and luciferase reporter assays, identified YAP1 as a direct target of miR-484 in GCs. Additionally, reintroduction of YAP1 rescued the effects of miR-484 in GCs. The present study indicates that miR-484 can directly target the mRNA of YAP1, induce mitochondrial dysfunction, and consequently reduce the viability and promote the apoptosis of granulosa cells, which contributes to the pathogenesis of DOR.
| S-EPMC8771835 | biostudies-literature
Project description:Follicular atresia is one of the main processes for the loss of granulosa cells and oocytes from the mammalian ovary and any impairment to premature ovarian failure. Large numbers of studies have demonstrated that granulosa cell apoptosis causes follicular atresia, yet the rescue of these cells remains elusive. We aimed to use Esculentoside A (3-O-b-D-glucopyranosyl-1, 4-b-D-xylopyranosyl) phytolaccagenin, a saponin extracted from Phytolacca esculenta roots, as a potential rescue agent for the apoptosis of granulosa cells. Our results revealed the rescue of normal body and ovary weights, normal ovarian histo-architecture of ovaries, and hormones levels with regular estrus cycle. Consistently, the expression of proliferating and anti-apoptotic markers, i.e. KI67 and BCL-2 in granulosa cells, was enhanced. Meanwhile, the expressions of pro-apoptotic markers, which were BAX and CASPASEs (CASPASE-9 and CASPASE-3), were prominently reduced in Esculentoside A-induced premature ovarian failure mice. Additionally, PPAR?, a potential therapeutic target, has also rescued its expression by treating the premature ovarian failure mice with Esculentoside A. Our results advocated that Esculentoside A could restore folliculogenesis in premature ovarian failure mice. Furthermore, it has the potential to be investigated as a therapeutic agent for premature ovarian failure.
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Project description:PurposePolycystic ovary syndrome (PCOS) is a leading cause of anovulatory infertility and closely correlated with apoptosis in ovarian granulosa cells (GCs). Programmed cell death protein-1/programmed cell death ligand-1 (PD-1/PD-L1) pathway plays an important role throughout gestation and the pathogenesis of adverse pregnancy outcomes, but its mechanism in PCOS remains unclear.MethodsThe RNA sequencing data for PCOS patients were downloaded from the Gene Expression Omnibus (GEO) databases. Bioinformatics analysis was conducted to identify differentially expressed PD-1/PD-L1 pathway genes (DEPPGs) and related signaling pathways. PCOS mouse model was established by injecting dehydroepiandrosterone (DHEA), apoptosis of ovarian GCs in PCOS mouse were detected by TUNEL staining. The main genes and proteins in the PI3K/AKT signaling pathway and apoptosis were detected by qRT-PCR and Western blot analyses after PD-L1 intervention in GCs. Finally, the hub gene of differentially expressed PI3K/AKT pathway genes (DEPAGs) in GCs was evaluated in PCOS patients.ResultsThe DEPPGs in GCs and oocyte were identified, showing enrichment in Th1 and Th2 cell differentiation, apoptosis, and PI3K/AKT signaling pathway. More apoptosis was observed in ovarian GCs of PCOS mice. In vitro experiments showed that PI3K/AKT pathway was activated and the apoptosis of GCs was suppressed after PD-L1 intervention. The hub gene COL1A1 was upregulated in the GCs of PCOS patients.ConclusionsPD-L1 may reduce the apoptosis of GCs through PI3K/AKT signaling pathway activation, providing a novel strategy for inhibiting the apoptosis of GCs in PCOS.
| S-EPMC11871242 | biostudies-literature
Project description:Several studies have indicated that mutations of LARS2 are associated with premature ovarian insufficiency (POI). However, the pathogenic mechanism of LARS2 in POI has not been reported yet. In the present study, the expression levels of LARS2 and E2F1 in granulosa cells (GCs) of POI patients were examined. CCK-8 and Edu assay were performed to determine the effect of LARS2 on cell proliferation. Apoptosis rate, mitochondrial membrane potential, reactive oxygen species (ROS), and cytoplasm Ca2+ levels were analyzed by flow cytometry. Western blot was conducted to evaluate the expression level of genes affected by LARS2. Transmission electron microscopy (TEM) was used to observe mitochondrial structure in GCs. Chromatin immunoprecipitation (ChIP) was used to evaluate the regulatory effect of E2F1 on Mfn-2 expression. Our results showed that LARS2 expression was downregulated in GCs of POI patients. Silencing of LARS2 inhibited cell proliferation and promoted the apoptosis of GCs. Meanwhile, LARS2 knockdown could induce mitochondrial dysfunction and accumulation of ROS levels. Moreover, ROS was found to be involved in the antiproliferation, proapoptotic, and endoplasmic reticulum (ER) stress effects of LARS2 knockdown. Furthermore, we also found that the expression level of E2F1 was positively correlated with LARS2. In addition, E2F1 could bind at the -61/-46 region of Mfn-2 promoter and regulated MFN-2 transcription. These findings demonstrated that LARS2 could promote the expression of E2F1. E2F1 mediated the effect of LARS2 on Mfn-2 expression via targeting the promoter region of Mfn-2, in which subsequently regulated cell proliferation and apoptosis, which resulted in the etiology of POI. This study will provide useful information for further investigations on the LARS2 in the occurrence of POI.
| S-EPMC9110257 | biostudies-literature
Project description:Folliculogenesis is a complex biological process involving a central oocyte and its surrounding somatic cells. Three-dimensional chromatin architecture is an important transcription regulator; however, little is known about its dynamics and role in transcriptional regulation of granulosa cells during chicken folliculogenesis. We investigate the transcriptomic dynamics of chicken granulosa cells over ten follicular stages and assess the chromatin architecture dynamics and how it influences gene expression in granulosa cells at three key stages: the prehierarchical small white follicles, the first largest preovulatory follicles, and the postovulatory follicles. Our results demonstrate the consistency between the global reprogramming of chromatin architecture and the transcriptomic divergence during folliculogenesis, providing ample evidence for compartmentalization rearrangement, variable organization of topologically associating domains, and rewiring of the long-range interaction between promoter and enhancers. These results provide key insights into avian reproductive biology and provide a foundational dataset for the future in-depth functional characterization of granulosa cells.
| S-EPMC8748434 | biostudies-literature
Project description:Hexokinase-II (HK2) is a key enzyme involved in glycolysis, which is required for breast cancer progression. However, the underlying post-translational mechanisms of HK2 activity are poorly understood. Here, we showed that Proviral Insertion in Murine Lymphomas 2 (PIM2) directly bound to HK2 and phosphorylated HK2 on Thr473. Biochemical analyses demonstrated that phosphorylated HK2 Thr473 promoted its protein stability through the chaperone-mediated autophagy (CMA) pathway, and the levels of PIM2 and pThr473-HK2 proteins were positively correlated with each other in human breast cancer. Furthermore, phosphorylation of HK2 on Thr473 increased HK2 enzyme activity and glycolysis, and enhanced glucose starvation-induced autophagy. As a result, phosphorylated HK2 Thr473 promoted breast cancer cell growth in vitro and in vivo. Interestingly, PIM2 kinase inhibitor SMI-4a could abrogate the effects of phosphorylated HK2 Thr473 on paclitaxel resistance in vitro and in vivo. Taken together, our findings indicated that PIM2 was a novel regulator of HK2, and suggested a new strategy to treat breast cancer.
| S-EPMC6224402 | biostudies-literature
Project description:Premature ovarian insufficiency (POI) is characterized by early loss of ovarian function before the age of 40 years. It is confirmed to have a strong and indispensable genetic component. Caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP) is a key inducer of mitochondrial protein quality control for the clearance of misfolded or damaged proteins, which is necessary to maintain mitochondrial function. Previous findings have shown that the variation in CLPP is closely related to the occurrence of POI, which is consistent with our findings. This study identified a novel CLPP missense variant (c.628G > A) in a woman with POI who presented with secondary amenorrhea, ovarian dysfunction, and primary infertility. The variant was located in exon 5 and resulted in a change from alanine to threonine (p.Ala210Thr). Importantly, Clpp was mainly localized in the cytoplasm of mouse ovarian granulosa cells and oocytes, and was relatively highly expressed in granulosa cells. Moreover, the overexpression of c.628G > A variant in human ovarian granulosa cells decreased the proliferative capacity. Functional experiments revealed that the inhibition of CLPP decreased the content and activity of oxidative respiratory chain complex IV by affecting the degradation of aggregated or misfolded COX5A, leading to the accumulation of reactive oxygen species and reduction of mitochondrial membrane potential, ultimately activating the intrinsic apoptotic pathways. The present study demonstrated that CLPP affected the apoptosis of granulosa cells, which might be one of the mechanisms by which CLPP aberrations led to the development of POI.
| S-EPMC10065195 | biostudies-literature
Project description:Rationale: Fructose-1, 6-bisphosphatase 1 (FBP1), a rate-limiting enzyme in gluconeogenesis, was recently shown to be a tumor suppressor and could mediate the activities of multiple transcriptional factors via its non-canonical functions. However, the underlying mechanism of posttranscriptional modification on the non-canonical functions of FBP1 remains elusive. Methods: We employed immunoaffinity purification to identify binding partner(s) and used co-immunoprecipitation to verify their interactions. Kinase reaction was used to confirm PIM2 could phosphorylate FBP1. Overexpression or knockdown proteins were used to assess the role in modulating p65 protein stability. Mechanistic analysis was involved in protein degradation and polyubiquitination assays. Nude mice and PIM2-knockout mice was used to study protein functions in vitro and in vivo. Results: Here, we identified Proviral Insertion in Murine Lymphomas 2 (PIM2) as a new binding partner of FBP1, which could phosphorylate FBP1 on Ser144. Surprisingly, phosphorylated FBP1 Ser144 abrogated its interaction with NF-κB p65, promoting its protein stability through the CHIP-mediated proteasome pathway. Furthermore, phosphorylation of FBP1 on Ser144 increased p65 regulated PD-L1 expression. As a result, phosphorylation of FBP1 on Ser144 promoted breast tumor growth in vitro and in vivo. Moreover, the levels of PIM2 and pSer144-FBP1 proteins were positively correlated with each other in human breast cancer and PIM2 knockout mice. Conclusions: Our findings revealed that phosphorylation noncanonical FBP1 by PIM2 was a novel regulator of NF-κB pathway, and highlights PIM2 inhibitors as breast cancer therapeutics.
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Project description:Targeted disruption of the inhibin alpha gene (Inha(-)(/)(-)) in mice results in an ovarian phenotype of granulosa cell tumors that renders the animals infertile. Little is known about the reproductive defects prior to tumor development. Here, we report novel data on early follicle dynamics in Inha(-)(/)(-) mice, which demonstrate that inhibin alpha has important consequences upon follicle development. Morphological changes in both germ and somatic cells were evident in postnatal day 12 ovaries, with Inha(-/-) mice exhibiting numerous multilayered follicles that were far more advanced than those observed in age-matched controls. These changes were accompanied by alterations in follicle dynamics such that Inha(-/-) ovaries had fewer follicles in the resting pool and more committed in the growth phase. Absence of inhibin alpha resulted in advanced follicular maturation as marked by premature loss of anti-Müllerian hormone (AMH) in secondary follicles. Additionally, gene expression analysis revealed changes in factors known to be vital for oocyte and follicle development. Together, these data provide key evidence to suggest that regulation of the inhibin/activin system is essential for early folliculogenesis in the prepubertal mouse ovary.
| S-EPMC2753717 | biostudies-literature
Project description:Although silica nanoparticles (SNPs) are generally thought to be biocompatible and safe, the adverse effects of SNPs were also reported in previous studies. SNPs cause follicular atresia via the induction of ovarian granulosa cell apoptosis. However, the mechanisms for this phenomenon are not well understood. This study focuses on exploring the relationship between autophagy and apoptosis induced by SNPs in ovarian granulosa cells. Our results showed that 25.0 mg/kg body weight (b.w.)/intratracheal instillation of 110 nm in diameter spherical Stöber SNPs caused ovarian granulosa cell apoptosis in follicles in vivo. We also found that SNPs mainly internalized into the lumens of the lysosomes in primary cultured ovarian granulosa cells in vitro. SNPs induced cytotoxicity via a decrease in viability and an increase in apoptosis in a dose-dependent manner. SNPs increased BECLIN-1 and LC3-II levels, leading to the activation of autophagy and increased P62 level, resulting in the blockage of autophagic flux. SNPs increased the BAX/BCL-2 ratio and cleaved the caspase-3 level, resulting in the activation of the mitochondrial-mediated caspase-dependent apoptotic signaling pathway. SNPs enlarged the LysoTracker Red-positive compartments, decreased the CTSD level, and increased the acidity of lysosomes, leading to lysosomal impairment. Our results reveal that SNPs cause autophagy dysfunction via lysosomal impairment, resulting in follicular atresia via the enhancement of apoptosis in ovarian granulosa cells.
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