scholarly journals Dynamics and Regulations of BimEL Ser65 and Thr112 Phosphorylation in Porcine Granulosa Cells during Follicular Atresia

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 402
Author(s):  
Feng Yang ◽  
Yanhong Chen ◽  
Qiang Liu ◽  
Shizhen Dai ◽  
Shenming Zeng
Keyword(s):  
Heat Stress ◽  
Granulosa Cells ◽  
Kinase Inhibitors ◽  
Phosphorylation Sites ◽  
Follicular Atresia ◽  
Dynamic Changes ◽  
Jnk Pathway ◽  
Regulatory Pathways ◽  
Porcine Granulosa Cells ◽  
Specific Inhibitors

BimEL protein is involved in follicular atresia by regulating granulosa cell apoptosis, but the dynamic changes of BimEL phosphorylation during follicular atresia are poorly understood. The aim of this study was to explore the changes of key BimEL phosphorylation sites and their upstream regulatory pathways. First, the levels of BimEL-Ser65 and BimEL-Thr112 phosphorylation (p-BimEL-S65, p-BimEL-T112) in granulosa cells (GC) from healthy (H), slightly-atretic (SA), and atretic (A) follicles and in cultured GC after different treatments were detected by Western blotting. Next, the effects of the corresponding site mutations of BIM on apoptosis of GC were investigated. Finally, the pathways of two phosphorylation sites were investigated by kinase inhibitors. The results revealed that p-BimEL-S65 levels were higher in GC from H than SA and A, whereas p-BimEL-T112 was reversed. The prosurvival factors like FSH and IGF-1 upregulated the level of p-BimEL-S65, while the proapoptotic factor, heat stress, increased the level of p-BimEL-T112 in cultured GC. Compared with the overexpression of wild BimEL, the apoptotic rate of the GC overexpressed BimEL-S65A (replace Ser65 with Ala) mutant was significantly higher, but the apoptotic rate of the cells overexpressing BimEL-T112A did not differ. In addition, inhibition of the ERK1/2 or JNK pathway by specific inhibitors reduced the levels of p-BimEL-S65 and p-BimEL-T112. In conclusion, the levels of p-BimEL-S65 and p-BimEL-T112 were reversed during follicular atresia. Prosurvival factors promote p-BimEL-S65 levels via ERK1/2 to inhibit GC apoptosis, whereas proapoptotic factor upregulates the level of p-BimEL-T112 via JNK to induce GC apoptosis.

scholarly journals Integrative Proteomic and Phosphoproteomic Analyses of Granulosa Cells During Follicular Atresia in Porcine

2021 ◽  
Vol 8 ◽  
Author(s):  
Feng Yang ◽  
Qiang Liu ◽  
Yanhong Chen ◽  
Huizhen Ye ◽  
Han Wang ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Beta Catenin ◽  
Phosphorylation Sites ◽  
Differentially Expressed Proteins ◽  
Follicular Atresia ◽  
Dynamic Changes ◽  
Phosphorylated Proteins ◽  
Apoptosis Regulation ◽  
Quality Categories ◽  
Regulation Of Actin Cytoskeleton

Ovarian follicular atresia is a natural physiological process; however, the mechanism is not fully understood. In this study, quantitative proteomic and phosphoproteomic analyses of granulosa cells (GCs) in healthy (H), slightly atretic (SA), and atretic follicles (A) of porcine were performed by TMT labeling, enrichment of phosphopeptides, and liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis. In total, 6,201 proteins were quantified, and 4,723 phosphorylation sites of 1,760 proteins were quantified. In total, 24 (11 up, 13 down) and 50 (29 up, 21 down) proteins with a fold change (FC) > 5 were identified in H/SA and H/A, respectively. In addition, there were 20 (H/SA, up) and 39 (H/A, up) phosphosites with an FC > 7 that could serve as potential biomarkers for distinguishing different quality categories of follicles. Western blotting and immunofluorescence confirmed the reliability of the proteomic analysis. Some key proteins (e.g., MIF, beta catenin, integrin β2), phosphosites (e.g., S76 of caspase6, S22 and S636 of lamin A/C), pathways (e.g., apoptosis, regulation of actin cytoskeleton pathway), transcription factors (e.g., STAT5A, FOXO1, and BCLAF1), and kinases (e.g., PBK, CDK5, CDK12, and AKT3) involved in the atresia process were revealed via further analysis of the differentially expressed proteins (DEPs) and phosphorylated proteins (DEPPs). Further study showed that mutant caspase6 Ser76 to Ala increased the ratios of cleaved caspase6/caspase6 and cleaved caspase3/caspase3 and dephosphorylation of caspase6 at Ser76 increased cell apoptotic rate, a new potential pathway of follicular atresia. Collectively, the proteomic and phosphoproteomic profiling and functional research in the current study comprehensively analyzed the dynamic changes in protein expression and phosphorylation during follicular atresia and provided some new explanations regarding the regulation of this process.

scholarly journals Integrative proteomic and phosphoproteomic analysis of granulosa cells during follicular atresia in porcine

2020 ◽  
Author(s):  
Feng Yang ◽  
Qiang Liu ◽  
Yanhong Chen ◽  
Huizhen Ye ◽  
Han Wang ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Beta Catenin ◽  
Phosphorylation Sites ◽  
Differentially Expressed Proteins ◽  
Follicular Atresia ◽  
Dynamic Changes ◽  
Phosphorylated Proteins ◽  
Apoptosis Regulation ◽  
Quality Categories ◽  
Regulation Of Actin Cytoskeleton

AbstractOvarian follicular atresia is a natural physiological process; however, the mechanism is not fully understood. In this study, quantitative proteomic and phosphoproteomic analyses of granulosa cells (GC) in healthy (H), slightly atretic (SA), and atretic follicles (A) of porcine were performed by TMT labeling, enrichment of phosphopeptides and LC-MS/MS analysis. In total, 6,201 proteins were quantified and 4,723 phosphorylation sites of 1,760 proteins were quantified. In total, 24 (11 up, 13 down) and 50 (29 up, 21 down) proteins with a fold change (FC) > 5 were identified in H/SA and H/A, respectively. In addition, there were 20 (H/SA, up) and 39 (H/A, up) phosphosites with an FC > 7, that could serve as potential biomarkers for distinguishing different quality categories of follicles. Western blotting and immunofluorescence confirmed the reliability of the proteomic analysis. Some key proteins (e.g., MIF, beta catenin, integrin β2), phosphosites (e.g., S76 of caspase6, S22 and S636 of lamin A/C), pathways (e.g., apoptosis, regulation of actin cytoskeleton pathway), transcription factors (e.g., STAT5A, FOXO1, and BCLAF1), and kinases (e.g., PBK, CDK5, CDK12, AKT3) involved in atresia process were revealed via further analysis of the differentially expressed proteins (DEPs) and phosphorylated proteins (DEPPs). Collectively, the proteomic and phosphoproteomic profiling and functional research in the current study comprehensively analyzed the dynamic changes in protein expression and phosphorylation during follicular atresia and provided some new explanations regarding the regulation of this process.

scholarly journals Apoptosis in Porcine Granulosa Cells During Follicular Atresia Is Regulated by Anti-Apoptotic cFLIP.

2008 ◽  
Vol 78 (Suppl_1) ◽  
pp. 289-289 ◽  
Author(s):  
Noboru Manabe ◽  
Fuko Minehata ◽  
Yasufumi Goto ◽  
Akihisa Maeda ◽  
Yuang Cheng ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Follicular Atresia ◽  
Porcine Granulosa Cells

scholarly journals Transcriptome Analysis of Porcine Granulosa Cells in Healthy and Atretic Follicles: Role of Steroidogenesis and Oxidative Stress

Antioxidants ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 22
Author(s):  
Li Meng ◽  
Zhenfang Wu ◽  
Kun Zhao ◽  
Jian Tao ◽  
Tam Chit ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Granulosa Cells ◽  
Oxidative Dna Damage ◽  
Molecular Mechanisms ◽  
Glutathione Metabolism ◽  
Follicular Atresia ◽  
Rna Seq ◽  
Antioxidant Genes ◽  
Porcine Granulosa Cells ◽  
Apoptotic Factors

One of the main causes of female infertility is a deregulated antral follicular atresia, a process of which the underlying molecular mechanisms are largely unknown. Our objective was therefore to characterize the complex transcriptome changes in porcine granulosa cells of healthy antral (HA) and advanced antral atretic (AA) follicles, using ELISA and RNA-Seq followed by qRT-PCR and immunohistochemistry. Granulosa cell RNA-Seq data revealed 2160 differentially expressed genes, 1483 with higher and 677 with lower mRNA concentrations in AA follicles. Bioinformatic analysis showed that the upregulated genes in AA follicles were highly enriched in inflammation and apoptosis processes, while the downregulated transcripts were mainly highlighted in the steroid biosynthesis pathway and response to oxidative stress processes including antioxidant genes (e.g., GSTA1, GCLC, GCLM, IDH1, GPX8) involved in the glutathione metabolism pathway and other redox-related genes (e.g., RRM2B, NDUFS4). These observations were confirmed by RT-qPCR and immunohistochemistry. Additionally, the granulosa cells of AA follicles express significantly stronger 8-OHdG immunostaining, a marker of oxidative DNA damage, implicating that oxidative stress may participate in follicular atresia. We hypothesize that the decrease in anti-apoptotic factors and steroid hormones coincides with increased oxidative stress markers and the expression of pro-apoptotic factors, all contributing to antral follicular atresia.

scholarly journals Typing FGFR2 translocation determines the response to targeted therapy of intrahepatic cholangiocarcinomas

2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Xiaohong Pu ◽  
Qing Ye ◽  
Jing Cai ◽  
Xin Yang ◽  
Yao Fu ◽  
...  
Keyword(s):  
Targeted Therapy ◽  
Bile Duct ◽  
Kinase Inhibitors ◽  
Fibroblast Growth Factor Receptor ◽  
Growth Factor Receptor ◽  
Chromosomal Translocations ◽  
Small Bile Duct ◽  
Druggable Targets ◽  
Inhibitor Treatment ◽  
Specific Inhibitors

AbstractChromosomal translocations involving fibroblast growth factor receptor 2 (FGFR2) gene at the breakpoints are common genetic lesions in intrahepatic cholangiocarcinoma (ICC) and the resultant fusion protein products have emerged as promising druggable targets. However, predicting the sensitivity of FGFR2 fusions to FGFR kinase inhibitors is crucial to the prognosis of the ICC-targeted therapy. Here, we report identification of nine FGFR2 translocations out of 173 (5.2%) ICC tumors. Although clinicopathologically these FGFR2 translocation bearing ICC tumors are indistinguishable from the rest of the cohort, they are invariably of the mass-forming type originated from the small bile duct. We show that the protein products of FGFR2 fusions can be classified into three subtypes based on the breaking positions of the fusion partners: the classical fusions that retain the tyrosine kinase (TK) and the Immunoglobulin (Ig)-like domains (n = 6); the sub-classical fusions that retain only the TK domain without the Ig-like domain (n = 1); and the non-classical fusions that lack both the TK and Ig-like domains (n = 2). We demonstrate that cholangiocarcinoma cells engineered to express the classical and sub-classical fusions show sensitivity to FGFR-specific kinase inhibitors as evident by the suppression of MAPK/ERK and AKT/PI3K activities following the inhibitor treatment. Furthermore, the kinase-deficient mutant of the sub-classical fusion also lost its sensitivity to the FGFR-specific inhibitors. Taken together, our study suggests that it is essential to determine the breakpoint and type of FGFR2 fusions in the small bile duct subtype of ICC for the targeted treatment.

scholarly journals Genome-wide identification and analysis of the heat shock transcription factor family in moso bamboo (Phyllostachys edulis)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Bin Huang ◽  
Zhinuo Huang ◽  
Ruifang Ma ◽  
Jialu Chen ◽  
Zhijun Zhang ◽  
...  
Keyword(s):  
Heat Stress ◽  
Stress Response ◽  
Heat Shock ◽  
Gene Family ◽  
Regulatory Elements ◽  
Moso Bamboo ◽  
Naphthalene Acetic Acid ◽  
Plant Responses ◽  
Heat Stress Response ◽  
Regulatory Pathways

AbstractHeat shock transcription factors (HSFs) are central elements in the regulatory network that controls plant heat stress response. They are involved in multiple transcriptional regulatory pathways and play important roles in heat stress signaling and responses to a variety of other stresses. We identified 41 members of the HSF gene family in moso bamboo, which were distributed non-uniformly across its 19 chromosomes. Phylogenetic analysis showed that the moso bamboo HSF genes could be divided into three major subfamilies; HSFs from the same subfamily shared relatively conserved gene structures and sequences and encoded similar amino acids. All HSF genes contained HSF signature domains. Subcellular localization prediction indicated that about 80% of the HSF proteins were located in the nucleus, consistent with the results of GO enrichment analysis. A large number of stress response–associated cis-regulatory elements were identified in the HSF upstream promoter sequences. Synteny analysis indicated that the HSFs in the moso bamboo genome had greater collinearity with those of rice and maize than with those of Arabidopsis and pepper. Numerous segmental duplicates were found in the moso bamboo HSF gene family. Transcriptome data indicated that the expression of a number of PeHsfs differed in response to exogenous gibberellin (GA) and naphthalene acetic acid (NAA). A number of HSF genes were highly expressed in the panicles and in young shoots, suggesting that they may have functions in reproductive growth and the early development of rapidly-growing shoots. This study provides fundamental information on members of the bamboo HSF gene family and lays a foundation for further study of their biological functions in the regulation of plant responses to adversity.

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