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Gonadotropin releasing hormone activation of the mTORC2/Rictor complex regulates actin remodeling and ERK activity in L?T2 cells.

ABSTRACT: The mammalian target of rapamycin (mTOR) assembles into two different multi-protein complexes, mTORC1 and mTORC2. The mTORC2 complex is distinct due to the unique expression of the specific core regulatory protein Rictor (rapamycin-insensitive companion of mTOR). mTORC2 has been implicated in regulating actin cytoskeletal reorganization but its role in gonadotrope function is unknown. Using the gonadotrope-derived L?T2 cell line, we find that the GnRH agonist buserelin (GnRHa) phosphorylates both mTOR and Rictor. Interestingly, inhibition of mTORC2 blunts GnRHa-induced cyto-architectural rearrangements. Coincident with blunting of actin reorganization, inhibition of mTORC2 also attenuates GnRHa-mediated activation of both protein kinase C (PKC) and extracellular signal regulated kinase (ERK). Collectively, our data suggests that GnRHa-mediated mTORC2 activation is important in facilitating actin reorganization events critical for initiating PKC activity and subsequent ERK phosphorylation in the gonadotrope-derived L?T2 cell line.

SUBMITTER: Edwards BS

PROVIDER: S-EPMC5123956 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Gonadotropin releasing hormone activation of the mTORC2/Rictor complex regulates actin remodeling and ERK activity in LβT2 cells.

Edwards Brian S BS Isom William J WJ Navratil Amy M AM

Molecular and cellular endocrinology 20160920

The mammalian target of rapamycin (mTOR) assembles into two different multi-protein complexes, mTORC1 and mTORC2. The mTORC2 complex is distinct due to the unique expression of the specific core regulatory protein Rictor (rapamycin-insensitive companion of mTOR). mTORC2 has been implicated in regulating actin cytoskeletal reorganization but its role in gonadotrope function is unknown. Using the gonadotrope-derived LβT2 cell line, we find that the GnRH agonist buserelin (GnRHa) phosphorylates bot ...[more]

PMID: 27663077

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Project description:The L?T2 mouse pituitary cell line has many characteristics of a mature gonadotrope and is a widely used model system for studying the developmental processes and the response to gonadotropin-releasing hormone (GnRH). The global epigenetic landscape, which contributes to cell-specific gene regulatory mechanisms, and the single-cell transcriptome response variation of L?T2 cells have not been previously investigated. Here, we integrate the transcriptome and genome-wide chromatin accessibility state of L?T2 cells during GnRH stimulation. In addition, we examine cell-to-cell variability in the transcriptional response to GnRH using Gel bead-in-Emulsion Drop-seq technology. Analysis of a bulk RNA-seq data set obtained 45?min after exposure to either GnRH or vehicle identified 112 transcripts that were regulated >4-fold by GnRH (FDR?<?0.05). The top regulated transcripts constitute, as determined by Bayesian massive public data integration analysis, a human pituitary-relevant coordinated gene program. Chromatin accessibility [assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq)] data sets generated from GnRH-treated L?T2 cells identified more than 58,000 open chromatin regions, some containing notches consistent with bound transcription factor footprints. The study of the most prominent open regions showed that 75% were in transcriptionally active promoters or introns, supporting their involvement in active transcription. Lhb, Cga, and Egr1 showed significantly open chromatin over their promoters. While Fshb was closed over its promoter, several discrete significantly open regions were found at -40 to -90?kb, which may represent novel upstream enhancers. Chromatin accessibility determined by ATAC-seq was associated with high levels of gene expression determined by RNA-seq. We obtained high-quality single-cell Gel bead-in-Emulsion Drop-seq transcriptome data, with an average of >4,000 expressed genes/cell, from 1,992 vehicle- and 1,889 GnRH-treated cells. While the individual cell expression patterns showed high cell-to-cell variation, representing both biological and measurement variation, the average expression patterns correlated well with bulk RNA-seq data. Computational assignment of each cell to its precise cell cycle phase showed that the response to GnRH was unaffected by cell cycle. To our knowledge, this study represents the first genome-wide epigenetic and single-cell transcriptomic characterization of this important gonadotrope model. The data have been deposited publicly and should provide a resource for hypothesis generation and further study.

Project description:BackgroundThe main objective of the present work was to compare the effects of the gonadotropin-releasing hormone agonist (GnRH-a) and GnRH antagonist (GnRH-ant) on the gene expression profiles of oocytes obtained from Iranian infertile couples undergoing in vitro fertilization (IVF).MethodsFifty infertile couples who underwent IVF between June 2012 and November 2013 at the Infertility Center of Tehran Women General Hospital, Tehran University of Medical Sciences, were included in this study. We included women that had undergone IVF treatment because of male factor, tubal factor, or unexplained infertility. The women randomly underwent controlled ovarian stimulation (COS) with either the GnRH-a (n = 26) or the GnRH-ant (n = 24). We obtained 50 germinal vesicle (GV) oocytes donated by women in each group. After the sampling, pool of 50 GV oocytes for each group was separately analyzed by quantitative polymerase chain reaction (qPCR).ResultThe expression levels of Adenosine triphosphatase 6 (ATPase 6), Bone morphogenetic protein 15 (BMP15), and Neuronal apoptosis inhibitory protein (NAIP) genes were significantly upregulated in the GnRH-ant group compared to the GnRH-a group, with the fold change of 3.990 (SD ± 1.325), 6.274 (SD ± 1.542), and 2.156 (SD ± 1.443), respectively, (P < 0.001). Growth differentiation factor 9 (GDF9) mRNA did not have any expression in the GnRH-a group; however, GDF9 mRNA was expressed in the GnRH-ant group. Finally, it was found that the genes involved in the DNA repairing and cell cycle checkpoint did not have any expression in either group.ConclusionThe present study showed, for the first time, the expression levels of genes involved in the cytoplasmic maturity (BMP15, GDF9), adenosine triphosphate production (ATPase 6), and antiapoptotic process (NAIP), in human GV oocytes were significantly higher in the GnRH-anta group than in the GnRH-a group in COS. Higher expression level of these genes when GnRH-ant protocol is applied, this protocol seems to be a more appropriate choice for women with poly cystic ovarian syndrome, because it can probably improve the expression of the aforementioned genes.Trial registrationCurrent Controlled Trials: IRCT 2014031112307 N3.

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Gonadotropin releasing hormone activation of the mTORC2/Rictor complex regulates actin remodeling and ERK activity in L?T2 cells.

Publications

Gonadotropin releasing hormone activation of the mTORC2/Rictor complex regulates actin remodeling and ERK activity in LβT2 cells.

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