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Gonadotropin-releasing hormone induces miR-132 and miR-212 to regulate cellular morphology and migration in immortalized LbetaT2 pituitary gonadotrope cells.

ABSTRACT: GnRH is central to the regulation of reproductive function. It acts on pituitary gonadotropes to stimulate LH and FSH synthesis and secretion. We had previously presented evidence for translational control of LH? synthesis; therefore we investigated whether micro-RNAs might play a role in GnRH regulation in L?T2 cells. We show here that GnRH strongly induces the AK006051 gene transcript that encodes two micro-RNAs, miR-132 and miR-212, within the first intron. We show furthermore that the AK006051 promoter region is highly GnRH responsive. We verify that the p250Rho GTPase activating protein (GAP) is a target of miR-132/212 and show that GnRH treatment leads to a decrease in mRNA and protein expression. This reduction is blocked by an anti-miR to miR-132/212 and mimicked by a pre-miR-132. GnRH inhibits p250RhoGAP expression through a miR-132/212 response element within the 3'-untranslated region. The loss of p250RhoGAP expression leads to activation of Rac and marked increases in both the number and length of neurite-like processes extending from the cell. Knockdown of p250RhoGAP by small interfering RNA induces the same morphological changes observed with GnRH treatment. In addition, loss of p250RhoGAP causes an increase in cellular motility. Our findings suggest a novel pathway regulating long-term changes in cellular motility and process formation via the GnRH induction of miR-132/212 with the subsequent down-regulation of p250RhoGAP.

SUBMITTER: Godoy J

PROVIDER: S-EPMC3082323 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Gonadotropin-releasing hormone induces miR-132 and miR-212 to regulate cellular morphology and migration in immortalized LbetaT2 pituitary gonadotrope cells.

Godoy Joseph J Nishimura Marin M Webster Nicholas J G NJ

Molecular endocrinology (Baltimore, Md.) 20110303 5

GnRH is central to the regulation of reproductive function. It acts on pituitary gonadotropes to stimulate LH and FSH synthesis and secretion. We had previously presented evidence for translational control of LHβ synthesis; therefore we investigated whether micro-RNAs might play a role in GnRH regulation in LβT2 cells. We show here that GnRH strongly induces the AK006051 gene transcript that encodes two micro-RNAs, miR-132 and miR-212, within the first intron. We show furthermore that the AK0060 ...[more]

PMID: 21372146

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Project description:The LβT2 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 LβT2 cells have not been previously investigated. Here, we integrate the transcriptome and genome-wide chromatin accessibility state of LβT2 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 LβT2 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.

Dataset Information

Gonadotropin-releasing hormone induces miR-132 and miR-212 to regulate cellular morphology and migration in immortalized LbetaT2 pituitary gonadotrope cells.

Publications

Gonadotropin-releasing hormone induces miR-132 and miR-212 to regulate cellular morphology and migration in immortalized LbetaT2 pituitary gonadotrope cells.

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