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. 2016 Oct;30(10):1081-1091.
doi: 10.1210/me.2016-1085. Epub 2016 Sep 7.

GnRH Stimulates Peptidylarginine Deiminase Catalyzed Histone Citrullination in Gonadotrope Cells

Shaihla A Khan  1 , Brian S Edwards  1 , Aaron Muth  1 , Paul R Thompson  1 , Brian D Cherrington  1 , Amy M Navratil  1
Affiliations

GnRH Stimulates Peptidylarginine Deiminase Catalyzed Histone Citrullination in Gonadotrope Cells

Shaihla A Khan et al. Mol Endocrinol. 2016 Oct.

Abstract

Peptidylarginine deiminase (PAD) enzymes convert histone tail arginine residues to citrulline resulting in chromatin decondensation. Our previous work found that PAD isoforms are expressed in female reproductive tissues in an estrous cycle-dependent fashion, but their role in the anterior pituitary gland is unknown. Thus, we investigated PAD expression and function in gonadotrope cells. The gonadotrope-derived LβT2 cell line strongly expresses PAD2 at the protein level compared with other PAD isoforms. Consistent with this, PAD2 protein expression is highest during the estrous phase of the estrous cycle and colocalizes with the LH β-subunit in the mouse pituitary. Using the GnRH agonist buserelin (GnRHa), studies in LβT2 and mouse primary gonadotrope cells revealed that 30 minutes of stimulation caused distinct puncta of PAD2 to localize in the nucleus. Once in the nucleus, GnRHa stimulated PAD2 citrullinates histone H3 tail arginine residues at sites 2, 8, and 17 within 30 minutes; however, this effect and PAD2 nuclear localization was blunted by incubation of the cells with the pan-PAD inhibitor, biphenyl-benzimidazole-Cl-amidine. Given that PAD2 citrullinates histones in gonadotropes, we next analyzed the functional consequence of PAD2 inhibition on gene expression. Our results show that GnRHa stimulates an increase in LHβ and FSHβ mRNA and that this response is significantly reduced in the presence of the PAD inhibitor biphenyl-benzimidazole-Cl-amidine. Overall, our data suggest that GnRHa stimulates PAD2-catalyzed histone citrullination in gonadotropes to epigenetically regulate gonadotropin gene expression.

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Figures

Figure 1.
Figure 1.. PAD2 is expressed in the gonadotrope-derived LβT2 cell line.
Protein concentration of LβT2 cell lysates was determined and equal amounts examined by Western blotting. Membranes were probed with anti-PAD1, anti-PAD2, anti-PAD3, and anti-PAD4 antibodies or anti-β-actin for a loading control. Positive controls (+) were generated by transient overexpression of PAD1–PAD4 plasmids in LβT2 cells.
Figure 2.
Figure 2.. PAD2 expression in mouse gonadotrope cells is highest during estrus.
A, Female mice were estrous cycle staged by vaginal cytology and pituitary glands were collected on the morning of diestrus, proestrus, estrus, and metestrus. Pituitaries were fixed, sectioned frozen (16 μm) and probed with appropriate anti-PAD2 (green) and anti-LHβ (red) antibodies and stained with DAPI (blue) to label nuclei. Tissues were imaged with a Zeiss LSM710 confocal microscope at ×40 resolution. B, Three independent pituitary tissue sections from each stage of the estrous cycle were examined for corrected total fluorescence micrometer square (μm2) of PAD2 in gonadotropes using the region of interest (ROI) feature in ImageJ software. Means were separated using Tukey's HSD, asterisks indicate significant differences (*, P < .05), and error bars are SEM. C. Mice were estrous cycle staged by vaginal cytology and pituitary glands collected during estrus. Protein concentrations of female mouse pituitary lysates were determined and equal concentrations loaded and examined by Western blotting. Membranes were probed with anti-PAD1, anti-PAD2, anti-PAD3, and anti-PAD4 antibodies or with anti-β-tubulin as a loading control.
Figure 3.
Figure 3.. GnRHa stimulation of L-type calcium channel activity induces citrullination of arginine residues on histone H3 tails.
A, Cells were serum starved for 6 hours, then treated with either vehicle or 10nM GnRHa for 0, 30, 60, or 180 minutes. After treatment, histones were purified by acid extraction and quantified, and equal concentrations were examined by Western blotting. Membranes were probed with an antihistone H3 arginine residue 2, 8, and 17 antibody or antihistone H3 total antibody as a loading control. Bar graphs on the right show quantitative analysis of the Western blottings (n = 3) conducted using Bio-Rad ImageLab software and normalized to total histone H3 levels. Means were separated using Tukey's HSD, asterisks indicate significant differences (P < .05), whereas error bars are SEM. B, Cells were serum starved for 6 hours, then treated with either vehicle, GnRHa (10nM), FPL 64176 (500nM), or Thapsigargin (50nM) for 30 minutes followed by stimulation with vehicle or 10nM GnRHa. After treatment, histones were purified and equal concentrations examined by Western blotting as described above. Bar graphs on the right show quantitative analysis of the Western blottings (n = 3) conducted using Bio-Rad ImageLab software and normalized to total histone H3 levels. Means were separated using Tukey's HSD, asterisks indicate significant differences (**, P < .01), and error bars are SEM.
Figure 4.
Figure 4.. GnRHa induces PAD2 nuclear localization in LβT2 cells.
A, LβT2 cells were plated on glass bottom confocal dishes, serum starved for 6 hours, and then treated with either vehicle or 10nM GnRHa for 0, 30, or 60 minutes. Cells were then fixed in 4% PFA and probed with an anti-PAD2 antibody with the appropriate fluorescently labeled secondary antibody and stained with DAPI. Cells were imaged with a confocal microscope using a ×40 objective. B, LβT2 cells were serum starved for 6 hours, then treated with either vehicle or 10nM GnRHa for 0, 30, or 60 minutes. The chromatin-associated protein fraction was isolated and quantified, and equal concentrations were examined by Western blotting. Membranes were probed with an anti-PAD2 and with antihistone H3 total as a loading control.
Figure 5.
Figure 5.. PAD inhibition decreases GnRHa-induced histone citrullination in LβT2 cells.
A, LβT2 cells were pretreated for 12 hours with DMSO or 1μM BB-ClA, then treated with either vehicle or 10nM GnRHa for 0 and 30 minutes. After treatment, histones were purified by acid extraction and quantified, and equal concentrations were examined by Western blotting. Membranes were probed with an antihistone H3 antibody that detects citrullination at arginine residues 2, 8, and 17 and antihistone H3 as a loading control. Bar graphs on the right show quantitative analysis of the Western blottings (n = 3) conducted using Bio-Rad ImageLab software and normalized to total histone H3 levels. Means were separated using Tukey's HSD, asterisks indicate significant differences (P < .05), and error bars are SEM. B, Mice were estrous cycle staged by vaginal cytology and pituitary glands collected during estrus. Pituitaries were explanted, dispersed, and cultured for 12 hours with DMSO or 1μM BB-ClA. Cells were next treated with vehicle or 10nM GnRHa for 30 minutes, then fixed and probed with anti-PAD2 (green) and anti-LHβ (red) antibodies and stained with DAPI (blue). Tissues were imaged with a Zeiss LSM710 confocal microscope using a ×40 objective.
Figure 6.
Figure 6.. Inhibition of PAD-catalyzed citrullination reduces LHβ and FSHβ mRNA expression.
LβT2 cells were pretreated for 12 hours with DMSO or 1μM BB-ClA, then treated with either vehicle or 10nM GnRHa for 0 and 180 minutes. Total RNA was purified from cells, reverse transcribed, and resulting cDNA examined by TaqMan probe qPCR for LHβ, FSHβ, CGA, and GAPDH as the reference gene control. All data values were normalized to vehicle treated to yield fold change, and data are expressed as mean ± SEM. Means were separated using Tukey's HSD, and asterisks indicate significant differences (*, P < .05 and **, P < .01).
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References

    1. Naor Z. Signaling by G-protein-coupled receptor (GPCR): studies on the GnRH receptor. Front Neuroendocrinol. 2009;30:10–29. - PubMed
    1. Thompson IR, Kaiser UB. GnRH pulse frequency-dependent differential regulation of LH and FSH gene expression. Mol Cell Endocrinol. 2014;385:28–35. - PMC - PubMed
    1. Pernasetti F, Vasilyev VV, Rosenberg SB, et al. Cell-specific transcriptional regulation of FSHb by activin and GnRH in the LbT2 pituitary gonadotrope cell model. Endocrinology. 2001;142:2284–2295. - PubMed
    1. Ferris HA, Walsh HE, Stevens J, Fallest PC, Shupnik MA. Luteinizing hormone β promoter stimulation by adenylyl cyclase and cooperation with gonadotropin-releasing hormone 1 in transgenic mice and LβT2 Cells. Biol Reprod. 2007;77:1073–1080. - PubMed
    1. Mouillet JF, Sonnenberg-Hirche C, Yan X, Sadovsky Y. p300 regulates the synergy of steroidogenic factor-1 and early growth response-1 in activating luteinizing hormone-β subunit gene. J Biol Chem. 2004;279:7832–7839. - PubMed