Relevance of GnRHR expression level for TEY phosphorylation unattributable nuclear localization of ERK2-GFP.

<p>For panels A and B, HeLa cells transfected with ERK siRNAs were transduced with Ad ERK2-GFP (WT) and with Ad mGnRHR at varied titre (0, 0.03, 0.06, 0.125, 0.25 or 0.5 pfu/nl) in order to vary GnRHR number. They were then stimulated for the indicated period before being fixed and stained for ppERK2-GFP and DAPI. Imaging and analysis of single cells was carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040077#s2" target="_blank">Methods</a>. The plots show cell population averaged ppERK2-GFP levels for cells stimulated with 100 nM GnRH plotted against time (A), or for cells stimulated 15 min with 0 (Ctrl.) or 100 nM GnRH, plotted against Ad mGnRHR titre (B). These data are from a single experiment with duplicate wells, and are representative of those from 2 similar experiments. Panel C show ERK2-GFP N:C ratios calculated for data from the same experiment, binned according to ppERK2-GFP levels (80 AFU per bin, accepting a minimum of 50 cells per bin in each experiment). Data are shown for cells transduced with 0.25 pfu/nl Ad mGnRHR and stimulated for 0 (Ctrl.), 15 or 30 min with 100 nM GnRH. Panel D shows ppERK2-GFP values (AFU) and ERK2-GFP N:C ratios calculated for cells within a single ppERK2-GFP bin (120–160 AFU), plotted against Ad mGnRHR titre. ANOVAs of the ppERK2-GFP data in panels B and C both revealed Ad mGnRHR titre as a significant variable with significant elevation (P<0.01 compared to control without Ad mGnRHR) at all titres.</p

GnRH-stimulated MEK-ERK regulation in cell populations.

<p>HeLa cells were cultured, transduced, stimulated with 0, 1 nM or 100 nM GnRH and then fixed and stained as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040077#pone-0040077-g001" target="_blank">Fig. 1</a>. Images of endogenous ppERK, ERK and DAPI stains were analysed (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040077#s2" target="_blank">Methods</a>), using 9 images for each fluorophore and in each well, with cells in duplicate wells for each experiment. Graphs represent population average values for ppERK intensity (in arbitrary fluorescence units (AFU), left panels) and ERK N:C ratio (derived from AFU measures of total ERK stain intensity in the nuclear and cytoplasmic compartments) derived from 8 (A) and 14 (B) separate experiments ± SEM. Two way ANOVAs of data in panel A revealed GnRH as a significant source of variation (P<0.01) and post-hoc Bonferroni tests revealed significant differences between control and GnRH-treated cells as indicated (*P<0.05, **P<0.01). One way ANOVA of data in panel B revealed GnRH as a significant source of variation (P<0.01) and post-hoc Bonferroni tests revealed significant differences to control as indicated (*P<0.05, **P<0.01).</p

GnRH-stimulated MEK-ERK measured by western blotting or high content imaging.

<p>(A) HeLa cells were seeded in 12 well plates, transduced with Ad mGnRHR and kept in reduced (0.1%) serum for 16 hours prior to stimulation for varied periods with 100 nM GnRH or 1 µM PDBu. Whole cell protein extracts were then immunoblotted for phospho Ser217/221 MEK1/2 (ppMEK1/2), phospho Thr183/Tyr185 ERK (ppERK) and for total ERK (ERK1 and/or 2) as indicated. (B) HeLa cells were seeded in 96-well imaging plates, transduced with Ad mGnRHR and kept in reduced (0.1%) serum for 16 hours prior to stimulation for 5 min with 1 or 100 nM GnRH as indicated. Control cells (Ctrl.) were treated with medium alone. The cells were then fixed and stained for endogenous ppERK, ERK and DAPI before image acquisition and analysis (as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040077#s2" target="_blank">Methods</a>). The figure shows representative images and the lower images show “segmented” ERK staining, with lines indicating the perimeters of the nuclei and cells obtained using automated image analysis algorithms, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040077#s2" target="_blank">Methods</a>. Each of the image panels corresponds to a width of approximately 250 µm and represents approximately 1/20^th of the area captured in each field of view.</p

GnRH-mediated uncoupling of ERK phosphorylation from nuclear localization.

<p>Cells were treated, imaged and analysed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040077#pone-0040077-g002" target="_blank">Fig. 2</a>, except that the stimulation was for 5 min with varied concentrations of GnRH (as indicated). (A) Frequency histograms of individual cells (pooled from 2 independent experiments) were plotted according to ppERK stain intensity in AFU (left panel) and ERK N:C ratio (right panel) using the same cell population for both graphs. (B) The left panel shows direct comparison of ppERK levels to ERK N:C in Ctrl and 100 nM GnRH-stimulated samples. Individual cells were sorted into bins of ppERK staining intensity (80 AFU per bin, using a minimum bin size of 50 cells per experiment). The average ERK N:C ratio within each defined bin of ppERK staining intensity was calculated and is shown plotted against average ppERK stain intensity. Note that this plot effectively obscures the effect of the stimulus on ERK phosphorylation because the major effect of PDBu is to increase the number of cells in the higher ppERK bins (as shown in A). In doing so it reveals the TEY phosphorylation unattributable effect of PDBu: that is, the increase in ERK N:C under conditions matched for indistinguishable ppERK levels. The right hand panel illustrates the concentration-dependence of this effect with cells binned to ensure comparable levels of ppERK (240–320 AFU), plotting GnRH concentration against ERK N:C ratio (left y-axis)) and ppERK stain intensity (AFU, right y-axis). Data are shown from 13 separate experiments (mean ± SEM). Two way ANOVA of data in the lower left panel revealed both ppERK bin and GnRH concentration as significant sources of variation (P<0.01) and post-hoc Bonferroni tests revealed significant differences between control and GnRH-treated cells in the 200, 280, 360 and 440 AFU bins (**P<0.01). Since this analysis does not permit unpaired data, only data from the first six ppERK bins were included. One way ANOVA of the ppERK data in the lower right panel revealed GnRH concentration as a significant source of variation (P<0.01) and post-hoc Bonferroni tests revealed a significant difference between control and 10 or 100 nM GnRH-treated cells (**P<0.01).</p

PKC inhibition reduces GnRH-induced nuclear localization of ERK even at comparable levels of ERK TEY phosphorylation.

<p>Cells were treated, imaged and analysed as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040077#pone-0040077-g005" target="_blank">fig. 5</a>, except that stimulation was for 5 min with 100 nM GnRH (panel A) or with GnRH at varied concentration (panel B). (A) The individual imaged cells were sorted into bins of ppERK staining intensity (80 AFU per bin, using a minimum bin size of 50 cells per experiment). The average ERK N:C ratio within each defined bin of ppERK staining intensity was then calculated and is shown plotted against average ppERK stain intensity (in AFU). The data are shown are means ± SEM (n = 6) and two way ANOVAs revealed ppERK bin, PD032501 and Ro31-8425 as significant sources of variation (P<0.01), whereas AG1478 was not (P>0.05). Post-hoc Bonferroni tests revealed significant differences between ERK N:C values in control and Ro31-8425 treated cells in 4 of the ppERK bins (*P<0.05). Only paired data (i.e. bins where data are available with and without inhibitor) were used in this analysis. (B) To reveal whether inhibitor effects in cells at matched ppERK levels were dependent on GnRH dose, we used data from single cells to comparing ERK N:C ratio (left y-axis) in cells within a comparable range (240–320 AFU) of ppERK staining intensity (in AFU, right y-axis) for each GnRH concentration tested. Data are means ± SEM (n = 6). Two way ANOVAs of the ERK N:C data revealed GnRH, Ro31-8425 and PD0325901 as significant sources of variation (P<0.01) whereas AG1478 was not (P>0.05). Post-hoc Bonferroni tests revealed significant differences between ERK N:C values in control and Ro31-8425 treated cells at 10 and 100 nM GnRH (*P<0.05).</p

Time-dependent changes in GnRH-induced ERK localization occur at matched levels of TEY phosphorylation.

<p>Cells were treated, imaged and analysed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040077#pone-0040077-g002" target="_blank">Fig. 2</a>, except that the GnRH concentration was 100 nM. The top graph shows population average values for ppERK intensity in AFU (right y-axis) and ERK N:C ratio (left y-axis). The lower graph shows the same time-course, but comparing ERK N:C ratio (left y-axis) in cells within a comparable range (240–320 AFU) of ppERK staining intensity (right y-axis). Data are shown from 3 separate experiments (mean ± SEM). One way ANOVAs and post-hoc Bonferroni tests revealed significant effects of GnRH on ERK N:C (**P<0.01 compared to t = 0) at 5 and 15 min for the population average data (upper panel) and at 5 min for the binned data (lower panel).</p

data_sheet_1_Glial Cells Missing 1 Regulates Equine Chorionic Gonadotrophin Beta Subunit via Binding to the Proximal Promoter.DOCX

<p>Equine chorionic gonadotrophin (eCG) is a placental glycoprotein critical for early equine pregnancy and used therapeutically in a number of species to support reproductive activity. The factors in trophoblast that transcriptionally regulate eCGβ-subunit (LHB), the gene which confers the hormones specificity for the receptor, are not known. The aim of this study was to determine if glial cells missing 1 regulates LHB promoter activity. Here, studies of the LHB proximal promoter identified four binding sites for glial cells missing 1 (GCM1) and western blot analysis confirmed GCM1 was expressed in equine chorionic girdle (ChG) and surrounding tissues. Luciferase assays demonstrated endogenous activity of the LHB promoter in BeWo choriocarcinoma cells with greatest activity by a proximal 335 bp promoter fragment. Transactivation studies in COS7 cells using an equine GCM1 expression vector showed GCM1 could transactivate the proximal 335 bp LHB promoter. Chromatin immunoprecipitation using primary ChG trophoblast cells showed GCM1 to preferentially bind to the most proximal GCM1-binding site over site 2. Mutation of site 1 but not site 2 resulted in a loss of endogenous promoter activity in BeWo cells and failure of GCM1 to transactivate the promoter in COS-7 cells. Together, these data show that GCM1 binds to site 1 in the LHB promoter but also requires the upstream segment of the LHB promoter between −119 bp and −335 bp of the translation start codon for activity. GCM1 binding partners, ETV1, ETV7, HOXA13, and PITX1, were found to be differentially expressed in the ChG between days 27 and 34 and are excellent candidates for this role. In conclusion, GCM1 was demonstrated to drive the LHB promoter, through direct binding to a predicted GCM1-binding site, with requirement for another factor(s) to bind the proximal promoter to exert this function. Based on these findings, we hypothesize that ETV7 and HOXA13 act in concert with GCM1 to initiate LHB transcription between days 30 and 31, with ETV1 partnering with GCM1 to maintain transcription.</p

image_1_Glial Cells Missing 1 Regulates Equine Chorionic Gonadotrophin Beta Subunit via Binding to the Proximal Promoter.TIFF

<p>Equine chorionic gonadotrophin (eCG) is a placental glycoprotein critical for early equine pregnancy and used therapeutically in a number of species to support reproductive activity. The factors in trophoblast that transcriptionally regulate eCGβ-subunit (LHB), the gene which confers the hormones specificity for the receptor, are not known. The aim of this study was to determine if glial cells missing 1 regulates LHB promoter activity. Here, studies of the LHB proximal promoter identified four binding sites for glial cells missing 1 (GCM1) and western blot analysis confirmed GCM1 was expressed in equine chorionic girdle (ChG) and surrounding tissues. Luciferase assays demonstrated endogenous activity of the LHB promoter in BeWo choriocarcinoma cells with greatest activity by a proximal 335 bp promoter fragment. Transactivation studies in COS7 cells using an equine GCM1 expression vector showed GCM1 could transactivate the proximal 335 bp LHB promoter. Chromatin immunoprecipitation using primary ChG trophoblast cells showed GCM1 to preferentially bind to the most proximal GCM1-binding site over site 2. Mutation of site 1 but not site 2 resulted in a loss of endogenous promoter activity in BeWo cells and failure of GCM1 to transactivate the promoter in COS-7 cells. Together, these data show that GCM1 binds to site 1 in the LHB promoter but also requires the upstream segment of the LHB promoter between −119 bp and −335 bp of the translation start codon for activity. GCM1 binding partners, ETV1, ETV7, HOXA13, and PITX1, were found to be differentially expressed in the ChG between days 27 and 34 and are excellent candidates for this role. In conclusion, GCM1 was demonstrated to drive the LHB promoter, through direct binding to a predicted GCM1-binding site, with requirement for another factor(s) to bind the proximal promoter to exert this function. Based on these findings, we hypothesize that ETV7 and HOXA13 act in concert with GCM1 to initiate LHB transcription between days 30 and 31, with ETV1 partnering with GCM1 to maintain transcription.</p