Selective Estrogen Receptor Down-Regulator and Selective Estrogen Receptor Modulators Differentially Regulate Lactotroph Proliferation

occupation, differentially modulates the biological outcome of anti-estrogens. expression and release, as well as ERE-mediated transcriptional activity. expression

Epidermal growth factor receptor cross-talks with ligand-occupied estrogen receptor-α to modulate both lactotroph proliferation and prolactin gene expression

Both estrogen (E2) and EGF regulate lactotrophs, and we recently demonstrated that EGF phosphorylates S118 on estrogen receptor-α (ERα) and requires ERα to stimulate prolactin (PRL) release. However, the interactions between ligand-occupied ERα and activated ErbB1 and its impact on lactotroph function are unknown. Using rat GH3 lactotrophs, we found that both E2 and EGF independently stimulated proliferation and PRL gene expression. Furthermore, their combination resulted in an enhanced stimulatory effect on both cell proliferation and PRL gene expression. Inhibitors of ER as well as ErbB1 blocked the combined effects of E2 and EGF. Pretreatment with UO126 abolished the combined effects, demonstrating Erk1/2 requirement. Although bidirectionality in ER-ErbB1 cross-talk is a well-accepted paradigm, interestingly in lactotrophs, ErbB1 kinase inhibitor failed to block the effect of E2 on proliferation and stimulation of PRL gene expression, suggesting that ER does not require ErbB1 to mediate its effects. Furthermore, E2 did not affect the ability of EGF to induce c-Fos expression or modulate AP-1 activity. However, both E2 and EGF combine to enhance S118 phosphorylation of ERα, leading to enhanced E2-mediated estrogen response element transactivation. Taken together, our results suggest that, in lactotrophs, activated ErbB1 phosphorylates ERα to enhance the stimulatory effect of E2, thereby providing the molecular basis by which EGF amplifies the response of E2

MPP and ICI 182780 inhibit ERE transcriptional activity.

<p>GH3 cells, transiently co-transfected with an ERE reporter gene and a control reporter gene, were treated with the indicated concentrations of ICI or MPP for 24 hrs, and normalized luciferase activity was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010060#s4" target="_blank">methods</a>. Data was calculated as % of vehicle control (100%). Each value is the mean ±SEM of 4 separate experiments each performed in triplicates. * indicates significant difference from control (<i>p<0.05</i>).</p

Differential effects of ERα antagonist on cell proliferation and ERα degradation.

<p><i>A]</i> GH3 cells were treated with either vehicle, ICI (10 nM) or MPP (100 nM) for 5 days. Cell lysates were subjected to western blotting with an anti-ERα Ab (<i>top panel</i>). Equal loading was verified by using the anti-actin Ab (<i>lower panel</i>). Results shown are from a single experiment and it is a representative of 3 independent experiments yielding similar results. <i>B]</i> GH3 cells were treated either with 10 nM ICI for 1 hr, or pretreated with a broad-spectrum proteosome inhibitor MG132 (10 µM) for 1 hr, followed by ICI for 1 hr. Western blotting using anti-ERα Ab <i>(Upper Panel)</i>, or anti-actin Ab <i>(Lower Panel)</i>, was performed. Data is from a single experiment and is representative of 2 independent experiments yielding similar results. <i>C]</i> GH3 cells were treated with either vehicle or ICI (0.001, 0.01, 0.1, 1, 10 and 100 nM) or MPP (0.001, 0.01, 0.1, 1, 10 and 100 nM) for 5 days, and cell proliferation was determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010060#s4" target="_blank">materials and methods</a>. Data is expressed as % of Control and it is the mean ± SEM of 3 separate experiments. * indicates significant differences from control (<i>p<0.05</i>). <i>D]</i> GH3 cells were treated with either vehicle, ICI (1 and 10 nM) or pretreated with MPP (100 nM for 1 hr) followed by treatment with ICI (1 and 10 nM) for 5 days. Cell proliferation was determined, and data is expressed as optical density, and it is the mean ± SEM of 4–6 determinations from a single experiment, which is a representative of 3 separate experiments with similar results. * designates significant differences from control (<i>p<0.05</i>).</p

ICI 182780 but not MPP inhibits anchorage independent growth of GH3 cells.

<p>GH3 cells were cultured on soft agar in complete medium containing ICI (10 nM) or (MPP 10 nM) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010060#s4" target="_blank">materials and methods</a>. <i>A]</i> Bright field microscopy shows GH3 cell colonies in soft agar. <i>B]</i> Quantitative analysis of GH3 cell colonies in soft agar following anti-estrogen treatment. Colonies were counted in at least 3 independent fields. Data is calculated as % of control and it is the mean ± SEM of 4 separate experiments. * indicates significant differences from control (<i>p<0.05</i>).</p

Genomic reconstruction of the SARS-CoV-2 epidemic in England

AbstractThe evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus leads to new variants that warrant timely epidemiological characterization. Here we use the dense genomic surveillance data generated by the COVID-19 Genomics UK Consortium to reconstruct the dynamics of 71 different lineages in each of 315 English local authorities between September 2020 and June 2021. This analysis reveals a series of subepidemics that peaked in early autumn 2020, followed by a jump in transmissibility of the B.1.1.7/Alpha lineage. The Alpha variant grew when other lineages declined during the second national lockdown and regionally tiered restrictions between November and December 2020. A third more stringent national lockdown suppressed the Alpha variant and eliminated nearly all other lineages in early 2021. Yet a series of variants (most of which contained the spike E484K mutation) defied these trends and persisted at moderately increasing proportions. However, by accounting for sustained introductions, we found that the transmissibility of these variants is unlikely to have exceeded the transmissibility of the Alpha variant. Finally, B.1.617.2/Delta was repeatedly introduced in England and grew rapidly in early summer 2021, constituting approximately 98% of sampled SARS-CoV-2 genomes on 26 June 2021.</jats:p