O corpo lúteo (CL) canino é responsável pela síntese de E2 durante o diestro. O E2 atua de forma autócrina e/ou parácrina sobre essa glândula. O mecanismo de atuação do E2 depende da razão entre receptor alfa (ERα) e receptor beta (ERβ). A ligação ao ERα tem efeito proliferativo e ao ERβ antiproliferativo. O objetivo deste trabalho foi entender a sinalização mediada pelo ERα e ERβ no processo de formação e regressão do CL. CLs foram obtidos de cadelas não prenhes (n=30) nos dias 10, 20, 30, 40, 50 e 60 (n=5/grupo) após a ovulação (po). No dia da ovariossalpingohisterectomia foi colhido sangue para mensuração das concentrações de P4 e E2. Dezoito CLs (n=3/grupo) foram submetidos ao sequenciamento de RNA (RNAseq). Os genes diferencialmente expressos (DE) identificados pelo RNAseq foram submetidos ao software oPOSSUM3 para identificação das regiões de ligação mais representadas nos fatores de transcrição relacionados aos genes do ERα (ESR1) e ERβ (ESR2). A análise de expressão temporal revelou a presença de 5.116 diferencialmente expressos (DE) em pelo menos uma comparação e 1.106 não foram anotados ainda no genoma canino, destes genes DE 295 apresentavam regiões de ligação de fatores de transcrição em comum com ESR1 e ESR2. Dez genes DE foram selecionados para validação dos resultados de RNAseq através do qPCR; usando o GAPDH como gene de referência. Desses genes, quatro apresentaram regioes em comum com ESR2 (LEF-1; PAPPA; NDGR2 ATP1A1) e um com ESR1 (CAV1) e os demais controlam proliferação celular (CTNNB1; CCND1; IGFBP 3, 4 e 5). A expressão proteíca de PAPPA e IGFBP 3, 4 e 5 (componentes do sistema IGF) foi avaliada também por imunohistoquímica. Durante a primeira metade do diestro, nossos resultados indicam que a sinalização mediada pelo E2 ocorre via interação do ERα com CAV-1 (sinalização não genômica), ativando as vias de sinalização IGF e WNT/βcatenina, regulando o processo de proliferação celular. Enquanto na segunda metade, o ERβ regularia a expressão gênica de NDGR2 e ATP1A1, contribuindo para a regressão do CL. Assim nos resultados sugerem que o E2 atue tanto como um fator luteotrófico e quanto regulador da regressão do CL canino. / The canine corpus luteum (CL) is responsible for E2 synthesis during diestrus, which acts in an autocrine and / or paracrine manner in this gland. The E2 mechanism of action depends on the ratio between alpha (ERα) and beta (ERβ) receptor The binding to ERα has a proliferative effect whereas to ERβ an antiproliferative. The objective of this study was to understand the signaling mediated by ERα and ERβ in the formation and regression of the CL. CLs were obtained from non-pregnant bitches (n = 30) on days 10, 20, 30, 40, 50 and 60 (n = 5 / group) post-ovulation (po). On the day of ovariohysterectomy blood was collected for measurement of P4 and E2 concentrations. Eighteen CLs (n = 3 / group) were subjected to RNA sequencing (RNA-Seq). Genes differentially expressed (DE) identified by RNA-Seq were submitted to oPOSSUM3 software for detection of over-represented transcription factors binding sites (TFBS) related to the ERα (ESR1) and ERβ (ESR2) genes. The temporal expression analysis revealed the presence of 5116 differentially expressed (DE) genes in at least one comparison, and 1106 genes, which have not been recorded to the canine genome yet From these, 295 genes showed TFBS in common with ESR1 and ESR2. Ten DE genes were selected to validate the results of RNA-Seq by qPCR; using GAPDH as reference gene. Of these genes, four had TFBS in common with ESR2 (LEF-1; PAPPA; NDGR2; ATP1A1) and one with ESR1 (CAV1) and others genes were chosen because they control cell proliferation (CTNNB1; CCND1, IGFBP 3, 4 and 5). Protein expression of the IGF related genes (PAPPA and IGFBP 3, 4 and 5) was also evaluated by immunohistochemistry. During the first half of diestrus, it appears that E2 mediated signaling occurs via interaction of ERα with CAV-1 (non-genomic signaling), activating signaling pathways of IGF and WNT / βcatenin, then regulating the cell proliferation process. Whereas in the second half, ERβ appears to regulate NDGR2 and ATP1A1 gene expression, contributing to the regression of the CL. Thus our results suggest that E2 may act as a luteotrophic and also a luteolytic factor in the canine CL.

O corpo lúteo (CL) canino é responsável pela síntese de E2 durante o diestro. O E2 atua de forma autócrina e/ou parácrina sobre essa glândula. O mecanismo de atuação do E2 depende da razão entre receptor alfa (ERα) e receptor beta (ERβ). A ligação ao ERα tem efeito proliferativo e ao ERβ antiproliferativo. O objetivo deste trabalho foi entender a sinalização mediada pelo ERα e ERβ no processo de formação e regressão do CL. CLs foram obtidos de cadelas não prenhes (n=30) nos dias 10, 20, 30, 40, 50 e 60 (n=5/grupo) após a ovulação (po). No dia da ovariossalpingohisterectomia foi colhido sangue para mensuração das concentrações de P4 e E2. Dezoito CLs (n=3/grupo) foram submetidos ao sequenciamento de RNA (RNAseq). Os genes diferencialmente expressos (DE) identificados pelo RNAseq foram submetidos ao software oPOSSUM3 para identificação das regiões de ligação mais representadas nos fatores de transcrição relacionados aos genes do ERα (ESR1) e ERβ (ESR2). A análise de expressão temporal revelou a presença de 5.116 diferencialmente expressos (DE) em pelo menos uma comparação e 1.106 não foram anotados ainda no genoma canino, destes genes DE 295 apresentavam regiões de ligação de fatores de transcrição em comum com ESR1 e ESR2. Dez genes DE foram selecionados para validação dos resultados de RNAseq através do qPCR; usando o GAPDH como gene de referência. Desses genes, quatro apresentaram regioes em comum com ESR2 (LEF-1; PAPPA; NDGR2 ATP1A1) e um com ESR1 (CAV1) e os demais controlam proliferação celular (CTNNB1; CCND1; IGFBP 3, 4 e 5). A expressão proteíca de PAPPA e IGFBP 3, 4 e 5 (componentes do sistema IGF) foi avaliada também por imunohistoquímica. Durante a primeira metade do diestro, nossos resultados indicam que a sinalização mediada pelo E2 ocorre via interação do ERα com CAV-1 (sinalização não genômica), ativando as vias de sinalização IGF e WNT/βcatenina, regulando o processo de proliferação celular. Enquanto na segunda metade, o ERβ regularia a expressão gênica de NDGR2 e ATP1A1, contribuindo para a regressão do CL. Assim nos resultados sugerem que o E2 atue tanto como um fator luteotrófico e quanto regulador da regressão do CL canino. / The canine corpus luteum (CL) is responsible for E2 synthesis during diestrus, which acts in an autocrine and / or paracrine manner in this gland. The E2 mechanism of action depends on the ratio between alpha (ERα) and beta (ERβ) receptor The binding to ERα has a proliferative effect whereas to ERβ an antiproliferative. The objective of this study was to understand the signaling mediated by ERα and ERβ in the formation and regression of the CL. CLs were obtained from non-pregnant bitches (n = 30) on days 10, 20, 30, 40, 50 and 60 (n = 5 / group) post-ovulation (po). On the day of ovariohysterectomy blood was collected for measurement of P4 and E2 concentrations. Eighteen CLs (n = 3 / group) were subjected to RNA sequencing (RNA-Seq). Genes differentially expressed (DE) identified by RNA-Seq were submitted to oPOSSUM3 software for detection of over-represented transcription factors binding sites (TFBS) related to the ERα (ESR1) and ERβ (ESR2) genes. The temporal expression analysis revealed the presence of 5116 differentially expressed (DE) genes in at least one comparison, and 1106 genes, which have not been recorded to the canine genome yet From these, 295 genes showed TFBS in common with ESR1 and ESR2. Ten DE genes were selected to validate the results of RNA-Seq by qPCR; using GAPDH as reference gene. Of these genes, four had TFBS in common with ESR2 (LEF-1; PAPPA; NDGR2; ATP1A1) and one with ESR1 (CAV1) and others genes were chosen because they control cell proliferation (CTNNB1; CCND1, IGFBP 3, 4 and 5). Protein expression of the IGF related genes (PAPPA and IGFBP 3, 4 and 5) was also evaluated by immunohistochemistry. During the first half of diestrus, it appears that E2 mediated signaling occurs via interaction of ERα with CAV-1 (non-genomic signaling), activating signaling pathways of IGF and WNT / βcatenin, then regulating the cell proliferation process. Whereas in the second half, ERβ appears to regulate NDGR2 and ATP1A1 gene expression, contributing to the regression of the CL. Thus our results suggest that E2 may act as a luteotrophic and also a luteolytic factor in the canine CL.

Estrogen is an important regulator and promoter of epithelial wound healing. This is facilitated by increased keratinocyte and fibroblast migration and proliferation, as well as promotion of angiogenesis, matrix deposition and inflammatory response dampening. The potential to target this pathway for therapeutics is highlighted by observations that post-menopausal women on hormone replacement therapy have a significantly lower incidence of venous ulcers. Previous work from this laboratory identified four SNPs (single nucleotide polymorphisms) in the 5’UTR of estrogen receptor beta (ERβ) gene that are associated with venous ulcer predisposition. Disease association is further supported by the identification of ERβ as the main conduit of the beneficial effects of estrogen signalling on wound healing. SNP’s of the 5’UTR can affect transcriptional expression through the modification of transcription factor binding sites, epigenetic modifications and translational efficiency via mRNA localisation and secondary structure alterations. To investigate the possible biological function of these SNPs, we have developed disease relevant cell based assays where primary keratinocyte and fibroblast cells were selected harbouring disease-associated SNPs. We demonstrate that the presence of venous ulcer-associated ERβ SNPs reduced the expression of ERβ in skin cells and reduced their migration and proliferative capabilities. Evidence gathered here suggests that ERβ expression is curtailed by a change in transcription factor binding, likely facilitated by the change in nucleotide sequence brought about by the rs2987983 SNP. Further, we demonstrate that SNP-induced changes in fibroblast expression of growth factors and inflammatory mediators can hinder keratinocyte migration and induce a pro-inflammatory phenotype in human monocytes. Lastly, RNAseq analysis of keratinocytes reveals a SNP-dependant gene expression profile that is detrimental to wound healing. This work provides the first evidence of a direct functional link between venous ulcer-associated ERβ SNPs and dysfunctional wound healing. Investigating ERβ SNPs has provided insight into novel mechanisms of estrogen signalling that can be applied for therapeutic development to treat venous ulcers.

Background. Endocrine disruptors (EDC) are exogenous compounds that mimic the action of natural hormones and alter the normal endocrine system. Life-long chronic exposure to Bisphenol A (BPA), a putative EDC, has been linked with risk of metabolic disorders in epidemiological studies. Objectives. The aim was to study the human health effects of exposure to BPA, using an integrated approach combining environmental epidemiology and toxicology. Methods. Urinary levels of BPA exposure were measured in participants of the InChianti longitudinal study, a representative population-based study of Italian adults, at the Baseline (1998-00) and nine years later (3rd Wave, 2007-09). Hormones levels and the gene expression of specific target genes were the end points considered. Results were validated in laboratory studies on a human leukemic T-cell line (Jurkat cells). Results. In general, urinary BPA (uBPA) concentrations were higher among men and younger respondents, and within subjects uBPA concentrations were correlated (r=0.58; p=0.013, model adjusted for age, sex, urinary creatinine). At baseline, uBPA concentration were associated with higher total testosterone concentrations in men (β = 0.05; 95% CI, 0.02–0.08). In the 3rd wave, gene expression analysis revealed positive associations between uBPA concentrations and ESR2 (estrogen receptor beta) expression (β=0.18; 95% CI: 0.04, 0.32) and ESRRA (estrogen related receptor alpha) expression (β= 0.17; 95% CI: 0.02, 0.32). In a following in vitro study, BPA exposure (0.001-1 micro molar) led to enhanced expression of ESRRA and ESR2 in Jurkat cells over a period of 72 hours. Conclusions. Results indicate that BPA is bioactive in the human body and is able to alter circulating hormone concentrations and estrogen receptor/estrogen-related receptr gene expression. In particular, given the role of ERRα as a major control point for oxidative metabolism and heart development, this research provides indications on the possible molecular mechanisms of action of BPA in metabolic diseases.

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