Proliferation of bovine endometrial epithelial cells is promoted by prostaglandin E2-PTGER2 signaling through cell cycle regulation.

It is known that prostaglandin E2 (PGE2) induces proliferation of epithelia in bovine endometrial explants, however, the detailed mechanism of regulation of PGE2 in inducing bovine endometrial epithelial cell (bEEC) proliferation is unclear. In this study, we determined whether proliferation of bEECs is promoted by PGE2-prostaglandin E receptor 2 (PTGER2) signaling activation through cell cycle regulation. The results demonstrated that bEECs proliferation was induced by treatment of PGE2 and PTGER2 agonist butaprost. These processes were down-regulated by PTGER2 antagonist AH6809 and CDK inhibitors (LEE011, CDK2 Inhibitor II and Ro 3306). PGE2 and butaprost induced cyclins (A, B1, D1, D3 and E2), cyclin-dependent kinases (CDKs, 1, 2, 4 and 6), and epidermal growth factor (EGF) expression were inhibited by AH6809 treatment in bEECs. Moreover, proliferating cell nuclear antigen (PCNA), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and PTGER2 expression in bEECs were up-regulated by PGE2 and butaprost treatment. Our data demonstrate that PGE2-PTGER2 signaling activation has a direct molecular association with cell cycle regulation and cell proliferation in bEECs. Collectively, these findings will improve our understanding of the roles for PGE2-PTGER2 signaling activation in the physiological and pharmacological processes of bovine endometrium.

Live S. aureus and heat-killed S. aureus induce different inflammation-associated factors in bovine endometrial tissue in vitro.

Staphylococcus aureus is majorly involved in bovine mastitis; however, it weakly induces pro-inflammatory factors in mammary gland epithelial cells. We aimed to clarify the involvement of S. aureus in other inflammation types and its relationship with inflammatory factor secretion in bovine endometritis. We used live S. aureus (LSA)- and heat-killed S. aureus (HK-SA)-treated bovine endometrial tissue in vitro. The HK-SA-treated group showed significantly higher IL-6, IL-1ß, TNF-α, CXCL1/2 and TLR2 expression than the LSA-infected group. Contrastingly, the LSA-infected group showed significantly higher PTGS2, mPGES-1, and EP4 expression than the HK-SA treated group. There was no significant between-group difference in hyaluronan-binding protein 1 expression, which suggested similar inflammatory responses. H&E results indicated that LSA and HK-SA induced shedding of endometrial gland epithelial cells. The LSA-infected group showed higher high-mobility group box 1 protein expression than the HK-SA treated groups, which indicated differences in signaling pathway activation. Further, the LSA-treated group had higher JNK and p38 MAPK levels while the HK-SA-treated group had higher IκB-α levels. There was no significant between-group difference in the ERK signaling pathway. Our findings indicate that the pathogen-associated molecular patterns (PAMPs) of S. aureus activate pro-inflammatory factor expression via the TLR2-ERK-NF-κB signaling pathway. Contrastingly, LSA induced PGE2 accumulation via the TLR2/MAPKs signaling pathway. This is the first report that S. aureus and the PAMPs of S. aureus activate different signaling pathways and that LSA mainly induce PGE2 accumulation rather than cytokine secretion.

Lipopolysaccharide stimulates bovine endometrium explants through toll­like receptor 4 signaling and PGE2 synthesis.

Bovine endometrium infection with gram-negative bacteria commonly causes uterine diseases. Previous studies indicate that prostaglandin E2 (PGE2) is an inflammatory mediator in bacterial endometritis. However, the mechanism underlying lipopolysaccharide (LPS)-induced inflammatory response regulation in bovine endometrial explants remains elusive. In the present study, bovine explants were pre-treated with 15-hydroxyprostaglandin dehydrogenase (15-PGDH) inhibitors before LPS stimulation. PGE2 secretion, prostaglandin synthetase, pro-inflammatory factor, damage-associated molecular pattern (DAMP), and related signaling pathway factor levels were evaluated. Using 15-PGDH inhibitors pre-treatment, LPS-treated bovine endometrial explants exhibited augmentation of PGE2 and DAMP expression, and upregulation of various signaling pathway factors. Protein kinase A (PKA), extracellular-signal-regulated kinase, and c-Jun N-terminal kinase phosphorylation and degradation of nuclear transcription factor-κB (NF-κB) inhibitors were induced in the pre-treated endometrial explants. The mechanism underlying LPS-induced PGE2 accumulation acting as a pro-inflammatory mediator through toll-like receptor 4 signaling in bovine explants could involve the PKA, mitogen-activated protein kinase, and NF-κB pathways.

17ß-estradiol regulates prostaglandin E2 and F2α synthesis and function in endometrial explants of cattle.

Prostaglandins (PG) have primary functions in the reproductive tract, however, the mechanism of regulation of PG secretion in the endometrium is unclear. Estrogen as a predominant regulator of uterine functions during the mammalian estrous cycle and effects of estrogen on synthesis of PG and function in uterine tissues of cattle are not fully understood. In this study, there was evaluation of the concentration- and time-effects of 17ß-estradiol on PG synthesis in endometrial explants of cattle, focusing on the secretion of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) as well as relative abundance of mRNA transcript and protein for both the enzymes responsible for PGE2 and PGF2α synthesis, including prostaglandin-endoperoxide synthase 1 and 2 (PTGS1, PTGS2), PGE2 synthase (PGES), PGF2α synthase (PGFS), and carbonyl reductase (CBR1), and the receptors responsible for downstream PGE2 (PTGER2, PTGER4) and PGF2α (PTGFR) signaling. Results indicated that 17ß-estradiol increased PGE2 and PGF2α production at concentrations ranging from 10-11 to 10-8 M. Furthermore, abundances of PTGS1, PTGS2, PGES, PGFS, PTGER2, PTGER4, and PTGFR mRNA transcripts and protein were greater immediately after 17ß-estradiol treatment at almost all the concentrations, while these CBR1 abundances were less as a result of treatments with 17ß-estradiol. These data support the hypothesis that estradiol modulates the synthesis and function of PG in the endometrium of cattle.

Prostaglandin F2α-PTGFR signaling promotes proliferation of endometrial epithelial cells of cattle through cell cycle regulation.

There is production of prostaglandin F2α (PGF2α) and there is PGF2α receptor (PTGFR) mRNA transcript in endometrial epithelial cells of cattle. The aims of the present study were to (1) determine whether PGF2α-PTGFR signaling modulates the proliferation of endometrial epithelial cells and (2) increase knowledge of PGF2α-PTGFR signaling on the physiological and pharmacological processes in the endometrium of cattle. Amount of cellular proliferation was determined using real-time cell analysis and cell proliferation reagent WST-1 procedures. Abundance of cyclins, cyclin-dependent kinases (CDKs), cyclin-kinase inhibitors, proliferating cell nuclear antigen (PCNA), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), PTGFR, epidermal growth factor (EGF) mRNA and protein abundances were evaluated using real-time RT-PCR and western blot analyses. The PGF2α-PTGFR signaling promoted the proliferation of endometrial epithelial cells by inducing changes in abundance of mRNA transcript and protein that resulted in an increase in the abundance for the cyclins (A, B1, D1, D3), CDKs (1, 2, 4, 6), and PCNA; decrease in abundance for p21; and increase in abundance for EGF, COX-1, COX-2, and PTGFR. There was a direct molecular association between PGF2α-PTGFR signaling and cell cycle regulation in endometrial epithelial cells of cattle. In addition, findings improve the understanding of PGF2α-PTGFR signaling in the physiological and pharmacological processes of the endometrium of cattle.

Regulatory roles of PGE2 in LPS-induced tissue damage in bovine endometrial explants.

Bovine endometritis is the most common uterine disease following parturition. The role of prostaglandin E2 (PGE2) in regulating normal physiological function in the bovine endometrium has been clearly established. Although PGE2 accumulation is observed in multiple inflammatory diseases, such as endometritis, its association with pathogen-induced inflammatory damage in the endometrium is unclear. To clarify the role of PGE2 in lipopolysaccharide (LPS)-induced endometritis in cultured bovine endometrial explants, the levels of PGE2 secretion, prostaglandin synthetases, pro-inflammatory factors, and damage-associated molecular patterns (DAMPs) were evaluated in the present study. Significant PGE2 accumulation in response to LPS stimulation, up-regulation of prostaglandin-endoperoxide synthase-2 (PTGS-2), microsomal prostaglandin E synthase-1 (mPGES-1), pro-inflammatory factors including interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and induced nitric oxide synthase (iNOS)/nitric oxide (NO) and DAMPs including hyaluronan binding protein 1 (HABP1) and high mobility group box-1 (HMGB1), were observed compared to the control group. LPS induced distinct damage in the bovine endometrium, characterized by morphological changes and increases in HABP1 and HMGB1 expression. PTGS-2 inhibitors CAY10404 and NS398 effectively decreased the secretion of PGE2 and the expression of prostaglandin synthetases, pro-inflammatory factors and DAMPs, and alleviated LPS-induced tissue damage. These results indicate that PGE2 accumulates via PTGS-2 and mPGES-1 and induces tissue damage by upregulating pro-inflammatory factors and DAMPs in LPS-treated bovine endometrial explants. These findings provide a basis for the effect of PGE2 on LPS-treated bovine endometrium, and suggest a potential target for curing endometritis.

LP induced/mediated PGE2 synthesis through activation of the ERK/NF-κB pathway contributes to inflammatory damage triggered by Escherichia coli-infection in bovine endometrial tissue.

The bovine endometrium is constantly challenged with pathogenic bacteria, especially with Escherichia coli. In previous studies, we showed that prostaglandin E2 (PGE2) synthesis was increased in E. coli-infected bovine endometrial tissue, which promoted the development of inflammatory damage. However, the molecular mechanism underlying this accumulation of PGE2 remained undefined. Lipoprotein (LP) is one of critical outer membrane protein in E. coli, which regulates inflammatory response. In this study, we determined the role of LP in PGE2 accumulation in bovine endometrial tissue by infecting the tissue with wild endometrial pathogenic E. coli and E. coli LP deletion mutant (JE5505) strains. We demonstrate that JE5505 was less effective than pathogenic E. coli in inducing the production of PGE2,IL-6, TNF-α, HMGB-1, and HABP1 and that the induction of cytokines was dependent on the activation of MAPKs, as revealed by rapid phosphorylation of ERK1/2/NF-κB in the endometrial tissues, furthermore, LP also induced PGE2 synthessis and cytokine secretion. Additionally, ERK and NF-κB inhibitors significantly inhibited PGE2 production and cytokine secretion and reduced or attenuated tissue damage in JE5505-infected and LP induced endometrial tissues. What is more important, we reported PGE2 introduction increased the expression of pro-inflammatory factors and DAMPs in E. coli-infected bovine endometrial tissue. Taken together, these results indicate that LP is involved in the accumulation of PGE2 through the activation of the ERK/NF-κB pathway that induces the production of pro-inflammatory factors and damage-associated molecular patterns (DAMPs) in E. coli-infected bovine endometrial tissue. These results should help in better understanding and management of postpartum inflammatory diseases in dairy cows.

PTGFR activation promotes the expression of PTGS-2 and growth factors via activation of the PKC signaling pathway in bovine endometrial epithelial cells.

The endometrium of domestic animals has a remarkable capacity to self-repair. Prostaglandin F2α (PGF2α) is one of the major prostaglandins secreted from the endometrium. The role of PGF2α in endometrial repair, however, is still unknown. In the present study, it was investigated whether prostaglandin F2α receptor (PTGFR) activation could induce expression of prostaglandin-endoperoxide synthase 2 (PTGS-2) and growth factors associated with endometrial repair via activation of protein kinase C (PKC) signaling in endometrial epithelial cells (bEECs) of cattle. Results of the present study indicated that the treatment with the PTGFR agonist, fluprostenol, resulted in an increase in abundance of proteins for PTGS-2, vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), transforming growth factor beta 1 (TGF-ß1), and interleukin-8 (IL-8). The increased abundances of these proteins were suppressed by the treatment with the PTGFR antagonist, AL8810.Furthermore, fluprostenol treatment also induced PKC phosphorylation. Subsequently, treatment with AL8810 inhibited the fluprostenol-induced PKC phosphorylation. Additionally, treatment with the PKC inhibitor, chelerythrine, reduced the fluprostenol-induced increase in the relative abundance of VEGF, CTGF, TGF-ß1, and IL-8 mRNA in bEECs. Taken together, these results suggest that PTGFR activation may induce endometrial repair by upregulating PTGS-2 gene expression and stimulating VEGF, CTGF, TGF-ß1, and IL-8 gene expression via activation of the PKC signaling pathway.

PGE2 downregulates LPS-induced inflammatory responses via the TLR4-NF-κB signaling pathway in bovine endometrial epithelial cells.

Postpartum bacterial infections of the uterus cause endometritis in dairy cows. Inflammatory responses to bacterial infections in the bovine uterus were generated through pattern recognition receptors (PRRs) that bind to pathogen-associated molecules such as lipopolysaccharide (LPS) from Escherichia coli. Among these PRRs, Toll-like receptor 4 (TLR4) is primarily responsible for LPS recognition, which triggers inflammatory responses via mitogen-activated protein kinases (MAPKs) and NF-κB signaling activation, resulting in the expression of inflammatory mediators in mammals such as IL-8 and IL-6. Previous studies indicate that PGE2 plays an important role in bacterial endometritis, although details on the mechanism underlying how it regulates LPS-induced inflammatory responses in bovine endometrial epithelial cells (bEECs) remain elusive. In the present study, bEECs were pre-treated with exogenous PGE2 and/or PGF2α prior to LPS stimulation. With PGE2 pre-treatment, we observed an augmentation in LPS-stimulated PKA, ERK, and IκBα phosphorylation and cyclooxygenase-2 (COX-2) and anti-inflammatory cytokine IL-6 expression and downregulation of prostaglandin E2 receptor 4 (EP4) and TLR4 in bEECs. These results indicate that LPS-induced inflammatory responses through TLR4 signaling in bEECs could be downregulated by exogenous PGE2 pre-treatment, but not PGF2α.

PTGER2 activation induces PTGS-2 and growth factor gene expression in endometrial epithelial cells of cattle.

The prostaglandin E2 receptor 2 (PTGER2) is present in the endometrium and its gene expression is accompanied with endometrial growth, however, it is unknown whether there is endometrial repair through stimulation of growth factor gene expression that is promoted by PTGER2 activation in cattle. The aim of this study was to investigate whether PTGER2 activation can induce prostaglandin-endoperoxide synthase-2 (PTGS-2) and growth factor gene expression by activating PKA and ERK signaling pathways in endometrial epithelial cells of cattle. Results demonstrated that the PTGER2 agonist, butaprost, induced cAMP/PKA and ERK activation and up-regulated PTGS-2, VEGF, CTGF, TGF-ß1 and IL-8 gene expression. These activations were less after PTGER2 antagonist, AH6809, treatment. Data suggested that PTGS-2 gene expression was induced by PTGER2 activation through the PKA and ERK pathways. Furthermore, PTGER2 activation promoted several growth factor gene expressions in endometrial epithelial cells. One potential implication of this finding is that PTGER2 activation in the endometrium of cattle could induce endometrial repair by stimulating VEGF, CTGF, TGF-ß1 and IL-8 gene expression.

The effect of prostaglandin E2 receptor (PTGER2) activation on growth factor expression and cell proliferation in bovine endometrial explants.

The domestic animal endometrium undergoes regular periods of regeneration and degeneration during cycles of oestrus and dioestrus. If blastocyst implantation occurs in the uterus, the endometrium will prepare for pregnancy by changing its pattern of development to build a connection with the embryo to nourish it. Prostaglandin E2 (PGE2) secretion synchronized with endometrial growth in these processes and could be involved in endometrial growth. One of the PGE2 receptors (PTGER2) is present in endometrium and its increased expression accompanies with endometrial growth in above processes. However, the association between PTGER2 and endometrial growth remains unclear. Endometrial growth factors and cell proliferation is the foundation for endometrial growth. Therefore, in this study, the response of growth factors and cell proliferation essential for endometrial growth to PTGER2 activation were investigated in bovine endometrium. The results indicated that mRNA and protein expression of connective tissue growth factor (CTGF), fibroblast growth factor-2 (FGF-2), interleukin-8 (IL-8), transforming growth factor-ß1 (TGF-ß1), matrix metalloproteinase-2 (MMP-2), and vascular endothelial growth factor A (VEGFA) were up-regulated after PTGER2 activation by corresponding agonist butaprost (P < 0.05), and proliferation of endometrial epithelia and fibroblasts were induced in response to increased levels of proliferating cell nuclear antigen (PCNA), cytokeratin-18 (CK-18) and fibroblast-specific protein 1 (FSP-1) expression in bovine endometrial explants in vitro (P < 0.05).

Prostaglandin F2α-PTGFR signalling activation, growth factor expression and cell proliferation in bovine endometrial explants.

The endometrium of domestic animals undergoes regular periods of regeneration and degeneration and exhibits a remarkable capacity for self-repair during the oestrous cycle. The endometrial growth pattern is also observed during in the implantation period and early pregnancy, but the mechanism underlying endometrial growth in these processes remains unclear. A positive correlation between endometrial growth in these processes and prostaglandin (PG) F2α secretion has been reported, but the roles that PGF2α plays in endometrial growth is less studied. In the present study, cell proliferation and the responses of a series of growth factors essential for endometrial growth to PGF2α receptor (PTGFR) activation were investigated in bovine endometrial explants in vitro. Using real-time reverse transcription-polymerase chain reaction and western blotting, mRNA and protein expression of connective tissue growth factor, fibroblast growth factor2, interleukin8, matrix metalloproteinase2, transforming growth factor ß1 and vascular endothelial growth factor A was increased (P<0.05) and cell proliferation, including epithelial and fibroblast proliferation, was induced in response to increased levels of proliferating cell nuclear antigen, cytokeratin-18 and fibroblast-specific protein-1 (P<0.05) following PTGFR activation by adding fluprostenol (10-9-10-5 M) into culture medium of bovine endometrial explants. However, caspase-3 protein expression was reduced following treatment of explants with fluprostenol (10-9-10-5 M, P<0.05). These results may help define the possible roles the PGF2α-PTGFR signalling pathway plays in endometrial growth.