Transcriptome analysis of adipose tissue in grazing cattle: Identifying key regulators of fat metabolism.

The taste and tenderness of meat are the main determinants of carcass quality in many countries. This study aimed to discuss the mechanisms of intramuscular fat deposition in grazing and house-breeding cattle. We performed transcriptome analysis to characterize messenger RNA and microRNA (miRNA) expression profiles. A total of 456 and 66 differentially expressed genes (DEGs) and differentially expressed (DE) miRNAs were identified in the adipose tissue of grazing and house-breeding cattle. Kyoto Encyclopedia of Genes and Genomes pathway analysis identified the association of DEGs with fatty acid metabolism, fatty acid degradation, peroxisome proliferator-activated receptors signaling pathway, adenosine monophosphate-activated protein kinase signaling pathway, adipocytokine signaling pathway, and the association of DE miRNAs with mitogen-activated protein kinase signaling pathway. Apolipoprotein L domain containing 1, pyruvate dehydrogenase kinase 4, and sphingosine-1-phosphate lyase 1 genes may be the key regulators of fat metabolism in grazing cattle. Finally, we found that miR-211 and miR-331-5p were negatively correlated with the elongation of very long-chain fatty acids protein 6 (ELOVL6), and miR-331-5p might be the new regulator involved in fat metabolism. The results indicated that ELOVL6 participated in various functions and pathways related to fat metabolism. Meanwhile, miR-331-5p, as a new regulator, might play an essential role in this process. Our findings laid a more in-depth and systematic research foundation for the formation mechanism and characteristics of adipose tissue in grazing cattle.

Prostaglandin E2 accumulation is closely associated with S. aureus-infected bovine endometritis.

S. aureus isolated from bacterial bovine endometritis is common in epidemiological reports, but is often ignored as a subclinical pathogenic microorganism. In a previous study, we showed that live S. aureus (LSA) and heat killed S. aureus (HK-SA) induce different inflammatory responses in bovine endometrial tissue, and possibly being associated with the accumulation of prostaglandin E2 (PGE2). Thus, in this study, we varied PGE2 concentrations using inhibitors or agonists in HK-SA-treated bovine endometrial tissues. The results demonstrated that PGE2 has a positive relationship with IL-6, TNF-α, and damage-associated molecular patterns (DAMPs; e.g., HMGB-1 and HABP-1) expression and tissues damage, and is regulated by the EP4-p38 MAPK pathway. We concluded that lipoproteins of S. aureus are associated with PGE2 generation. To further explore the relationship between LSA and PGE2 accumulation, we used the S. aureus strain SA113 lipoprotein knockout (SA113Δlpl) to infect bovine endometrial epithelial cells (BECs). LSA decreased PGE2, cAMP, EP4, IL-6, IL-8, cAMP secretion, and the MAPK and PKA signaling pathways when infected with SA113Δlpl, as compared with SA113-infected groups. Moreover, the adhesion and invasion of BECs were similarly downregulated when lipoproteins in S. aureus were knocked out. The results of this study show that PGE2 is involved in both HK-SA- and LSA-induced inflammatory responses in the bovine endometrium. We suggest that S. aureus infection is associated with bovine endometritis, and although HK-SA and LSA induce different inflammatory responses, the strategy of decreasing PGE2 accumulation is helpful in reducing the inflammation stage caused by S. aureus.

Anti-inflammatory effects of the prostaglandin D2/prostaglandin DP1 receptor and lipocalin-type prostaglandin D2 synthase/prostaglandin D2 pathways in bacteria-induced bovine endometrial tissue.

Dairy cows often develop different degrees of endometritis after calving and this is attributed to pathogenic bacterial infections such as by Escherichia coli and Staphylococcus aureus. Infection of the bovine endometrium causes tissue damage and increases the expression of prostaglandin D2 (PGD2), which exerts anti-inflammatory effects on lung inflammation. However, the roles of PGD2 and its DP1 receptor in endometritis in cows remain unclear. Here, we examined the anti-inflammatory roles of the lipocalin-type prostaglandin D2 synthase (L-PGDS)/PGD2 and DP1 receptor regulatory pathways in bovine endometritis. We evaluated the regulatory effects of PGD2 on inflammation and tissue damage in E. coli- and S. aureus-infected bovine endometrial cells cultured in vitro. We found that the secretion of pro-inflammatory cytokines interleukin (IL)-6, IL-1ß, and tumour necrosis factor (TNF)-α as well as expression of matrix metalloproteinase (MMP)-2, platelet-activating factor receptor (PAFR), and high mobility group box (HMGB)-1 were suppressed after DP1 receptor agonist treatment. In contrast, IL-6, IL-1ß, and TNF-α release and MMP-2, PAFR, and HMGB-1 expression levels were increased after treatment of bovine endometrial tissue with DP1 receptor antagonists. DP1-induced anti-inflammatory effects were dependent on cellular signal transduction. The L-PGDS/PGD2 pathway and DP1 receptor induced anti-inflammatory effects in bovine endometrium infected with S. aureus and E. coli by inhibiting the mitogen-activated protein kinase and nuclear factor-κB signalling pathways, thereby reducing tissue damage. Overall, our findings provide important insights into the pathophysiological roles of PGD2 in bovine endometritis and establish a theoretical basis for applying prostaglandins or non-steroidal anti-inflammatory drugs for treating endometrial inflammatory infertility in bovines.

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.

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.

Prostaglandin E2 promotes nitric oxide synthase 2, platelet-activating factor receptor, and matrix metalloproteinase-2 expression in Escherichia coli-challenged ex vivo endometrial explants via the prostaglandin E2 receptor 4/protein kinase a signaling pathway.

Prostaglandin E2 (PGE2) is an inflammatory mediator involved in the pathogenesis of several chronic inflammatory conditions, including endometritis. Previous studies have shown that PGE2 accumulates in Escherichia coli-challenged ex vivo endometrial explants, increasing the expression of pro-inflammatory factors and aggravating tissue damage; these alterations are linked to key enzymes involved in the synthesis of PGE2, including cyclooxygenases-2 (COX-2) and microsomal PGES-1 (mPGES-1). In this study, we aimed to investigate whether administration of PGE2 modulated the activities of nitric oxide synthase 2 (NOS2), platelet-activating factor receptor (PAFR), and matrix metalloproteinase (MMP)-2 in E. coli-challenged ex vivo bovine endometrial explants. Our findings showed that COX-2 and mPGES-1 inhibitors significantly reduced NOS2, PAFR, and MMP-2 expression in the E. coli-challenged ex vivo endometrial explants. In addition, NOS2, PAFR, and MMP-2 expression levels were strongly increased in response to treatment with 15-prostaglandin dehydrogenase inhibitors in the E. coli-challenged ex vivo endometrial explants. However, these stimulatory effects could be blocked by PGE2 receptor 4 (EP4) and protein kinase A (PKA) inhibitors. Overall, these findings show that pathogenic PGE2 upregulated NOS2, PAFR, and MMP-2 expression, which may enhance inflammatory damage via the EP4/PKA signaling pathway in E. coli-challenged ex vivo endometrial explants.

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.

Prostaglandin E2 promotes Pam3CSK4-induced inflammation in endometrial epithelial cells of cattle.

Bacterial contamination often impairs uterine function in cattle leading to uterine diseases such as endometritis. Inflammatory responses to bacterial infections in the uterus of cattle are generated through pattern recognition receptors, including Toll-like receptor 2 (TLR2), which is responsible for Pam3CSK4 recognition. This cellular response induces inflammatory responses through stimulation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB signaling activation, stimulating the expression of inflammatory mediators. Prostaglandin (PG) E2 has important actions in bacterial endometritis, although details through which these mechanisms regulate Pam3CSK4-induced inflammatory responses in cattle endometrial epithelial cells (bEECs) remain unclear. In the present study there was examination of the actions of exogenous PGE2 in Pam3CSK4-induced inflammatory responses. The bEECs pre-treated with exogenous PGE2 prior to Pam3CSK4 treatment had an augmented Pam3CSK4-stimulated phosphorylation of protein kinase A (PKA), extracellular signal-regulated kinase (ERK), and IκB-α; stimulation of TLR2, cyclooxygenase-2, and interleukin-6 functions; and suppression of the activation of PGE2 receptor 4. Thus, Pam3CSK4-induced inflammatory responses through TLR2 signaling in bEECs were enhanced by exogenous PGE2 pre-treatment.

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.