Dynamics of DNA methylation during osteogenic differentiation of porcine synovial membrane mesenchymal stem cells from two metabolically distinct breeds.

Mesenchymal stem cells (MSCs), with the ability to differentiate into osteoblasts, adipocytes, or chondrocytes, show evidence that the donor cell's metabolic type influences the osteogenic process. Limited knowledge exists on DNA methylation changes during osteogenic differentiation and the impact of diverse donor genetic backgrounds on MSC differentiation. In this study, synovial membrane mesenchymal stem cells (SMSCs) from two pig breeds (Angeln Saddleback, AS; German Landrace, DL) with distinct metabolic phenotypes were isolated, and the methylation pattern of SMSCs during osteogenic induction was investigated. Results showed that most differentially methylated regions (DMRs) were hypomethylated in osteogenic-induced SMSC group. These DMRs were enriched with genes of different osteogenic signalling pathways at different time points including Wnt, ECM, TGFB and BMP signalling pathways. AS pigs consistently exhibited a higher number of hypermethylated DMRs than DL pigs, particularly during the peak of osteogenesis (day 21). Predicting transcription factor motifs in regions of DMRs linked to osteogenic processes and donor breeds revealed influential motifs, including KLF1, NFATC3, ZNF148, ASCL1, FOXI1, and KLF5. These findings contribute to understanding the pattern of methylation changes promoting osteogenic differentiation, emphasizing the substantial role of donor the metabolic type and epigenetic memory of different donors on SMSC differentiation.

Effect of metabolically divergent pig breeds and tissues on mesenchymal stem cell expression patterns during adipogenesis.

BACKGROUND: Unraveling the intricate and tightly regulated process of adipogenesis, involving coordinated activation of transcription factors and signaling pathways, is essential for addressing obesity and related metabolic disorders. The molecular pathways recruited by mesenchymal stem cells (MSCs) during adipogenesis are also dependent on the different sources of the cells and genetic backgrounds of donors, which contribute to the functional heterogeneity of the stem cells and consequently affect the developmental features and fate of the cells. METHODS: In this study, the alteration of transcripts during differentiation of synovial mesenchymal stem cells (SMSCs) derived from fibrous synovium (FS) and adipose synovial tissue (FP) of two pig breeds differing in growth performance (German Landrace (DL)) and fat deposition (Angeln Saddleback (AS)) was investigated. SMSCs from both tissues and breeds were stimulated to differentiate into adipocytes in vitro and sampled at four time points (day 1, day 4, day 7 and day 14) to obtain transcriptomic data. RESULTS: We observed numerous signaling pathways related to the cell cycle, cell division, cell migration, or cell proliferation during early stages of adipogenesis. As the differentiation process progresses, cells begin to accumulate intracellular lipid droplets and changes in gene expression patterns in particular of adipocyte-specific markers occur. PI3K-Akt signaling and metabolic pathways changed most during adipogenesis, while p53 signaling and ferroptosis were affected late in adipogenesis. When comparing MSCs from FS and FP, only a limited number of differentially expressed genes (DEGs) and enriched signaling pathways were identified. Metabolic pathways, including fat, energy or amino acid metabolism, were highly enriched in the AS breed SMSCs compared to those of the DL breed, especially at day 7 of adipogenesis, suggesting retention of the characteristic metabolic features of their original source, demonstrating donor memory in culture. In contrast, the DL SMSCs were more enriched in immune signaling pathways. CONCLUSIONS: Our study has provided important insights into the dynamics of adipogenesis and revealed metabolic shifts in SMSCs associated with different cell sources and genetic backgrounds of donors. This emphasises the critical role of metabolic and genetic factors as important indications and criteria for donor stem cell selection.

Transcriptome changes during osteogenesis of porcine mesenchymal stem cells derived from different types of synovial membranes and genetic background.

Synovial membrane mesenchymal stem cells (SMSCs) often serve as in vitro model for bone disease, but the molecular mechanisms driving osteogenesis in SMSCs from different donor cells of various sources and breeds remain unclear. In this study, porcine SMSCs isolated from adipose synovium (FP) and fibrous synovium (FS) of Angeln Saddleback (AS) and German Landrace (DL) were used to discover the signaling network change after osteogenic induction. During osteogenic differentiation, mineral deposition was first observed at day 14 and further increased until day 21. Transcriptional changes between day 1 and day 21 were enriched in several signaling pathways, including Wnt, PI3K-Akt, and TGF-beta pathway. Certain pathways related to osteogenesis, including osteoblast differentiation, regulation of bone mineralization, and BMP signaling pathway, were enriched at late time points, as confirmed by the osteogenic markers ALPL, COL1A1, and NANOG. A fraction of differentially expressed genes (DEGs) were found between FP and FS, while DEGs between AS and DL increased during the differentiation phase until day 7 and then decreased from day 14 to day 21. These genes are involved in several important signaling pathways, including TGF-beta, Wnt, and lipid-related signaling pathways, suggesting that SMSCs from these two breeds have different osteogenic capabilities.

Central Suppression of the GH/IGF Axis and Abrogation of Exercise-Related mTORC1/2 Activation in the Muscle of Phenotype-Selected Male Marathon Mice (DUhTP).

The somatotropic axis is required for a number of biological processes, including growth, metabolism, and aging. Due to its central effects on growth and metabolism and with respect to its positive effects on muscle mass, regulation of the GH/IGF-system during endurance exercise is of particular interest. In order to study the control of gene expression and adaptation related to physical performance, we used a non-inbred mouse model, phenotype-selected for high running performance (DUhTP). Gene expression of the GH/IGF-system and related signaling cascades were studied in the pituitary gland and muscle of sedentary males of marathon and unselected control mice. In addition, the effects of three weeks of endurance exercise were assessed in both genetic groups. In pituitary glands from DUhTP mice, reduced expression of Pou1f1 (p = 0.002) was accompanied by non-significant reductions of Gh mRNA (p = 0.066). In addition, mRNA expression of Ghsr and Sstr2 were significantly reduced in the pituitary glands from DUhTP mice (p ≤ 0.05). Central downregulation of Pou1f1 expression was accompanied by reduced serum concentrations of IGF1 and coordinated downregulation of multiple GH/IGF-signaling compounds in muscle (e.g., Ghr, Igf1, Igf1r, Igf2r, Irs1, Irs2, Akt3, Gskb, Pik3ca/b/a2, Pten, Rictor, Rptor, Tsc1, Mtor; p ≤ 0.05). In response to exercise, the expression of Igfbp3, Igfbp 4, and Igfbp 6 and Stc2 mRNA was increased in the muscle of DUhTP mice (p ≤ 0.05). Training-induced specific activation of AKT, S6K, and p38 MAPK was found in muscles from control mice but not in DUhTP mice (p ≤ 0.05), indicating a lack of mTORC1 and mTORC2 activation in marathon mice in response to physical exercise. While hormone-dependent mTORC1 and mTORC2 pathways in marathon mice were repressed, robust increases of Ragulator complex compounds (p ≤ 0.001) and elevated sirtuin 2 to 6 mRNA expression were observed in the DUhTP marathon mouse model (p ≤ 0.05). Activation of AMPK was not observed under the experimental conditions of the present study. Our results describe coordinated downregulation of the somatotropic pathway in long-term selected marathon mice (DUhTP), possibly via the pituitary gland and muscle interaction. Our results, for the first time, demonstrate that GH/IGF effects are repressed in a context of superior running performance in mice.

Jejunal transcriptomic profiling of two layer strains throughout the entire production period.

The jejunum plays crucial roles for the digestion and absorption of nutrients and minerals and for barrier functions that are essential for a healthy, productive life cycle of farm animals, including laying hens. Accordingly, knowledge of the molecular pathways that emerge in the intestine during development, and particularly at the beginning of laying activity, will help to derive strategies for improving nutrient efficiency in laying hens. In this study, jejunal samples were obtained from two high-yielding layer strains at five developmental stages (weeks 10, 16, 24, 30 and 60 of life) for RNA-sequencing, alongside the profiling of blood plasma parameters to approximate the dynamics of mineral homeostasis. The results reflected a marked distinction between the pre-laying and laying phase as inferred from levels of parathyroid hormone, triiodothyronine, estradiol, vitamin D, and calcium. Moreover, the expression patterns of the intestinal mucosa responded directly to the changing metabolic and nutritional profiles at the beginning of the laying phase in maturing high-yielding strains of laying hens. These comprise signaling events namely RANK/RANKL signaling and cellular senescence. Taken together, the timing of sexual maturity of laying hens demands closer examination to unravel metabolic requirements and associated endogenous mechanisms.

Identification of the Key Molecular Drivers of Phosphorus Utilization Based on Host miRNA-mRNA and Gut Microbiome Interactions.

Phosphorus is an essential mineral for all living organisms and a limited resource worldwide. Variation and heritability of phosphorus utilization (PU) traits were observed, indicating the general possibility of improvement. Molecular mechanisms of PU, including host and microbial effects, are still poorly understood. The most promising molecules that interact between the microbiome and host are microRNAs. Japanese quail representing extremes for PU were selected from an F2 population for miRNA profiling of the ileal tissue and subsequent association with mRNA and microbial data of the same animals. Sixty-nine differentially expressed miRNAs were found, including 21 novel and 48 known miRNAs. Combining miRNAs and mRNAs based on correlated expression and target prediction revealed enrichment of transcripts in functional pathways involved in phosphate or bone metabolism such as RAN, estrogen receptor and Wnt signaling, and immune pathways. Out of 55 genera of microbiota, seven were found to be differentially abundant between PU groups. The study reveals molecular interactions occurring in the gut of quail which represent extremes for PU including miRNA-16-5p, miR-142b-5p, miR-148a-3p, CTDSP1, SMAD3, IGSF10, Bacteroides, and Alistipes as key indicators due to their trait-dependent differential expression and occurrence as hub-members of the network of molecular drivers of PU.

Cross-talk between energy metabolism and epigenetics during temperature stress response in C2C12 myoblasts.

Objective: Environmental stress induces disturbances in cell energy metabolism and may cause epigenetic modifications. This study aimed to understand the possible impact of temperature stress (35 °C, 39 °C and 41 °C, compared to control 37 °C) on energy metabolism and epigenetic modifications, such as DNA methylation and histone H4 acetylation, as well as its effects on the expression of genes responsible for epigenetic changes, in mouse skeletal myoblasts (C2C12 cells). Methods: The results showed significantly reduced maximal respiration and spare respiratory capacity under heat stress (39 °C and 41 °C), suggesting that mitochondrial functions were compromised under these conditions. The glycolytic capacity and glycolysis markedly increased following low-temperature stress (35 °C). The results suggested that, under cold stress, cells prefer glycolysis as a rapid compensatory mechanism to meet energy requirements for adaptive thermogenic response. Results: Epigenetic changes (histone H4 acetylation and global DNA methylation) were observed under both heat and cold stress. Among the genes coding for DNA methyltransferases, the Dnmt3a was significantly increased under high-temperature conditions (39 °C and 41 °C), while Dnmt1 expression was significantly increased at low temperature (35 °C), indicating that under these conditions the cells preferred maintenance of methylation to de novo methylation activity. An expression pattern similar to Dnmt3a was observed for Gcn5, encoding for a histone acetyltransferase. The study revealed that temperature stress induced changes in the metabolic profiles, as well as epigenetic modifications, including the dynamics of the key enzymes. Conclusion: The results indicated the existence of crosstalk mechanisms between energy metabolism and epigenetics during cell stress response.

Transcriptome Responses to Dexamethasone Depending on Dose and Glucocorticoid Receptor Sensitivity in the Liver.

Tissue sensitivity to glucocorticoids is a key factor dictating outcome of their homeostatic and therapeutic action, whereby liver represents one of the major peripheral targets. Here, we used pigs carrying a natural gain-of-function glucocorticoid receptor (GR) variant Ala610Val (GRAla610Val) as a model to identify genes and pathways related to differential glucocorticoid sensitivity. Animals with different GRAla610Val genotypes were treated either with saline or two different doses of dexamethasone. Genome-wide transcriptional responses depending on treatment, genotype, and their interaction in the liver were investigated using mRNA sequencing. Dexamethasone induced vast transcriptional responses, with more than 30% of present genes being affected. Functional annotation of genes differentially expressed due to dexamethasone treatment suggested that genes related to inflammation respond more sensitively, despite absence of an immune stimulus. In contrast, genes involved in glucose metabolism and cancer appeared to be less sensitive. Analysis of genotype and genotype × treatment interaction revealed that clustered protocadherins, particularly PCDHB7, are most prominently affected by GRAla610Val, mainly depending on dose. GRAla610Val influenced also expression of a set of glucose metabolism related genes, including PPARGC1A and CEBPB, in the absence of dexamethasone though no differences in basal plasma glucose level were observed. This might represent an adaptive response, keeping balance between receptor sensitivity, and level of circulating endogenous glucocorticoids. Administration of low dexamethasone dose changed their expression pattern and induced higher glucose response in carriers of the hypersensitive Val receptor. Our findings suggest that GRAla610Val modulates tissue responses to glucocorticoids dynamically, depending on their circulating level.

Mitochondrial-nuclear crosstalk, haplotype and copy number variation distinct in muscle fiber type, mitochondrial respiratory and metabolic enzyme activities.

Genes expressed in mitochondria work in concert with those expressed in the nucleus to mediate oxidative phosphorylation (OXPHOS), a process that is relevant for muscle metabolism and meat quality. Mitochondrial genome activity can be efficiently studied and compared in Duroc and Pietrain pigs, which harbor different mitochondrial haplotypes and distinct muscle fiber types, mitochondrial respiratory activities, and fat content. Pietrain pigs homozygous-positive for malignant hyperthermia susceptibility (PiPP) carried only haplotype 8 and showed the lowest absolute mtDNA copy number accompanied by a decrease transcript abundance of mitochondrial-encoded subunits ND1, ND6, and ATP6 and nuclear-encoded subunits NDUFA11 and NDUFB8. In contrast, we found that haplotype 4 of Duroc pigs had significantly higher mitochondrial DNA (mtDNA) copy numbers and an increase transcript abundance of mitochondrial-encoded subunits ND1, ND6, and ATP6. These results suggest that the variation in mitochondrial and nuclear genetic background among these animals has an effect on mitochondrial content and OXPHOS system subunit expression. We observed the co-expression pattern of mitochondrial and nuclear encoded OXPHOS subunits suggesting that the mitochondrial-nuclear crosstalk functionally involves in muscle metabolism. The findings provide valuable information for understanding muscle biology processes and energy metabolism, and may direct use for breeding strategies to improve meat quality and animal health.

Differentiating Staphylococcus aureus from Escherichia coli mastitis: S. aureus triggers unbalanced immune-dampening and host cell invasion immediately after udder infection.

The etiology determines quality and extent of the immune response after udder infection (mastitis). Infections with Gram negative bacteria (e.g. Escherichia coli) will quickly elicit strong inflammation of the udder, fully activate its immune defence via pathogen receptor driven activation of IκB/NF-κB signaling. This often eradicates the pathogen. In contrast, Gram-positive bacteria (e.g. Staphylococcus aureus) will slowly elicit a much weaker inflammation and immune response, frequently resulting in chronic infections. However, it was unclear which immune regulatory pathways are specifically triggered by S. aureus causing this partial immune subversion. We therefore compared in first lactating cows the earliest (1-3 h) udder responses against infection with mastitis causing pathogens of either species. Global transcriptome profiling, bioinformatics analysis and experimental validation of key aspects revealed as S. aureus infection specific features the (i) failure to activating IκB/NF-κB signaling; (ii) activation of the wnt/ß-catenin cascade resulting in active suppression of NF-κB signaling and (iii) rearrangement of the actin-cytoskeleton through modulating Rho GTPase regulated pathways. This facilitates invasion of pathogens into host cells. Hence, S. aureus mastitis is characterized by eliciting unbalanced immune suppression rather than inflammation and invasion of S. aureus into the epithelial cells of the host causing sustained infection.

MicroRNA-mRNA regulatory networking fine-tunes the porcine muscle fiber type, muscular mitochondrial respiratory and metabolic enzyme activities.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in diverse biological processes via regulation of gene expression including in skeletal muscles. In the current study, miRNA expression profile was investigated in longissimus muscle biopsies of malignant hyperthermia syndrome-negative Duroc and Pietrain pigs with distinct muscle metabolic properties in order to explore the regulatory role of miRNAs related to mitochondrial respiratory activity and metabolic enzyme activity in skeletal muscle. RESULTS: A comparative analysis of the miRNA expression profile between Duroc and Pietrain pigs was performed, followed by integration with mRNA profiles based on their pairwise correlation and computational target prediction. The identified target genes were enriched in protein ubiquitination pathway, stem cell pluripotency and geranylgeranyl diphosphate biosynthesis, as well as skeletal and muscular system development. Next, we analyzed the correlation between individual miRNAs and phenotypical traits including muscle fiber type, mitochondrial respiratory activity, metabolic enzyme activity and adenosine phosphate concentrations, and constructed the regulatory miRNA-mRNA networks associated with energy metabolism. It is noteworthy that miR-25 targeting BMPR2 and IRS1, miR-363 targeting USP24, miR-28 targeting HECW2 and miR-210 targeting ATP5I, ME3, MTCH1 and CPT2 were highly associated with slow-twitch oxidative fibers, fast-twitch oxidative fibers, ADP and ATP concentration suggesting an essential role of the miRNA-mRNA regulatory networking in modulating the mitochondrial energy expenditure in the porcine muscle. In the identified miRNA-mRNA network, a tight relationship between mitochondrial and ubiquitin proteasome system at the level of gene expression was observed. It revealed a link between these two systems contributing to energy metabolism of skeletal muscle under physiological conditions. CONCLUSIONS: We assembled miRNA-mRNA regulatory networks based on divergent muscle properties between different pig breeds and further with the correlation analysis of expressed genes and phenotypic measurements. These complex networks relate to muscle fiber type, metabolic enzyme activity and ATP production and may contribute to divergent muscle phenotypes by fine-tuning the expression of genes. Altogether, the results provide an insight into a regulatory role of miRNAs in muscular energy metabolisms and may have an implication on meat quality and production.

Advanced Running Performance by Genetic Predisposition in Male Dummerstorf Marathon Mice (DUhTP) Reveals Higher Sterol Regulatory Element-Binding Protein (SREBP) Related mRNA Expression in the Liver and Higher Serum Levels of Progesterone.

Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice.

Molecular changes in mitochondrial respiratory activity and metabolic enzyme activity in muscle of four pig breeds with distinct metabolic types.

Skeletal muscles are metabolically active and have market value in meat-producing farm animals. A better understanding of biological pathways affecting energy metabolism in skeletal muscle could advance the science of skeletal muscle. In this study, comparative pathway-focused gene expression profiling in conjunction with muscle fiber typing were analyzed in skeletal muscles from Duroc, Pietrain, and Duroc-Pietrain crossbred pigs. Each breed type displayed a distinct muscle fiber-type composition. Mitochondrial respiratory activity and glycolytic and oxidative enzyme activities were comparable among genotypes, except for significantly lower complex I activity in Pietrain pigs homozygous-positive for malignant hyperthermia syndrome. At the transcriptional level, lactate dehydrogenase B showed breed specificity, with significantly lower expression in Pietrain pigs homozygous-positive for malignant hyperthermia syndrome. A similar mRNA expression pattern was shown for several subunits of oxidative phosphorylation complexes, including complex I, complex II, complex IV, and ATP synthase. Significant correlations were observed between mRNA expression of genes in focused pathways and enzyme activities in a breed-dependent manner. Moreover, expression patterns of pathway-focused genes were well correlated with muscle fiber-type composition. These results stress the importance of regulation of transcriptional rate of genes related to oxidative and glycolytic pathways in the metabolic capacity of muscle fibers. Overall, the results further the breed-specific understanding of the molecular basis of metabolic enzyme activities, which directly impact meat quality.

Muscle Transcriptional Profile Based on Muscle Fiber, Mitochondrial Respiratory Activity, and Metabolic Enzymes.

Skeletal muscle is a highly metabolically active tissue that both stores and consumes energy. Important biological pathways that affect energy metabolism and metabolic fiber type in muscle cells may be identified through transcriptomic profiling of the muscle, especially ante mortem. Here, gene expression was investigated in malignant hyperthermia syndrome (MHS)-negative Duroc and Pietrian (PiNN) pigs significantly differing for the muscle fiber types slow-twitch-oxidative fiber (STO) and fast-twitch-oxidative fiber (FTO) as well as mitochondrial activity (succinate-dependent state 3 respiration rate). Longissimus muscle samples were obtained 24 h before slaughter and profiled using cDNA microarrays. Differential gene expression between Duroc and PiNN muscle samples were associated with protein ubiquitination, stem cell pluripotency, amyloid processing, and 3-phosphoinositide biosynthesis and degradation pathways. In addition, weighted gene co-expression network analysis within both breeds identified several co-expression modules that were associated with the proportion of different fiber types, mitochondrial respiratory activity, and ATP metabolism. In particular, Duroc results revealed strong correlations between mitochondrion-associated co-expression modules and STO (r = 0.78), fast-twitch glycolytic fiber (r = -0.98), complex I (r=0.72) and COX activity (r = 0.86). Other pathways in the protein-kinase-activity enriched module were positively correlated with STO (r=0.93), while negatively correlated with FTO (r = -0.72). In contrast to PiNN, co-expression modules enriched in macromolecule catabolic process, actin cytoskeleton, and transcription activator activity were associated with fiber types, mitochondrial respiratory activity, and metabolic enzyme activities. Our results highlight the importance of mitochondria for the oxidative capacity of porcine muscle and for breed-dependent molecular pathways in muscle cell fibers.

Identification and Functional Characterization of Cis-Regulatory Elements Controlling Expression of the Porcine ADRB2 Gene.

The beta-2 adrenergic receptor (beta-2 AR) modulates metabolic processes in skeletal muscle, liver, and adipose tissue in response to catecholamine stimulation. We showed previously that expression of the porcine beta-2 AR gene (ADRB2) is affected by cis-regulatory polymorphisms. These are most likely responsible for the association of ADRB2 with economically relevant muscle-related traits in pigs. The present study focused on characterization of promoter elements involved in basal transcriptional regulation of the porcine ADRB2 in different cell types to aid identification of its cis-regulatory polymorphisms. Based on in silico analysis, luciferase reporter gene assays and gel shift assays were performed using COS-7, HepG2, C2C12, and 3T3-L1 cells. Deletion mapping of the 5´ flanking region (-1324 to +33) of ADRB2 revealed the region between -307 and -269 to be the minimal promoter, including regulatory elements essential for the basal transcriptional activity in all four tested cell types. Directly upstream (-400 to -323) we identified an important enhancer element required for maximal promoter activity. In silico analysis and gel shift assays revealed that this GC-rich element harbors two evolutionarily conserved binding sites of Sp1, a constitutive transcriptional activator. Significant transcriptional activation of the porcine ADRB2 promoter was demonstrated by overexpression of Sp1. Our results demonstrate, for the first time, an important role of Sp1 and of the responsive enhancer element in the regulation of ADRB2 expression. Polymorphisms located in this domain of the porcine ADRB2 promoter represent candidate causal cis-regulatory variants.

Gene expression profiling of porcine mammary epithelial cells after challenge with Escherichia coli and Staphylococcus aureus in vitro.

Postpartum Dysgalactia Syndrome (PDS) represents a considerable health problem of postpartum sows, primarily indicated by mastitis and lactation failure. The poorly understood etiology of this multifactorial disease necessitates the use of the porcine mammary epithelial cell (PMEC) model to identify how and to what extent molecular pathogen defense mechanisms prevent bacterial infections at the first cellular barrier of the gland. PMEC were isolated from three lactating sows and challenged with heat-inactivated potential mastitis-causing pathogens Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) for 3 h and 24 h, in vitro. We focused on differential gene expression patterns of PMEC after pathogen challenge in comparison with the untreated control by performing microarray analysis. Our results show that a core innate immune response of PMEC is partly shared by E. coli and S. aureus. But E. coli infection induces much faster and stronger inflammatory response than S. aureus infection. An immediate and strong up-regulation of genes encoding cytokines (IL1A and IL8), chemokines (CCL2, CXCL1, CXCL2, CXCL3, and CXCL6) and cell adhesion molecules (VCAM1, ICAM1, and ITGB3) was explicitly obvious post-challenge with E. coli inducing a rapid recruitment and activation of cells of host defense mediated by IL1B and TNF signaling. In contrast, S. aureus infection rather induces the expression of genes encoding monooxygenases (CYP1A1, CYP3A4, and CYP1B1) initiating processes of detoxification and pathogen elimination. The results indicate that the course of PDS depends on the host recognition of different structural and pathogenic profiles first, which critically determines the extent and effectiveness of cellular immune defense after infection.

MicroRNAs Regulate Cellular ATP Levels by Targeting Mitochondrial Energy Metabolism Genes during C2C12 Myoblast Differentiation.

In our previous study, we identified an miRNA regulatory network involved in energy metabolism in porcine muscle. To better understand the involvement of miRNAs in cellular ATP production and energy metabolism, here we used C2C12 myoblasts, in which ATP levels increase during differentiation, to identify miRNAs modulating these processes. ATP level, miRNA and mRNA microarray expression profiles during C2C12 differentiation into myotubes were assessed. The results suggest 14 miRNAs (miR-423-3p, miR-17, miR-130b, miR-301a/b, miR-345, miR-15a, miR-16a, miR-128, miR-615, miR-1968, miR-1a/b, and miR-194) as cellular ATP regulators targeting genes involved in mitochondrial energy metabolism (Cox4i2, Cox6a2, Ndufb7, Ndufs4, Ndufs5, and Ndufv1) during C2C12 differentiation. Among these, miR-423-3p showed a high inverse correlation with increasing ATP levels. Besides having implications in promoting cell growth and cell cycle progression, its function in cellular ATP regulation is yet unknown. Therefore, miR-423-3p was selected and validated for the function together with its potential target, Cox6a2. Overexpression of miR-423-3p in C2C12 myogenic differentiation lead to decreased cellular ATP level and decreased expression of Cox6a2 compared to the negative control. These results suggest miR-423-3p as a novel regulator of ATP/energy metabolism by targeting Cox6a2.

Differential expression of miRNAs and their target mRNAs in endometria prior to maternal recognition of pregnancy associates with endometrial receptivity for in vivo- and in vitro-produced bovine embryos.

Endometrial receptivity is a prerequisite for successful embryo implantation and pregnancy. Receptivity involves complex processes promoted by many transcripts that are key components of molecular pathways that depend on ovarian hormones and that contribute to shaping structural, metabolic, and communication properties of endometrial cells toward reception of embryos. MicroRNAs (miRNAs) are important regulators of the expression of these transcripts encoding effector molecules. We acquired miRNA and mRNA signatures, miRNA-mRNA pairs, and regulatory networks linked with the emergence and maintenance of postimplantation pregnancy. Endometrial tissue samples were obtained at Days 3 and 7 of the estrous cycle of cows that did or did not become pregnant after transfer of either in vivo-produced (IVV) or in vitro-produced (IVT) embryos in the next cycle following the biopsy. We report a list of endometrial miRNAs that were differentially expressed between Day 3 and Day 7 of the bovine estrous cycle (including miR-1290, miR-3437, miR-1246, miR-486, miR-3107, and miR-382), that differed with high or low endometrial receptivity (miR-3902-3p, miR-1825, miR-H14-3p, miR-885-3p, miR-504-3p, and miR-186), or that differed among the IVT and IVV transfers (miR-449a/b/c, miR-138, miR-874, miR-4342, miR-2231, and miR-2751). Moreover, mRNA transcripts were also analyzed, and pairs of negatively correlated miRNAs and mRNAs were predicted in silico. The miRNA-mRNA target pairs had roles in response to hormonal stimuli and oxidative stress, chromatin organization, miRNA-mediated epigenetic histone changes, cell proliferation, p53 signaling, and apoptosis. Overall, we identified significant miRNAs, miRNA-mRNA pairs, and functional networks that are associated with the state of pregnancy at Day 28 as a parameter of endometrial receptivity and that are affected by estrous cycle and embryo culture systems.

Gene expression and DNA-methylation of bovine pretransfer endometrium depending on its receptivity after in vitro-produced embryo transfer.

Embryonic implantation to establish a pregnancy is a complex process that requires appropriate communication between the embryo and the maternal endometrium. Inadequate uterine receptivity may contribute to the majority of implantation failures. To provide a comprehensive inventory of genes and functional networks that represent the maternal input of the embryo-maternal cross-talk, a longitudinal, holistic study of the endometrial transcriptome in relation to the days of estrous and to the receptivity of the endometrium was performed in bovine. At day 3 of estrous, genes related to cell communication and mitochondrial energy metabolism were differentially expressed among high- and low-receptive endometria (HR, LR); at day 7, transcripts functioning in immune and inflammatory pathways, oxidative stress, and angiogenesis had different abundances. Additionally, temporal transcriptional changes between days 3 and 7 differed considerably among HR and LR. Further, several transcription factors were predicted as relevant for receptivity because they were either differentially expressed among HR and LR animals or are known to be associated with genes we detected to have differential expression. Finally, global DNA methylation varied according to the interaction of receptivity group and day of estrous, and a divergent trend, which correlated with abundance of DNMT1 transcript, was observed in LR and HR along the estrous cycle days. The study revealed that, even in early estrous, transcripts related to cell communication and response to exogenous stimuli, vascularization, and energy supply show divergent expression and longitudinal temporal regulation in HR and LR. Key components of these molecular pathways are known to be dependent on ovarian hormones that promote uterine receptivity.

Gene regulation of intestinal porcine epithelial cells IPEC-J2 is dependent on the site of deoxynivalenol toxicological action.

The intestinal epithelial cell layer represents the border between the luminal and systemic side of the gut. The decision between absorption and exclusion of substances is the quintessential function of the gut and varies along the gut axis. Consequently, potentially toxic substances may reach the basolateral domain of the epithelial cell layer via blood stream. The mycotoxin deoxynivalenol (DON) is a Fusarium derived secondary metabolite known to enter the blood stream and displaying a striking toxicity on the basolateral side of polarised epithelial cell layers in vitro. Here we analysed potential mechanisms of apical and basolateral DON toxicity reflected in the gene expression. We used the jejunum-derived, polarised intestinal porcine epithelial cell line IPEC-J2 as an in vitro cell culture model. Luminal and systemic DON challenge of the epithelial cell layer was mimicked by a DON application from the apical or basolateral compartment of membrane inserts for 72 h. We compared the genome-wide gene expression of untreated and DON-treated IPEC-J2 cells with the GeneChip® Porcine Genome Array of Affymetrix. Low basolateral DON (200 ng/mL) application triggered 10 times more gene transcripts in comparison to the corresponding apical application (2539 versus 267) despite the intactness of the challenged cell layer as measured by transepithelial electrical resistance. Analysis of the regulated genes by bioinformatic resource DAVID identified several groups of biochemical pathways modulated by concentration and orientation of DON application. Selected genes representing pathways of the cellular metabolism, information processing and structural design were analysed in detail by quantitative PCR. Our findings clearly show that apical and basolateral challenge of epithelial cell layers trigger different gene response profiles paralleled with a higher susceptibility towards basolateral challenge. The evaluation of toxicological potentials of mycotoxins should take this difference in gene regulation dependent on route of application into account.