Knockdown of phosphatase and tensin homolog (PTEN) inhibits fatty acid oxidation and reduces very low density lipoprotein assembly and secretion in calf hepatocytes.

Dairy cows with fatty liver exhibit hepatic lipid accumulation and disturbances in fatty acid oxidation and lipid transport. Phosphatase and tensin homolog (PTEN), a lipid phosphatase, regulates intrahepatic fatty acid oxidation and lipid transport in mice. Whether PTEN play a role in fatty acid oxidation and very low density lipoprotein (VLDL) assembly in calf hepatocytes are unknown. Hepatocytes isolated from 3 healthy female Holstein calves (1 d old, 30-40 kg) were infected with empty adenovirus with green fluorescent protein for 48 h (Ad-GFP group) or infected with PTEN knockdown adenovirus for 48 h (Ad-shPTEN group), or cultured in RPMI-1640 without Ad-shPTEN or Ad-GFP (control group). Compared with the Ad-GFP group, PTEN knockdown decreased mRNA and protein abundance and the activity of fatty acid oxidation-related molecules, including acyl-coA synthetase long-chain 1, carnitine palmitoyltransferase 1, carnitine palmitoyltransferase 2, and 3-hydroxy acyl-coA dehydrogenase. Furthermore, PTEN knockdown decreased mRNA and protein abundance of VLDL assembly-related molecules, including apolipoprotein B100, apolipoprotein E, microsomal triglyceride transfer protein, and low density lipoprotein receptor. Importantly, PTEN knockdown promoted triglyceride accumulation in hepatocytes and reduced the VLDL content in culture medium. A subsequent study was conducted on the following 4 groups: cells infected with Ad-GFP for 48 h and then treated with 2% BSA for another 24 h (Ad-GFP + BSA); cells infected with Ad-GFP for 48 h and then treated with 1.2 mM free fatty acids (FFA) and 2% BSA for another 24 h (Ad-GFP + 1.2 mM FFA); cells infected with Ad-shPTEN for 48 h and then treated with 2% BSA for another 24 h (Ad-shPTEN + BSA); cells infected with Ad-shPTEN for 48 h and then treated with 1.2 mM FFA and 2% BSA for another 24 h (Ad-shPTEN + 1.2 mM FFA). Compared with Ad-GFP + BSA, the abundances of PTEN and of fatty acid oxidation- and VLDL assembly-related proteins were lower in the Ad-GFP + 1.2 mM FFA group. Importantly, PTEN knockdown heightened the increase in triglyceride accumulation of hepatocytes and the decrease in VLDL content in culture medium induced by FFA. Overall, these in vitro data indicate that FFA inhibits PTEN expression, leading to triglyceride accumulation and the inhibition of VLDL assembly in calf hepatocytes. These findings suggest that PTEN may be a potential therapeutic target for FFA-induced hepatic steatosis in dairy cows.

Interferon-stimulated genes inhibit caprine parainfluenza virus type 3 replication in Madin-Darby bovine kidney cells.

Caprine parainfluenza virus type 3 (CPIV3) is the one of most common causative agents of caprine respiratory infection, resulting in significant economic losses in the goat and sheep industries. However, the molecular mechanisms and host genes involved in the pathogenesis of and immunity against CPIV3 infection remain poorly understood. In this study, we used RNA-Seq to understand the responses of madin-darby bovine kidney (MDBK) cells to CPIV3 infection. A total of 261 differentially-expressed genes (DEGs) were identified in CPIV3-infected compared with mock-infected MDBK cells at 24 h post-infection (hpi). The DEGs were mainly involved in immune system processes, metabolic processes, and signal transduction. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that the most significantly enriched signaling pathways were MAPK, Wnt, PI3K-Akt, tumor necrosis factor, Toll-like receptor and ubiquitin-mediated proteolysis. STRING analysis revealed that seven interferon-stimulated genes (ISGs) were upregulated (IFI6, ISG15, OAS1Y, OAS1Z, MX1, MX2 and RSAD2) and may play a pivotal role during CPIV3 infection. Moreover, overexpression of these ISGs significantly reduced CPIV3 replication in vitro, while siRNA silencing markedly improved CPIV3 replication 24 and 48 hpi. Ours is the first study to profile the gene expression of CPIV3-infected MDBK cells. We identified seven ISGs that could be targeted in novel antiviral strategies against CPIV3.

The interleukin-6 regulates the function of monocytes/macrophages (MO/MФ) via the interleukin-6 receptor ß in ayu (Plecoglossus altivelis).

Interleukin-6 (IL-6) is one of the most pleiotropic cytokines because of its wide range of effects on cells of the immune and non-immune systems in the body. However, the role of IL-6 in fish monocytes/macrophages (MO/MФ) is poorly understood. In this study, we cloned the cDNA sequence of the IL-6 gene from ayu (Plecoglossus altivelis) and demonstrated using a tissue distribution assay that ayu interleukin-6 (PaIL-6) mRNA is expressed in all tested tissues. Changes in expression were observed in immune tissues as well as in MO/MФ after a Vibrio anguillarum infection; subsequently, PaIL-6 was expressed and purified to prepare anti-PaIL-6 antibodies. Recombinant PaIL-6 protein (rPaIL-6) treatment enhanced pro-inflammatory cytokine expression. Ayu interleukin-6 receptor ß (PaIL-6Rß) knockdown resulted in decreased pro-inflammatory cytokine expression in MO/MФ treated with rPaIL-6, whereas no significant changes were observed after ayu interleukin-6 receptor α (PaIL-6Rα) knockdown in MO/MФ. PaIL-6 and PaIL-6Rß knockdown in MO/MФ inhibited the phosphorylation of signal transducer and activator of transcription 1. Moreover, PaIL-6Rß knockdown inhibited the phagocytic and bactericidal ability of ayu MO/MФ treated with rPaIL-6. These data indicate that PaIL-6 may be able to regulate the function of ayu MO/MФ.

Effect of EZH2 knockdown on preimplantation development of porcine parthenogenetic embryos.

The EZH2 protein endows the polycomb repressive complex 2 (PRC2) with histone lysine methyltransferase activity that is associated with transcriptional repression. Recent investigations have documented crucial roles for EZH2 in mediating X-inactivation, stem cell pluripotency and cancer metastasis. However, there is little evidence demonstrating the maternal effect of EZH2 on porcine preimplantation development. Here, we took parthenogenetic activation embryos to eliminate the confounding paternal influence. We showed that the dynamic expression of EZH2 during early development was accompanied by changes in H3K27me3 levels. Depletion of EZH2 in MII oocytes by small interfering RNA not only impaired embryonic development at the blastocyst stage (P < 0.05), but also disrupted the equilibrium of H3K4me3 and H3K27me3 in the embryo. Interestingly, the expression of TET1, a member of Ten-Eleven Translocation gene family for converting 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5hmC), was decreased after EZH2 knockdown, in contrast to the increase of the other two members, TET2 and TET3 (P < 0.05). These results indicate a correlation between histone methylation and DNA methylation, and between EZH2 and TET1. Along with the downregulation of TET1, the expression of the pluripotency gene NANOG was decreased (P < 0.05), which is consistent with a previous finding in mouse ES cells. Meanwhile, the abundance of OCT4 and SOX2 were also down-regulated. Moreover, EZH2 knockdown reduced the capacity of cells in the blastocysts to resist apoptosis. Taken together, our data suggest that EZH2 is integral to the developmental program of porcine parthenogenetic embryos and exerts its function by regulating pluripotency, differentiation and apoptosis.

Role of AMPK in the expression of tight junction proteins in heat-treated porcine Sertoli cells.

Hyperthermia can cause dysfunction of the tight junctions (TJs) in testes. Adenosine 5'-monophosphate-activated protein kinase (AMPK) participates in the regulation of TJs in testis. However, whether AMPK regulates the expression of TJ proteins in the response of Sertoli cells to heat treatment remains unknown. We subjected Sertoli cells from 3-week-old piglets to heat treatment (43 °C, 30 min), which decreased cell viability, and increased the early apoptosis rate. These effects were reversible and the cells gradually recovered to normal viability at 48 h post-heat treatment. Expression of TJ proteins (claudin 11, JAMA, occludin, and ZO1) was detected in immature porcine Sertoli cells. The mRNA and protein levels of TJ proteins significantly decreased at 1 h after heat exposure, but recovered with increasing recovery time. Additionally, the expression of claudin 11 in the cytoplasm was also markedly decreased by heat treatment. AMPK phosphorylation, the cellular ATP level, and Ca2+/calmodulin-dependent protein kinase kinase B (CaMKKB) level, but not the liver kinase B1 (LKB1) level, were downregulated by heat treatment. More importantly, activation or overexpression of AMPK, which is a regulator of the assembly of TJs, partially rescued the heat treatment-induced downregulation of TJ proteins. By contrast, AMPK knockdown using small interfering RNA (siRNA) further decreased the expression levels of TJ proteins. In addition, claudin 11 was almost undetectable post heat treatment. Collectively, this study demonstrated that heat treatment could reversibly perturb the expression of TJ proteins in immature porcine Sertoli cells by inhibiting the AMPK signaling pathway.

Knockout of the Gnrh genes in zebrafish: effects on reproduction and potential compensation by reproductive and feeding-related neuropeptides.

Gonadotropin-releasing hormone (GNRH) is known as a pivotal upstream regulator of reproduction in vertebrates. However, reproduction is not compromised in the hypophysiotropic Gnrh3 knockout line in zebrafish (gnrh3-/-). In order to determine if Gnrh2, the only other Gnrh isoform in zebrafish brains, is compensating for the loss of Gnrh3, we generated a double Gnrh knockout zebrafish line. Surprisingly, the loss of both Gnrh isoforms resulted in no major impact on reproduction, indicating that a compensatory response, outside of the Gnrh system, was evoked. A plethora of factors acting along the reproductive hypothalamus-pituitary axis were evaluated as possible compensators based on neuroanatomical and differential gene expression studies. In addition, we also examined the involvement of feeding factors in the brain as potential compensators for Gnrh2, which has known anorexigenic effects. We found that the double knockout fish exhibited upregulation of several genes in the brain, specifically gonadotropin-inhibitory hormone (gnih), secretogranin 2 (scg2), tachykinin 3a (tac3a), and pituitary adenylate cyclase-activating peptide 1 (pacap1), and downregulation of agouti-related peptide 1 (agrp1), indicating the compensation occurs outside of Gnrh cells and therefore is a noncell autonomous response to the loss of Gnrh. While the differential expression of gnih and agrp1 in the double knockout line was confined to the periventricular nucleus and hypothalamus, respectively, the upregulation of scg2 corresponded with a broader neuronal redistribution in the lateral hypothalamus and hindbrain. In conclusion, our results demonstrate the existence of a redundant reproductive regulatory system that comes into play when Gnrh2 and Gnrh3 are lost.

Retinoid X Receptor Activation Alters the Chromatin Landscape To Commit Mesenchymal Stem Cells to the Adipose Lineage.

Developmental exposure to environmental factors has been linked to obesity risk later in life. Nuclear receptors are molecular sensors that play critical roles during development and, as such, are prime candidates to explain the developmental programming of disease risk by environmental chemicals. We have previously characterized the obesogen tributyltin (TBT), which activates the nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor (RXR) to increase adiposity in mice exposed in utero. Mesenchymal stem cells (MSCs) from these mice are biased toward the adipose lineage at the expense of the osteoblast lineage, and MSCs exposed to TBT in vitro are shunted toward the adipose fate in a PPARγ-dependent fashion. To address where in the adipogenic cascade TBT acts, we developed an in vitro commitment assay that permitted us to distinguish early commitment to the adipose lineage from subsequent differentiation. TBT and RXR activators (rexinoids) had potent effects in committing MSCs to the adipose lineage, whereas the strong PPARγ activator rosiglitazone was inactive. We show that activation of RXR is sufficient for adipogenic commitment and that rexinoids act through RXR to alter the transcriptome in a manner favoring adipogenic commitment. RXR activation alters expression of enhancer of zeste homolog 2 (EZH2) and modifies genome-wide histone 3 lysine 27 trimethylation (H3K27me3) in promoting adipose commitment and programming subsequent differentiation. These data offer insights into the roles of RXR and EZH2 in MSC lineage specification and shed light on how endocrine-disrupting chemicals such as TBT can reprogram stem cell fate.

Fatty acid elongase 6 plays a role in the synthesis of long-chain fatty acids in goat mammary epithelial cells.

In nonruminants, it is well established that elongation of very long-chain fatty acid-like fatty acid elongase 6 (ELOVL6) catalyzes the synthesis of C18:0 from C16:0 in lipogenic tissues like adipose and liver. However, the role of ELOVL6 in regulating lipid metabolism in ruminant mammary gland remains unknown. In the present study, ELOVL6 was overexpressed or knocked down via adenoviral transfection to assess its role in goat mammary epithelial cells. Results revealed that ELOVL6 overexpression had a weak effect on the expression of genes related to triacylglycerol (TAG) synthesis and desaturation. Overexpression of ELOVL6 increased the content of C18:0 at the expense of C16:0, and increased the elongation index of C16:0. Overexpression of ELOVL6 had no significant effect on the elongation index of C16:1n-7 and the desaturation indices of C16:0 and C18:0. Knockdown of ELOVL6 had a negative effect on mRNA expression of the esterification genes GPAM and diacylglycerolacyltransferase 2 (DGAT2) and TAG concentration; however, it increased the concentration of C16:0 and decreased C18:1n-7 and C18:1n-9 in goat mammary epithelial cells. Accordingly, downregulation of ELOVL6 significantly decreased the elongation indices of C16:0 and C16:1n-7. The lack of change in the desaturation indices of C16:0 and C18:0 upon knockdown of ELOVL6 was consistent with the minor change in SCD1 expression. In conclusion, these are the first results highlighting an important role of ELOVL6 in long-chain fatty elongation and TAG synthesis in ruminant mammary cells.

Adipocyte differentiation-related protein promotes lipid accumulation in goat mammary epithelial cells.

Milk fat originates from the secretion of cytosolic lipid droplets (CLD) synthesized within mammary epithelial cells. Adipocyte differentiation-related protein (ADRP; gene symbol PLIN2) is a CLD-binding protein that is crucial for synthesis of mature CLD. Our hypothesis was that ADRP regulates CLD production and metabolism in goat mammary epithelial cells (GMEC) and thus plays a role in determining milk fat content. To understand the role of ADRP in ruminant milk fat metabolism, ADRP (PLIN2) was overexpressed or knocked down in GMEC using an adenovirus system. Immunocytochemical staining revealed that ADRP localized to the surface of CLD. Supplementation with oleic acid (OA) enhanced its colocalization with CLD surface and enhanced lipid accumulation. Overexpression of ADRP increased lipid accumulation and the concentration of triacylglycerol in GMEC. In contrast, morphological examination revealed that knockdown of ADRP decreased lipid accumulation even when OA was supplemented. This response was confirmed by the reduction in mass of cellular TG when ADRP was knocked down. The fact that knockdown of ADRP did not completely eliminate lipid accumulation at a morphological level in GMEC without OA suggests that some other compensatory factors may also aid in the process of CLD formation. The ADRP reversed the decrease of CLD accumulation induced by adipose triglyceride lipase. This is highly suggestive of ADRP promoting triacylglycerol stability within CLD by preventing access to adipose triglyceride lipase. Collectively, these data provide direct in vitro evidence that ADRP plays a key role in CLD formation and stability in GMEC.

Identification of the STAC3 gene as a skeletal muscle-specifically expressed gene and a novel regulator of satellite cell differentiation in cattle.

Recent studies in mice and zebrafish suggest that the unannotated Src homology 3 and cysteine rich domain 3 (STAC3) gene plays an important role in skeletal muscle development and contraction. The objective of this study was to determine the tissue specificity of the bovine STAC3 gene and its potential role in the proliferation and differentiation of bovine satellite cells. The STAC3 mRNA was detected only in skeletal muscle among 18 bovine tissues examined by reverse transcription PCR. Western blotting revealed the expression of STAC3 protein in bovine skeletal muscle and the absence of it in 6 bovine tissues analyzed. Transfection of the bovine satellite cells with a pool of 2 STAC3 small interfering RNA (siRNA) caused a 90% reduction in STAC3 mRNA. Cell proliferation assays revealed that STAC3 knockdown had no effect on the proliferation rate of bovine satellite cells. Approximately 60% of bovine satellite cells transfected with STAC3 siRNA formed myotubes by 72 h of differentiation, whereas that percentage was 40% for those transfected with negative control siRNA (P < 0.05). At 24, 48, and 72 h of differentiation, bovine satellite cells transfected with STAC3 siRNA had greater mRNA expression of myogenin, myosin heavy chain 3, and myosin heavy chain 7, markers of myotubes, than those transfected with negative control siRNA (P < 0.05). These results suggest that the STAC3 gene is a negative regulator of the differentiation and fusion of bovine satellite cells into myotubes. However, STAC3 expression was increased during the differentiation of bovine satellite cells into myotubes. This suggests that STAC3 might have different functions in bovine myotubes than in bovine satellite cells.

Targeted knockdown of canine KIT (stem cell factor receptor) using RNA interference.

Canine mast cell tumours often express KIT mutations that result in constitutive activation of the c-kit receptor and which are associated with more aggressive disease. The aim of the current study was to determine whether small inhibitory RNA (SiRNA) molecules could specifically target canine KIT mRNA for knock-down. Canine beta-2 microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and KIT sequences were cloned into the psiCHECK™-2 vector. SiRNA molecules, designed to target gene-specific sequences, were co-transfected with plasmid DNA into Chinese hamster ovary (CHO) cells. Renilla and firefly luciferase activity was measured using the Dual-GLO(®) Luciferase Assay (Promega). Using this reporter system, canine housekeeping gene-specific SiRNA molecules demonstrated knockdown of their targets (72.0% knockdown for B2M and 94.5% knockdown for GAPDH). An SiRNA molecule targeting exon 2 of canine KIT successfully knocked-down reporter gene expression of a KIT(26-407) construct (90.8% knockdown). An SiRNA molecule targeting a 48 base-pair in-tandem duplication mutation in KIT exon 11 selectively knocked down expression of the KIT(1569-1966mutant) construct (93.1% knockdown) but had no effect on the KIT(1569-1918wild-type) construct. The results show that RNA interference can be used to inhibit canine KIT mRNA expression and has the potential to selectively target the mutant version of KIT that is expressed by some malignant mast cells.

Knocking down a Taura syndrome virus (TSV) binding protein Lamr is lethal for the whiteleg shrimp Penaeus vannamei.

A cDNA encoding a laminin receptor protein (Lamr) has been isolated from hemocytes of the Pacific white shrimp Penaeus (Litopenaeus) vannamei (Pv), based on primers designed from a previously published Lamr sequence of a Taura syndrome virus (TSV) binding protein of the black tiger shrimp Penaeus monodon (Pm). The deduced amino acid sequence of PvLamr shares 97% identity with PmLamr and has significant homology to laminin receptors and ribosomal protein p40 from various organisms. Tissue distribution analysis by RT-PCR revealed that Lamr transcripts were widely expressed in all tested tissues of P. monodon and Penaeus vannamei. PmLamr was constructed and expressed in Escherichia coli, and the recombinant protein was purified and used to raise a polyclonal antibody. The antiserum reacted with purified recombinant PmLamr and crude muscle tissue proteins from both P. monodon and P. vannamei, but not with hemocyte-free shrimp hemolymph. Examination of protein localization by immunohistochemical analysis revealed the presence of Lamr positive cytoplasm in subcuticular epithelial cells, hematopoietic tissues, epithelial cells of the stomach, epithelial cells of the anterior midgut cecum, antennal gland epithelial cells, F cells of the hepatopancreas, cells in the ovarian zone of proliferation and spheroid cells in the lymphoid organ. RNA interference-mediated silencing of the messenger from Lamr in P. vannamei led to shrimp mortality and indicated an essential function of Lamr for shrimp viability. A negative consequence was that the effect of Lamr knockdown on shrimp infection by Taura syndrome virus could not be assessed.

Genetically modified mouse models in cancer studies.

Genetically modified animals represent a resource of immense potential for cancer research. Classically, genetic modifications in mice were obtained through selected breeding experiments or treatments with powerful carcinogens capable of inducing random mutagenesis. A new era began in the early 1980s when genetic modifications by inserting foreign DNA genes into the cells of an animal allowed for the development of transgenic mice. Since that moment, genetic modifications have been able to be made in a predetermined way. Gene targeting emerged later as a method of in vivo mutagenesis whereby the sequence of a predetermined gene is selectively modified within an intact cell. In this review we focus on how genetically modified mice can be created to study tumour development, and how these models have contributed to an understanding of the genetic alterations involved in human cancer. We also discuss the strengths and weaknesses of the different mouse models for identifying cancer genes, and understanding the consequences of their alterations in order to obtain the maximum benefit for cancer patients.