Haruo Nakano

Gene expression and maintenance of pregnancy in bovine: roles of trophoblastic binucleate cell-specific molecules

Cell to cell interaction plays a pivotal role in the regulation of placentogenesis and exchange of stage-specific developmental signals between the fetal and maternal units. Specifically, these interactions are paramount for programmed fetal growth, maternal adaptation to pregnancy and coordination of parturition. However, little is known about the precise regulation of placentation and maintenance of gestation in cattle. Therefore, the aim of the present study was to decipher the complex networks ofcell communication to gain an insight into the multifaceted developmental process and understand the profound consequences of flawed communication. In the ruminant, the binucleate cell plays a central role in forming the structures and secretions at the fetomaternal interface that are crucial in establishing and maintaining pregnancy. Herein, we summarise differences in the abundance of specific RNA transcripts in the bovine cotyledon and caruncle using global gene expression profiling and further investigate the relationship of mRNA abundance for selected pregnancy-specific genes of interest (identified from microarray studies) that are localised exclusively to the binucleate cell, such as placental lactogen, prolactin-related proteins and pregnancy-associated glycoproteins. The results suggest that a well-orchestrated transcriptional command from binucleate cells is pivotal to the establishment and progression of pregnancy in cattle.

Expression of placental lactogen and cytokeratin in bovine placental binucleate cells in culture.

Abstract. Binucleate cells are present in ruminant placenta and play an endocrine role in the production of many hormones during pregnancy. We isolated and cultured binucleate cells from bovine placenta at middle to late gestation and characterized these cells using immunofluorescence techniques. Enriched preparations of binucleate cells were obtained using Percoll density gradient centrifugation following collagenase digestion. Binucleate cells in culture preferentially attached to collagen-coated dishes rather than to noncoated plastic dishes. The cells gradually extended their edges on collagen substrata, and finally assumed a flattened morphology. Antibodies to placental lactogen (PL) and pregnancy-associated glycoprotein-1 (PAG-1) specifically stained the majority of round binucleate cells, but not the flat cells. We found that PL-positive binucleate cells were consistently devoid of cytokeratin. In contrast, cytokeratin was expressed in PL-negative binucleate cells as well as mononuclear epithelial cells. Furthermore, the PL-negative flat binucleate cells also developed intense cytokeratin networks in the cytoplasm. These results indicate that cytokeratin expression is inversely proportionate to that of PL in cultured binucleate cells. We conclude that downregulation of cytokeratin in binucleate cells is a function of the state of cellular differentiation.

Differential Regulation of the Expression of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases by Cytokines and Growth Factors in Bovine Endometrial Stromal Cells and Trophoblast Cell Line BT-1 In Vitro

Abstract Degradation and reconstitution of extracellular matrix in uterine endometrium is a crucial event for embryonic implantation and is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). In the present study, we investigated the regulation of MMP and TIMP expression in cultured bovine endometrial stromal cells (BESCs) and a bovine trophoblast cell line BT-1 (BT-1 cells). The production of proMMP-9 was induced by transforming growth factor β (TGFβ) and 12-O-tetradecanoylphorbol 13-acetate in the stromal cells. The treatment of BESCs with TGFβ, insulin-like growth factor-I, and hepatocyte growth factor (HGF) resulted in a significant increase in the level of TIMP-1 in the culture medium. In addition, a significant increase of TIMP-2 production was observed in interleukin (IL)-1α and HGF-treated BESCs. However, the expression of TIMP-1 and TIMP-2 mRNA was not augmented by these factors. The treatment of BESCs with 12-O-tetradecanoylphorbol 13-acetate resulted in a significant increase in the level of TIMP-1 but a significant decrease in the level of TIMP-2 in the stromal cells. Membrane type-1 MMP mRNA expression in the stromal cells was augmented by tumor necrosis factor α (TNFα), IL-6, HGF, and 12-O-tetradecanoylphorbol 13-acetate. On the other hand, BT-1 cells constitutively produced proMMP-9 and proMMP-2, and the treatment of BT-1 cells with TNFα, HGF, and 12-O-tetradecanoylphorbol 13-acetate resulted in a significant increase in the level of proMMP-9 but not in the level of proMMP-2. The production of TIMP-1 in BT-1 cells was also augmented by IL-1α, TNFα, and HGF at the level of translation and was transcriptionally increased by 12-O-tetradecanoylphorbol 13-acetate. However, the level of TIMP-2 mRNA in BT-1 cells was not affected by any of the treatments. These results suggest that the expression of MMPs and TIMPs is differentially regulated by cytokines and growth factors and that the production of TIMP-1 and TIMP-2 may not be accompanied by changes in their mRNA expression in bovine endometrium and trophoblasts. Furthermore, as in humans and rodents, MMPs and TIMPs may contribute to the control of degradation and reconstitution of extracellular matrix in bovine endometrium during embryonic implantation and early placentation.

Bovine Trophoblast Cell Culture Systems

Bovine trophoblastic cells are the first cells to differentiate during embryogenesis and play pivotal role in morphological and physiological development of the placenta. We have developed culture systems for bovine trophoblast stem cells isolated from in vitro fertilized blastocysts in the absence of feeder cells. These cells continuously proliferate in Dulbeccos modified Eagles/F12 culture medium supplemented with bovine endometrial fibroblast-conditioned medium. The cells possess epithelial morphology, express cytokeratin, and form dome-like structures (vesicles). Methods for the maintenance, subculture, storage, and measurement of bovine trophoblast stem cell growth are described. The cells exhibit characteristics of bovine trophoblastic stem cells and possess the ability to differentiate into binucleate cells and express placental lactogen, prolactin-related protein-1, pregnancy-associated glycoprotein-1, and interferon tau.

The 5′‐untranslated region regulates ATF5 mRNA stability via nonsense‐mediated mRNA decay in response to environmental stress

We previously reported that activating transcription factor 5 (ATF5) mRNA increases in response to amino acid limitation, and that this increase is dependent on mRNA stabilization. The ATF5 gene allows transcription of mRNAs with two alternative 5′‐UTRs, 5′‐UTRα and 5′‐UTRβ, derived from exon 1α and exon 1β. 5′‐UTRα contains the upstream open reading frames uORF1 and uORF2. Phosphorylation of eukaryotic initiation factor 2α during the integrated stress response had been previously shown to lead to bypassing of uORF2 translation and production of ATF5 protein. Translation of uORF2 is expected to result in translational termination at a position 125 nucleotides upstream of the exon junction, and this fits the criterion of a nonsense‐mediated decay target mRNA. We investigated the potential role of 5′‐UTRα in the control of mRNA stabilization, and found that 5′‐UTRα reduced the stability of ATF5 mRNA. 5′‐UTRα‐regulated destabilization of mRNA was suppressed by knockdown of the nonsense‐mediated decay factors Upf1 and Upf2. Mutation of the downstream AUG (uAUG2) rendered mRNA refractory to Upf1 and Upf2 knockdown. Moreover, 5′‐UTRα‐regulated down‐regulation was hindered by amino acid limitation and tunicamycin treatment, and stress‐induced phosphorylation of eukaryotic initiation factor 2α was involved in stabilization of ATF5 mRNA. These studies show that ATF5 mRNA is a naturally occurring normal mRNA target of nonsense‐mediated decay, and provide evidence for linkage between stress‐regulated translational regulation and the mRNA decay pathway. This linkage constitutes a mechanism that regulates expression of stress response genes.

Activating transcription factor 5 (ATF5) is essential for the maturation and survival of mouse basal vomeronasal sensory neurons

Activating transcription factor 5 (ATF5) is a member of the CREB/ATF family of transcription factors, which is highly expressed in olfactory chemosensory tissues, the main olfactory epithelium and vomeronasal epithelium (VNE) in mice. The vomeronasal sensory neurons in the VNE detect pheromones in order to regulate social behaviors such as mating and aggression; however, the physiological role of ATF5 in the vomeronasal sensory system remains unknown. In this study, we found that the differentiation of mature vomeronasal sensory neurons, assessed by olfactory marker protein expression, was inhibited in ATF5-deficient VNE. In addition, many apoptotic vomeronasal sensory neurons were evident in ATF5-deficient VNE. The vomeronasal sensory neurons consist of two major types of neuron expressing either vomeronasal 1 receptor (V1r)/Gαi2 or vomeronasal 2 receptor (V2r)/Gαo. We demonstrated that the differentiation, survival and axonal projection of V2r/Gαo-type rather than V1r/Gαi2-type vomeronasal sensory neurons were severely inhibited in ATF5-deficient VNE. These results suggest that ATF5 is one of the transcription factors crucial for the vomeronasal sensory formation.

Activating transcription factor 5 is required for mouse olfactory bulb development via interneuron

Activating transcription factor 5 (ATF5) is a stress response transcription factor of the cAMP-responsive element-binding/ATF family. Earlier, we reported that ATF5 expression is up-regulated in response to stress, such as amino acid limitation or arsenite exposure. Although ATF5 is widely expressed in the brain and the olfactory epithelium, the role of ATF5 is not fully understood. Here, the olfactory bulbs (OBs) of ATF5-deficient mice are smaller than those of wild-type mice. Histological analysis reveals the disturbed laminar structure of the OB, showing the thinner olfactory nerve layer, and a reduced number of interneurons. This is mainly due to the reduced number of bromodeoxyuridine-positive proliferating cells in the subventricular zone, where the interneuron progenitors are formed and migrate to the OBs. Moreover, the olfaction-related aggressive behavior of ATF5-deficient mice is reduced compared to wild-type mice. Our data suggest that ATF5 plays a crucial role in mouse OB development via interneuron. Graphical abstract ATF5-deficient mice (ATF5-/-) have smaller olfactory bulbs (OBs) showing the thinner olfactory nerve layer and a reduced number of interneurons.

Comprehensive Behavioral Analysis of Activating Transcription Factor 5-Deficient Mice

Activating transcription factor 5 (ATF5) is a member of the CREB/ATF family of basic leucine zipper transcription factors. We previously reported that ATF5-deficient (ATF5-/-) mice demonstrated abnormal olfactory bulb development due to impaired interneuron supply. Furthermore, ATF5-/- mice were less aggressive than ATF5+/+ mice. Although ATF5 is widely expressed in the brain, and involved in the regulation of proliferation and development of neurons, the physiological role of ATF5 in the higher brain remains unknown. Our objective was to investigate the physiological role of ATF5 in the higher brain. We performed a comprehensive behavioral analysis using ATF5-/- mice and wild type littermates. ATF5-/- mice exhibited abnormal locomotor activity in the open field test. They also exhibited abnormal anxiety-like behavior in the light/dark transition test and open field test. Furthermore, ATF5-/- mice displayed reduced social interaction in the Crawley’s social interaction test and increased pain sensitivity in the hot plate test compared with wild type. Finally, behavioral flexibility was reduced in the T-maze test in ATF5-/- mice compared with wild type. In addition, we demonstrated that ATF5-/- mice display disturbances of monoamine neurotransmitter levels in several brain regions. These results indicate that ATF5 deficiency elicits abnormal behaviors and the disturbance of monoamine neurotransmitter levels in the brain. The behavioral abnormalities of ATF5-/- mice may be due to the disturbance of monoamine levels. Taken together, these findings suggest that ATF5-/- mice may be a unique animal model of some psychiatric disorders.

Two Alkaloids from Bulbs of Lycoris sanguinea MAXIM. Suppress PEPCK Expression by Inhibiting the Phosphorylation of CREB.

In the fasting state, gluconeogenesis is upregulated by glucagon. Glucagon stimulates cyclic adenosine monophosphate production, which induces the expression of key enzymes for gluconeogenesis, such as cytosolic phosphoenolpyruvate carboxykinase (PEPCK‐C), which are involved in gluconeogenesis through the protein kinase A/cAMP response element‐binding protein (CREB) pathway. Using a luciferase reporter gene assay, a methanol extract of the bulbs of Lycoris sanguinea MAXIM. var. kiushiana Makino was found to suppress cAMP‐enhanced PEPCK‐C promoter activity. In addition, two alkaloids, lycoricidine and lycoricidinol, in the extract were identified as active constituents. In forskolin‐stimulated human hepatoma cells, these alkaloids suppressed the expression of a reporter gene under the control of cAMP response element and also prevented increases in the endogenous levels of phosphorylated CREB and PEPCK mRNA expression. These results suggest that lycoricidine and lycoricidinol suppress PEPCK‐C expression by inhibiting the phosphorylation of CREB and may thus have the potential to prevent excessive gluconeogenesis in type 2 diabetes. Copyright

N-terminal hydrophobic amino acids of activating transcription factor 5 (ATF5) protein confer interleukin 1β (IL-1β)-induced stabilization.

Background: The N-terminal region of ATF5 is responsible for its CdCl2- and NaAsO2-induced expression. Results: IL-1β stabilizes ATF5 protein and elevates the translation efficiency of ATF5 mRNA. Conclusion: The N-terminal hydrophobic amino acids of ATF5 are important for protein stabilization and responsiveness to IL-1β. Significance: This study provides new insights about the roles of ATF5 in immune response. Activating transcription factor 5 (ATF5) is a stress-response transcription factor that responds to amino acid limitation and exposure to cadmium chloride (CdCl2) and sodium arsenite (NaAsO2). The N-terminal amino acids contribute to the destabilization of the ATF5 protein in steady-state conditions and serve as a stabilization domain in the stress response after CdCl2 or NaAsO2 exposure. In this study, we show that interleukin 1β (IL-1β), a proinflammatory cytokine, increases the expression of ATF5 protein in HepG2 hepatoma cells in part by stabilizing the ATF5 protein. The N-terminal domain rich in hydrophobic amino acids that is predicted to form a hydrophobic network was responsible for destabilization in steady-state conditions and served as an IL-1β response domain. Furthermore, IL-1β increased the translational efficiency of ATF5 mRNA via the 5′ UTRα and phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α). ATF5 knockdown in HepG2 cells up-regulated the IL-1β-induced expression of the serum amyloid A 1 (SAA1) and SAA2 genes. Our results show that the N-terminal hydrophobic amino acids play an important role in the regulation of ATF5 protein expression in IL-1β-mediated immune response and that ATF5 is a negative regulator for IL-1β-induced expression of SAA1 and SAA2 in HepG2 cells.