Corrigendum to "Expression of miRNA-122 Induced by Liver Toxicants in Zebrafish".

[This corrects the article DOI: 10.1155/2016/1473578.].

Expression of miRNA-122 Induced by Liver Toxicants in Zebrafish.

MicroRNA-122 (miRNA-122), also known as liver-specific miRNA, has recently been shown to be a potent biomarker in response to liver injury in mammals. The objective of this study was to examine its expression in response to toxicant treatment and acute liver damage, using the zebrafish system as an alternative model organism. For the hepatotoxicity assay, larval zebrafish were arrayed in 24-well plates. Adult zebrafish were also tested and arrayed in 200 mL cages. Animals were exposed to liver toxicants (tamoxifen or acetaminophen) at various doses, and miRNA-122 expression levels were analyzed using qRT-PCR in dissected liver, brain, heart, and intestine, separately. Our results showed no significant changes in miRNA-122 expression level in tamoxifen-treated larvae; however, miRNA-122 expression was highly induced in tamoxifen-treated adults in a tissue-specific manner. In addition, we observed a histological change in adult liver (0.5 µM) and cell death in larval liver (5 µM) at different doses of tamoxifen. These results indicated that miRNA-122 may be utilized as a liver-specific biomarker for acute liver toxicity in zebrafish.

IFT46 plays an essential role in cilia development.

Cilia are microtubule-based structures that project into the extracellular space. Ciliary defects are associated with several human diseases, including polycystic kidney disease, primary ciliary dyskinesia, left-right axis patterning, hydrocephalus and retinal degeneration. However, the genetic and cellular biological control of ciliogenesis remains poorly understood. The IFT46 is one of the highly conserved intraflagellar transport complex B proteins. In zebrafish, ift46 is expressed in various ciliated tissues such as Kupffer׳s vesicle, pronephric ducts, ears and spinal cord. We show that ift46 is localized to the basal body. Knockdown of ift46 gene results in multiple phenotypes associated with various ciliopathies including kidney cysts, pericardial edema and ventral axis curvature. In ift46 morphants, cilia in kidney and spinal canal are shortened and abnormal. Similar ciliary defects are observed in otic vesicles, lateral line hair cells, olfactory pits, but not in Kupffer׳s vesicle. To explore the functions of Ift46 during mouse development, we have generated Ift46 knock-out mice. The Ift46 mutants have developmental defects in brain, neural tube and heart. In particular Ift46(-/-) homozygotes displays randomization of the embryo heart looping, which is a hallmark of defective left-right (L/R) axis patterning. Taken together, our results demonstrated that IFT46 has an essential role in vertebrate ciliary development.

Induction of clusterin expression by neuronal cell death in Zebrafish.

Clusterin, a protein associated with multiple functions, is expressed in a wide variety of mammalian tissues. Although clusterin is known to be involved in neurodegenerative diseases, ageing, and tumorigenesis, a detailed analysis of the consequences of gain- or loss-of-function approaches has yet to be performed to understand the underlying mechanisms of clusterin functions. Since clusterin levels change in neurological diseases, it is likely that clusterin contributes to cell death and degeneration in general. Zebrafish was investigated as a model system to study human diseases. During development, zebrafish clusterin was expressed in the notochord and nervous system. Embryonic overexpression of clusterin by mRNA microinjection did not affect axis formation, whereas its knock-down by anti-sense morpholino treatment resulted in neuronal cell death. To analyze the function of clusterin in neurodegeneration, a transgenic zebrafish was investigated, in which nitroreductase expression is regulated under the control of a neuron-specific huC promoter which is active between the stages of early neuronal precursors and mature neurons. Nitroreductase turns metronidazole into a cytotoxic agent that induces cell death within 12 h. After metronidazole treatment, transgenic zebrafish showed neuron-specific cell death. Interestingly, we also observed a dramatic induction of clusterin expression in the brain and spinal cord in these fish, suggesting a direct or indirect role of clusterin in neuronal cell death and thus, more generally, in neurodegeneration.

Establishment of a bone-specific col10a1:GFP transgenic zebrafish.

During skeletal development, both osteogenic and chondrogenic programs are initiated from multipotent mesenchymal cells, requiring a number of signaling molecules, transcription factors, and downstream effectors to orchestrate the sophisticated process. Col10a1, an important downstream effector gene, has been identified as a marker for maturing chondrocytes in higher vertebrates, such as mammals and birds. In zebrafish, this gene has been shown to be expressed in both osteoblasts and chondrocytes, but no study has reported its role in osteoblast development. To initially delineate the osteogenic program from chondrogenic lineage development, we used the zebrafish col10a1 promoter to establish a transgenic zebrafish expressing a GFP reporter specifically in osteoblast-specific bone structures that do not involve cartilaginous programs. A construct harboring a -2.2-kb promoter region was found to be sufficient to drive the reporter gene in osteoblast-specific bone structures within the endogenous col10a1 expression domain, confirming that separable cis-acting elements exist for distinct cell type-specific expression of col10a1 during zebrafish skeletal development. The -2.2-kb col10a1:GFP transgenic zebrafish marking only bone structures derived from osteoblasts will undoubtedly be an invaluable tool for identifying and characterizing molecular events driving osteoblast development in zebrafish, which may further provide a differential mechanism where col10a1 is involved in the development of chondrocytes undergoing maturation in other vertebrate systems.

Establishment of a transgenic zebrafish EF1α:Kaede for monitoring cell proliferation during regeneration.

Zebrafish is considered as a versatile experimental animal for various research models from development to diseases. In this study, we report the development of transgenic zebrafish line named as Tg(EF1α:Kaede) that expresses translation elongation factor 1 subunit alpha (EF1α) promoter linked to a fluorescent protein (FP), Kaede for monitoring proliferating cells in during regeneration. It was revealed that about 1.4 kb 5'-flanking region of the EF1α was sufficient for its promoter activity. Expression of Kaede with a property of photo-conversion from green to red was detected in different embryonic stages as well as various organs such as brain, heart, pancreas, intestine, ovary, and fins of adult fish. Cell proliferation pattern during fin regeneration was monitored after amputation of Tg(EF1α:Kaede) caudal fin and results shown that this system is simple and efficient method for detecting proliferating cells during tissue regeneration. Developed Tg(EF1α:Kaede) line has potential to investigate the cell proliferation, regeneration, wound healing capacities after tissue damage and evaluate the therapeutic power of wound healing drugs.

Evolution of the tbx6/16 subfamily genes in vertebrates: insights from zebrafish.

In any comparative studies striving to understand the similarities and differences of the living organisms at the molecular genetic level, the crucial first step is to establish the homology (orthology and paralogy) of genes between different organisms. Determination of the homology of genes becomes complicated when the genes have undergone a rapid divergence in sequence or when the involved genes are members of a gene family that has experienced a differential gain or loss of its constituents in different taxonomic groups. Organisms with duplicated genomes such as teleost fishes might have been especially prone to these problems because the functional redundancies provided by the duplicate copies of genes would have allowed a rapid divergence or loss of genes during evolution. In this study, we will demonstrate that much of the ambiguities in the determination of the homology between fish and tetrapod genes resulting from the problems like these can be eliminated by complementing the sequence-based phylogenies with nonsequence information, such as the exon-intron structure of a gene or the composition of a gene's genomic neighbors. We will use the Tbx6/16 subfamily genes of zebrafish (tbx6, tbx16, tbx24, and mga genes), which have been well known for the ambiguity of their evolutionary relationships to the Tbx6/16 subfamily genes of tetrapods, as an illustrative example. We will show that, despite the similarity of sequence and expression to the tetrapod Tbx6 genes, zebrafish tbx6 gene is actually a novel T-box gene more closely related to the tetrapod Tbx16 genes, whereas the zebrafish tbx24 gene, hitherto considered to be a novel gene due to the high level of sequence divergence, is actually an ortholog of tetrapod Tbx6 genes. We will also show that, after their initial appearance by the multiplication of a common ancestral gene at the beginning of vertebrate evolution, the Tbx6/16 subfamily of vertebrate T-box genes might have experienced differential losses of member genes in different vertebrate groups and gradual pooling of member gene's functions in surviving members, which might have prevented the revelation of the true identity of member genes by way of the comparison of sequence and function.

Protein tyrosine phosphatase profiling analysis of HIB-1B cells during brown adipogenesis.

A number of evidence have been accumulated that the regulation of reversible tyrosine phosphorylation, which can be regulated by the combinatorial activity of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), plays crucial roles in various biological processes including differentiation. There are a total of 107 PTP genes in the human genome, collectively referred to as the "PTPome." In this study, we performed PTP profiling analysis of the HIB-1B cell line, a brown preadipocyte cell line, during brown adipogenesis. Through RT-PCR and real-time PCR, several PTPs showing differential expression pattern during brown adipogenesis were identified. In the case of PTP-RE, it was shown to decrease significantly until 4 days after brown adipogenic differentiation, followed by a dramatic increase at 6 days. The overexpression of PTP-RE led to decreased brown adipogenic differentiation via reducing the tyrosine phosphorylation of the insulin receptor, indicating that PTP-RE functions as a negative regulator at the early stage of brown adipogenesis.

TRIM32 protein sensitizes cells to tumor necrosis factor (TNFα)-induced apoptosis via its RING domain-dependent E3 ligase activity against X-linked inhibitor of apoptosis (XIAP).

TRIM32, which belongs to the tripartite motif (TRIM) protein family, has the RING finger, B-box, and coiled-coil domain structures common to this protein family, along with an additional NHL domain at the C terminus. TRIM32 reportedly functions as an E3 ligase for actin, a protein inhibitor of activated STAT y (PIASy), dysbindin, and c-Myc, and it has been associated with diseases such as muscular dystrophy and epithelial carcinogenesis. Here, we identify a new substrate of TRIM32 and propose a mechanism through which TRIM32 might regulate apoptosis. Our overexpression and knockdown experiments demonstrate that TRIM32 sensitizes cells to TNFα-induced apoptosis. The RING domain is necessary for this pro-apoptotic function of TRM32 as well as being responsible for its E3 ligase activity. TRIM32 colocalizes and directly interacts with X-linked inhibitor of apoptosis (XIAP), a well known cancer therapeutic target, through its coiled-coil and NHL domains. TRIM32 overexpression enhances XIAP ubiquitination and subsequent proteasome-mediated degradation, whereas TRIM32 knockdown has the opposite effect, indicating that XIAP is a substrate of TRIM32. In vitro reconstitution assay reveals that XIAP is directly ubiquitinated by TRIM32. Our novel results collectively suggest that TRIM32 sensitizes TNFα-induced apoptosis by antagonizing XIAP, an anti-apoptotic downstream effector of TNFα signaling. This function may be associated with TRIM32-mediated tumor suppressive mechanism.

Neuron-specific expression of scratch genes during early zebrafish development.

Scratch (scrt) genes are neural-specific in mammals, but their homologues have not been well studied in non-mammalian vertebrates. In this report, we isolated three zebrafish scrt genes, scratch1a (scrt1a), scratch1b (scrt1b), and scratch2 (scrt2), which belong to the Snail superfamily of zinc finger transcription factors. Spatiotemporal expression analysis revealed that scrt1a and scrt2 were initially detected in the central nervous system (CNS) during early somitogenesis while scrt1b was first detectable in neuronal clusters in the brain during late somitogenesis. Interestingly, scrt-expressing cells largely overlapped with huC-positive differentiating neurons and partially with neurogenin1-positive neuronal precursor cells. In addition, scrt-expressing cells were dramatically increased in mind bomb, a neurogenic mutant. Taken together, these results suggest that each zebrafish scrt gene is specifically expressed in neuronal cells and may be involved in differentiation of distinct neuronal populations in the vertebrate nervous system.

Bombyx mori transferrin: genomic structure, expression and antimicrobial activity of recombinant protein.

Transferrin (Tf) is a multifunctional, iron binding protein found in both vertebrates and invertebrates. Although transferrin has been suggested to play a role in innate immunity, its immunological function during infection has not been characterized. In this study, we identified and characterized Bombyx mori transferrin (BmTf). The promoter region of BmTf has numerous putative NF-kappaB binding sites, suggesting its possible function in innate immunity. Analysis of BmTf gene expression shows that it is highly inducible in response to a wide variety of pathogens including bacteria, fungus, and viruses. Recombinant BmTf protein produced in a baculovirus system exhibits iron binding capacity and antibacterial activity against various Gram-positive and -negative bacteria. Taken together, our results indicate that BmTf is an inducible immune effector molecule that may play an important role in pathogen clearance of insect innate immunity.

Prediction and evaluation of protein-protein interaction in keratinocyte differentiation.

Terminal differentiation of skin keratinocytes is a vertically directed multi-step process that is tightly controlled by the sequential expression of a variety of genes. We previously investigated the gene expression profile and found that many of differentiation-related genes expressed in a temporally regulated manner. In this study, we attempted to find the hub-molecules and their intracellular signaling networks during keratinocyte differentiation using in silico analysis of data obtained from previous studies. We used protein-protein interaction prediction software called PSIMAP, and drew a hypothetical signaling network. We chose one candidate hub-molecule SHC1 that were predicted to link EGFR and MAPK signal, and then evaluated the protein-protein interactions experimentally. As predicted, SHC1 bound to the MEK1 in an EGF-regulated manner. Furthermore, SHC1 bound to the MEK1 and p38 MAPK in a keratinocyte differentiation dependent manner. These results demonstrate that in silico protein-protein interaction prediction system can be used to efficiently and cost-effectively select the experimental candidates.

Drosophila short neuropeptide F signalling regulates growth by ERK-mediated insulin signalling.

Insulin and insulin growth factor have central roles in growth, metabolism and ageing of animals, including Drosophila melanogaster. In Drosophila, insulin-like peptides (Dilps) are produced by specialized neurons in the brain. Here we show that Drosophila short neuropeptide F (sNPF), an orthologue of mammalian neuropeptide Y (NPY), and sNPF receptor sNPFR1 regulate expression of Dilps. Body size was increased by overexpression of sNPF or sNPFR1. The fat body of sNPF mutant Drosophila had downregulated Akt, nuclear localized FOXO, upregulated translational inhibitor 4E-BP and reduced cell size. Circulating levels of glucose were elevated and lifespan was also extended in sNPF mutants. We show that these effects are mediated through activation of extracellular signal-related kinases (ERK) in insulin-producing cells of larvae and adults. Insulin expression was also increased in an ERK-dependent manner in cultured Drosophila central nervous system (CNS) cells and in rat pancreatic cells treated with sNPF or NPY peptide, respectively. Drosophila sNPF and the evolutionarily conserved mammalian NPY seem to regulate ERK-mediated insulin expression and thus to systemically modulate growth, metabolism and lifespan.

Macrophage inhibitory cytokine-1 activates AKT and ERK-1/2 via the transactivation of ErbB2 in human breast and gastric cancer cells.

Macrophage inhibitory cytokine-1 (MIC-1) is a member of the transforming growth factor-beta superfamily, which is overexpressed in a variety of human cancers, including breast and gastric cancer. The function of MIC-1 in cancer remains controversial and its signaling pathways remain poorly understood. In this study, we demonstrate that MIC-1 induces the transactivation of ErbB2 in SK-BR-3 breast and SNU-216 gastric cancer cells. MIC-1 induced a significant phosphorylation of Akt and ERK-1/2, and also effected an increase in the levels of tyrosine phosphorylation of ErbB1, ErbB2 and ErbB3 in SK-BR-3 and SNU-216 cells. The treatment of these cells with AG825 and AG1478, inhibitors specific for ErbB2 tyrosine kinase, resulted in the complete abolition of MIC-1-induced Akt and ERK-1/2 phosphorylation. Furthermore, the small-interfering RNA-mediated downregulation of ErbB2 significantly reduced not only the phosphorylation of Akt and ERK-1/2 but also the invasiveness of the cells induced by MIC-1. Our results show that ErbB2 activation performs a crucial function in MIC-1-induced signaling pathways. Further investigations revealed that MIC-1 induced the expression of the hypoxia inducible factor-1alpha protein and the expression of its target genes, including vascular endothelial growth factor, via the activation of the mammalian target of rapamycin (mTOR) signaling pathway. Stimulation of SK-BR-3 with MIC-1 profoundly induces the phosphorylation of mTOR and its downstream substrates, including p70S6K and 4E-BP1. Collectively, these results show that MIC-1 may participate in the malignant progression of certain human cancer cells that overexpress ErbB2 through the transactivation of ErbB2 tyrosine kinase.

Clusterin, a novel modulator of TGF-beta signaling, is involved in Smad2/3 stability.

Clusterin (CLU) is known as a multifunctional protein involved in a variety of physiological processes including lipid transport, epithelial cell differentiation, tumorigenesis, and apoptosis. It is known that CLU interacts with TGF-beta type ll receptor (TbetaRll). However, the relationship of CLU and TGF-beta signaling is unclear. Here we present that CLU is a novel modulator of TGF-beta signaling by regulating Smad2/3 proteins. Overexpression of CLU enhanced TGF-beta-induced transcriptional activity and increased the amount of Smad2/3 proteins, while CLU siRNA repressed TGF-beta-induced transcriptional activity and decreased the amount of Smad2/3 proteins in Hep3B cells. We also found that CLU was involved in Smad2/3 stability at the protein level. These findings suggest that CLU regulates TGF-beta signaling pathway by modulating the stability of Smad2/3 proteins.

Effects of KR-31378, a novel ATP-sensitive potassium channel activator, on hypertrophy of H9c2 cells and on cardiac dysfunction in rats with congestive heart failure.

The present study was performed to evaluate the effects of (2S, 3S, 4R)-N"-cyano-N-(6-amino-3, 4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-2H-1-benzopyran-4yl)-N'-benzylguanidine (KR-31378), a novel mitochondrial ATP-sensitive potassium channel activator, on hypertrophy of H9c2 cells and on cardiac dysfunction in rats with congestive heart failure. In rat heart-derived H9c2 cells treated with hypertrophic agonists, such as angiotensin II, phenylephrine, isoproterenol, and urotensin II, cell size was significantly increased by 27-47%. The increases in cell size induced by the hypertrophic agonists were inhibited by treatment of KR-31378 in a concentration-dependent manner. This was confirmed by the results showing that KR-31378 inhibited the angiotensin II-induced increase in cell protein content. The effect of KR-31378 on the angiotensin II-induced increase in cell size was reversed by mitochondrial ATP-sensitive potassium channel blockers, 5-hydroxydecanoate or glibenclamide. In rats with congestive heart failure, induced by permanent coronary artery occlusion for 8 weeks, KR-31378 significantly reversed the cardiac dysfunction (increase in ratios of stroke volume or cardiac output to body weight) induced by myocardial infarction without reducing infarct size. In addition, KR-31378 significantly inhibited atrial hypertrophy (decrease in ratio of right atrium to body weight) and decreased the serum pro-atrial natriuretic peptide level, a biochemical marker of heart failure. These results suggest that KR-31378 suppresses hypertrophy induced by hypertrophic agonists in H9c2 cells and improves cardiac dysfunction in rats with congestive heart failure induced by myocardial infarction, and that the effects may be mediated by the activation of mitochondrial ATP-sensitive potassium channels.

Molecular cloning and expression of human keratinocyte proline-rich protein (hKPRP), an epidermal marker isolated from calcium-induced differentiating keratinocytes.

We isolated a human gene encoding keratinocyte proline-rich protein (hKPRP). hKPRP gene is located in the region of epidermal differentiation complex on chromosome 1q21, and its approximately 2.5 kb mRNA encodes 579 amino acid protein with high proline content (18%). The mRNA level of hKPRP was markedly increased at both 7 and 14 d after treatment with 1.2 mM calcium in cultured normal human epidermal keratinocytes. In situ hybridization demonstrated that hKPRP was expressed in upper granular layer of normal epidermis with characteristic intermittent pattern. In psoriatic lesion, hKPRP expression was increased as compared with normal skin and showed continuous pattern. Immunohistochemical analysis also confirmed the expression of hKPRP at the protein level. Western blot analysis showed that hKPRP protein of approximately 70 kDa size was significantly increased by calcium in a time-dependent manner. In mouse tissue blot assays, the expression of KPRP was detected in stomach and skin tissues, and began at 17.5 embryonic days. Additionally, hKPRP expression was detected in the periderm of human fetal skin from 16 wk estimated gestational age. Together, these results suggest that hKPRP is an epidermal marker expressed in stratified squamous epithelia and has a potential role in keratinocytes differentiation.

Tumor suppressor VDUP1 increases p27(kip1) stability by inhibiting JAB1.

Vitamin D3 up-regulated protein 1 (VDUP1) is a stress-response gene that is up-regulated by 1,25(OH)2D3 in many cells. It has been reported that VDUP1 expression is reduced in many tumor cells and the enforced expression of VDUP1 inhibits cell proliferation by arresting cell cycle progression. Here, we found that VDUP1-/- fibroblast cells proliferated more rapidly compared with wild-type cells with reduced expression of p27(kip1), a cyclin-dependent kinase inhibitor. JAB1 is known to interact with p27(kip1) and to decrease the stability of p27(kip1). VDUP1 interacted with JAB1 and restored JAB1-induced suppression of p27(kip1) stability. In this process, VDUP1 blocked the JAB1-mediated translocation of p27(kip1) from the nucleus to the cytoplasm. In addition, VDUP1 inhibited JAB1-mediated activator protein-1 activation and cell proliferation. Taken together, these results indicate that VDUP1 is a novel factor of p27(kip1) stability via regulating JAB1.

Overexpression of ERp29 in the thyrocytes of FRTL-5 cells.

It was previously reported that the up-regulation of ERp29 mRNA depends on the levels of thyroid stimulating hormone (TSH) in the thyrocytes of FRTL-5 cells. In order to investigate the putative new function of ERp29 as an endoplasmic molecular (ER) chaperone, an ERp29-overexpressing FRTL-5 cell line was established. This cell line had approximately three times the levels of ERp29 protein and an enhanced level of thyroglobulin (Tg) secretion. The results showed both enhanced ERp29 expression and an interaction with the other ER chaperones such as GRP94, BiP, ERp72 and calnexin. In addition, ERp29 enhanced the expression of PKR-like ER kinase (PERK), which is a transmembrane protein located in the ER membrane. These findings suggest that ERp29 assists in protein folding as well as in the secretion of the secretory/plasma membrane proteins under close co-operation with other ER chaperones and the ER stress signaler, PERK.

Drosophila short neuropeptide F regulates food intake and body size.

Neuropeptides regulate a wide range of animal behavior including food consumption, circadian rhythms, and anxiety. Recently, Drosophila neuropeptide F, which is the homolog of the vertebrate neuropeptide Y, was cloned, and the function of Drosophila neuropeptide F in feeding behaviors was well characterized. However, the function of the structurally related short neuropeptide F (sNPF) was unknown. Here, we report the cloning, RNA, and peptide localizations, and functional characterizations of the Drosophila sNPF gene. The sNPF gene encodes the preprotein containing putative RLRF amide peptides and was expressed in the nervous system of late stage embryos and larvae. The embryonic and larval localization of the sNPF peptide in the nervous systems revealed the larval central nervous system neural circuit from the neurons in the brain to thoracic axons and to connective axons in the ventral ganglion. In the adult brain, the sNPF peptide was localized in the medulla and the mushroom body. However, the sNPF peptide was not detected in the gut. The sNPF mRNA and the peptide were expressed during all developmental stages from embryo to adult. From the feeding assay, the gain-of-function sNPF mutants expressed in nervous systems promoted food intake, whereas the loss-of-function mutants suppressed food intake. Also, sNPF overexpression in nervous systems produced bigger and heavier flies. These findings indicate that the sNPF is expressed in the nervous systems to control food intake and regulate body size in Drosophila melanogaster.