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.

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.

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.

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.

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.

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.

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.

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

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

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.

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.

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.

CXC chemokine receptor 4 expression and function in human anaplastic thyroid cancer cells.

Anaplastic thyroid carcinomas (ATCs) are highly aggressive, extremely lethal human cancers with poor therapeutic response. Chemokines are a superfamily of small cytokine-like proteins that induce, through their interaction with G protein-coupled receptors, cytoskeletal rearrangement, firm adhesion to endothelial cells, and directional migration. In this study, we characterized the expression of CXC chemokine receptor 4 (CXCR4) and analyzed its functions in ARO cells, a human ATC cell. The normal primary cultured thyroid cells and ATC cell lines expressed CXCR4 and stromal cell-derived factor (SDF)-1 alpha transcripts, detected by RT-PCR. Fluorescence activated cell sorting analysis of CXCR4 expression in normal and ATC cells showed that ARO cells expressed significant levels of CXCR4. FRO, NPA, and normal thyroid cells did not express membrane CXCR4, as determined by fluorescence activated cell sorting analysis. To identify the functional role of CXCR4 in ARO cells, we treated ARO cells with SDF-1 alpha and analyzed the signaling pathways, cellular migration, and proliferation. SDF-1alpha enhanced the migration but did not affect the proliferation of ARO cells or activate the Janus kinase/signal transducer and activator of transcription signaling pathways. However, SDF-1 alpha/CXCR4 activation resulted in phosphorylation of the p70S6 kinase and its target protein, ribosomal S6 protein, and also activation of the ERK1/ERK2 signaling pathways. Furthermore, SDF-1 alpha/CXCR4- mediated activation of the p70S6 kinase and phosphorylation of the S6 protein were inhibited by treatment with an mTOR/FRAP inhibitor. The specificity of the CXCR4-mediated migration of ARO cells was demonstrated by the dose-dependent inhibition of migration by neutralizing anti-CXCR4. The ATC cells, FRO and NPA, which do not express CXCR4, did not demonstrate significant SDF-1 alpha-mediated migration in vitro. In addition, the CXCR4-mediated migration of ARO cells was inhibited by treatment with pertussis toxin (a Gi-protein inhibitor) and PD 98059 (a mitogen-activated ERK kinase inhibitor) but not by LY294002 and wortmanin, phosphatidylinositol 3-kinase inhibitors. These findings suggest that a subset of ATC cells expresses functional CXCR4, which may be important in tumor cell migration and local tumor invasion.

Leptin receptor isoform expression in rat osteoblasts and their functional analysis.

The genetic defect in producing the adipose hormone leptin results among others in a drastic increase of bone mass. The current understanding is that under normal circumstances, osteoblast activity is indirectly suppressed by a hypothalamic relay induced by leptin-signalling in the brain. To investigate whether leptin might also regulate osteoblast activity in a direct manner, expression of leptin receptors in rat osteoblasts was determined and their functionality was analyzed upon recombinant leptin treatment. Reverse transcription-PCR confirmed the expression of four among the six currently described receptor isoforms, which were also able to transduce cell signalling as shown by STAT3 phosphorylation after activation.

Clusterin mRNA expression in apoptotic and activated rat thymocytes.

Clusterin is a 75-80 kDa heterodimeric glycoprotein, that is produced in most tissues but which exact biological role is still not clear. Particularly, its role in protection or promotion of apoptosis is heavily disputed, since data supporting both views have been reported in several independent studies. To clarify this issue, and also to determine whether clusterin expression itself might be affected by apoptosis, in the present study, rat thymocytes were treated with dexamethasone, -a synthetic glucocorticoid that elicits apoptosis in thymocytes-, and clusterin mRNA expression was analyzed by semi-quantitative RT-PCR before and after induction of apoptosis. Interestingly, neither the treatment with dexamethasone in vitro nor triggering of apoptosis in vivo up- regulated clusterin expression, opposing the view that clusterin is involved in apoptotic processes. On the other hand, a new clusterin mRNA isoform was detected and isolated, whose expression was restricted to freshly isolated thymocytes. This novel isoform lacks the post-translational proteolytic cleavage site and is therefore predicted to encode a monomeric protein. The biological function under normal circumstances, however, will need further investigations for clarification. While apoptosis could not modulate clusterin expression, activation of thymocytes with concanavalin A and interleukin-2 resulted in up-regulation of clusterin mRNA level, indicating that clusterin expression is rather under the control of cell activation-mediated rather than apoptosis-induced signals.

Molecular characterization of a Bombyx mori protein disulfide isomerase (bPDI).

We have isolated a complementary deoxyribonucleic acid clone that encodes the protein disulfide isomerase of Bombyx mori (bPDI). This protein has a putative open reading frame of 494 amino acids and a predicted size of 55.6 kDa. In addition, 2 thioredoxin active sites, each with a CGHC sequence, and an endoplasmic reticulum (ER) retention signal site with a KDEL motif were found at the C-terminal. Both sites are typically found in members of the PDI family of proteins. The expression of bPDI messenger ribonucleic acid (mRNA) was markedly increased during ER stress induced by stimulation with calcium ionophore A23187, tunicamycin, and dithiothreitol, all of which are known to cause an accumulation of unfolded proteins in the ER. We also examined the tissue distribution of bPDI mRNA and found pronounced expression in the fat body of insects. Hormonal regulation studies showed that juvenile hormone, insulin, and a combination of juvenile hormone and transferrin (although not transferrin alone) affected bPDI mRNA expression. A challenge with exogenous bacteria also affected expression, and the effect peaked 16 hours after infection. These results suggest that bPDI is a member of the ER-stress protein group, that it may play an important role in exogenous bacterial infection of the fat body, and that its expression is hormone regulated.

Expression of the endoplasmic reticulum chaperone GRP94 gene in ischemic gerbil brain.

GRP94 (glucose regulated protein 94) gene expression in the ischemic-hippocampus of gerbils, which was induced by a temporary occlusion of the bilateral common carotid arteries (CCAs), was tested by Northern blot analysis. The maximum GRP94 gene expression level was detected at the occipital lobe 10 min after the induction of ischemia. In the hippocampus, GRP94 gene expression reached a maximum 15 min after inducing ischemia. Following reperfusion, the maximum expression level was shown at 12 h and continuing thereafter.

Clusterin overexpression is responsible for the anti-apoptosis effect in a mouse neuroblastoma cell line, B103.

The functional role of clusterin in apoptosis was examined using flow cytometry. Clusterin cDNA was transfected into the mouse neuroblastoma cell line, B103, in order to determine if clusterin overexpression inhibits apoptosis. The increased clusterin expression level in the B103 cells tended to suppress the apoptotic index. This suggests an association of clusterin gene expression with apoptosis inhibition. These results support the conclusion that clusterin expression in B103 cells has an anti-apoptotic influence.

Phylogenetic relationship of Bombyx mori protein disulfide isomerase.

A cDNA that encodes protein disulfide isomerase was isolated from Bombyx mori (bPDI), in which an open reading frame of 494 amino acids contained two PDI-typical thioredoxin active sites of WCGHCK and an ER retention signal of the KDEL motif at its C-terminal. The bPDI protein shared less than 55% of the amino acid sequence homology with other reported PDIs. bPDI is most genetically similar to the D. melanogaster PDI. The most serious evolutional diversity was observed between the metazoa and nematoda through PDI evolutional processing. Although bPDI shows a relatively low amino acid homology with other PDIs, in which both sites of the two thioredoxin active sites and the endoplasmic reticulum (ER) retention signal are completely conserved, it was successfully recognized by anti-rat PDI antibodies. This suggests that bPDI may have the activity of a protein isomerase and a chaperone.

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.