Hyunju Ro

Lrig3 regulates neural crest formation in Xenopus by modulating Fgf and Wnt signaling pathways.

Leucine-rich repeats and immunoglobulin-like domains 3 (Lrig3) was identified by microarray analysis among genes that show differential expression during gastrulation in Xenopus laevis. Lrig3 was expressed in the neural plate and neural crest (NC) at neurula stages, and in NC derivatives and other dorsal structures during tailbud stages. A prominent consequence of the morpholino-induced inhibition of Lrig3 expression was impaired NC formation, as revealed by the suppression of marker genes, including Slug, Sox9 and Foxd3. In the NC induction assay involving Chordin plus Wnt3a-injected animal caps, Lrig3 morpholino inhibited expression of Slug, Sox9 and Foxd3, but not of Pax3 and Zic1. In line with this, Lrig3 knockdown prevented NC marker induction by Pax3 and Zic1, suggesting that Lrig3 acts downstream of these two genes in NC formation. Injection of Lrig3 and Wnt3a led to low-level induction of NC markers and enhanced induction of Fgf3, Fgf4 and Fgf8 in animal caps, suggesting a positive role for Lrig3 in Wnt signaling. Lrig3 could attenuate Fgf signaling in animal caps, did interact with Fgf receptor 1 in cultured cells and, according to context, decreased or increased the induction of NC markers by Fgf. We suggest that Lrig3 functions in NC formation in Xenopus by modulating the Wnt and Fgf signaling pathways.

PHF20 regulates NF-κB signalling by disrupting recruitment of PP2A to p65

Constitutive NF-κB activation in cancer cells is caused by defects in the signalling network responsible for terminating the NF-κB response. Here we report that plant homeodomain finger protein 20 (PHF20) maintains NF-κB in an active state in the nucleus by inhibiting the interaction between PP2A and p65. We show that PHF20 induces canonical NF-κB signalling by increasing the DNA-binding activity of NF-κB subunit p65. In PHF20 overexpressing cells, the termination of tumour necrosis factor-induced p65 phosphorylation is impaired whereas upstream signalling events triggered by tumour necrosis factor are unaffected. This effect strictly depends on the interaction between PHF20 and methylated lysine residues of p65, which hinders recruitment of PP2A to p65, thereby maintaining p65 in a phosphorylated state. We further show that PHF20 levels correlate with p65 phosphorylation levels in human glioma specimens. Our work identifies PHF20 as a novel regulator of NF-κB activation and suggests that elevated expression of PHF20 may drive constitutive NF-κB activation in some cancers.

Inhibitory Effect of Arctigenin from Fructus Arctii Extract on Melanin Synthesis via Repression of Tyrosinase Expression

To identify the active compound arctigenin in Fructus Arctii (dried seed of medicinal plant Arctium lappa) and to elucidate the inhibitory mechanism in melanogenesis, we analyzed melanin content and tyrosinase activity on B16BL6 murine melanoma and melan-A cell cultures. Water extracts of Fructus Arctii were shown to inhibit tyrosinase activity in vitro and melanin content in α-melanocyte stimulating hormone-stimulated cells to similar levels as the well-known kojic acid and arbutin, respectively. The active compound arctigenin of Fructus Arctii displayed little or no cytotoxicity at all concentrations examined and decreased the relative melanin content and tyrosinase activity in a dose-dependent manner. Melanogenic inhibitory activity was also identified in vivo with zebrafish embryo. To determine the mechanism of inhibition, the effects of arctigenin on tyrosinase gene expression and tyrosinase promoter activity were examined. Also in addition, in the signaling cascade, arctigenin dose dependently decreased the cAMP level and promoted the phosphorylation of extracellular signal-regulated kinase. This result suggests that arctigenin downregulates cAMP and the tyrosinase enzyme through its gene promoter and subsequently upregulates extracellular signal-regulated kinase activity by increasing phosphorylation in the melanogenesis signaling pathway, which leads to a lower melanin content.

Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas

Significance Pancreas differentiation is of interest as an example of organogenesis and for its medical implications. We report here that regulation of protein stability is a player in the differentiation of certain cell lineages in the early zebrafish pancreas. The related E3 ubiquitin ligases Ligand of Numb protein-X (Lnx)2a and Lnx2b affect differentiation of exocrine cells, apparently by destabilizing Numb in exocrine progenitor cells. Numb is an inhibitor of Notch, a key regulator of pancreatic development. We suggest that Lnx2a/b destabilize Numb to derepress Notch, allowing precursors to proliferate and support subsequent differentiation. This study also highlights the fact that detailed analysis of morpholino versus mutant phenotypes may be required for a full understanding of their relationship. The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A frame shifting null mutation in lnx2a did not mimic this phenotype, but a mutation that removed the exon 2 splice donor site did. We found that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb, and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction in the number of Notch active cells in the pancreas. We suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. Further, the complex relationships among genotype, phenotype, and morpholino effect in this case may be instructive in the ongoing consideration of morpholino use.

3-Methoxy-catalposide inhibits inflammatory effects in lipopolysaccharide-stimulated RAW264.7 macrophages.

HIGHLIGHTS3‐Methoxy‐catalposide inhibits LPS‐increased expression of COX‐2 and iNOS.3‐Methoxy‐catalposide inhibits LPS‐increased expression of TNF‐&agr;, IL1&bgr; and IL‐6.3‐Methoxy‐catalposide inhibits both LPS‐activated MAP kinases and NF‐&kgr;B translocation into cell nucleus. ABSTRACT Pseudolysimachion rotundum var. subintegrum is utilized as a traditional herbal remedy to treat cough, bronchitis, and asthma in Korea, Russia, China, and Europe. Here, we show that 3‐methoxy‐catalposide, a novel iridoide glycoside isolated from P. rotundum var. subintegrum has the anti‐inflammatory activity in lipopolysaccharide (LPS)‐stimulated macrophages. The chemical structure of 3‐methoxy‐catalposide was determined by NMR, optical rotation and HRESIMS. In in vitro experiment, RAW264.7 cells were treated with 3‐methoxy‐catalposide for 2 h before exposure to LPS for different times. Inflammatory gene and protein expressions were assayed using RT‐PCR and ELISA. Activities of signal proteins were examined using western analysis. Our results demonstrated that 3‐methoxy‐catalposide significantly inhibits the expression of cyclooxygenase (COX)‐2 and inducible nitric oxide synthase (iNOS) in RAW264.7 cells stimulated by LPS, thereby suppressing the release of prostaglandin E2 (PGE2) and nitric oxide (NO). Moreover, 3‐methoxy‐catalposide markedly reduced the LPS‐induced expression of pro‐inflammatory genes, such as interleukin (IL)‐6, IL‐1&bgr;, and TNF‐&agr;. Further, 3‐methoxy‐catalposide inhibited both LPS‐induced activation of three MAP kinases (ERK 1/2, JNK, and p38) and the nuclear translocation of NF‐&kgr;B and AP‐1. These results support that 3‐methoxy‐catalposide may be a promising candidate for inflammation treatment.

Siah Ubiquitin Ligases Modulate Nodal Signaling during Zebrafish Embryonic Development

Siah2 is a zebrafish homologue of mammalian Siah family. Siah acts as an E3 ubiquitin ligase that binds proteins destined for degradation. Extensive homology between siah and Drosophila Siah homologue (sina) suggests their important physiological roles during embryonic development. However, detailed functional studies of Siah in vertebrate development have not been carried out. Here we report that Siah2 specifically augments nodal related gene expression in marginal blastomeres at late blastula through early gastrula stages of zebrafish embryos. Siah2 dependent Nodal signaling augmentation is confirmed by cell-based reporter gene assays using 293T cells and 3TP-luciferase reporter plasmid. We also established a molecular hierarchy of Siah as a upstream regulator of FoxH1/Fast1 transcriptional factor in Nodal signaling. Elevated expression of nodal related genes by overexpression of Siah2 was enough to override the inhibitory effects of atv and lft2 on the Nodal signaling. In particular, E3 ubiquitin ligase activity of Siah2 is critical to limit the duration and/or magnitude of Nodal signaling. Additionally, since the embryos injected with Siah morpholinos mimicked the atv overexpression phenotype at least in part, our data support a model in which Siah is involved in mesendoderm patterning via modulating Nodal signaling.

Dynamic expression patterns of zebrafish 1G5 (1G5z), a calmodulin kinase‐like gene in the developing nervous system

Evolutionarily well‐conserved Ca2+/calmodulin‐dependent protein kinase (CaMK) proteins are known for their role as Ca2+ signaling mediators. 1G5 encodes a CaMK like protein, which belongs to a calmodulin (CaM) kinase gene family. Here, we report the isolation of zebrafish homologue of mammalian 1G5, which we named 1G5z. 1G5z is composed of three major domains: (1) an N‐terminal serine/threonine kinase domain, (2) a central calmodulin‐binding domain, and (3) a C‐terminal alanine‐rich domain, the 1G5z‐specific domain. 1G5z shares 83∼84% homology with other vertebrate 1G5 proteins. Spatiotemporal expression studies found that 1G5z is expressed by means of zygotic transcription and appears in various neuronal tissues from the 20‐somite stage. 1G5z transcripts are more regionalized in the brain and spinal cord at 24 hr postfertilization (hpf). At 35 hpf, 1G5z transcripts are exclusively present in the anterior trunk spinal cord as well as in the hindbrain, tegmentum, hypothalamus, and telencephalon. This expression pattern lasts until 48 hpf but ceases in the trunk. At 72 hpf, 1G5z is abundantly transcribed particularly in the specific region of the tectum and eye. We further observed that the number of 1G5z‐positive cells is dramatically increased in the mindbomb mutant embryos but abolished in the trigeminal ganglion and caudal trunk sensory neuron of the neurogenin1 morphant at 24 hpf. In addition, bromodeoxyuridine staining further confirmed that the 1G5z‐positive cells were postmitotic sensory and interneurons. Developmental Dynamics 235:835–842, 2006.

Traditional herbal prescription LASAP-C inhibits melanin synthesis in B16F10 melanoma cells and zebrafish

BackgroundIn this study, the anti-melanogenesis efficacy of clinically used herbal prescription LASAP-C, which consists of four herbal medicines—Rehmanniae Radix Crudus, Lycii Fructus, Scutellariae Radix, and Angelicae Dahuricae Radix, was investigated.MethodsThe chemical profile of LASAP-C was established by conducting ultra-performance liquid chromatography-electrospray ionization-mass spectrometry. Anti-melanogenic efficacy was evaluated by tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 expression in B16F10 melanoma cells. In vivo evaluation was performed by using zebrafish model.ResultsMolecular evidences suggested that melanin synthesis was inhibited via the down-regulation of tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 expression in B16F10 melanoma cells treated with LASAP-C. The anti-melanogenesis efficacy was also confirmed in vivo by using the zebrafish model.ConclusionThe results of this study provide strong evidences that LASAP-C can be used as an active component in cosmeceutical products for reducing excess pigmentation in the human skin.

Lnx2b, an E3 ubiquitin ligase, in dorsal forerunner cells and Kupffer's vesicle is required for specification of zebrafish left–right laterality

The establishment of left–right (LR) axis in zebrafish embryos relies on numerous genes expressed in the cluster of dorsal forerunner cells (DFCs) that form Kupffers vesicle (KV), the transient cilia-rich organ with functional similarity to mouse node in the case of LR axis determination. Even though several genes in the DFCs and KV have been identified to be implicated in LR body patterning so far, the underlying regulatory mechanisms in particular dependent upon ubiquitin (Ub)– proteasome system have not yet been identified. In this study, we report that Lnx2b, a RING domain containing E3 Ub ligase, specifically expressed in migratory DFCs and developing KV, plays a critical role in the establishment of LR laterality. Depletion of Lnx2b using antisense morpholino oligonucleotides (MOs) inhibited the left-side biased expression of southpaw and resulted in the randomization of the heart jogging and looping in zebrafish embryos. A DFCs-specific Lnx2b loss of function approach showed that the randomization of LR patterning caused by the depletion of Lnx2b was not simply due to the early dorsoventral body patterning defects or the MO toxicity, but the loss of its function in the DFCs and KV. Collectively, our data showed that Lnx2b is the first analyzed E3 Ub ligase, which is involved in LR laterality during zebrafish embryogenesis.

Ottogi Inhibits Wnt/β-catenin Signaling by Regulating Cell Membrane Trafficking of Frizzled8

Wnt signaling controls critical developmental processes including tissue/body patterning. Here we report the identification of a novel regulator of Wnt signaling, OTTOGI (OTG), isolated from a large-scale expression screening of human cDNAs in zebrafish embryos. Overexpression of OTG in zebrafish embryos caused dorso-anteriorized phenotype, inhibited the expression of Wnt target genes, and prevented nuclear accumulation of β-catenin. Conversely, knockdown of zebrafish otg using specific antisense morpholino promoted nuclear accumulation of β-catenin and caused ventralization. However, OTG failed to rescue headless-like phenotype induced by inhibition of GSK-3β activity, suggesting that OTG acts upstream of GSK-3β. OTG bound specifically to Frizzled8 (Fz8) receptor and caused retention of Fz8 in the endoplasmic reticulum possibly by preventing N-linked glycosylation of Fz8. Taken together, our data indicate that OTG functions as a novel negative regulator of Wnt signaling during development by the modulation of cell surface expression of Fz receptor.