Shyh-Jye Lee

Probing lectin and sperm with carbohydrate-modified quantum dots.

We report the encapsulation of quantum dots with biologically important β‐N‐acetylglucosamine (GlcNAc) in different ratios, together with studies of their specific/sensitive multivalent interactions with lectins and sperm by fluorimetry, transmission electron microscopy, dynamic light scattering microscopy, confocal imaging techniques, and flow cytometry. These GlcNAc‐encapsulated quantum dots (QDGLNs) specifically bind to wheat germ agglutinin, and cause fluorescence quenching and aggregation. Further studies of QDGLNs and the mannose‐encapsulated QDs (QDMANs) with sperm revealed site‐specific interactions, in which QDGLNs bind to the head of the sperm, while QDMANs spread over the whole sperm body.

Protein tyrosine kinase‐dependent release of intracellular calcium in the sea urchin egg

The aminoguanide, methylglyoxal bis(guanylhydrazone) (MGBG), was shown to stimulate phosphorylation of RR‐SRC, a synthetic protein tyrosine kinase (PTK) substrate, and different levels of tyrosyl phosphorylation of endogenous proteins in a sea urchin egg membrane‐cortex preparation. Stimulating protein tyrosine kinase activity in the sea urchin egg stimulated intracellular Ca2+ release, because microinjection of 1–5 mM of MGBG into unfertilized eggs triggered a transient rise in intracellular Ca2+ activity ([Ca2+]i) after a brief latent period. Pretreating eggs with PTK‐specific inhibitors, genistein or tyrphostin B42+, significantly inhibited the MGBG‐induced rise in [Ca2+]i. Methylglyoxal bis(guanylhydrazone) stimulation of PTK activities in the unfertilized sea urchin egg appeared to trigger Ca2+ release through phospholipase C (PLC)‐dependent inositol 1,4,5‐trisphosphate (InsP3) production. The MGBG‐induced Ca2+ response could be suppressed in eggs preloaded with the InsP3 receptor antagonist, heparin, and was reduced in eggs pretreated with U7312+, a PLC inhibitor. However, the response was unchanged in eggs treated with nicotinamide, an inhibitor of ADP‐ribosyl cyclase, or nifedipine, an inhibitor of nicotinic acid adenine dinucleotide phosphate activity. These results suggest that MGBG may be useful as a chemical agonist of PTK in sea urchin eggs and allow direct testing of the PTK requirement for the transient rise in [Ca2+]i in sea urchin eggs during fertilization. Although genistein was observed to significantly delay the onset, the sperm‐induced Ca2+ response in PTK inhibitor‐loaded eggs otherwise appeared normal. Therefore, it was concluded that sea urchin eggs contain a PTK‐dependent pathway that can mediate intracellular Ca2+ release, but PTK activity does not appear to be required for the fertilization response.

Lysophosphatidic acid upregulates vascular endothelial growth factor-C and tube formation in human endothelial cells through LPA1/3, COX-2, and NF-κB activation- and EGFR transactivation-dependent mechanisms

Lysophosphatidic acid (LPA) is a lipid bioactive mediator which binds to G-protein-coupled receptors and activates a variety of cellular functions. LPA modulates multiple behaviors in endothelial cells, including cell proliferation and migration, capillary-like tube formation in vitro, activation of proteases, interactions with leukocytes, and expressions of inflammation-related genes, thereby regulating vessel formation. LPA has been reported to modulate the angiogenesis process. However, the role of LPA in the lymphangiogenesis process has not been studied. In this study, we showed that LPA upregulated vascular endothelial growth factor-C (VEGF-C) mRNA expression in human umbilical vein endothelial cells (HUVECs) and subsequent endothelial cell tube formation in vitro and in vivo. These enhancement effects were LPA(1)- and LPA(3)-dependent and required cyclooxygenase-2 (COX-2), endothelial growth factor receptor (EGFR) transactivation and activation of nuclear factor kappaB (NF-kappaB). Moreover, LPA induced the protein expressions of the lymphatic markers, Prox-1, LYVE-1, and podoplanin, in HUVECs, and these enhancement effects were dependent on LPA(1) and LPA(3) activation and EGFR transactivation. Our results demonstrated that LPA might regulate VEGF-C and lymphatic marker expression in endothelial cells, which contributes to endothelial cell tube formation in vitro and in vivo, thus facilitating endothelial cell participation in the lymphangiogenesis process. This study clarifies the signaling mechanism of LPA-regulated VEGF-C expression and lymphatic marker expressions in endothelial cells, which suggest that LPA may be a suitable target for generating therapeutics against lymphangiogenesis and tumor metastasis.

LPA1 is essential for lymphatic vessel development in zebrafish

Lysophosphatidic acid (LPA) has long been implicated in regulating vascular development via endothelial cell‐expressed G protein‐coupled receptors. However, because of a lack of notable vascular defects reported in LPA receptor knockout mouse studies, the regulation of vasculature by LPA receptors in vivo is still uncertain. Using zebrafish as a model, we studied the gene expression patterns and functions of an LPA receptor, LPA1, during embryonic development, in particular, vascular formation. Whole‐mount in situ hybridization experiments revealed that zebrafish lpa1 (zlpa1) was ubiquitously expressed early in development, and its expression domains were later localized to the head region and the vicinity of the dorsal aorta. The expression of zlpa1 surrounding the dorsal aorta suggests its role in vasculature development. Knocking down of zLPA1 by injecting morpholino (MO) oligonucleotides at 0.625–1.25 ng per embryo resulted in the absence of thoracic duct and edema in pericardial sac and trunk in a dose‐dependent manner. These zlpa1‐MO‐resulted defects could be specifically rescued by ectopic expression of zlpa1. In addition, overexpression of vegf‐c, a well‐known lymphangiogenic factor, also partially ameliorated the inhibition of thoracic duct development. Taken together, these results demonstrate that LPA1 is necessary for lymphatic vessel formation during embryonic development in zebrafish.—Lee, S.‐J., Chan, T.‐H., Chen, T.‐C., Liao, B.‐O., Hwang, P.‐P., Lee, H. LPA1 is essential for lymphatic vessel development in zebrafish. FASEB J. 22, 3706–3715 (2008)

Diaphanous-related formin 2 and profilin I are required for gastrulation cell movements.

Intensive cellular movements occur during gastrulation. These cellular movements rely heavily on dynamic actin assembly. Rho with its associated proteins, including the Rho-activated formin, Diaphanous, are key regulators of actin assembly in cellular protrusion and migration. However, the function of Diaphanous in gastrulation cell movements remains unclear. To study the role of Diaphanous in gastrulation, we isolated a partial zebrafish diaphanous-related formin 2 (zdia2) clone with its N-terminal regulatory domains. The GTPase binding domain of zDia2 is highly conserved compared to its mammalian homologues. Using a yeast two-hybrid assay, we showed that zDia2 interacts with constitutively-active RhoA and Cdc42. The zdia2 mRNAs were ubiquitously expressed during early embryonic development in zebrafish as determined by RT-PCR and whole-mount in situ hybridization analyses. Knockdown of zdia2 by antisense morpholino oligonucleotides (MOs) blocked epiboly formation and convergent extension in a dose-dependent manner, whereas ectopic expression of a human mdia gene partially rescued these defects. Time-lapse recording further showed that bleb-like cellular processes of blastoderm marginal deep marginal cells and pseudopod-/filopod-like processes of prechordal plate cells and lateral cells were abolished in the zdia2 morphants. Furthermore, zDia2 acts cell-autonomously since transplanted zdia2-knockdown cells exhibited low protrusive activity with aberrant migration in wild type host embryos. Lastly, co-injection of antisense MOs of zdia2 and zebrafish profilin I (zpfn 1), but not zebrafish profilin II, resulted in a synergistic inhibition of gastrulation cell movements. These results suggest that zDia2 in conjunction with zPfn 1 are required for gastrulation cell movements in zebrafish.

Establishment of a Transgenic Zebrafish Line for Superficial Skin Ablation and Functional Validation of Apoptosis Modulators In Vivo

Background Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish. Methodology/Principal Findings This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)cy17 (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR+ fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR+ signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR+ fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR+ fluorescent signaling. Conclusion/Significance The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo.

Autotaxin in embryonic development

Autotaxin (ATX) is a secreted lysophospholipase D that generates the multifunctional lipid mediator lysophosphatidic acid (LPA). LPA signals through six distinct G protein-coupled receptors, acting alone or in concert to activate multiple effector pathways. The ATX-LPA signaling axis is implicated in a remarkably wide variety of physiological and pathological processes and plays a vital role in embryonic development. Disruption of the ATX-encoding gene (Enpp2) in mice results in intrauterine death due to vascular defects in the extra-embryonic yolk sac and embryo proper. In addition, Enpp2 (-/-) embryos show impaired neural development. The observed angiogenic defects are attributable, at least in part, to loss of LPA signaling through the Gα(12/13)-linked RhoA-ROCK-actin remodeling pathway. Studies in zebrafish also have uncovered a dual role for ATX in both vascular and neural development; furthermore, they point to a key role for ATX-LPA signaling in the regulation of left-right asymmetry. Here we discuss our present understanding of the role of ATX-LPA signaling in vertebrate development. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Lysophosphatidic acid induces erythropoiesis through activating lysophosphatidic acid receptor 3.

Lysophosphatidic acid (LPA), an extracellular lipid mediator, exerts multiple bioactivities through activating G protein‐coupled receptors. LPA receptor 3 (LPA3) is a member of the endothelial differentiation gene family, which regulates differentiation and development of the circulation system. However, the relationship among the LPA receptors (LPARs) and erythropoiesis is still not clear. In this study, we found that erythroblasts expressed both LPA1 and LPA3, and erythropoietic defects were observed in zLPA3 antisense morpholino oligonucleotide‐injected zebrafish embryos. In human model, our results showed that LPA enhanced the erythropoiesis in the cord blood‐derived human hematopoietic stem cells (hHSCs) with erythropoietin (EPO) addition in the plasma‐free culture. When hHSCs were treated with Ki16425, an antagonist of LPA1 and LPA3, erythropoietic process of hHSCs was also blocked, as detected by mRNA and protein expressions of CD71 and GlyA. In the knockdown study, we further demonstrated that specific knockdown of LPA3, not LPA1, blocked the erythropoiesis. The translocation of β‐catenin into the nucleus, a downstream response of LPAR activation, was blocked by Ki16425 treatment. In addition, upregulation of erythropoiesis by LPA was also blocked by quercetin, an inhibitor of the β‐catenin/T‐cell factor pathway. Furthermore, the enhancement of LPA on erythropoiesis was diminished by blocking c‐Jun‐activated kinase/signal transducer and activator of transcription and phosphatidylinositol 3‐kinase/AKT activation, the downstream signaling pathways of EPO receptor, suggested that LPA might play a synergistic role with EPO to regulate erythropoietic process. In conclusion, we first reported that LPA participates in EPO‐dependent erythropoiesis through activating LPA3. STEM CELLS 2011;29:1763–1773

Lysophosphatidic acid up-regulates vascular endothelial growth factor-C and lymphatic marker expressions in human endothelial cells

Abstract.Lysophosphatidic acid (LPA) is a low-molecular-weight lipid growth factor, which binds to G-protein-coupled receptors. Previous studies have shown that LPA enhances vascular endothelial growth factor-A (VEGF-A) expression in cancer cells and promotes angiogenesis process. However, the roles of LPA in lymphatic vessel formation and lymphangiogenesis have not been investigated. Here, we demonstrated that LPA up-regulated VEGF-C mRNA and protein expressions in human umbilical vein endothelial cells (HUVECs). Furthermore, the expression levels of lymphatic markers, including Prox-1, LYVE-1 and podoplanin, were enhanced in LPA-stimulated tube forming endothelial cells in vitro and in vivo. Moreover, we showed that pretreatment with MAZ51, a VEGFR-3 kinase inhibitor, and introduction of VEGFR-3 siRNA suppressed LPA-induced HUVEC tube formation and lymphatic marker expressions. These results demonstrated that LPA enhances expression of lymphatic markers through activating VEGF-C receptors in endothelial cells. This study provides basic information that LPA might be a target for therapeutics against lymphangiogenesis and tumor metastasis.

Autotaxin/Lpar3 signaling regulates Kupffer's vesicle formation and left-right asymmetry in zebrafish

Left-right (L-R) patterning is essential for proper organ morphogenesis and function. Calcium fluxes in dorsal forerunner cells (DFCs) are known to regulate the formation of Kupffers vesicle (KV), a central organ for establishing L-R asymmetry in zebrafish. Here, we identify the lipid mediator lysophosphatidic acid (LPA) as a regulator of L-R asymmetry in zebrafish embryos. LPA is produced by Autotaxin (Atx), a secreted lysophospholipase D, and triggers various cellular responses through activation of specific G protein-coupled receptors (Lpar1-6). Knockdown of Atx or LPA receptor 3 (Lpar3) by morpholino oligonucleotides perturbed asymmetric gene expression in lateral plate mesoderm and disrupted organ L-R asymmetries, whereas overexpression of lpar3 partially rescued those defects in both atx and lpar3 morphants. Similar defects were observed in embryos treated with the Atx inhibitor HA130 and the Lpar1-3 inhibitor Ki16425. Knockdown of either Atx or Lpar3 impaired calcium fluxes in DFCs during mid-epiboly stage and compromised DFC cohesive migration, KV formation and ciliogenesis. Application of LPA to DFCs rescued the calcium signal and laterality defects in atx morphants. This LPA-dependent L-R asymmetry is mediated via Wnt signaling, as shown by the accumulation of β-catenin in nuclei at the dorsal side of both atx and lpar3 morphants. Our results suggest a major role for the Atx/Lpar3 signaling axis in regulating KV formation, ciliogenesis and L-R asymmetry via a Wnt-dependent pathway.