Interaction of ZFPIP with PBX1 is crucial for proper expression of neural genetic markers during Xenopus development

ZFPIP/Zfp462 has been recently identified as a new vertebrate zinc finger encoding gene whose product interacts with Pbx1. Previous work indicates that ZFPIP is maternally expressed in Xenopus laevis oocytes and plays a key role during the cleavage phase of embryogenesis. This early expression is followed by a zygotic expression which overlaps with the neural Pbx1 expression pattern, suggesting an interaction between these two partners during Xenopus neurogenesis. In order to test the physiological interaction between ZFPIP and Pbx1, we carried out a dominant negative assay in which the Pbx1 interacting domain of ZFPIP (ZFPIPp) was overexpressed in Xenopus laevis embryos. We observed that ZFPIPp ectopic expression led to abnormal en2 and N‐cam expression patterns, whereas krox‐20 expression was not affected. Furthermore, we showed that while ZFPIPp alone was localized in the nucleus of Cos‐7 cells, additional expression of Pbx1 induced a location of ZFPIPp at the perinuclear region of the cells. These overall data suggest that ZFPIP and Pbx1 could be partners and cooperate in the regulation of essential neural genes during Xenopus development.     

Different signaling pathway between sphingosine-1-phosphate and lysophosphatidic acid in Xenopus oocytes: Functional coupling of the sphingosine-1-phosphate receptor to PLC-xβ in Xenopus oocytes

In Xenopus oocytes, both sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) activate Ca²⁺-dependent oscillatory Cl⁻ currents by acting through membrane-bound receptors. External application of 50 μM S1P elicited a long-lasting oscillatory current that continued over 30 min from the beginning of oscillation, with 300 nA (n = 11) as a usual maximum peak of current, whereas 1-μM LPA treatment showed only transiently oscillating but more vigorous current responses, with 2,800 nA (n = 18) as a maximum peak amplitude. Both phospholipid-induced Ca²⁺-dependent Cl⁻ currents were observed in the absence of extracellular Ca²⁺, were blocked by intracellular injection of the Ca²⁺ chelator, EGTA, and could not be elicited by treatment with thapsigargin, an inhibitor of endoplasmic reticulum (ER) Ca²⁺ ATPase. Intracellular Ca²⁺ release appeared to be from inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ store, because Cl⁻ currents were blocked by heparin injection. Pretreatment with the aminosteroid, U-73122, an inhibitor of G protein-mediated phospholipase C (PLC) activation, to oocytes inhibited the current responses evoked both by S1P and LPA. However, when they were injected with 10 ng of antisense oligonucleotide (AS-ODN) against Xenopus phospholipase C (PLC-xβ), oocytes could not respond to S1P application, whereas they responded normally to LPA, indicating that the S1P signaling pathway goes through PLC-xβ, whereas LPA signaling goes through another unknown PLC. To determine the types of G proteins involved, we introduced AS-ODNs against four types of G-protein α subunits that were identified in Xenopus laevis; G_qα, G₁₁α, G₀α, and G_i1α. Among AS-ODNs against the Gαs tested, AS-G_qα and AS-G_i1α to S1P and AS-G_qα and AS-G₁₁α to LPA specifically reduced current responses, respectively, to about 20–30% of controls. These results demonstrate that LPA and S1P, although they have similar structural features, release intracellular Ca²⁺ from the IP₃-sensitive pool, use different components in their signal transduction pathways in Xenopus oocytes. J. Cell. Physiol. 176:412–423, 1998. © 1998 Wiley-Liss, Inc.     

Purification and characterization of a germ cell-specific form of elongation factor 1 alpha (EF-1 alpha) from Xenopus laevis.

Elongation factor 1 alpha (EF-1 alpha) was purified to homogeneity from full-grown oocytes of Xenopus laevis. This protein is encoded by a gene previously shown to be expressed in male and female germ cells, and repressed in somatic cells. The purified protein was identified with EF-1 alpha on criteria of molecular mass, cross-reaction with antibodies raised against Artemia salina EF-1 alpha, affinity for guanine nucleotides, and ability to promote the mRNA-dependent binding of aminoacyl tRNA to 80S ribosomes.     

Intrinsic ligand binding properties of the human and bovine α,-interferon receptors

The Type I interferon receptor (IFN-αR) interacts with all IFN-αs, IFN-β and IFN-ω, and seems to be a multisubunit receptor. To investigate the role of a cloned receptor subunit (IFN-αR1), we have examined the intrinsic ligand binding properties of the bovine and human IFN-αR1 polypeptides expressed in Xenopus laevis oocytes. Albeit with different efficiencies, Xenopus oocytes expressing either the human or bovine IFN-αR1 polypeptide exhibit significant binding and formation of crosslinked complexes with human IFN-αA and IFN-αB. Thus, the IFN-αR1 polypeptide most likely plays a direct role in ligand binding.     

PLCγ1–PKCγ Signaling‐Mediated Hsp90α Plasma Membrane Translocation Facilitates Tumor Metastasis

The 90‐kDa heat shock protein (Hsp90α) has been identified on the surface of cancer cells, and is implicated in tumor invasion and metastasis, suggesting that it is a potentially important target for tumor therapy. However, the regulatory mechanism of Hsp90α plasma membrane translocation during tumor invasion remains poorly understood. Here, we show that Hsp90α plasma membrane expression is selectively upregulated upon epidermal growth factor (EGF) stimulation, which is a process independent of the extracellular matrix. Abrogation of EGF‐mediated activation of phospholipase (PLCγ1) by its siRNA or inhibitor prevents the accumulation of Hsp90α at cell protrusions. Inhibition of the downstream effectors of PLCγ1, including Ca²⁺ and protein kinase C (PKCγ), also blocks the membrane translocation of Hsp90α, while activation of PKCγ leads to increased levels of cell‐surface Hsp90α. Moreover, overexpression of PKCγ increases extracellular vesicle release, on which Hsp90α is present. Furthermore, activation or overexpression of PKCγ promotes tumor cell motility in vitro and tumor metastasis in vivo, whereas a specific neutralizing monoclonal antibody against Hsp90α inhibits such effects, demonstrating that PKCγ‐induced Hsp90α translocation is required for tumor metastasis. Taken together, our study provides a mechanistic basis for the role for the PLCγ1–PKCγ pathway in regulating Hsp90α plasma membrane translocation, which facilitates tumor cell motility and promotes tumor metastasis.     

Three genes under different developmental control encode elongation factor 1-alpha in Xenopus laevis.

We have cloned cDNAs encoding two variants of the elongation factor for protein synthesis in Xenopus laevis, called EF-1 alpha. One of these (42Sp50) is expressed exclusively in immature oocytes. It is one of two protein components of a 42S RNP particle that is very abundant in previtellogenic oocytes. The 42S RNP particle consists of various tRNAs, 5S RNA, 42Sp50 and a 5S RNA binding protein (42Sp43). A major function served by 42Sp50 appears to be the storage of tRNAs for later use in oogenesis and early embryogenesis. The second EF-1 alpha variant (EF-1 alpha O) is expressed mainly in oocytes but transiently in early embryogenesis as well. Its mRNA cannot be detected after neurulation in somatic cells. EF-1 alpha O is closely related to a third EF-1 alpha (EF-1 alpha S), discovered originally by Krieg et al. (1). EF-1 alpha S is expressed at low levels in oocytes but actively in somatic cells. The latter two proteins are very similar to known eukaryotic EF-1 alpha from other organisms and presumably function in their respective cell types to support protein synthesis.     

Xenopus lipovitellin 1 is a Zn2+- and Cd2+-binding protein

This report discusses the identification of a Zn²⁺- and Cd²⁺-binding protein of Xenopus laevis that is abundant in vitellogenic oocytes and in embryos from fertilization to stage 46. Oocyte or embryo homogenates were fractionated by SDS-PAGE, blotted onto nitrocellulose, and probed with ⁶⁵Zn²⁺ or ¹⁰⁹Cd²⁺. The resulting autoradiograms showed binding of both radio-nuclides to a protein, designated pCdZn. Freon extraction of oocyte and embryo homogenates showed pCdZn to be a yolk protein. When pCdZn was isolated from oocyte homogenates by ammonium sulfate precipitation, delipidation, and chromatography, it co-purified with lipovitellin 1. The amino acid composition of pCdZn closely resembled the reported composition of lipovitellin 1 and the molecular weight of purified pCdZn (～115 kD) corresponded to reported values for lipovitellin 1 (111–121 kD). Amino acid sequence analyses of five peptides derived from pCdZn yielded 94% identity to the reported sequence of lipovitellin 1, deduced from the DNA sequence of the Xenopus vitellogenin A2 precursor gene. Based on these findings, pCdZn was identified as lipovitellin 1. This study suggests that lipovitellin 1 is the major storage protein for zinc in mature oocytes and developing embryos of Xenopus laevis. © 1995 wiley-Liss, Inc.     

Lipovitellin 2β is the 31 kD Ni2+-binding protein (pNiXb) in Xenopus oocytes and embryos

An Ni²⁺-binding protein (pNiXb, 31 kD) present in mature Xenopus laevis oocytes and in embryos from fertilization in N/F stage 42, was isolated and characterized. After oocytes or embryos were fractionated by PAGE, electroblotted onto nitrocellulose, and probed with ⁶³Ni²⁺, pNiXb was detected by autoradiography. pNiXb, a yolk protein located in the embryonic gut, was purified from yolk platelets by ammonium sulfate precipitation, delipidation, gel filtration chromatography, and HPLC analysis. During these steps, pNiXb copurified with lipovitellin 2. The N-terminal sequence of purified pNiXb exactly matched that of Xenopus lipovitellin 2β, deduced from the DNA sequence of the Xenopus vitellogenin A2 precursor gene. Since pNiXb and lipovitellin 2β agree in N-terminal sequence, amino acid composition, and apparent molecular weight, they appear to be identical. Based on a metalblot competition assay, the abilities of metal ions to compete with ⁶³Ni²⁺ for binding to pNiXb were ranked: Zn²⁺ ≈ Cu²⁺ ≈ Co²⁺ > Cd²⁺ ≈ Mn²⁺ > Sn²⁺. This study shows that Xenopus lipovitellin 2β is a metal-binding protein in vitro, and raises the possibility that it may function similarly in vivo. © 1994 Wiley-Liss, Inc.     

42Sp48 in previtellogenic Xenopus oocytes is structurally homologous to EF-1 alpha and may be a stage-specific elongation factor

We have isolated the cDNA for 42Sp48 and EF-1 alpha from mixed stage oocytes and tailbud (stage 22) Xenopus laevis cDNA libraries by use of the cDNA for human elongation factor-1 alpha (EF-1 alpha) as probe. The nucleotide and deduced amino acid sequences of the entire coding region of 42Sp48 and EF-1 alpha cDNA were established. The proposed functional homology of the proteins is reflected in highly conserved amino acid sequences (91% identity), while the large number of silent mutations at the gene level may serve to prevent recombination at their loci. 42Sp48 is apparently encoded by two genes in Xenopus, while no sequences corresponding to 42Sp48 could be found in murine or human genomic DNA. 42Sp48 has been proposed to act as a stage-specific elongation factor in Xenopus. Comparison of the deduced amino acid sequences of 42Sp48 and EF-1 alpha with that of elongation factor Tu from E. coli, for which the three-dimensional structure including that of the GTP binding sites have been determined, supports this hypothesis.     

Elongation factor 1 alpha (EF-1 alpha) is concentrated in the Balbiani body and accumulates coordinately with the ribosomes during oogenesis of Xenopus laevis

In Xenopus laevis oocytes two distinct systems catalyze the mRNA-dependent binding of aminoacyl tRNA to the A site of ribosomes. These systems are elongation factor 1 alpha (EF-1 alpha) and the 42S nucleoprotein particle. This particle is also implicated in the long-term storage of 5S RNA and aminoacyl tRNA during early oogenesis. We report here that the ribosomes and the storage particles are distributed uniformly in the cytoplasm of previtellogenic (stage I) oocytes. In contrast, EF-1 alpha is concentrated in a small region of the cytoplasm, known as the mitochondrial mass or Balbiani body. When the Balbiani body disperses in early vitellogenic oocytes (stage II), EF-1 alpha becomes evenly distributed in the cytoplasm. The main phase of EF-1 alpha accumulation follows the disappearance of the 42S particles (stage II), but coincides with the main phase of ribosome accumulation (stages III and IV).     

Biochemical research on oogenesis: Oocytes of Xenopus laevis synthesize but do not accumulate 5S RNA of somatic type

The oocytes of amphibians and teleosts begin to accumulate 5S RNA several months before other components of the ribosomes become available. Two types of genes coding for 5S RNA are active during oogenesis of these animals. One type of genes is expressed only in oocytes. The other type is expressed in both oocytes and somatic cells. In this paper, we show that the oocytes of Xenopus laevis do not accumulate 5S RNA of somatic type. We conclude that the products of the two types of genes behave differently during oogenesis. One product is stored by the oocytes, whereas the other is not. The heterogeneity of 5S genes in Xenopus laevis might have arisen because oocytes and somatic cells needed different kinds of 5S RNA. These needs are met by molecules having different primary structures, different conformations, and different metabolic stabilities in vivo. We do not understand how these properties are related to one another.     

Hypoxia‐inducible factors 1α and 2α exert both distinct and overlapping functions in long bone development

The hypoxia‐inducible factors have recently been identified as critical regulators of angiogenic–osteogenic coupling. Mice overexpressing HIFα subunits in osteoblasts produce abundant VEGF and develop extremely dense, highly vascularized long bones. In this study, we investigated the individual contributions of Hif‐1α and Hif‐2α in angiogenesis and osteogenesis by individually disrupting each Hifα gene in osteoblasts using the Cre‐loxP method. Mice lacking Hif‐1α demonstrated markedly decreased trabecular bone volume, reduced bone formation rate, and altered cortical bone architecture. By contrast, mice lacking Hif‐2α had only a modest decrease in trabecular bone volume. Interestingly, long bone blood vessel development measured by angiography was decreased by a similar degree in both ΔHif‐1α and ΔHif‐2α mice suggesting a common role for these Hifα subunits in skeletal angiogenesis. In agreement with this idea, osteoblasts lacking either Hif‐1α or Hif‐2α had profound reductions in VEGF mRNA expression but only the loss of Hif‐1α impaired osteoblast proliferation. These findings indicate that expression of both Hif‐1α and Hif‐2α by osteoblasts is required for long bone development. We propose that both Hif‐1α and Hif‐2α function through cell non‐autonomous modes to promote vascularization of bone and that Hif‐1α also promotes bone formation by exerting direct actions on the osteoblast. J. Cell. Biochem. 109: 196–204, 2010. © 2009 Wiley‐Liss, Inc.