Activation of autophagy by FOXO3 regulates redox homeostasis during osteogenic differentiation.

Bone remodeling is a continuous physiological process that requires constant generation of new osteoblasts from mesenchymal stem cells (MSCs). Differentiation of MSCs to osteoblast requires a metabolic switch from glycolysis to increased mitochondrial respiration to ensure the sufficient energy supply to complete this process. As a consequence of this increased mitochondrial metabolism, the levels of endogenous reactive oxygen species (ROS) rise. In the current study we analyzed the role of forkhead box O3 (FOXO3) in the control of ROS levels in human MSCs (hMSCs) during osteogenic differentiation. Treatment of hMSCs with H2O2 induced FOXO3 phosphorylation at Ser294 and nuclear translocation. This ROS-mediated activation of FOXO3 was dependent on mitogen-activated protein kinase 8 (MAPK8/JNK) activity. Upon FOXO3 downregulation, osteoblastic differentiation was impaired and hMSCs lost their ability to control elevated ROS levels. Our results also demonstrate that in response to elevated ROS levels, FOXO3 induces autophagy in hMSCs. In line with this, impairment of autophagy by autophagy-related 7 (ATG7) knockdown resulted in a reduced capacity of hMSCs to regulate elevated ROS levels, together with a reduced osteoblast differentiation. Taken together our findings are consistent with a model where in hMSCs, FOXO3 is required to induce autophagy and thereby reduce elevated ROS levels resulting from the increased mitochondrial respiration during osteoblast differentiation. These new molecular insights provide an important contribution to our better understanding of bone physiology.

Syntenin-mediated regulation of Sox4 proteasomal degradation modulates transcriptional output.

The transcription factor Sox4 is aberrantly expressed in many human tumors and can modulate tumorigenesis and metastases of murine tumors in vivo. However, mechanisms that control Sox4 function remain poorly defined. It has recently been observed that DNA damage increases Sox4 protein expression independently of Sox4 mRNA levels, suggesting an as yet undefined post-transcriptional mechanism regulating Sox4 expression and functionality. Here, we show that Sox4 protein is rapidly degraded by the proteasome as indicated by pharmacological inhibition with Mg132 and epoxymycin. Sox4 half-life was found to be less than 1 h as evident by inhibition of protein synthesis using cycloheximide. Ectopic expression of Sox4 deletion mutants revealed that the C-terminal 33 residues of Sox4 were critical in modulating its degradation in a polyubiquitin-independent manner. Syntenin, a Sox4 binding partner, associates with this domain and was found to stabilize Sox4 expression. Syntenin-induced stabilization of Sox4 correlated with Sox4-syntenin relocalization to the nucleus, where both proteins accumulate. Syntenin overexpression or knockdown in human tumor cell lines was found to reciprocally modulate Sox4 protein expression and transcriptional activity implicating its role as a regulator of Sox4. Taken together, our data demonstrate that the Sox4 C-terminal domain regulates polyubiquitin-independent proteasomal degradation of Sox4 that can be modulated by interaction with syntenin. As aberrant Sox4 expression has been found associated with many human cancers, modulation of Sox4 proteasomal degradation may impact oncogenesis and metastatic properties of tumors.

Vasa protein expression and localization in the zebrafish.

Primordial germ cell (PGC) development in the zebrafish is poorly understood. The expression of vasa RNA, the only molecular marker so far found to be expressed in fish PGCs, suggests its function in the establishment of the germline. The protein product of vasa is present throughout the life cycle in the germline of Drosophila, Caenorhabditis and Xenopus. The expression pattern of the Vasa protein in zebrafish, is still unresolved. We generated an anti-Vasa polyclonal antibody and show that it is maternally expressed initially throughout the embryo. Interestingly, from the two-cell- to the 1000-cell stage the protein is highly concentrated in two 'dots' near the center of the blastomeres and as such remains longest detectable in the animal pole blastomeres. The first distinct cell-specific expression occurs at 60% epiboly on one side of the blastoderm margin. The Vasa protein in the PGCs is organized in a subcellular granular-like conformation which is dynamic throughout development.

Characterization of zebrafish primordial germ cells: morphology and early distribution of vasa RNA.

Research into germ line development is of conceptual and biotechnologic importance. In this study, we used morphology at the level of light and electron microscope to characterize the primordial germ cells (PGCs) of the zebrafish throughout embryonic and larval development. The study was complemented by the detailed analysis of mRNA expression of a putative germ line marker vasa. By morphology alone PGCs were identified at the earliest at the 5-somite stage in the peripheral endoderm in contact with the yolk syncytial layer. Subsequently, they move from lateral to medial positions into the median mesoderm and from there by means of the dorsal mesentery into the gonadal anlage at day 5 postfertilization (pf), to establish gonads with mesenchymal cells by day 9 pf. Ultrastructural analysis of the 4-day-old zebrafish larvae demonstrates the presence of the germ line-specific structures, nuage, and annulate lamellae. vasa RNA-positive cells can be followed during zebrafish embryogenesis from the 32-cell stage onward (Yoon et al., 1997). Upon completion of gastrulation, the RNA is exclusively present in the cells of the hypoblast, which as a consequence of convergence and extension movements first arrange themselves in a V-shaped string-like conformation to end up, by late somitogenesis, as a string of cells on each side of the midline. We show that the localization of maternal vasa RNA in the ovary changes from cytoplasmic, in the previtellogenic oocytes, to cortical in the vitellogenic oocytes, to concentrate at the boundary of the yolk and cytoplasm in the one cell stage zygote. These results demonstrate that the cortical vasa RNA localization precedes its cleavage furrow-associated localization in the embryos and is presumably cytoskeleton dependent. vasa RNA localization changes from asymmetric subcellular at the sphere stage, to become entirely cytoplasmic at the dome stage. These data suggest a close resemblance in modes of segregation of the germ plasma in the frog and vasa mRNA in the fish during cleavage stages. Based on the significantly larger size and the stereotype and similar position of morphologically distinct cells, presumed to be PGCs, and their vasa RNA-positive counterparts, we conclude that vasa RNA-positive cells are the PGCs and vasa RNA represents a definitive germ line marker in the fish. Dev Dyn 1999;216:153-167.

Cloning and expression of the zebrafish germ cell nuclear factor.

Nuclear orphan receptors are DNA-binding proteins that share the domain structure of the nuclear hormone receptor superfamily, although their ligands are unknown. Members of the nuclear receptor family are involved in the regulation of various developmental and reproductive processes. We have identified such a nuclear orphan receptor in the zebrafish and named it zebrafish germ cell nuclear factor (zfGCNF) based on its high sequence homology to previously described mouse, human, and Xenopus laevis GCNF. Detailed sequence comparison of zfGCNF with mouse, human, and frog GCNF revealed high homologies in the domains conserved in the nuclear receptor family. Homology in the DNA-binding domain is 97% for frog and even 98.5% for mouse and human when compared to the zebrafish sequence. Homology in the E III subdomain of the transactivation/ligand-binding E domain is 100% when compared to the mouse and human sequences. Transcripts of different size were detected by Northern blot analysis in the zebrafish ovary, whereas, in the testis only one transcript was present. In situ hybridization revealed that zfGCNF was predominantly expressed in previtellogenic oocytes in the ovary and in spermatocytes in the testis.

Germ line development in fishes.

Classical work on germ cells in fishes has dealt with three main issues; their embryonic origin, the proliferation, and migration pathway during embryonic and larval development. Until recently, primordial germ cells (PGCs) have been studied in a number of fishes using morphological criteria only. The identification of the Drosophila vasa homolog gene of zebrafish now allows comparison of these morphological data with vasa RNA expression patterns in zebrafish. Teleost PGCs can be distinguished from somatic cells by their distinct morphology, at the earliest during gastrulation, and in most fishes their number varies between 10 and 30 during pregonial development. Mitosis is generally not observed in PGCs at extragonadal locations, whereas they are mitotically active once at the gonadal ridges. During gastrulation, PGCs appear to translocate from the epiblast to the hypoblast and during somitogenesis they are found associated with the most peripheral yolk syncitial layer (YSL). From the peripheral YSL they migrate through the median mesoderm into the dorsal mesoderm and then to the dorsal mesentery, where they establish the gonad primordia with mesenchymal cells. Vasa RNA positive cells, the PGCs of the zebrafish conform to these general observations. Interestingly, classical descriptive and experimental data can now be reevaluated using vasa as a molecular marker of the fish germ line. The power of zebrafish genetics together with possibilities of experimental embryology should accelerate research on aspects of vertebrate germ line development such as PGC migration, division and apoptosis, as well as (in) fertility. The present review summarizes some of the classical data on germ line development in fishes in relation to recent data on vasa expression in zebrafish and compares these findings, where appropriate, with those in other model organisms. Special emphasis is placed on vasa gene expression as a potential universal germ line marker and suggestions are made for novel, zebrafish specific approaches to investigate the vertebrate germ line.

Development, characterization, and biological properties of meningococcal immunotype L3,7,(8),9-specific monoclonal antibodies.

In this study, we characterize the properties of nine monoclonal antibodies (MAbs) that recognize meningococcal lipopolysaccharides (LPS). The following three specific MAbs that had not been described previously were elicited in BALB/c mice by using an immunotype L3,7,9 oligosaccharide-tetanus toxoid conjugate in combination with Quil A: 4D1-B3, 3A12-E1, and 4A8-B2. These MAbs reacted with L3,7,9 LPS on immunoblots and in the LPS enzyme-linked immunosorbent assay (ELISA) and recognised strains containing L3, L3,7, L8 (except 3A12-E1), or L9 LPS in the whole-cell ELISA. The six other MAbs have been described in previous studies (K. Saukkonen, M. Leinonen, H. Abdillahi, and J.T. Poolman, Vaccine 7:325-328, 1989; R.J.P.M. Scholten, B. Kuipers, H.A. Valkenburg, J. Danjert, W.D. Zollinger, and J.T. Poolman, J. Med. Microbiol., in press) and were obtained after immunization with outer membrane protein complexes containing LPS: MN15A11, MN15A8-1, MN15A17-1, MN11A11G, MN14F20-11, and MN14F21-11. MN15A11 was specific for L3,7,9 LPS and displayed properties similar to those of 3A12-E1. MN15A17-1, MN14F20-1, and MN11A11G were cross-reactive, and MN14F21-11 was specific for the L1,8 immunotype. Epitope specificities of MAbs reacting with L3,7,(8),9 strains were analyzed. MAbs 4D1-B3, 3A12-E1, and 4A8-B2 recognized phosphoethanolamine group-containing oligosaccharide-specific epitopes. MN15A11 and MN15A17-1 were probably directed against a conformational epitope, although for MN5A11 recognition of an unknown L3,7,9-specific epitope in the 2-keto-3-deoxyoctulosonic acid (KDO)-lipid A region cannot be excluded. MN15A8-1, a strongly cross-reactive MAb, recognized a determinant which included the KDO-lipid A region and the more terminal saccharides.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation, characterization, and immunogenicity of meningococcal immunotype L2 and L3,7,9 phosphoethanolamine group-containing oligosaccharide-protein conjugates.

A method was developed for the well-defined coupling of phosphoethanolamine group (PEA)- and carboxylic acid group-containing polysaccharides and oligosaccharides to proteins without the need for extensive modification of the carbohydrate antigens. The carboxylic acid group of the terminal 2-keto-3-deoxyoctulosonic acid moiety was utilized to introduce a thiol function in meningococcal immunotype L2 and L3,7,9 lipopolysaccharide-derived oligosaccharides. The thiol group-containing oligosaccharides were subsequently coupled to bromoacetylated proteins. Immunotype L2 and L3,7,9 PEA group-containing oligosaccharide-tetanus toxoid conjugates were prepared, and their immunogenicities were studied in rabbits. Both the immunotype L2 and immunotype L3,7,9 conjugates evoked high immunoglobulin G (IgG) antibody titers after the first booster injection. These conjugates also displayed an ability to induce long-lasting IgG antibody levels which could be detected until 9 months after one booster injection at week 3. The adjuvant Quil A enhanced the immune response to all the conjugates to a minor extent, which is in contrast with reported adjuvant effects of Quil A on these types of antigens in mice. A conjugate prepared from the dephosphorylated L3,7,9 oligosaccharides evoked a significantly lower IgG response than a similar PEA-containing conjugate, and enzyme-linked immunosorbent assay inhibition studies indicated a different epitope specificity. Furthermore, antisera elicited with the complete bacteria contained antibodies directed against PEA-containing epitopes, which stresses the importance of the presence of unmodified PEA groups in meningococcal lipopolysaccharide-derived oligosaccharide-protein conjugates. The procedure developed offers an elegant solution for the specific coupling of meningococcal PEA-containing oligosaccharides to proteins and may therefore be a very useful tool in the development of a vaccine against group B meningococci.