[Show abstract][Hide abstract] ABSTRACT: BTB-kelch proteins can elicit diverse biological functions but very little is known about the physiological role of these proteins in vivo. Kelch-like protein 6 (KLHL6) is a BTB-kelch protein with a lymphoid tissue-restricted expression pattern. In the B-lymphocyte lineage, KLHL6 is expressed throughout ontogeny, and KLHL6 expression is strongly upregulated in germinal center (GC) B cells. To analyze the role of KLHL6 in vivo, we have generated mouse mutants of KLHL6. Development of pro- and pre-B cells was normal but numbers of subsequent stages, transitional 1 and 2, and mature B cells were reduced in KLHL6-deficient mice. The antigen-dependent GC reaction was blunted (smaller GCs, reduced B-cell expansion, and reduced memory antibody response) in the absence of KLHL6. Comparison of mutants with global loss of KLHL6 to mutants lacking KLHL6 specifically in B cells demonstrated a B-cell-intrinsic requirement for KLHL6 expression. Finally, B-cell antigen receptor (BCR) cross-linking was less sensitive in KLHL6-deficient B cells compared to wild-type B cells as measured by proliferation, Ca2+ response, and activation of phospholipase Cgamma2. Our results strongly point to a role for KLHL6 in BCR signal transduction and formation of the full germinal center response.
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial cells are structurally and functionally heterogeneous. However, the molecular basis of this heterogeneity remains poorly defined. We used subtractive and differential screening to identify genes that exhibit heterogeneous expression patterns among vascular endothelial cells. One such gene is cellular retinol binding protein III (CRBP-III/Rbp7). Analysis of the lacZ knockin line for this gene (CRBP-III:lacZ) revealed a novel organ-specific vascular endothelial expression pattern. LacZ was expressed in vascular endothelial cells in heart, skeletal muscle, adipose tissues, thymus, and salivary gland. However, it was not detected in other tissues such as brain, liver, and lung. Furthermore, the expression within each organ was primarily restricted to small capillary endothelial cells, but could not be detected in larger vessels. This organ-specific vascular endothelial expression of CRPB:lacZ is relatively resistant to the changes of organ microenvironment. However, the level of expression can be modified by vitamin A deficiency. Therefore, our results provide novel molecular evidence for the heterogeneity of vascular endothelial cells.
[Show abstract][Hide abstract] ABSTRACT: Junctional adhesion molecule-A (JAM-A) is a transmembrane adhesive protein expressed at endothelial junctions and in leukocytes. In the present work, we found that DCs also express JAM-A. To evaluate the biological relevance of this observation, Jam-A(-/-) mice were generated and the functional behavior of DCs in vitro and in vivo was studied. In vitro, Jam-A(-/-) DCs showed a selective increase in random motility and in the capacity to transmigrate across lymphatic endothelial cells. In vivo, Jam-A(-/-) mice showed enhanced DC migration to lymph nodes, which was not observed in mice with endothelium-restricted deficiency of the protein. Furthermore, increased DC migration to lymph nodes was associated with enhanced contact hypersensitivity (CHS). Adoptive transfer experiments showed that JAM-A-deficient DCs elicited increased CHS in Jam-A(+/+) mice, further supporting the concept of a DC-specific effect. Thus, we identified here a novel, non-redundant role of JAM-A in controlling DC motility, trafficking to lymph nodes, and activation of specific immunity.
[Show abstract][Hide abstract] ABSTRACT: "Protein-trap" is a method that allows epitope-tagging of endogenous proteins. This method allows for the identification of endogenously expressed proteins that exhibit specific localization of interest. This method has been recently reported for its application in the study of Drosophila development by using a relatively large epitope, green-fluorescent-protein (GFP).
Herein, we report a new "protein-trap" vector for mammalian cells. This new method utilizes a much smaller epitope-tag and also allows for drug-selection prior to the epitope-tagging. Pre-selection by drug resulted in the highly efficient protein-trapping frequency.
The "protein-trap" method based on this new vector is expected to serve as a complimentary approach to the previously reported GFP-based method.
[Show abstract][Hide abstract] ABSTRACT: Vascular development is a complex process. In the past decade, significant advances have been made in the understanding of molecular mechanisms underlying vascular system development. Most recently, a model describing a signaling pathway that governs the differentiation of arteries and veins has emerged. Furthermore, the idea that angiogenesis of arteries and veins are coordinately but also differentially regulated has been proposed based on several pieces of molecular evidence. This article discusses the historical background leading to these most recent discoveries of signaling pathways that dictate arterial-venous formation and proposes a model that describes how arterial and venous systems are specified and established. The article also discusses future perspectives of this exciting area of vascular development in concluding remarks.
Current Opinion in Hematology 04/2003; 10(2):131-5. DOI:10.1097/00062752-200303000-00006 · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Flk1 is one of the specific cell surface receptors for vascular endothelial growth factor and one of the most specific markers highlighting the earliest stage of hematopoietic and vascular lineages. However, recent new evidence suggests that these Flk1(+) mesodermal progenitor cells also contribute to muscle lineages. All evidence is based on the experiments using in vitro differentiation and in vivo transplantation systems. Although this approach revealed a differentiation potential range of Flk1(+) cells that is wider than previously expected, it fails to determine whether Flk1(+) cells contribute to muscle lineage as part of the normal developmental process. To obtain direct evidence for the fate of Flk1(+) cells in development, we used a knock-in mouse line where Cre is expressed in Flk1(+) cells. Studies with these Cre lines provide direct evidence that Flk1(+) cells are progenitors for muscles, in addition to hematopoietic and vascular endothelial cells.
[Show abstract][Hide abstract] ABSTRACT: We report a transgenic mouse line in which Akt/protein kinase B (PKB) pathway can be activated in an inducible manner in defined cell types. In this transgenic mouse line, Cre expression allows the expression of a tamoxifen-activatable form of Akt/PKB in a defined cell type. Subsequent injection of tamoxifen triggers the transient activation of Akt/PKB in mice. Thus, this transgenic line allows the transient activation of Akt/PKB pathway in a predefined cell type. We expect that this transgenic system will provide a unique tool to study the roles of Akt/PKB pathway in mice.
[Show abstract][Hide abstract] ABSTRACT: Multiple classes of factors contribute to angiogenesis. In past years, the primary focus has been to understand the functions of individual classes of angiogenic factors. However, few studies have focused on the combinatorial roles of multiple classes of factors in angiogenesis. In this report, we have investigated the in vivo angiogenic processes regulated by two major classes of angiogenic factors, the angiopoietins and vascular endothelial growth factor (VEGF). Here we show that angiopoietin-1, a factor previously considered to be proangiogenic, can offset VEGF-induced angiogenesis in vivo. We also provide direct in vivo evidence for the synergistic effect of angiopoietin-2 and VEGF on the induction of angiogenesis. Furthermore, we show that these two classes of factors control the ratio of arterial and venous blood vessel types during angiogenesis. We believe that our study is a step toward understanding how multiple classes of factors harmonize angiogenesis and blood vessel types.
Proceedings of the National Academy of Sciences 07/2002; 99(12):8219-24. DOI:10.1073/pnas.122109599 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The angiopoietin ligands and Tie receptors belong to a novel class of ligand/receptor families, which play critical roles in blood vessel formation. They are considered to control numerous signaling pathways that are involved in diverse cellular processes, such as cell migration, proliferation and survival, and reorganization of the actin cytoskeleton. In this review, we summarize the important biochemical and biological properties of this interesting ligand/receptor family. Particular emphasis will be made on potential downstream targets and consequences of the endothelial cell behavior, due to regulation by the angiopoietin/Tie pathway.
[Show abstract][Hide abstract] ABSTRACT: Vascular polarity is a fundamental feature of angiogenesis and left-right asymmetry of the vascular network. Contrary to this importance, the molecular basis of vascular polarity is completely unknown. In this report, we show that the combinatorial function of angiopoietin-1 and the orphan receptor TIE1 is critical specifically for the development of the right-hand side venous system but is dispensable for the left-hand side venous system. Furthermore, our current finding reveals the existence of a distinct genetic program for the establishment of the right-hand side and left-hand side vascular networks well before the network asymmetry becomes morphologically discernible.
[Show abstract][Hide abstract] ABSTRACT: We report the generation and characterization of transgenic mouse and zebrafish expressing green fluorescent protein (GFP) specifically in vascular endothelial cells in a relatively uniform fashion. These reporter lines exhibit fluorescent vessels in developing embryos and throughout adulthood, allowing visualization of the general vascular patterns with single cell resolution. Furthermore, we show the ability to purify endothelial cells from whole embryos and adult organs by a single step fluorescence activated cell sorting. We expect that these transgenic reporters will be useful tools for imaging vascular morphogenesis, global gene expression profile analysis of endothelial cells, and high throughput screening for vascular mutations.