The transcription factor Grainyhead like 3 (GRHL3) affects endothelial cell apoptosis and migration in a NO-dependent manner

Migratory capacity and resistance to apoptosis are crucial for proper endothelial function. In a screen for anti-apoptotic genes in a breast cancer cell line, we identified Grainyhead like 3 (GRHL3). Therefore, the aim of our study was to investigate whether GRHL3 is expressed in endothelial cells and moreover, to determine its role in migration, apoptosis and senescence. GRHL3 is expressed in human endothelial cells. GRHL3 is required for endothelial cell migration. The underlying mechanism is independent of vascular endothelial growth factor. GRHL3 induces Akt and endothelial nitric oxide synthase phosphorylation and its expression is increased by physiological concentrations of nitric oxide. Nitric oxide dependent migration is completely dependent on GRHL3 expression. Moreover, GRHL3 inhibits apoptosis of endothelial cells in an eNOS-dependent manner. Thus, loss of GRHL3 may result in endothelial dysfunction in vivo. One may consider new therapeutic strategies with the aim to conserve GRHL3 expression in the vasculature.     

Melanoma cells produce multiple laminin isoforms and strongly migrate on α5 laminin(s) via several integrin receptors

Melanoma cells express and interact with laminins (LMs) and other basement membrane components during invasion and metastasis. In the present study we have investigated the production and migration-promoting activity of laminin isoforms in melanoma. Immunohistochemistry of melanoma specimens and immunoprecipitation/western blotting of melanoma cell lines indicated expression of laminin-111/121, laminin-211, laminin-411/421, and laminin-511/521. Laminin-332 was not detected. In functional assays, laminin-111, laminin-332, and laminin-511, but not laminin-211 and laminin-411, strongly promoted haptotactic cell migration either constitutively or following stimulation with insulin-like growth factors. Both placenta and recombinant laminin-511 preparations were highly active, and the isolated recombinant IVa domain of LMα5 also promoted cell migration. Function-blocking antibodies in cell migration assays revealed α6β1 integrin as the major receptor for laminin-111, and both α3β1 and α6β1 integrins for laminin-332 and laminin-511. In contrast, isolated LMα5 IVa domain-promoted melanoma cell migration was largely mediated via αVβ3 integrin and inhibited by RGD peptides. Given the ubiquitous expression of α5 laminins in melanoma cells and in melanoma-target tissues/anatomical structures, as well as the strong migration-promoting activity of these laminin isoforms, the α5 laminins emerge as putative primary extracellular matrix mediators of melanoma invasion and metastasis via α3β1 and other integrin receptors.     

Genes that control the development of migrating muscle precursor cells

Skeletal muscles in vertebrates, despite their functional and biochemical similarities, are generated via diverse developmental mechanisms. A major subclass of hypaxial muscle groups is derived from long-range migrating progenitor cells that delaminate from the dermomyotome. The development of this lineage is controlled by Pax3, the c-Met tyrosine kinase receptor, its ligand SF/HGF (scatter factor/hepatocyte growth factor) and the homeobox factor Lbx1. These molecules are essential for establishment of the precursor pool, delamination, migration and target finding. Progress has been made in understanding patterning of the muscles, which requires a precise control of proliferation and differentiation of myogenic precursor cells.     

Recombinant vascular basement membrane derived multifunctional peptide blocks endothelial cell angiogenesis and neovascularization

Angiogenesis is an innovative target in the therapy of cancer and other diseases, but the effects of anti‐angiogenic drugs have been rather modest in clinical trials. We have developed a small peptide, recombinant vascular basement membrane derived multifunctional peptide (rVBMDMP), which significantly inhibits endothelial cells in vitro. Here we test the mechanisms of rVBMDMP in angiogenesis balance in assays of tubule formation, colony formation, and apoptosis in HUVE‐12 endothelial cells. We also analyzed the differential expression of phosphorylation proteins and related genes in a protein phosphorylation chip and extracellular matrix adhesion molecule cDNA microarray, and validated changes with Western blot or real‐time quantitative PCR, respectively. rVBMDMP dose‐dependently inhibited colony formation, induced apoptosis, and inhibited in vitro tubule formation. rVBMDMP increased the phosphorylation of 88 signal proteins, including caspase‐3, death receptor 3, 4, and 5, and integrin αV, β1, and β3, and down‐regulated 41 signal proteins, including EGFR, pEGFR, VEGFR‐1, and survivin versus control. rVBMDMP upregulated 14 genes, including collagen 4, 7, and 27, and down‐regulated 21 genes, including integrin αVβ3, MMP10, and MMP12. Our study suggests that rVBMDMP inhibits angiogenesis and may be a viable drug candidate in anti‐angiogenesis and anticancer therapies. J. Cell. Biochem. 111: 453–460, 2010. © 2010 Wiley‐Liss, Inc.     

The pattern of gene expression in mouse Gr-1(+) myeloid progenitor cells

To understand the pattern of gene expression in mouse myeloid progenitor cells, we carried out a genome-wide analysis of gene expression in mouse bone marrow Gr-1(+) cells using SAGE and GLGI techniques. We identified 22,033 unique SAGE tags with quantitative information from 73,869 collected SAGE tags. Among these unique tags, 64% match known sequences, including many genes important for myeloid differentiation, and 36% have no matches to known sequences and are likely to represent novel genes. We compared the expression of mouse Gr-1(+) and human CD15(+) myeloid progenitor cells and showed that the pattern of gene expression of these two cell populations had some similarities. We also compared the expression of mouse Gr-1(+) myeloid progenitor cells with that of mouse brain tissue and found a highly tissue-specific manner of gene expression in these two samples. Our data provide a basis for studying altered gene expression in myeloid disorders using mouse models.     

Conserved signaling through vascular endothelial growth (VEGF) receptor family members in murine lymphatic endothelial cells

Lymphatic vessels guide interstitial fluid, modulate immune responses by regulating leukocyte and antigen trafficking to lymph nodes, and in a cancer setting enable tumor cells to track to regional lymph nodes. The aim of the study was to determine whether primary murine lymphatic endothelial cells (mLECs) show conserved vascular endothelial growth factor (VEGF) signaling pathways with human LECs (hLECs). LECs were successfully isolated from murine dermis and prostate. Similar to hLECs, vascular endothelial growth factor (VEGF) family ligands activated MAPK and pAkt intracellular signaling pathways in mLECs. We describe a robust protocol for isolation of mLECs which, by harnessing the power of transgenic and knockout mouse models, will be a useful tool to study how LEC phenotype contributes to alterations in lymphatic vessel formation and function.     

Oxygen and cytokine-dependent changes in choline phospholipid saturation in hematopoietic progenitor cells detected by MALDI-TOF mass spectrometry

The adaptation of cells to a changing environment is normally accompanied by rapid and/or chronic remodeling of membrane lipids. In order to understand the role played by membrane lipid metabolism in such responses, it is necessary to characterize in more detail the changes in membrane composition occurring in response to defined stimuli. There has been intense interest in characterizing the “stem cell niche” in recent years and an emerging consensus that stem cells are located in regions of low oxygen tension and probably well-isolated from the blood supply.We report here the use of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry to monitor changes in the composition and saturation degree of choline phospholipids of hematopoietic progenitor (FDCPmix) cells under standard nutrient-rich culture conditions and at low oxygen and low glucose concentrations. We found that the increase in proliferation rate driven by high concentrations of interleukin-3 (IL-3) is associated with a decrease in membrane phosphatidylcholine (PC) 18:0/20:4 and sphingomyelin (SM) together with an increase in PC 18:0/18:2 and dihydro SM. Furthermore, this effect is most pronounced under low oxygen and low glucose conditions, independent of cell proliferation rates.     

Methylmercury-injury effect on tube formation by cultured human vascular endothelial cells

The effect of methylmercury chloride (MeHg) on growth and tube formation by cultured human umbilical vein endothelial cells (HUVECs) was investigated. HUVECs were collected by enzymatic digestion with collagenase. Precultivation of HUVECs with MeHg at concentrations of 1.0–50.0 mol/L exerted negligible effects on the viable cell number, while the viable cell number was slightly reduced at 100 mol/L and fell to zero at concentrations exceeding 500.0 mol/L MeHg. The viable cell number was depressed in a concentration-dependent manner. Tube formation was studied by culturing the cells on gelled basement membrane matrix (Matrigel). Treatment of HUVECs with 0.1–5.0 mol/L MeHg for 24 h inhibited tube formation dose-dependently. Fetal bovine serum (FBS) increased tube formation in a dose-dependent manner, with half-maximum stimulation of tube formation at approximately 3.4% FBS. The length of tube formation decreased time-dependently at concentrations of 0.1 and 1.0 mol/L MeHg. Pretreatment of Matrigel with 1 mol/L MeHg before the cell seeding reduced the tube formation by HUVECs. These results suggest that the growth and tube formation by HUVECs is susceptible to MeHg cytotoxicity, and that MeHg could be injurious to endothelial cell function.Abbreviations MeHg methylmercury chloride - HUVECs human umbilical vein endothelial cells     

NKT ligand-loaded, antigen-expressing B cells function as long-lasting antigen presenting cells in vivo

We had previously shown that activated NKT cells licensed B cells to be immunogenic antigen-presenting cells and helped to elicit a wide spectrum of cancer targeted immune responses. In the current study, we sought to verify the safety of αGalCer-loaded, and adenovirus-transduced B cell-based vaccines, together with mechanism of action. Intravenously injected αGalCer-loaded, antigen-expressing B cells rapidly localized in the spleen and directly primed CD8⁺ T cells in an antigen-specific manner. The transferred antigen was sustained for at least 30 days. While some injected B cells produced nonspecific IgG, the antigen-specific IgG response was completely dependent on endogenous B cells. The liver was one of the main tissues where injected B cells were retained; however, we could not find the signs of liver toxicity. Our results demonstrate that αGalCer-loaded, antigen-expressing B cells behave as “antigen-presenting” cells that stimulate endogenous antigen-specific T cells and B cells in vivo without significant toxicity.     

Single-Nucleotide Polymorphism Alleles in the Insulin Receptor Gene Are Associated with Typical Migraine

We have identified a migraine locus on chromosome 19p13.3/2 using linkage and association analysis. We isolated 48 single-nucleotide polymorphisms within the locus, of which we genotyped 24 in a Caucasian population comprising 827 unrelated cases and 765 controls. Five single-nucleotide polymorphisms within the insulin receptor gene showed significant association with migraine. This association was independently replicated in a case-control population collected separately. We used experiments with insulin receptor RNA and protein to investigate functionality for the migraine-associated single-nucleotide polymorphisms. We suggest possible functions for the insulin receptor in migraine pathogenesis.     

Physical mapping of the mouse tilted locus identifies an association between human deafness loci DFNA6/14 and vestibular system development

The tilted (tlt) mouse carries a recessive mutation causing vestibular dysfunction. The defect in tlt homozygous mice is limited to the utricle and saccule of the inner ear, which completely lack otoconia. Genetic mapping of tlt placed it in a region orthologous with human 4p16.3-p15 that contains two loci, DFNA6 and DFNA14, responsible for autosomal dominant, nonsyndromic hereditary hearing impairment. To identify a possible relationship between tlt in mice and DFNA6 and DFNA14 in humans, we have refined the mouse genetic map, assembled a BAC contig spanning the tlt locus, and developed a comprehensive comparative map between mouse and human. We have determined the position of tlt relative to 17 mouse chromosome 5 genes with orthologous loci in the human 4p16.3-p15 region. This analysis identified an inversion between the mouse and human genomes that places tlt and DFNA6/14 in close proximity.     

Nucleotide Sequence, Genomic Organization, and Chromosomal Localization of Genes Encoding the Human NMDA Receptor Subunits NR3A and NR3B

The N-methyl-D-aspartate (NMDA) receptors are glutamate-regulated ion channels that are critically involved in important physiological and pathological functions of the mammalian central nervous system. We have identified and characterized the gene encoding the human NMDA receptor subunit NR3A (GRIN3A), as well as the gene (GRIN3B) encoding an entirely novel subunit that we named NR3B, as it is most closely related to NR3A (57.4% identity). GRIN3A localizes to chromosome 9q34, in the region 13-34, and consists of nine coding exons. The deduced protein contains 1115 amino acids and shows 92.7% identity to rat NR3A. GRIN3B localizes to chromosome 19p13.3 and contains, as does the mouse NR3B gene (Grin3b), eight coding exons. The deduced proteins of human and mouse NR3B contain 901 and 900 amino acid residues, respectively (81.6% identity). In situ hybridization shows a widespread distribution of Grin3b mRNA in the brain of the adult rat.     

A High-Resolution Genetic, Physical, and Comparative Gene Map of the Doublefoot (Dbf) Region of Mouse Chromosome 1 and the Region of Conserved Synteny on Human Chromosome 2q35

The mouse doublefoot (Dbf) mutant exhibits preaxial polydactyly in association with craniofacial defects. This mutation has previously been mapped to mouse chromosome 1. We have used a positional cloning strategy, coupled with a comparative sequencing approach using available human draft sequence, to identify putative candidates for the Dbf gene in the mouse and in homologous human region. We have constructed a high-resolution genetic map of the region, localizing the mutation to a 0. 4-cM (±0.0061) interval on mouse chromosome 1. Furthermore, we have constructed contiguous BAC/PAC clone maps across the mouse and human Dbf region. Using existing markers and additional sequence tagged sites, which we have generated, we have anchored the physical map to the genetic map. Through the comparative sequencing of these clones we have identified 35 genes within this interval, indicating that the region is gene-rich. From this we have identified several genes that are known to be differentially expressed in the developing mid-gestation mouse embryo, some in the developing embryonic limb buds. These genes include those encoding known developmental signaling molecules such as WNT proteins and IHH, and we provide evidence that these genes are candidates for the Dbf mutation.