The transcription factor EPAS-1/hypoxia-inducible factor 2α plays an important role in vascular remodeling

We have studied the role of the basic helix–loop–helix–PAS transcription factor EPAS-1/hypoxia-inducible factor 2α in vascular development by gene targeting. In ICR/129 Sv outbred background, more than half of the mutants displayed varying degrees of vascular disorganization, typically in the yolk sac, and died in utero between embryonic day (E)9.5 and E13.5. In mutant embryos directly derived from EPAS-1−/− embryonic stem cells (hence in 129 Sv background), all embryos developed severe vascular defects both in the yolk sac and embryo proper and died between E9.5 and E12.5. Normal blood vessels were formed by vasculogenesis but they either fused improperly or failed to assemble into larger vessels later during development. Our results suggest that EPAS-1 plays an important role at postvasculogenesis stages and is required for the remodeling of the primary vascular network into a mature hierarchy pattern.

Novel Nuclear Signaling Pathway Mediates Activation of Fibroblast Growth Factor-2 Gene by Type 1 and Type 2 Angiotensin II Receptors

In bovine adrenal medullary cells synergistically acting type 1 and type 2 angiotensin II (AII) receptors activate the fibroblast growth factor-2 (FGF-2) gene through a unique AII-responsive promoter element. Both the type 1 and type 2 AII receptors and the downstream cyclic adenosine 1′,3′-monophosphate- and protein kinase C-dependent signaling pathways activate the FGF-2 promoter through a novel signal-transducing mechanism. This mechanism, which we have named integrative nuclear FGF receptor-1 signaling, involves the nuclear translocation of FGF receptor-1 and its subsequent transactivation of the AII-responsive element in the FGF-2 promoter.

Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice

We previously reported the disruption of the murine gene encoding the transcription factor USF2 and its consequences on glucose-dependent gene regulation in the liver. We report here a peculiar phenotype of Usf2−/− mice that progressively develop multivisceral iron overload; plasma iron overcomes transferrin binding capacity, and nontransferrin-bound iron accumulates in various tissues including pancreas and heart. In contrast, the splenic iron content is strikingly lower in knockout animals than in controls. To identify genes that may account for the abnormalities of iron homeostasis in Usf2−/− mice, we used suppressive subtractive hybridization between livers from Usf2−/− and wild-type mice. We isolated a cDNA encoding a peptide, hepcidin (also referred to as LEAP-1, for liver-expressed antimicrobial peptide), that was very recently purified from human blood ultrafiltrate and from urine as a disulfide-bonded peptide exhibiting antimicrobial activity. Accumulation of iron in the liver has been recently reported to up-regulate hepcidin expression, whereas our data clearly show that a complete defect in hepcidin expression is responsible for progressive tissue iron overload. The striking similarity of the alterations in iron metabolism between HFE knockout mice, a murine model of hereditary hemochromatosis, and the Usf2−/− hepcidin-deficient mice suggests that hepcidin may function in the same regulatory pathway as HFE. We propose that hepcidin acts as a signaling molecule that is required in conjunction with HFE to regulate both intestinal iron absorption and iron storage in macrophages.

Tumor necrosis factor receptor associated factor 2 is a mediator of NF-kappa B activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein.

Latent infection membrane protein 1 (LMP1), the Epstein-Barr virus transforming protein, associates with tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) and TRAF3. Since TRAF2 has been implicated in TNFR-mediated NF-kappa B activation, we have evaluated the role of TRAF2 in LMP1-mediated NF-kappa B activation. TRAF2 binds in vitro to the LMP1 carboxyl-terminal cytoplasmic domain (CT), coprecipitates with LMP1 in B lymphoblasts, and relocalizes to LMP1 plasma membrane patches. A dominant negative TRAF2 deletion mutant that lacks amino acids 6-86 (TRAF/ delta 6-86) inhibits NF-kappa B activation from the LMP1 CT and competes with TRAF2 for LMP1 binding. TRAF2 delta 6-86 inhibits NF-kappa B activation mediated by the first 45 amino acids of the LMP1 CT by more than 75% but inhibits NF-kappa B activation through the last 55 amino acids of the CT by less than 40%. A TRAF interacting protein, TANK, inhibits NF-kappa B activation by more than 70% from both LMP1 CT domains. These data implicate TRAF2 aggregation in NF-kappa B activation by the first 45 amino acids of the LMP1 CT and suggest that a different TRAF-related pathway may be involved in NF-kappa B activation by the last 55 amino acids of the LMP1 CT.

Fibroblast growth factor 2 can replace ectodermal signaling for feather development.

The initiation and morphogenesis of cutaneous appendages depend on a series of reciprocal signaling events between the epithelium and mesenchyme of the embryonic skin. In the development of feather germs, early dermal signals induce the formation of epidermal placodes that in turn signal the mesoderm to form dermal condensations immediately beneath them. We find a spatially and temporally restricted pattern of transcription for the genes that encode fibroblast growth factor (FGF) 2 and FGF receptor (FGFR) 1 in developing feather germs of the chicken embryo. FGF-2 expression is restricted to the epidermal placodes, whereas FGFR-1 expression is limited to the dermal condensations. Transcription of these genes could not be detected in skins of scaleless (sc/sc) embryos that fail to develop feathers as a result of an ectodermal defect. Treatment of sc/sc skins with FGF-2 results in the formation of feathers at the site of application of the growth factor and the induced feathers express FGFR-1 in their dermal condensations. Thus, we have established FGF-2 as an epidermal signal in early feather germ formation. The observation that FGF-2 can rescue the mutant phenotype of sc/sc embryos suggests that FGF-2 either is, or is downstream from, the signal that the sc/sc mutant ectoderm fails to generate.

Myocyte-specific enhancer factor 2 acts cooperatively with a muscle activator region to regulate Drosophila tropomyosin gene muscle expression.

MEF2 (myocyte-specific enhancer factor 2) is a MADS box transcription factor that is thought to be a key regulator of myogenesis in vertebrates. Mutations in the Drosophila homologue of the mef2 gene indicate that it plays a key role in regulating myogenesis in Drosophila. We show here that the Drosophila tropomyosin I (TmI) gene is a target gene for mef2 regulation. The TmI gene contains a proximal and a distal muscle enhancer within the first intron of the gene. We show that both enhancers contain a MEF2 binding site and that a mutation in the MEF2 binding site of either enhancer significantly reduces reporter gene expression in embryonic, larval, and adult somatic body wall muscles of transgenic flies. We also show that a high level of proximal enhancer-directed reporter gene expression in somatic muscles requires the cooperative activity of MEF2 and a cis-acting muscle activator region located within the enhancer. Thus, mef2 null mutant embryos show a significant reduction but not an elimination of TmI expression in the body wall myoblasts and muscle fibers that are present. Surprisingly, there is little effect in these mutants on TmI expression in developing visceral muscles and dorsal vessel (heart), despite the fact that MEF2 is expressed in these muscles in wild-type embryos, indicating that TmI expression is regulated differently in these muscles. Taken together, our results show that mef2 is a positive regulator of tropomyosin gene transcription that is necessary but not sufficient for high level expression in somatic muscle of the embryo, larva, and adult.

Regulation of albumin endocytosis by PSD95/Dlg/ZO-1 (PDZ) scaffolds - Interaction of Na+-H+ exchange regulatory factor-2 with ClC-5

The constitutive reuptake of albumin from the glomerular filtrate by receptor-mediated endocytosis is a key function of the renal proximal tubules. Both the Cl- channel ClC-5 and the Na+-H+ exchanger isoform 3 are critical components of the macromolecular endocytic complex that is required for albumin uptake, and therefore the cell-surface levels of these proteins may limit albumin endocytosis. This study was undertaken to investigate the potential roles of the epithelial PDZ scaffolds, Na+-H+ exchange regulatory factors, NHERF1 and NHERF2, in albumin uptake by opossum kidney ( OK) cells. We found that ClC-5 co-immunoprecipitates with NHERF2 but not NHERF1 from OK cell lysate. Experiments using fusion proteins demonstrated that this was a direct interaction between an internal binding site in the C terminus of ClC-5 and the PDZ2 module of NHERF2. In OK cells, NHERF2 is restricted to the intravillar region while NHERF1 is located in the microvilli. Silencing NHERF2 reduced both cell-surface levels of ClC-5 and albumin uptake. Conversely, silencing NHERF1 increased cell-surface levels of ClC-5 and albumin uptake, presumably by increasing the mobility of NHE3 in the membrane and its availability to the albumin uptake complex. Surface biotinylation experiments revealed that both NHERF1 and NHERF2 were associated with the plasma membrane and that NHERF2 was recruited to the membrane in the presence of albumin. The importance of the interaction between NHERF2 and the cytoskeleton was demonstrated by a significant reduction in albumin uptake in cells overexpressing an ezrin binding-deficient mutant of NHERF2. Thus NHERF1 and NHERF2 differentially regulate albumin uptake by mechanisms that ultimately alter the cell-surface levels of ClC-5.

Adipose tissue engineering based on the controlled release of fibroblast growth factor-2 in a collagen matrix

Adipose tissue forms when basement membrane extract ( Matrigel (TM)) and fibroblast growth factor-2 (FGF-2) are added to our mouse tissue engineering chamber model. A mouse tumor extract, Matrigel is unsuitable for human clinical application, and finding an alternative to Matrigel is essential. In this study we generated adipose tissue in the chamber model without using Matrigel by controlled release of FGF-2 in a type I collagen matrix. FGF-2 was impregnated into biodegradable gelatin microspheres for its slow release. The chambers were filled with these microspheres suspended in 60 mu L collagen gel. Injection of collagen containing free FGF-2 or collagen containing gelatin microspheres with buffer alone served as controls. When chambers were harvested 6 weeks after implantation, the volume and weight of the tissue obtained were higher in the group that received collagen and FGF-2 impregnated microspheres than in controls. Histologic analysis of tissue constructs showed the formation of de novo adipose tissue accompanied by angiogenesis. In contrast, control groups did not show extensive adipose tissue formation. In conclusion, this study has shown that de novo formation of adipose tissue can be achieved through controlled release of FGF-2 in collagen type I in the absence of Matrigel.

The mitogenic signaling pathway for fibroblast growth factor-2 involves the tyrosine phosphorylation of cyclin D2 in MCF-7 human breast cancer cells

Fibroblast growth factor-2 (FGF-2) is mitogenic for the human breast cancer cell line MCF-7; here we investigate some of the signaling pathways subserving this activity. FGF-2 stimulation of MCF-7 cells resulted in a global increase of intracellular tyrosine phosphorylation of proteins, particularly FGF receptor substrate-2, the protooncogene product Src and the mitogen-activated protein kinase (MAP kinase) cascade, A major increase in the tyrosine phosphorylation of a 30-kDa protein species was also found. This protein was identified as cyclin D2 by mass spectrometry after trypsin digestion. Immunoprecipitation of cyclin D2 and immunoblotting with anti-phosphotyrosine antibodies confirmed that the tyrosine phosphorylation of cyclin D2 was indeed induced by FGF-2 stimulation. In addition, pharmacological inhibition of Src (with herbimycin A and PP2), and of the MAP kinase cascade (with PD98059), confirmed that Src activity is required for the FGF-2-induced phosphorylation of cyclin D2 whereas MAP kinase activity is not, Thus, tyrosine phosphorylation of cyclin D2 may be a hey regulatory target for FGF-2 signaling. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Increased expression of phosphorylated forms of RNA-dependent protein kinase and eukaryotic initiation factor 2 alpha may signal skeletal muscle atrophy in weight-losing cancer patients

Previous studies suggest that the activation (autophosphorylation) of dsRNA-dependent protein kinase (PKR) can stimulate protein degradation, and depress protein synthesis in skeletal muscle through phosphorylation of the translation initiation factor 2 (eIF2) on the alpha-subunit. To understand whether these mediators are important in muscle wasting in cancer patients, levels of the phospho forms of PKR and eIF2alpha have been determined in rectus abdominus muscle of weight losing patients with oesophago-gastric cancer, in comparison with healthy controls. Levels of both phospho PKR and phospho eIF2alpha were significantly enhanced in muscle of cancer patients with weight loss irrespective of the amount and there was a linear relationship between phosphorylation of PKR and phosphorylation of eIF2alpha (correlation coefficient 0.76, P=0.005). This suggests that phosphorylation of PKR led to phosphorylation of eIF2alpha. Myosin levels decreased as the weight loss increased, and there was a linear relationship between myosin expression and the extent of phosphorylation of eIF2alpha (correlation coefficient 0.77, P=0.004). These results suggest that phosphorylation of PKR may be an important initiator of muscle wasting in cancer patients.

Neuroprotection and regeneration of adult lesioned retinal ganglion cells by the modulation of fibroblast growth factor-2 and receptor tyrosine phosphatase-sigma

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Neuroprotection and regeneration of adult lesioned retinal ganglion cells by the modulation of fibroblast growth factor-2 and receptor tyrosine phosphatase-sigma

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.