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1.
Kidney Int ; 73(7): 797-9, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18340350

ABSTRACT

Changes in tissue oxygen levels trigger molecular signaling pathways that regulate cellular proliferation and differentiation in multiple cell types. The functional role of oxygen signaling in the immune system is not well understood. Rama and colleagues demonstrate that hypoxia induces dendritic-cell maturation; thus they provide a novel mechanistic link between hypoxia/ischemia and the activation of the immune system in the kidney.


Subject(s)
Cell Hypoxia/physiology , Immune System/physiology , Basic Helix-Loop-Helix Transcription Factors/physiology , Dendritic Cells/physiology , Hypoxia-Inducible Factor 1/physiology , Kidney Diseases/etiology
2.
Cell Death Differ ; 15(4): 650-9, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18219317

ABSTRACT

The von Hippel-Lindau tumor suppressor gene product, pVHL, functions as the substrate recognition component of an E3-ubiquitin ligase, which targets the oxygen-sensitive alpha-subunit of hypoxia-inducible factor (HIF) for rapid proteasomal degradation under normoxic conditions and as such plays a central role in molecular oxygen sensing. Mutations in pVHL can be found in familial and sporadic clear cell carcinomas of the kidney, hemangioblastomas of the retina and central nervous system, and pheochromocytomas, underscoring its gatekeeper function in the pathogenesis of these tumors. Tissue-specific gene targeting of VHL in mice has demonstrated that efficient execution of pVHL-mediated HIF proteolysis under normoxia is fundamentally important for survival, proliferation, differentiation and normal physiology of many cell types, and has provided novel insights into the biological function of individual HIF transcription factors. In this review, we discuss the role of HIF in the development of the VHL phenotype.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Neoplasms/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , von Hippel-Lindau Disease/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/metabolism , Gene Expression Regulation, Developmental , Gene Expression Regulation, Neoplastic , Genotype , Germ-Line Mutation , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Kidney Neoplasms/genetics , Kidney Neoplasms/metabolism , Mice , Mice, Knockout , Neoplasms/genetics , Neoplasms/pathology , Phenotype , Ubiquitin-Protein Ligases/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/genetics , von Hippel-Lindau Disease/genetics , von Hippel-Lindau Disease/pathology
3.
Oncogene ; 26(31): 4531-40, 2007 Jul 05.
Article in English | MEDLINE | ID: mdl-17297464

ABSTRACT

Individuals bearing germ line mutations in the Von Hippel-Lindau (VHL) tumor suppressor gene are predisposed to the development of highly angiogenic tumors. This is correlated with an increased expression of the angiogenic factor vascular endothelial growth factor (VEGF) in these tumors, which is in part caused by elevated expression of the HIF-1 hypoxia inducible transcription factors. We created malignant astrocytes with genetic deletions of the VHL gene and implanted them in subcutaneous and intracranial sites; these sites are respectively vessel poor and vessel-rich tissues. When grown in a vessel poor site, VEGF expression in VHL null cells was important for both vascularization and tumor growth. However, when the same cells are grown in the vessel-rich intracranial environment, loss of VEGF expression reduces vascularization, but does not affect tumor growth. This indicates that antiangiogenic therapies for tumors that express high levels of angiogenic factors such as VEGF may vary in their efficacy, with potentially lowered effectiveness in sites, such as the brain, that are inherently vessel rich.


Subject(s)
Astrocytoma/blood supply , Astrocytoma/metabolism , Brain Neoplasms/blood supply , Brain Neoplasms/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Neovascularization, Pathologic , Vascular Endothelial Growth Factor A/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Animals , Astrocytoma/pathology , Brain Neoplasms/pathology , Cell Line, Transformed , Mice , Neoplasm Transplantation
4.
Kidney Int ; 69(8): 1302-7, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16531988

ABSTRACT

Over the past decade major advances have been made in our understanding of the molecular machinery that mammalian cells use to sense and to adapt to a low-oxygen environment. A critical mediator of cellular adaptation to hypoxia is hypoxia-inducible factor (HIF), a basic helix-loop-helix transcription factor that consists of an oxygen-sensitive alpha-subunit, HIF-alpha and a constitutively expressed beta-subunit, HIF-beta. Under conditions of normal oxygen tension, the HIF-alpha subunit is hydroxylated by specific prolyl-hydroxylases and targeted for rapid proteasomal degradation by the von Hippel-Lindau (VHL) tumor suppressor, which is the substrate recognition component of an E3-ubiquitin ligase. In a hypoxic environment or in the absence of functional VHL tumor suppressor protein irrespective of oxygen concentration, HIF-alpha is not degraded and translocates to the nucleus, where it dimerizes with HIF-beta to form transcriptionally active HIF. As a transcription factor, HIF is involved in the regulation of many biological processes that facilitate both oxygen delivery and adaptation to oxygen deprivation by regulating genes that are involved in glucose uptake and energy metabolism, angiogenesis, erythropoiesis, cell proliferation and apoptosis, cell-cell and cell-matrix interactions, and barrier function. This review summarizes some of the most recent advances in the VHL/HIF field and discusses their relevance for pathogenesis and treatment of acute ischemic renal failure, renal fibrosis, and renal cancer.


Subject(s)
Carcinoma, Renal Cell/metabolism , Hypoxia-Inducible Factor 1/metabolism , Kidney Diseases/metabolism , Kidney Neoplasms/metabolism , Oxygen/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Acute Kidney Injury/metabolism , Carcinoma, Renal Cell/genetics , Chronic Disease , Humans , Kidney Neoplasms/genetics , Models, Biological , Von Hippel-Lindau Tumor Suppressor Protein/genetics , von Hippel-Lindau Disease/genetics , von Hippel-Lindau Disease/metabolism
5.
Blood ; 98(12): 3261-73, 2001 Dec 01.
Article in English | MEDLINE | ID: mdl-11719363

ABSTRACT

Erythropoietin (Epo) controls red cell production in the basal state and during stress. Epo binding to its receptor, EpoR, on erythroid progenitors leads to rapid activation of the transcription factor Stat5. Previously, fetal anemia and increased apoptosis of fetal liver erythroid progenitors were found in Stat5a(-/-)5b(-/-) mice. However, the role of Stat5 in adult erythropoiesis was not clear. The present study shows that some adult Stat5a(-/-)5b(-/-) mice have a near-normal hematocrit but are deficient in generating high erythropoietic rates in response to stress. Further, many adult Stat5a(-/-)5b(-/-) mice have persistent anemia despite a marked compensatory expansion in their erythropoietic tissue. Analysis of erythroblast maturation in Stat5a(-/-)5b(-/-) hematopoietic tissue shows a dramatic increase in early erythroblast numbers, but these fail to progress in differentiation. Decreased expression of bcl-x(L) and increased apoptosis in Stat5a(-/-)5b(-/-) early erythroblasts correlate with the degree of anemia. Hence, Stat5 controls a rate-determining step regulating early erythroblast survival.


Subject(s)
DNA-Binding Proteins/deficiency , Erythropoiesis/genetics , Milk Proteins , Trans-Activators/deficiency , Anemia/genetics , Animals , Apoptosis , Cell Differentiation , Cells, Cultured , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Erythroblasts/chemistry , Erythroblasts/pathology , Erythrocyte Count , Erythroid Precursor Cells/pathology , Erythropoietin/physiology , Fetal Diseases/genetics , Genotype , Hematocrit , Mice , Mice, Inbred C57BL , Polymerase Chain Reaction , Proto-Oncogene Proteins c-bcl-2/analysis , Receptors, Erythropoietin/physiology , STAT5 Transcription Factor , Spleen/chemistry , Spleen/pathology , Stress, Physiological/physiopathology , Trans-Activators/genetics , Trans-Activators/physiology , bcl-X Protein
6.
Proc Natl Acad Sci U S A ; 98(4): 1583-8, 2001 Feb 13.
Article in English | MEDLINE | ID: mdl-11171994

ABSTRACT

von Hippel-Lindau (VHL) disease is a pleomorphic familial tumor syndrome that is characterized by the development of highly vascularized tumors. Homozygous disruption of the VHL gene in mice results in embryonic lethality. To investigate VHL function in the adult we have generated a conditional VHL null allele (2-lox allele) and null allele (1-lox allele) by Cre-mediated recombination in embryonic stem cells. We show here that mice heterozygous for the 1-lox allele develop cavernous hemangiomas of the liver, a rare manifestation of the human disease. Histologically these tumors were associated with hepatocellular steatosis and focal proliferations of small vessels. To study the cellular origin of these lesions we inactivated VHL tissue-specifically in hepatocytes. Deletion of VHL in the liver resulted in severe steatosis, many blood-filled vascular cavities, and foci of increased vascularization within the hepatic parenchyma. These histopathological changes were similar to those seen in livers from mice heterozygous for the 1-lox allele. Hypoxia-inducible mRNAs encoding vascular endothelial growth factor, glucose transporter 1, and erythropoietin were up-regulated. We thus provide evidence that targeted inactivation of mouse VHL can model clinical features of the human disease and underline the importance of the VHL gene product in the regulation of hypoxia-responsive genes in vivo.


Subject(s)
Genes, Tumor Suppressor , Hemangioma/etiology , Ligases , Liver Neoplasms/etiology , Proteins/physiology , Tumor Suppressor Proteins , Ubiquitin-Protein Ligases , Vascular Neoplasms/etiology , Albumins , Alleles , Animals , Erythropoietin/blood , Heterozygote , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, Mutant Strains , Phenotype , Polycythemia , Proteins/genetics , Von Hippel-Lindau Tumor Suppressor Protein
7.
J Biol Chem ; 272(2): 1297-301, 1997 Jan 10.
Article in English | MEDLINE | ID: mdl-8995435

ABSTRACT

A lymphocyte-specific murine Ltk tyrosine kinase isoform was previously found to reside in the endoplasmic reticulum and to be potently activated upon treatment of cells with alkylating or thiol-oxidizing agents. Based on these observations, a unique role for Ltk was proposed as an endoplasmic reticulum-resident transmembrane kinase regulated by redox changes (Bauskin, A. R., Alkalay, I., and Ben-Neriah, Y. (1991) Cell 66, 685-696). To analyze why this Ltk isoform is retained in the endoplasmic reticulum, we investigated its behavior in over-expressing cells. Our results indicate that lymphoid Ltk exhibits a dual Nexo/Ccyt and Ncyt/Cexo transmembrane topology in transfected cells. This unusual behavior may be responsible for retention in the endoplasmic reticulum since mutants with an increased number of positive amino acids downstream of the transmembrane segment exhibit a conventional Nexo/Ccyt orientation and proceed to the cell surface. Endoplasmic reticulum-retained Ltk forms a prominent complex with the chaperone calnexin, suggesting that Ltk may be retained by the mechanism that prevents surface expression of inappropriately folded proteins or incompletely assembled protein complexes.


Subject(s)
Calcium-Binding Proteins/metabolism , Endoplasmic Reticulum/enzymology , Membrane Proteins/metabolism , Molecular Chaperones/metabolism , Phosphoproteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Immunologic/metabolism , Animals , COS Cells , Calnexin , Glycosylation , Humans , Mutagenesis, Site-Directed , Transfection
8.
Hum Mol Genet ; 3(3): 407-11, 1994 Mar.
Article in English | MEDLINE | ID: mdl-8012352

ABSTRACT

The recently isolated gene for neurofibromatosis type 2 (NF2) encodes a 595 amino acid protein, named merlin, which is related to the cytoskeleton-associated proteins moesin, ezrin and radixin. To identify evolutionarily conserved regions and to provide sequence information necessary for the establishment of a mouse model for NF2, we have determined the cDNA sequence of the mouse NF2 tumor suppressor gene, and mapped it in the mouse genome. Mouse merlin is a 596 amino acid protein, 98% identical to human merlin, but one amino acid longer due to the insertion of a proline residue near the C-terminus. Of the nine amino acid differences between mouse and humans, seven occur in the C-terminal 20% of the protein, far from the protein 4.1 domain that defines this family. Two of the NF2 cDNA clones reveal evidence of alternative splicing events that alter the predicted merlin product, one removing a 45 amino acid segment from the middle section of the protein and the other changing the C-terminus. The existence of several different forms of merlin potentially with different primary roles will complicate the identification of the precise function that must be disrupted to cause the NF2-associated tumors. The mouse NF2 homologue maps to Chr 11, in a region homologous to human Chr 22, but devoid of any mouse mutations which could be models of the human disorder.


Subject(s)
Alternative Splicing , Genes, Neurofibromatosis 2 , Membrane Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Biological Evolution , Chromosome Mapping , Conserved Sequence , DNA , Humans , Membrane Proteins/biosynthesis , Mice , Molecular Sequence Data , Neurofibromin 2 , Polymorphism, Genetic
9.
Oncogene ; 8(1): 27-35, 1993 Jan.
Article in English | MEDLINE | ID: mdl-8380920

ABSTRACT

Two alternatively spliced mouse lymphocyte and brain ltk cDNAs predict small transmembrane tyrosine kinases that use CUG translational start codons and that differ upstream of their transmembrane segment. A recently isolated human neuroblastoma ltk cDNA, in contrast, includes a regular AUG start codon and predicts a more conventional receptor kinase with a larger N-terminal segment. This raised the suggestion that previous mouse cDNAs may have been aberrantly spliced or incomplete and questioned the significance of a recent study that localized the lymphoid ltk protein to the endoplasmic reticulum. Here we show that mice tissue-specifically express four ltk mRNAs. In addition to the two previously described lymphoid and brain mRNAs, we now describe two mRNAs from C1300 neuroblastoma cells that start with five exons which are absent from lymphoid or brain transcripts. The pair of C1300 mRNAs differ by the same alternatively spliced exon that distinguishes brain from lymphoid mRNAs and predict much larger receptor-type kinases that use regular AUG start codons. Our results also show that at least one of the larger, more conventional C1300 ltk receptors shares the endoplasmic reticulum localization of the shorter lymphoid protein.


Subject(s)
Protein-Tyrosine Kinases/genetics , RNA, Messenger/analysis , Receptor Protein-Tyrosine Kinases , Receptors, Cell Surface/genetics , Amino Acid Sequence , Animals , Base Sequence , Endoplasmic Reticulum/enzymology , Mice , Molecular Sequence Data , Organ Specificity , Promoter Regions, Genetic , Protein-Tyrosine Kinases/analysis , Protein-Tyrosine Kinases/immunology , Transcription, Genetic
10.
DNA Cell Biol ; 11(10): 727-34, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1457041

ABSTRACT

Neurofibromatosis type 1 (NF1) is caused by mutations in a large gene on chromosome 17q11.2. Previously described partial cDNAs for this gene predicted a protein related to yeast IRA1/IRA2 and the mammalian RAS GTPase activator protein GAP. To initiate a detailed study of the role of this gene in NF1, we have characterized a set of overlapping cDNAs that represent its complete coding sequence. Our results show that two differentially expressed human NF1 mRNAs differ by a 63-bp insertion in the GAP-related domain. These mRNAs predict two 2,818- and 2,839-amino acid proteins with calculated molecular masses of approximately 317 and 319 kD. Extensive similarity to IRA proteins is evident in a 1,450-amino-acid central segment, roughly between amino acids 900 and 2,350. However, the remainder of the NF1 protein is not significantly similar to other proteins. Interestingly, the SK-N-SH human neuroblastoma line expresses no detectable NF1 mRNA, indicating that expression of NF1 is not essential for viability of this neural crest-derived tumor cell line.


Subject(s)
Alternative Splicing , Genes, Neurofibromatosis 1 , Neuroblastoma/genetics , RNA, Messenger/genetics , Amino Acid Sequence , Base Sequence , DNA , Gene Expression , Humans , Molecular Sequence Data , Mutation , Tumor Cells, Cultured
11.
Oncogene ; 6(12): 2319-25, 1991 Dec.
Article in English | MEDLINE | ID: mdl-1662793

ABSTRACT

Ltk is a new member of the ros/insulin receptor family of tyrosine kinases that is expressed in murine B-lymphocyte precursors and forebrain neurons. We previously reported that lymphoid ltk cDNAs predict a 69 kDa transmembrane glycoprotein, which uses a CUG translational start codon and has a 110 amino acid putative extracellular domain. We now show that the predominant ltk mRNA in brain is alternatively spliced and predicts a protein with a substantially larger extracellular part. The human ltk gene maps to chromosome 15, bands q13-21, a region containing the breakpoint of a recurring chromosomal abnormality in B-cell non-Hodgkin lymphomas.


Subject(s)
Lymphocytes/physiology , Neurons/physiology , Protein-Tyrosine Kinases/genetics , RNA Splicing , RNA, Messenger/genetics , Receptor, Insulin/genetics , Amino Acid Sequence , Animals , B-Lymphocytes/physiology , Base Sequence , Brain/physiology , Cells, Cultured , Chromosome Banding , Chromosomes, Human, Pair 15 , Cloning, Molecular , Codon/genetics , DNA/genetics , Exons , Genomic Library , Humans , Lymphocytes/cytology , Membrane Glycoproteins/genetics , Mice , Molecular Sequence Data , Neurons/enzymology , Restriction Mapping , Sequence Homology, Nucleic Acid
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