ABSTRACT
Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene predisposes to vascular tumor formation in several organs. VHL regulates two evolutionary conserved pathways: the targeting of hydroxylated hypoxia-inducible factor-alpha (HIF-alpha) for proteasomal degradation and the remodeling of extracellular matrix (ECM). The biochemical mechanisms of the ECM assembly pathway remain poorly defined. Here, we provide evidence supporting a biochemical role for VHL in ECM assembly. We show that VHL directly binds to the collagen IV alpha 2 (COL4A2) chain and that this interaction is necessary for its assembly into the ECM. The VHL-COL4A2 interaction is dependent on endoplasmic reticulum (ER)-mediated COL4A2 hydroxylation and independent of cytosolic, hypoxia regulated HIF-alpha-modifying enzymes. We find that the N-terminal tail of COL4A2 protrudes from the ER lumen into the cytosol where it is bound by VHL. Failure of VHL to interact with COL4A2 correlates with loss of collagen IV network formation in vitro and collagen IV remodeling in vivo. Our data suggest a HIF-alpha-independent role for the VHL-COL4A2 interaction in suppression of angiogenic tumor formation through collagen IV network assembly.
Subject(s)
Carcinoma, Renal Cell/metabolism , Collagen Type IV/metabolism , Extracellular Matrix/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Cell Hypoxia , Cell Membrane/metabolism , Collagen Type IV/genetics , Cytosol/metabolism , Endoplasmic Reticulum/metabolism , Fluorescent Antibody Technique , Humans , Hydroxylation , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Immunoprecipitation , Kidney Neoplasms/metabolism , Mixed Function Oxygenases/metabolism , Protein Binding , Von Hippel-Lindau Tumor Suppressor Protein/geneticsABSTRACT
Fibroblast growth factor (FGF) -10 is a new member of the FGF family initially reported in Japan. It is mainly synthesized by mesenchymal cells and acts on epithelial cells in a paracrine manner. FGF-10 actions are dependent on their binding to the iiib form of FGF receptor 2 (FGFR2) iiib, also known as keratinocyte growth factor receptor (KGFR). FGF-10 has high amino acid homology to keratinocyte growth factor (KGF) and plays an important role in fetal limb and lung development and skin wound healing. In the present study, the expression of FGF-10 and FGFR2 iiib messenger RNA (mRNA) in two different human uterine cervical cancer cell lines (CaSki and ME-180) were examined. Both CaSki and ME-180 cells expressed FGFR2 iiib mRNA, while only CaSki cells expressed FGF-10 mRNA and protein. Recombinant FGF-10 (1 ng/ml) increased the growth rate of ME-180 cells and also enhanced mitogen-activated protein kinase (MAPK) phosphorylation of the cells. These data indicate that FGF-10 may directly promote the growth of squamous cell cancer in the uterine cervix via the MAPK pathway.
Subject(s)
Fibroblast Growth Factors/biosynthesis , Signal Transduction/physiology , Uterine Cervical Neoplasms/metabolism , Cell Line , Female , Fibroblast Growth Factor 10 , Fibroblast Growth Factors/physiology , Humans , Tumor Cells, CulturedABSTRACT
The approach to annotating a genome critically affects the number and accuracy of genes identified in the genome sequence. Genome annotation based on stringent gene identification is prone to underestimate the complement of genes encoded in a genome. In contrast, over-prediction of putative genes followed by exhaustive computational sequence, motif and structural homology search will find rarely expressed, possibly unique, new genes at the risk of including non-functional genes. We developed a two-stage approach that combines the merits of stringent genome annotation with the benefits of over-prediction. First we identify plausible genes regardless of matches with EST, cDNA or protein sequences from the organism (stage 1). In the second stage, proteins predicted from the plausible genes are compared at the protein level with EST, cDNA and protein sequences, and protein structures from other organisms (stage 2). Remote but biologically meaningful protein sequence or structure homologies provide supporting evidence for genuine genes. The method, applied to the Drosophila melanogaster genome, validated 1,042 novel candidate genes after filtering 19,410 plausible genes, of which 12,124 matched the original 13,601 annotated genes. This annotation strategy is applicable to genomes of all organisms, including human.
Subject(s)
Drosophila melanogaster/genetics , Genes, Insect , Genome , Animals , Expressed Sequence Tags , Genetic Techniques , Humans , Insect Proteins/chemistry , Insect Proteins/genetics , Sequence Homology, Amino AcidABSTRACT
Our challenge in annotating the 2.91-Mb Adh region of the Drosophila melanogaster genome was to identify genetic and genomic features automatically, completely, and precisely within a 6-week period. To do so, we augmented the MAGPIE microbial genome annotation system to handle eukaryotic genomic sequence data. The new configuration required the integration of eukaryotic gene-finding tools and DNA repeat tools into the automatic data collection module. It also required us to define in MAGPIE new strategies to combine data about eukaryotic exon predictions with functional data to refine the exon predictions. At the heart of the resulting new eukaryotic genome annotation system is a reverse comparison of public protein and complementary DNA sequences against the input genome to identify missing exons and to refine exon boundaries. The software modules that add eukaryotic genome annotation capability to MAGPIE are available as EGRET (Eukaryotic Genome Rapid Evaluation Tool).
