Carbonic anhydrase IX (CA IX) is a transmembrane protein whose expression is strongly induced by hypoxia in a broad spectrum of human tumours. It is a highly active enzyme functionally involved in both pH control and cell adhesion. Its presence in tumours usually indicates poor prognosis. Ectodomain of CA IX is detectable in the culture medium and body fluids of cancer patients, but the mechanism of its shedding has not been thoroughly investigated. Here, we analysed several cell lines with natural and ectopic expression of CA IX to show that its ectodomain release is sensitive to metalloprotease inhibitor batimastat (BB-94) and that hypoxia maintains the normal rate of basal shedding, thus leading to concomitant increase in cell-associated and extracellular CA IX levels. Using CHO-M2 cells defective in shedding, we demonstrated that the basal CA IX ectodomain release does not require a functional TNFa-converting enzyme (TACE/ADAM17), whereas the activation of CA IX shedding by both phorbol-12-myristate-13-acetate and pervanadate is TACE-dependent. Our results suggest that the cleavage of CA IX ectodomain is a regulated process that responds to physiological factors and signal transduction stimuli and may therefore contribute to adaptive changes in the protein composition of tumour cells and their microenvironment.
CA IX is a hypoxia-induced, cancer-associated carbonic anhydrase isoform with functional involvement in pH control and cell adhesion. Here we describe an alternative splicing variant of the CA9 mRNA, which does not contain exons 8 -9 and is expressed in tumour cells independently of hypoxia. It is also detectable in normal tissues in the absence of the full-length transcript and can therefore produce false-positive data in prognostic studies based on the detection of the hypoxia-and cancer-related CA9 expression. The splicing variant encodes a truncated CA IX protein lacking the C-terminal part of the catalytic domain. It shows diminished catalytic activity and is intracellular or secreted. When overexpressed, it reduces the capacity of the full-length CA IX protein to acidify extracellular pH of hypoxic cells and to bind carbonic anhydrase inhibitor. HeLa cells transfected with the splicing variant cDNA generate spheroids that do not form compact cores, suggesting that they fail to adapt to hypoxic stress. Our data indicate that the splicing variant can functionally interfere with the full-length CA IX. This might be relevant particularly under conditions of mild hypoxia, when the cells do not suffer from severe acidosis and do not need excessive pH control. British Journal of Cancer (2008) Carbonic anhydrase IX (CA IX) is one of 12 enzymatically active carbonic anhydrase isoforms expressed in the human body. These zinc metalloenzymes catalyse a reversible conversion of carbon dioxide to bicarbonate and proton, and thereby contribute to modulation of ion transport and maintenance of acid-base balance. The CA isoforms show remarkable diversity in their enzyme activity, kinetic properties, distribution in tissues, localisation in subcellular compartments and sensitivity to inhibitors. This diversity enables them to fulfill specific roles in metabolically active organs and in various physiological situations .In contrast to the majority of CA isoforms that are mostly present in differentiated cells of the normal tissues, CA IX expression is predominantly associated with a broad range of tumours derived from cells, which contain no or low level of CA IX (Potter and Harris, 2003). The only normal tissues that show moderate-to-abundant expression of CA IX belong to gastrointestinal tract and comprise epithelia of the glandular stomach, small intestine, and gallbladder (Pastorekova et al, 1997).The tumour-related expression pattern of CA IX is principally determined by a strong activation of CA9 gene transcription via a hypoxia-inducible factor (HIF), which binds to hypoxia responsive element (HRE) localised in the minimal CA9 promoter proximal to transcription start site at À10/À3 position (Wykoff et al, 2000). The HIF transcription factor significantly changes the expression profile of weakly oxygenated tumour cells by the activation of genes that either support their survival and adaptation to hypoxic stress or lead to their death. As a result, hypoxia selects more aggressive tumour cells with increased capability...
Endosialin is a transmembrane glycoprotein selectively expressed in blood vessels and stromal fibroblasts of various human tumours. It has been functionally implicated in angiogenesis, but the factors that control its expression have remained unclear. As insufficient delivery of oxygen is a driving force of angiogenesis in growing tumours, we investigated whether hypoxia regulates endosialin expression. Here, we demonstrate that endosialin gene transcription is induced by hypoxia predominantly through a mechanism involving hypoxia-inducible factor-2 (HIF-2) cooperating with the Ets-1 transcription factor. We show that HIF-2 activates the endosialin promoter both directly, through binding to a hypoxia-response element adjacent to an Ets-binding site in the distal part of the upstream regulatory region, and indirectly, through Ets-1 and its two cognate elements in the proximal promoter. Our data also suggest that the SP1 transcription factor mediates responsiveness of the endosialin promoter to high cell density. These findings elucidate important aspects of endosialin gene regulation and provide a rational frame for future investigations towards better understanding of its biological significance.
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