Regulation & Roles of Carbonic Anhydrases IX & XII
By Heini Kallio
Tampere University Press
Distributed By Coronet Books
$92.50 Paper original
The carbonic anhydrases (CAs) are a family of metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton. The CAs are produced in a variety of tissues where they participate in several physiological processes, such as pH regulation, the production of body fluids, bone resorption, and metabolic processes. The CA family consists of 13 active isozymes in mammals: CA I, II, III, VII, and XIII are cytoplasmic; CA IV, IX, XII, XIV, and XV are anchored to plasma membranes; CA VA and VB are mitochondrial; and CA VI is the only secreted protein.
This study had four specific aims. The first aim was to identify new regulators of CA IX and CA XII. This aim was proposed because, besides hypoxia, the regulation of these proteins is poorly characterized. The second goal was to examine the expression profiles of these isozymes during mouse embryonic development. The third aim was to investigate the whole-genome expression changes that occur in response to CA IX deficiency in the mouse stomach. These mice exhibit gastric hyperplasia, but the exact mechanisms leading to this disturbed cell lineage phenotype remain unknown. The fourth and final goal was to elucidate further the properties of some selected proteins that are dysregulated in the gastric mucosa of Car9?/? mice.
Our studies demonstrate that the growth factors IGF-1, TGF-?, TGF-?1, and EGF increase CA9 and CA12 mRNA expression in cancer-derived cell lines as determined by quantitative real-time PCR (QRT-PCR). In the case of CA9, this effect was observed in only one cell line, whereas CA12 was elevated by growth factors in four cell lines. Thus, hypoxia may not be the primary regulator of CA12 in contrast to CA9.
Immunohistochemistry was used to study CA IX and CA XII expression during mouse organogenesis. These isozymes were detected in several tissues of which a portion are negative for expression in adult mice. Therefore, it is possible that CA IX and CA XII have specific roles in the assembly of certain tissues.
The changes caused by CA IX deficiency in the mouse stomach were explored using a cDNA microarray, which revealed the induction of 86 genes and the repression of 46 genes. Among the genes with altered regulation were those involved in developmental processes, cell differentiation and immune responses. These data and previous findings by others suggest that the primary role of CA IX in the stomach is not related to pH regulation or, alternatively, that its function in this tissue is compensated for by other CA isozymes.
The microarray data indicated significant upregulation of non-specific cytotoxic cell receptor protein 1 (Nccrp1), a poorly characterized gene that was selected for further analyses. Silencing of CA9 in HeLa cells did not affect NCCRP1 levels, suggesting that NCCRP1 is not directly regulated by CA9 and that its upregulation is secondary to Car9 gene disruption. Moreover, it was demonstrated that human recombinant NCCRP1 is expressed intracellularly. Bioinformatic analyses revealed that NCCRP1 belongs to the lectin-type subfamily of F-box (FBXO) proteins, which are components of the E3 ubiquitin ligase complex. In conclusion, our data clearly demonstrate that NCCRP1 is not a transmembrane protein responsible for the cytolytic function of nonspecific cytotoxic cells as has been previously suggested. Therefore, the name of Nccrp1 should be changed to FBXO50.
Acta Universitatis Tamperensis No. 1675
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