Expression of the hypoxia marker carbonic anhydrase IX is critically dependent on SP1 activity. Identification of a novel type of hypoxia-responsive enhancer.
In the present study, we further studied mechanisms of transcriptional regulation of the tumor-associated carbonic anhydrase IX (CAIX). We identified PR5 in the CA9 promoter as another SP1/SP3-binding site. As shown by electromobility shift assays and block-replacement mutagenesis, PR5 is functionally equivalent to the SP1/SP3-binding PR1 identified previously. However, there is a strong requirement for SP1/SP3 activity in the PR1 position, and SP1/SP3 activity from the PR5 position cannot compensate for this. In various cell lines, the expression of endogenous CAIX and activity of CA9 promoter constructs depend on SP1/SP3 activity as demonstrated by the dose-dependent inhibitory effect of the SP1 inhibitor mithramycin A. The two conditions of the induction of CAIX expression described previously differ in their sensitivity to mithramycin A inhibition; the hypoxia-mimic-induced expression is less sensitive than the cell density (mild hypoxia)-induced expression. Our present study highlights the importance of SP1/SP3 activity for CAIX expression and provides additional evidence for distinct mechanisms responsible for true and mild hypoxia-induced CAIX expression. The presence of a SP1/SP3-binding element in the PR1 position is absolutely required for mild hypoxia-induced activity, and it significantly up-regulates the true hypoxic induction. The SP1/SP3 and hypoxia-response element in the CA9 promoter thus may represent a novel type of enhancer capable of mounting responses to a wider range of hypoxic conditions.
Kaluz
Stefan
S
Department of Microbiology and Molecular Genetics, College of Medicine, University of California at Irvine, California 92717, USA.
Kaluzová
Milota
M
Stanbridge
Eric J
EJ
D000951:Antigens, Neoplasm / Q000096:biosynthesis* / Q000235:genetics; D001665:Binding Sites; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000096:biosynthesis* / Q000235:genetics; D015687:Cell Hypoxia / Q000502:physiology; D005354:Fibrosarcoma; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D010446:Peptide Fragments / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D016329:Sp1 Transcription Factor; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases