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.