A49
Mechanisms of functional interference between HIF-1 and β-catenin in the regulation of carbonic anhyrase-9 (CA9) expression
Natalie Burrows, Julia Resch, Georg Brabant, Kaye Williams
University of Manchester, Manchester, UK
Background
The transcriptional cascades regulated by the hypoxia induced transcription factor HIF-1 and the wnt/b- catenin signalling pathways are strongly implicated in tumour progression. Recent data have suggested that there is cross-talk between the two pathways. We have generated a dominant negative variant of HIF-1α (DN-HIF) that retains its ability to bind to HIF-1 α and DNA but is unable to activate gene transcription. Importantly DN-HIF retains the N-terminal regions thought to interact with β -catenin. We found that over-expression of DN-HIF in cell lines with varying levels of activation of wnt-signalling has differential effects on the expression of the HIF-target gene carbonic anhydrase-9 (CA9) and wished to explore this interaction further.
Method
An EGFP-DN-HIF construct was introduced into a panel of cell lines.CA9 expression was monitored by Western blotting and qPCR. Protein interactions were assessed by immunoprecipitation and protein localisation by immunofluorescence.
Results
HT1080 fibrosarcoma cell lines and thyroid cells with β-catenin activation show elevated CA9 expression when transfected with DN-HIF. qPCR revealed that the upregulation occurred at the transcriptional level. Immunofluorescence showed that whereas β-catenin is membrane associated in wildtype cells, in DN-HIF expressing cells there is marked cytoplasmic and nuclear expression. Immunoprecipitation confirmed that β-catenin can interact with both DN-HIF and HIF-1α proteins.
Conclusion
These data suggest that expression of DN-HIF can modify β-catenin subcellular localisation and that this may have consequential effect on the expression of genes such as CA9 that had previously been identified as “classical” HIF- targets, suggesting a novel regulatory mechanism.
