Investigating hypoxia inducible factor-1 alpha as a putative regulator of peroxidasin

Abstract

Peroxidasin is a mammalian haem-peroxidase that, unlike other mammalian haem peroxidases, contains additional motifs characteristic of extra-cellular matrix (ECM) proteins. This feature allows for the speculation that PXDN may have other functions within the ECM in addition to its catalytic activity. One such function is the crosslinking of collagen IV in the ECM – a crucial process for basement membrane integrity. Whilst normally expressed in the cardiovascular system and eye, dysregulated PXDN expression is observed in multiple human diseases including fibrosis and cancer. The role of hypoxia in the development and progression of such disease phenotypes has already been elucidated, therefore we hypothesised that pxdn may be regulated by HIF-1α is the master regulator of the cellular response to hypoxia, controlling the expression of approximately 200 genes once active. To accomplish this, localisation and protein quantification of PXDN and HIF-1ɑ were observed in HeLa cells treated with cobalt chloride (CoCl2) using immunofluorescence confocal microscopy and Western blot, respectively. Both HIF-1ɑ and PXDN show increased protein expression in response to treatment with 100µM CoCl2, a hypoxia mimetic, over a 24-hour period. PXDN expression was observed in the ECM and HIF-1ɑ in the nucleus, as expected. The interactions between HIF-1ɑ and the pxdn promoter region were then observed using chromatin immunoprecipitation and PCR. HIF-1 binds the pxdn promoter at a region 741bp and 801bp upstream of the transcription start site. This data shows that, in the presence of CoCl2, HIF-1α and PXDN expression is increased in cervical cancer cells and we also show that HIF-1α interacts with the pxdn promoter. However further work should be carried out to determine whether this interaction between HIF-1α and pxdn drives transcription. Understanding the interactions between HIF-1ɑ and PXDN may lead to the development of novel therapeutic targets for cancer and other diseases where PXDN and HIF-1ɑ are implicated.