Investigating hypoxia inducible factor-1 alpha as a putative regulator of peroxidasin
No Thumbnail Available
Date
2021
Authors
Niyobuntu, Eunice
Journal Title
Journal ISSN
Volume Title
Publisher
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.
Description
A dissertation submitted in fulfilment of the requirements for the degree of Master of Science to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2021