Investigating the regulation of PXDN expression by the early growth response 1 (EGR1) 

transcription factor in the context of human fibrotic diseases 

by 

Thokozile Makhanya 

(1064330) 

 

Dissertation  

Submitted in fulfilment of the requirements for the degree 

Master of Science 

in  

Molecular and Cell Biology 

in the Faculty of Science, University of the Witwatersrand, Johannesburg, South Africa 

 

Supervisor: Professor Demetra Mavri-Damelin 

August 2023 



i 
 

Declaration  

I, Thokozile Makhanya (1064430), am a student registered for the degree of Master of 

Science (Dissertation) in the academic year 2023. 

I hereby declare the following: 

• I am aware that plagiarism (the use of someone else‘s work without their permission 

and/or without acknowledging the original source) is wrong. 

• I confirm that the work submitted for assessment for the above degree is my own 

unaided work except where explicitly indicated otherwise and acknowledged. 

• I have not submitted this work before for any other degree or examination at this 

or any other University. 

• The information used in the thesis has not been obtained by me while employed by, 

working under the aegis of, any person or organisation other than the University. 

• I have followed the required conventions in referencing the thoughts and ideas of 

others. 

• I understand that the University of the Witwatersrand may take disciplinary action 

against me if there is a belief that this is not my own unaided work or that I have 

failed to acknowledge the source of the ideas or words in my writing. 

 

Signed    31/08/2023     



ii 
 

Abstract 

Peroxidasin (PXDN) is a novel member of the peroxidase-cyclooxygenase family of haem-

containing proteins that catalyze oxidative reactions. It consolidates the extracellular matrix 

(ECM) by using hydrogen peroxide (H2O2) as a substrate to generate hypohalous acid 

intermediates, which help catalyze the formation of sulfilimine bonds between collagen IV 

protomers. The aberrant expression of PXDN has been linked to the development of various 

diseases where the architecture of the ECM is compromised such as cardiovascular diseases, 

ocular diseases, cancer, and fibrosis. Fibrosis develops due to repetitive tissue injury which is 

followed by aberrant wound healing that causes the excessive deposition of ECM proteins 

such as collagen into the injured tissue. In turn, ECM crosslinking enzymes such as PXDN 

are upregulated, and the matrix becomes thick and heavily crosslinked. This study aims to 

elucidate whether early growth response 1 (EGR1), a zinc-finger transcription factor which 

regulates cell proliferation, differentiation, apoptosis, and a key pro-fibrotic protein, can drive 

PXDN expression. To address the aim, HEK293 cells were treated with TGF-β1, a master 

activator of fibrotic genes, and western blot and immunofluorescence microscopy were 

performed to detect EGR1 and PXDN and their cellular localization, respectively. Chromatin 

immunoprecipitation (ChIP) was performed to determine if EGR1 binds to the PXDN 

promoter and the luciferase reporter assay was employed to determine if the interaction 

resulted in an alteration in gene expression. Our western blot findings showed that for EGR1, 

there was a statistically insignificant increase in protein expression in response to the TGF-β1 

treatment. PXDN expression could not be quantified due to the high background on the blots. 

Further analysis by immunofluorescence microscopy showed that EGR1 expression was 

increased and was localised to the nuclei in response to the TGF-β1 treatment.  We also 

observed that PXDN was predominantly expressed extracellularly and showed a significant 

increase in protein expression with treatment. The bioinformatics analysis has identified two 

putative EGR1 binding sites in the PXDN promoter and ChIP-PCR showed that binding 

occurred at one of these sites. This site was cloned into the pGL4.10 vector to determine 

whether EGR1 drives PXDN expression. Due to unsuccessful transfection optimization, the 

luciferase assay could not be performed and therefore for future work this assay needs to be 

performed to verify if EGR1 can drive PXDN expression. In conclusion, we showed that 

PXDN is a TGF-β1-responsive gene and may be regulated by EGR1. Studying the interaction 

of EGR1 and PXDN may establish roles for PXDN in fibrosis and further consolidate PXDN 

as a possible anti-fibrotic therapeutic target. 



iii 
 

Acknowledgements 

This research project is the most exciting and challenging thing that I have ever done, and I 

was very blessed to have had support from various people and would like to relay my deepest 

gratitude to them. Firstly, I would like to thank my supervisor, Professor Demetra Mavri-

Damelin for designing the project and working tirelessly with me to bring it to fruition. Due 

to the pandemic, we experienced massive delays, but her expertise and calmness saw me 

through this tumultuous period. With her guidance and input, I have grown as a researcher 

and learned a great deal about molecular biology and for this, I am eternally grateful. I would 

also like to thank I would like to thank Dr Deran Reddy from the Wits Microscopy and 

Microanalysis Unit (MMU) for the extensive training in using the Olympus BX63 

Microscope and Dr Angela Botes from the school of Molecular and Cell Biology (MCB) for 

allowing me to use the Q-sonica in her lab.  

I would like to give a big thanks to my colleagues at the Functional Genetics Research 

Laboratory, starting with Dr Tebogo Marutha for teaching me how to culture cells, use the 

different equipment we have in the lab and perform the various experiments that were part of 

this project. He did everything with the outmost kindness and patience, and I hope that I may 

radiate these qualities and be a pillar of support to other students as he was for me. I would 

also like to thank Lebogang Moshupya, Kayleen Jegels, Jemma Falkov, Mistral Sabastian, 

and Jamie Fernandez for their support and assistance around the lab, I learnt so much through 

interacting with them and value the friendships that we made. A special thanks to my family 

for their unwavering love and support, more especially my mother Nomasonto Makhanya for 

her prayers. Everything that I do is through drawing strength from them, and I hope this work 

inspires and makes them proud. I am incredibly grateful to my friends, Mathabatha Ntjie, 

Keketso Muchichwa and Xolisiwe Mhlathi, for their continuous encouragement throughout 

this journey.  

Most importantly I would like to thank God for blessing me with the opportunity to do this 

degree and giving me the strength to see it through. Finally, I would like to thank the 

National Research Foundation (NRF) for the funding granted from the NRF Innovations 

Postgraduate Scholarship, without them this project would not have been possible. 

“The future belongs to those who believe in the beauty of their dreams” 

Eleanor Roosevelt 

 



iv 
 

 Table of contents 

 

Declaration .................................................................................................................................. i 

Abstract ...................................................................................................................................... ii 

Acknowledgements .................................................................................................................. iii 

Table of contents ....................................................................................................................... iv 

List of figures ......................................................................................................................... viii 

List of tables .............................................................................................................................. ix 

List of abbreviations .................................................................................................................. x 

List of symbols ....................................................................................................................... xiii 

Introduction ................................................................................................................................ 1 

1.1 PXDN ............................................................................................................................... 1 

1.1.1 Classification ............................................................................................................. 1 

1.1.2 Expression ................................................................................................................. 1 

1.1.3 Structure..................................................................................................................... 2 

1.1.4 Functions ................................................................................................................... 3 

1.1.4.1 ECM consolidation ............................................................................................. 3 

1.1.4.2 ECM-mediated cellular activities ....................................................................... 5 

1.1.4.3 Innate immunity .................................................................................................. 6 

1.1.5 The pathophysiological roles of PXDN ..................................................................... 6 

1.1.5.1 Developmental structural deformities ................................................................. 6 

1.1.5.2 Ocular diseases.................................................................................................... 7 

1.1.5.3 Cardiovascular diseases ...................................................................................... 8 

1.1.5.4 Cancer ................................................................................................................. 8 

1.1.5.5 Fibrosis ................................................................................................................ 9 

1.1.6 The regulation of PXDN expression .................................................................... 11 

1.2 Tissue fibrosis ................................................................................................................ 12 



v 
 

1.2.1 Pathogenesis ............................................................................................................ 12 

1.2.2 Type II EMT as a fibrosis pathway ......................................................................... 14 

1.2.3 TGF-β1 as a driver of fibrosis ................................................................................. 15 

1.2.4 Pharmacological treatments of fibrosis ................................................................... 17 

1.3 EGR1 .............................................................................................................................. 18 

1.3.1 Classification ........................................................................................................... 18 

1.3.2 Structure................................................................................................................... 19 

1.3.3 EGR1 expression ..................................................................................................... 20 

1.3.4 The transactivation of genes by EGR1 .................................................................... 21 

1.3.5 The pathophysiological roles EGR1 ........................................................................ 22 

1.4 Study rationale................................................................................................................ 23 

1.5 Aims and objectives ....................................................................................................... 23 

2. Materials and methods ......................................................................................................... 24 

2.1 Reagents ......................................................................................................................... 24 

2.2 Cell culture ..................................................................................................................... 24 

2.2.1 Cell counts ............................................................................................................... 24 

2.2.3 Treatments ............................................................................................................... 25 

2.3 Western blot ................................................................................................................... 25 

2.3.1. Protein extraction .................................................................................................... 25 

2.3.2 Bramhall assay ......................................................................................................... 25 

2.3.3 Discontinuous SDS-PAGE ...................................................................................... 26 

2.3.4 Wet electrophoretic transfer .................................................................................... 29 

2.3.5 Immunodetection ..................................................................................................... 29 

2.3.6 Chemiluminescence detection ................................................................................. 29 

2.3.7 Densitometry ........................................................................................................... 30 

2.4 Immunofluorescence microscopy .................................................................................. 30 

2.4.1 Sample preparation .................................................................................................. 30 



vi 
 

2.4.2 Indirect immunofluorescence .................................................................................. 30 

2.4.3 Microscope viewing and image analysis ................................................................. 31 

2.5 ChIP assay ...................................................................................................................... 31 

2.5.1 Identification of EBSs ............................................................................................. 32 

2.5.2 Primers design and testing ....................................................................................... 32 

2.5.3 Chromatin crosslinking............................................................................................ 32 

2.5.4 Sonication ................................................................................................................ 33 

2.5.5 Immunoprecipitation ............................................................................................... 33 

2.5.6 Reversal of the chromatin crosslinks ....................................................................... 33 

2.5.7 ChIP-PCR ................................................................................................................ 33 

2.6 Luciferase reporter assay ................................................................................................ 35 

2.6.1 Oligonucleotides design .......................................................................................... 35 

2.6.2 Annealing the oligonucleotides ............................................................................... 36 

2.6.3 Cloning vector ......................................................................................................... 36 

2.6.4 Restriction enzyme digests ...................................................................................... 36 

2.6.5 Ligation .................................................................................................................... 37 

2.6.6 Preparation of chemically competent bacterial cells ............................................... 37 

2.6.7 Bacterial transformation .......................................................................................... 37 

2.6.8 Colony PCR ............................................................................................................. 38 

2.6.9 Growing the transformed bacteria ........................................................................... 38 

2.6.10 Alkaline lysis ......................................................................................................... 39 

2.6.11 Post-alkaline lysis PCR ......................................................................................... 39 

2.6.12 Sanger sequencing ................................................................................................. 39 

2.6.13 Transfection optimization ...................................................................................... 39 

2.7 Statistical analysis .......................................................................................................... 40 

3. Results .................................................................................................................................. 41 

3.1 EGR1 and PXDN are expressed in HEK293 cells ......................................................... 41 



vii 
 

3.2 EGR1 localizes to the nucleus and PXDN to the ECM ................................................. 42 

3.3 EGR1 has two putative binding sites in the PXDN promoter ........................................ 44 

3.4 EGR1 interacts with EBS1 in the PXDN promoter ........................................................ 45 

3.5 EBS1 was successfully cloned into the pGL4.10 luciferase vector ............................... 47 

4. Discussion ............................................................................................................................ 48 

5. Conclusion ........................................................................................................................... 52 

6. References ............................................................................................................................ 53 

7. Appendices ........................................................................................................................... 69 

Appendix A .......................................................................................................................... 69 

Appendix B .......................................................................................................................... 72 

Appendix C .......................................................................................................................... 74 

Appendix D .......................................................................................................................... 75 

Appendix E ........................................................................................................................... 77 

Appendix F ........................................................................................................................... 79 

 

 

 

  



viii 
 

List of figures 

Figure 1.1: The structure of the PXDN. ..................................................................................... 3 

Figure 1. 2: The crosslinking of collagen IV protomers by PXDN ........................................... 5 

Figure 1.3: The phases of wound healing. ............................................................................... 14 

Figure 1.4: The structure of the EGR1 and its corresponding DNA binding site .................... 20 

Figure 3.1: EGR1 and PXDN expression in HEK293 cells..................................................... 41 

Figure 3.2: EGR1 expression and localization in HEK293 cells. ............................................ 42 

Figure 3.3: PXDN expression and localization in HEK293 cells. ........................................... 43 

Figure 3.4: Putative EBSs in the PXDN promoter ................................................................... 44 

Figure 3.5: EGR1 interacts with EBS1 in the PXDN promoter ............................................... 46 

Figure 3.6: Sanger sequencing of the recombinant pGL4.10 vectors ...................................... 47 

Figure 1E: Western blot optimization. ..................................................................................... 77 

Figure 2E: ChIP assay optimization. ....................................................................................... 77 

Figure 3E: Sanger sequencing showing sequences of cloned inserts ...................................... 78 

 

 

 

 

  



ix 
 

List of tables 

Table 2.1: SDS-PAGE percentages, antibody dilutions and transfer conditions for  EGR1, 

PXDN and β-actin .................................................................................................................... 27 

Table 2.2: Discontinuous SDS-PAGE recipes ......................................................................... 28 

Table 2.3: Antibodies used for immunofluorescence microscopy. .......................................... 31 

Table 2.4: ChIP-PCR primers. ................................................................................................. 34 

Table 2.5: Cloning oligonucleotides. ....................................................................................... 35 

Table 2.6: pGL4.10 vector amplifying primers. ...................................................................... 38 

Table 2.7: Transfection optimization conditions ..................................................................... 40 

Table A1: Recipes of chemical reagents and storage conditions ............................................. 69 

Table B1: Catalogue numbers of purchased reagents. ............................................................. 72 

Table C1: KAPA Taq ReadyMix PCR reaction mixture components. ................................... 74 

Table C2: KAPA Taq ReadyMix PCR cycling conditions. ..................................................... 74 

Table F1: EGR1 densitometry analysis ................................................................................... 79 

Table F2: EGR1 CTCF analysis .............................................................................................. 79 

Table F3: PXDN CTCF analysis ............................................................................................. 80 

 

 

 

  



x 
 

List of abbreviations 

APS - Ammonium persulfate 

Arg - Arginine  

ASD - Anterior segment dysgenesis  

BM - Basement membrane  

bp - Base pairs  

BSA - Bovine serum albumin  

CDK - Cyclin-dependent kinase 

ChIP - Chromatin immunoprecipitation 

CKD - Chronic kidney disease  

COX - Cyclooxygenase 

DAPI - 4’,6’-diamidino-2-phenylindole  

DBD - DNA binding domain  

DMEM - Dulbecco’s Modified Eagle Medium 

DNMT  - DNA methyltransferase 

dsDNA - Double-stranded DNA  

DUOX - Dual oxidase 

EBS - EGR binding site 

ECM - Extracellular matrix 

EDTA - Ethylenediaminetetraacetic acid  

EGF - Epidermal growth factor  

EGR1 - Early growth response 1 

EMT - Epithelial-to-mesenchymal transition 

EPO - Eosinophil peroxidase   

ERK - Extracellular signal-regulated kinase  



xi 
 

FBS  - Foetal bovine serum  

FEV - Forced expiratory volume 

FGF - Fibroblast growth factor 

FITC - Fluorescein isothiocyanate 

gDNA - Genomic DNA  

HEK293 - Human embryonic kidney cells 293 

HGF - Hepatocyte growth factor  

His - Histidine  

HOBr - Hypobromous acid  

HOCl - Hypochlorous acid  

HRP - Horseradish peroxidase  

HSC - Hepatic stellate cells 

IEG - Immediate early gene 

IFN-γ  - Interferon gamma 

IL - Interleukin 

JNK - Jun kinase 

KGF - Keratinocyte growth factor 

LDL - Low-density lipoprotein  

LOX - Lysl oxidases 

LPO - Lactoperoxidase 

LRRs - Leucine-rich repeats 

MCS - Multiple cloning site  

MMP - Matrix metalloproteinases  

MPO - Myeloperoxidase  



xii 
 

NAFLD - Non-alcoholic fatty liver disease 

NASH  - Non-alcoholic steatohepatitis  

NLS - Nuclear localization signal 

NOX - NADPH oxidases 

NTC - No template control  

NAC - No antibody control 

NPC - No primary antibody control  

PBS - Phosphate buffered saline  

PCR - Polymerase chain reaction 

PDGF - Platelet-derived growth factor  

PMSF - Phenylmethanesulfonyl fluoride  

PXDN - Peroxidasin 

PXDNL - Peroxidase-like 

POX - Peroxidase domain 

RIF - Radiation-induced fibrosis 

RIPA - Radioimmunoprecipitation assay 

ROS - Reactive oxygen species  

SDS - Sodium dodecyl sulfate 

SDS-PAGE - Sodium dodecyl sulfate polyacrylamide gel electrophoresis  

siRNA - Short interfering RNA 

TAE - Tris-acetate EDTA  

TBST - Tris-buffered saline with Tween-20  

TCA - Trichloroacetic acid   

TEMED - Tetramethylenediamine 



xiii 
 

TF - Transcription factor 

TGF-β - Transforming growth factor-β 

TLC  - Total lung capacity  

TNF-α - Tumour necrosis factor-α 

TPO - Thyroid peroxidase   

Tris - Tris(hydroxymethyl)aminomethane 

TSS - Transcription start site 

VEGF - Vascular endothelial growth factor 

vWFC domain - von Willebrand factor C 

ZF - Zinc finger 

List of symbols 

µg - Microgram 

µl - Microlitre 

℃ - Degrees Celsius 

G - Grams 

kDa  - Kilodaltons 

mA - Milliamperes 

mg - Milligram 

ml - Millilitre 

mM - Millimolar 

ng - Nanogram 

nm - Nanometre  

rpm - Revolutions per minute  



1 
 

Introduction  

1.1 PXDN 

1.1.1 Classification 

Peroxidasin (PXDN) is an animal haem peroxidase that catalyzes oxidative reactions needed 

in the formation of the extracellular matrix (ECM) (Nelson et al., 1994). Peroxidases are a 

large family of oxidoreductases that utilize hydrogen peroxide (H2O2) to oxidize substrates. 

They are classified into two broad groups, the haem-free peroxidase-catalases and the haem-

containing peroxidase-cyclooxygenase group (Zámocký et al., 2010). The peroxidase-

cyclooxygenase family is comprised of myeloperoxidase (MPO), lactoperoxidase (LPO), 

eosinophil peroxidase (EPO), thyroid peroxidase (TPO), cyclooxygenase (COX), PXDN and 

peroxidase-like (PXDNL) (Zámocký et al., 2010; Péterfi et al., 2014). MPO, LPO and EPO 

are involved in innate immune defences through their production of hypohalous acids, which 

destroy invading microorganisms (Davies et al., 2008). TPO is involved in thyroid hormone 

synthesis and COX in the synthesis of prostaglandins (Ruf and Carayon, 2006; Rouzer and 

Marnett, 2009). In comparison to its other protein family members, PXDN has a unique 

function in consolidating the matrix (Bhave et al., 2012). PXDNL on the other hand is also 

reported to be involved in ECM formation and promoting cell motility but does not seem to 

exhibit peroxidase function (Papageorgiou and Heymans, 2014).  

1.1.2 Expression  

PXDN is a unique haem-peroxidase expressed in vertebrates and invertebrates. In Drosophila 

melanogaster, PXDN is expressed by haemocytes during embryonic development where it 

functions in tissue formation and epidermal elongation by catalysing the formation of 

tyrosine bonds (Nelson et al., 1994). It is also found in species such as Caenorhabditis 

elegans, Chilo suppressalis and Cnidaria and plays similar roles to that in the fruit fly by 

being involved in the developmental stages of the organisms (Tindall et al., 2005; Gotenstein 

et al., 2010; Fidler et al., 2014; Ma et al., 2020). The human PXDN is expressed in the early 

stages of development and is found in all tissues supported by the ECM basement membrane 

(BM) but most notably the eyes, the heart, and the kidneys (Horikoshi et al., 1999; Péterfi et 

al., 2009; Khan et al., 2011). In these tissues, the protein is synthesized in the endoplasmic 

reticulum of fibroblasts, endothelial and epithelial cells, where it is subsequently secreted into 

extracellular spaces (Péterfi et al., 2009; Lázár et al., 2015).  PXDN expression is induced by 

various stimuli, including cytokines such as TGF-β1 and oxidative stress, which occur during 



2 
 

processes such as cell migration and tissue injury (Péterfi et al., 2009; Hanmer and Mavri-

Damelin, 2018).  

1.1.3 Structure 

The human PXDN gene is found on the 2p25.3 telomeric region and has 23 exons, the longest 

transcript of which gives rise to a multidomain protein (Figure 1.1) (Weiler et al., 1994; 

Cheng et al., 2008). The domains consist of a peroxidase domain (POX) that is conserved in 

mammalian peroxidases (Zámocký and Obinger, 2010). This domain is the catalytic site of 

peroxidases and contains a covalently linked haem prosthetic group, which enhances the 

catalytic activity of these enzymes and protects them from the highly reactive oxidant 

byproducts that they generate (Zederbauer et al., 2007). The haem is added during post-

translational modifications and is covalently attached with ester bonds via acidic aspartate 

(Asp) and glutamate (Glu) residues. With the exception of MPO, which has an additional 

covalent sulfonium ion bond, all the other haem-containing peroxidases have two ester bonds 

(Zederbauer et al., 2007). In PXDN, the ester bond is formed between Asp-826 and Glu-980 

(Paumann-Page et al., 2017). The iron in the porphyrin ring is linked to proximal and distal 

histidine (His) residues. The proximal His is linked to asparagine (Asp) and the distal one is 

attached to Glu and arginine (Arg). The His/Arg residues are responsible for the heterolytic 

cleavage of H2O2 whilst Glu binds the halide substrates (Soudi et al., 2015).  

In addition to the conserved POX domain, PXDN also contains non-catalytic domains which 

include leucine-rich repeats (LRRs), immunoglobulin (IgG) like domains and a C-terminal 

von Willebrand factor C (vWFC) domain (Figure 1.1) (Cheng et al., 2008). These domains 

are characteristic of ECM proteins and suggest that PXDN is involved in protein-protein 

interactions in the matrix and cell adhesion (Cheng et al., 2008; Lázár et al., 2015). The 

PXDN protein is formed through the trimerization of monomers at their N-terminal ends via 

disulfide bonds occurring between cysteine (Cys)-696 and 1412 and together, these form a 

protein with a molecular weight of 165 kDa (Cheng et al., 2008; Paumann-Page et al., 2020). 

Another important component of the PXDN protein is the N-terminal secretion signal 

peptide, which is used to transport the protein from the endoplasmic reticulum of cells to the 

ECM where it crosslinks collagen IV (Péterfi et al., 2009; Soudi et al., 2012). PXDN also 

undergoes N-glycosylation on its asparagine (Asn) groups, and this contributes about 12 kDa 

to the overall 165 kDa molecular weight of the protein (Cheng et al., 2008; Soudi et al., 

2015). The role of the glycan group has not been specifically described for PXDN; however, 



3 
 

this modification is generally reported to help with protein folding, stability, transport, 

secretion, and ECM organization (Varki, 2017; Schjoldager et al., 2020).  

 

Figure 1.1: The structure of the PXDN. PXDN contains multiple domains with the defining 

one being the haem-containing catalytic peroxidase domain (POX) which is conserved in the 

peroxidase protein family. The protein also has an N-terminal signal peptide (S), leucine-rich 

repeats (LRRs), immunoglobulin (IgG) like domains and a C-terminal von Willebrand factor 

C (vWFC) domain (Obtained from Ma et al., 2020).  

1.1.4 Functions 

1.1.4.1 ECM consolidation 

The most well-described role of PXDN is crosslinking collagen IV fibres found in the ECM 

BM with sulfilimine bonds (Bhave et al., 2012). The BM is a specialised sheet-like structure 

that anchors epithelial cells, forms physical barriers between different cells and supplies them 

with growth factors (LeBleu et al., 2007). The BM is primarily composed of collagen IV and 

it also contains laminin, perlecan, and nidogen and the modifying enzymes such as matrix 

metalloproteinases (MMPs), lysyl oxidase (LOX) and PXDN (Ramos-Lewis and Page-

McCaw, 2019). Type IV collagen fibres are exclusively found in the BM and encoded for by 

the COL4A1-COL4A6 genes. These genes encode for six homologous monomeric α-chains 

(α1-α6), which polymerize to form heterotrimeric protomers (Khoshnoodi et al., 2008). On 

the N-terminus of the trimer is the collagenous 7S domain and on the C-terminus is the non-

collagenous 1 (NC1) domain, with the collagen IV network assembled when protomers are 

joined end-to-end at these terminal domains. LOX forms disulfide bonds between four 

protomers at their 7S domains and this forms a dodecameric juncture. On the NC1 domains a 

hexamer is formed through the association of two protomers and this interaction is stabilized 

by sulfilimine bonds catalysed by PXDN (Bhave et al., 2012; Añazco et al., 2016; Brown et 

al., 2017).  

The assembly of the collagen IV network starts with the activation of PXDN through the 

proteolytic cleavage of its vWFC domain by proprotein convertase (PC) and this enhances 

the catalytic activity of PXDN (Colon and Bhave, 2016). Once PXDN is cleaved, it binds 

H2O2 in the catalytic site and starts to oxidize substrates. The H2O2 that is used by haem 



4 
 

peroxidases is primarily obtained from NADPH oxidases (NOX) and dual oxidases (DUOX) 

(Zhang et al., 2012; Sirokmány and Geiszt, 2019). For crosslinking collagen IV, however, 

recent evidence shows that PXDN functions independently of NOX/DUOX-derived H2O2. 

Sirokmány et al. (2018) showed that in the absence of NOX/DUOX isoforms sulfilimine 

bonds were still formed in the BM of the aorta, kidneys, thyroid, and bladder of model mice. 

This finding is consistent with the widespread tissue expression of PXDN in comparison to 

NOX/DUOX enzymes and the other peroxidases. The researchers instead postulate that the 

potential source of the H2O2 used by PXDN is derived from the mitochondria.  

PXDN  functions by cleaving the peroxidic bond of bound H2O2 molecules and this allows it 

to oxidize the halides bromine, chloride and iodide and the pseudohalide thiocyanate into 

hypobromous acid (HOBr), hypochlorous acid (HOCl) hypoiodous acid (HOI) and 

hypothiocyanous acid (HOSCN) (Paumann-Page et al., 2017). HOI and HOSCN have been 

shown to inhibit crosslinking and are therefore rarely generated by PXDN (McCall et al., 

2014). Between Cl- and Br- ions, PXDN preferentially halogenates Br- as its bromosulfonium 

intermediate has a lower activation energy than the chlorosulfonium intermediate, making the 

reaction more thermodynamically favourable for the catalysis of the sulfilimine bond 

(McCall et al., 2014). McCall et al. (2014) further describe Br- as having a 50,000-fold 

greater efficiency at crosslinking collagen compared to Cl− even at low plasma concentrations 

as it is a trace element in the body.   

To crosslink collagen IV, PXDN first catalyses the peroxidase cycle and here the iron (Fe3+) 

on the haem group reacts with H2O2 to form water and oxoiron intermediates. The oxorion 

molecules feed into the halogenation cycle and react with bromine (Br-) to form HOBr 

(Paumann-Page et al., 2017). The HOBr then binds to the NC1 domain of collagen IV 

protomers and on the first protomer it reacts with the sulphur on methionine-93 (Met-93) to 

form a bromosulfonium intermediate. The intermediate then reacts with the nitrogen on 

hydroxylysine-211 (Hyl-211) of the other protomer and a sulfilimine bond (S=N) is formed 

(Figure 1.2). The sulfilimine bond creates NC1 hexamers and these, along with the N-

terminal 7S bonds, form the collagen IV scaffold network which stabilizes the BM so it can 

support and anchor overlaying epithelial and endothelial cells, regulate cell polarity, 

proliferation, differentiation, and migration (LeBleu et al., 2007; Bhave et al., 2012; McCall 

et al., 2014; Bhave et al., 2017).  

https://en.wikipedia.org/wiki/Hypochlorous_acid
https://en.wikipedia.org/wiki/Hypoiodous_acid


5 
 

 

Figure 1. 2: The crosslinking of collagen IV protomers by PXDN. PXDN is found in the 

collagen IV scaffold of the basement membrane (BM) which underlies tissues. It catalyses 

the oxidation of bromine ion (Br-) by H2O2 to form hypobromous acid (HOBr) which binds to 

the NC1 interface of collagen IV protomers and crosslinks neighbouring Met-93 and Hyl-211 

residues to form a sulfilime bond (S=N) (Adapted from McCall et al., 2014). 

In addition to catalysing the formation of sulfilimine crosslinks, PXDN forms dityrosine 

bonds that stabilize the ECM (Cheng et al., 2011; Bathish, et al., 2020). In a recent study, it 

was further shown that the loss of PXDN not only compromised the collagen IV crosslinks 

but it affected the assembly of fibronectin and laminin networks (Lee et al., 2020). PXDN is 

therefore an important protein needed for the assembly and stability of the ECM.   

1.1.4.2 ECM-mediated cellular activities   

PXDN is involved in various ECM-mediated cellular activities including cell attachment, 

proliferation, and migration. Tauber et al. (2010) investigated the attachment of cells under 

haem oxygenase 1 (HO-1) signalling and showed that BeWo choriocarcinoma cells 

expressing the protein were more adherent as compared to those deficient in HO-1. PXDN 

was shown to facilitate this process by aiding the cells to adhere to the ECM proteins 

laminins, fibronectin, and collagen I. PXDN has also been shown to facilitate the 

proliferation of various cells such as ovarian cells, vascular smooth muscle cells and lens and 

corneal cells (Shi et al., 2011; Yan et al., 2014; Zheng et al., 2018). Lee et al. (2020) also 

recently showed that PXDN plays a role in the proliferation of vascular endothelial cells. 

Here, the siRNA knockdown of PXDN transcript reduced the cells proliferation and 

adherence and caspace3/7 levels increased, resulting in the reduced viability of the cells.  



6 
 

Another important role of PXDN is mediating cell migration and it does this through 

epithelial-to-mesenchymal transition (EMT), an ECM remodelling process that allows cells 

to be motile as they lose their cell-cell and cell-matrix attachments (Kalluri and Weinberg, 

2009). PXDN was shown to respond to transforming growth factor-β (TGF-β), a potent 

inducer of EMT and, under its signalling, PXDN levels decreased and this subsequently 

decreased cell attachment and increased their migration (Xu et al., 2009; Sitole and Mavri-

Damelin 2018). EMT also occurs under oxidative conditions and this is driven by the 

increased levels of reactive oxygen species (ROS), which are generated from cellular 

respiration and NOX (Schieber and Chandel, 2014; Jiang et al., 2017). Hanmer and Mavri-

Damelin (2018) showed that when cancerous cells are treated with H2O2, PXDN levels 

increase; the knockdown of PXDN during oxidative stress, however, led to a decrease in cell 

attachment, migration, and invasion. 

1.1.4.3 Innate immunity 

The haem-peroxidases MPO and LPO have well-described roles in host immunity and more 

recently PXDN has also been shown to participate in this protective function albeit to a lesser 

extent. Li et al. (2012) found that PXDN is secreted by endothelial cells into the plasma, and 

it can bind to bacteria at physiological pH. When E. Coli was incubated with PXDN, H2O2 

and Cl-, the bacteria died, but without the H2O2 and Cl- chemical substrates, the enzyme was 

unable to kill the bacteria, indicating that its oxidation of Cl into HOCl is the key in its 

bactericidal activity (Li et al., 2012). The study done by Shi et al. (2018) demonstrated that 

the bactericidal activity of PXDN is only directed towards gram-negative bacteria, which 

contain lipopolysaccharide (LPS) that activate the enzyme. In the study, PXDN was shown to 

bind to the bacteria at the LPS regions through the N-terminal LRR and IgG domains and was 

able to kill Escherichia coli and Pseudomonas aeruginosa in the presence of H2O2 and Cl- 

(Shi et al., 2018). The study further demonstrated that PXDN-deficient mice with gram-

negative pneumonia had a substantially lower survival rate than the wild-types and eventually 

died. These findings therefore highlight that PXDN is involved in lung defences (Shi et al., 

2018). 

1.1.5 The pathophysiological roles of PXDN 

1.1.5.1 Developmental structural deformities 

PXDN plays a key role in consolidating the structural integrity of the BM and this is key in 

organogenesis as it provides cells with structural support, anchorage, establishes their 

apicobasal polarity and mediates their proliferation and migration (Jayadev and Sherwood, 



7 
 

2017).  In invertebrates, the loss of PXDN results in structural deformities and early 

embryonic death. Mutations in the PXN gene of Drosophila result in the loss of collagen IV 

sulfilimine crosslinks, causing tearing in midgut visceral muscles and the larvae die at the 

third instar stage (Bhave et al., 2012). C. elegans has two PXDN homologues, PXN-1 and 

PXN-2 and mutations in the former do not cause developmental defects but in the latter, 

mutants have been shown to have a disorganized BM which deleteriously affects muscle-

epidermal attachment, epidermal elongation, egg laying and causes muscular dystrophy and 

early larval lethality (Gotenstein et al., 2010). These PXDN mutations are synonymous with 

collagen IV mutations where it is reported that the muscle tissue of invertebrates detach from 

the epidermis as the BM is not tensile enough to support their contractions (Borchiellini et 

al., 1996). This illustrates that collagen IV needs to be present and properly reinforced by 

PXDN for proper embryonic development.   

1.1.5.2 Ocular diseases 

PXDN mutations in vertebrates also cause pathophysiological conditions and in mice and 

humans, eye defects have been extensively described. Mice with a biallelic knockout of 

PXDN have decreased collagen IV sulfilimine crosslinks and this causes a range of ocular 

defects including severely reduced eye size, closed eyelids, cataracts, and abnormal eyeball 

formation which causes anophthalmia or microphthalmia (Kim et al., 2019). Interestingly 

these PXDN mutation must be homozygous as heterozygous mice had similar phenotypes to 

wildtypes indicating that the PXDN gene is haplosufficient (Kim et al., 2019). Anterior 

segment dysgenesis (ASD) is a pathology wherein the lens, cornea and iris develop 

abnormally. ASD is commonly associated with genes such as PAX6, PITX2, FOXC1, and 

COL4A1 but recently PXDN has been identified as a novel contributing gene (Reis and 

Semina, 2011; Choi et al., 2015). Homozygous nonsense mutations in mice were shown to 

result in the loss of the peroxidase and vWFC domains, and the PXDN knock-out mouse 

models developed ASD, microphthalmia, glaucoma and retinal dysgenesis (Yan et al., 2014). 

Similarly, in humans, homozygous mutations have been shown to cause ASD, congenital 

cataracts, corneal opacity, glaucoma, anophthalmia and microphthalmia (Khan et al., 2011; 

Choi et al., 2015). These ocular anomalies are thought to occur due to the destabilization of 

the BM, which affects the structural support to the developing anterior chamber structure and 

in addition, there is an increased accumulation of free radicals in the eye and these damage 

ocular structures causing inflammation and leading to impaired eye development and 

function (Khan et al., 2011).  

https://www.google.com/search?sxsrf=APwXEdfxVFR3kYm-GRs0NH4eybkQH3uXhA:1685012436486&q=C.+elegans&spell=1&sa=X&ved=2ahUKEwj15d69qJD_AhVjyLsIHSlQBA4QkeECKAB6BAgOEAE&cshid=1685012497598484


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1.1.5.3 Cardiovascular diseases  

In humans, PXDN is expressed quite ubiquitously throughout the body but studies have 

shown that it is more abundant in the heart and vascular tissues, hence the gene was 

alternatively named vascular peroxidase 1 (VPO1) (Cheng et al., 2008). VPO1 was shown to 

cause atherosclerosis through the oxidation of low-density-lipoproteins (LDL) and 

apolipoprotein E (ApoE) resulting in endothelial cell injury, lipid accumulation in the arteries 

which forms plaque thus initiating atherogenesis (Yang et al., 2013). Plaque formation is also 

exacerbated during inflammation as the high expression of tumour necrosis factor α (TNF-α) 

and lipopolysaccharides (LPS) increase VPO1 levels, which in turn oxidizes LDL and 

recruits foam cells to the arteries and atherosclerosis progresses (Yang et al., 2016).  Another 

role of VPO1 in cardiovascular diseases is linked to mitigating oxidized LDL (ox-LDL)-

induced apoptosis of endothelial cells. Increased intracellular levels of ox-LDL activate 

NOX2 and this increases ROS levels and then VPO1 and HOCl. These in turn increase p38 

MAPK and caspase-3 resulting in endothelial cell death which contributes to thrombosis and 

atherosclerosis (Bai et al., 2011).  

1.1.5.4 Cancer  

The earliest detection of PXDN in cancer was in melanoma cells where it was found to be 

highly expressed and it was consequentially termed melanoma-associated gene 50 (MG50) 

(Weiler et al., 1994). Paumann-Page et al. (2021) recently corroborated these early findings 

by showing that the mRNA and protein levels of PXDN were highly expressed in invasive 

melanoma cells compared to the non-invasive subtype, therefore PXDN enhances their 

migration. Ovarian cancer cells also have high levels of PXDN expression and when the gene 

is silenced, this decreases the proliferation, migration, and invasion of cells and this was 

attributed to a perturbation in the PI3K/Akt pathway (Zheng and Liang, 2018). In breast 

cancer, the oncogenic effects of PXDN are similarly linked to the PI3K/Akt pathway and this 

was shown by Young et al. (2015) where they found that PIK3CA mutant cells had high 

expression levels of genes which included PXDN, laminin, fibronectin, and thrombospondin 

1 (THBS1). The knock-down of these genes decreased proliferation. PXDN also contributes 

to carcinogenesis via the moderation of H2O2, which is a potent ROS. This was illustrated in 

prostate cancer, where the protein is overexpressed and it reduces the intracellular levels of 

ROS thus inhibiting apoptosis from being triggered and tumours survive (Dougan et al., 

2019). In colon cancer however, PXDN increases ROS levels thus triggering p53-mediated 



9 
 

apoptosis and from this PXDN was termed p53-responsive gene 2 protein (PRG2) (Horikoshi 

et al., 1999).  

PXDN also contributes to the development of cancer by being aberrantly expressed with 

other proteins such as HO-1 which promotes tumour growth and survival. The proteins were 

shown to co-localize in invasive trophoblasts and the knockdown of HO-1 in the cells 

decreased their adhesion, migration, and invasion. When the PXDN was instead silenced, the 

cells similarly showed decreased adhesion to laminins and fibronectin and were less invasive. 

This, therefore, showed that PXDN mediates the oncogenic effects of HO-1 in the ECM 

(Tauber et al., 2010). In bladder cancer, a hub of genes was identified using gene co-

expression networks and PXDN was among them along with COL1A1, COL1A2, COL5A1, 

COL8A1, COL11A1, MMP2 and TGFβ1I1, all of which were associated with poor prognosis 

(Di et al., 2019). In glioblastomas, PXDN is also overexpressed and protein-protein 

interaction networks identified COL4A1 and COL5A1 as some of the genes associated with 

promoting tumour proliferation, migration, and survival (Shi et al., 2022). In addition to 

aberrantly expressed proteins, PXDN is also co-expressed with the long non-coding RNAs 

(lncRNA) AC046143.1 and hepatocellular carcinoma up-regulated EZH2-associated long 

non-coding RNA (HEIH) which are known to promote tumour progression (Shi et al., 2022). 

Interestingly PXDN has also been shown to activate cancer hallmarks including EMT, 

glycolysis, hypoxia, and inflammation (Shi et al., 2022). In oral squamous cell carcinoma 

(OSCC), PXDN is highly expressed with concomitant increases in lactate and ATP, and this 

increases tumour metastasis and invasion (Kurihara-Shimomura et al., 2020). Collectively, 

these studies show that PXDN has both pro- and anti-tumour effects on cancers by 

moderating ROS levels, ECM remodelling, being co-expressed with other oncogenes and 

driving key cancer phenotypes. 

1.1.5.5 Fibrosis 

The aberrant expression of PXDN is also linked to the development and progression of 

fibroproliferative diseases. PXDN, along with MPO and EPO, have been shown to cause 

fibrosis through the production of hypohalous acids. These highly reactive compounds can 

oxidize proteins and lipids that can cause inflammation, which triggers abnormal wound 

healing (Davies et al., 2008). Fibrotic mouse models with unilateral ureteral obstruction 

(UUO) were shown to have high expressions of the PXDN, MPO and EPO with concomitant 

increases in renal inflammation and fibrosis. The knockout of these peroxidases identified 

PXDN and EPO as the proteins promoting fibrosis due to the synthesis of HOBr as there was 



10 
 

a markedly reduced collagen I deposition and α-smooth muscle actin (α-SMA) expression, in 

the renal tubules (Colon et al., 2019). α-SMA is an important protein which gives fibroblasts 

a contractile phenotype thus enabling them to migrate into injured and fibrotic tissue sites 

(Shinde et al., 2017). PXDN has been shown to participate in wound healing by modulating 

the architecture and stiffness of the matrix and it therefore plays a role in fibrosis where 

matrix stiffness is a hallmark (Thannickal et al., 2014). Bhave et al. (2017) demonstrated the 

role of PXDN in matrix stiffness by knocking out its gene in mouse models and this 

significantly reduced the renal tubule collagen IV sulfilimine crosslinks along with the 

tubular BM stiffness.  

Aberrant PXDN expression is also associated with the development of Goodpasture (GP) 

disease, which is an autoimmune disease wherein antibodies attack the α3 chain NC1 

domains of collagen IV molecules in BM of the alveoli and glomerulus (Kalluri et al., 1994). 

In a study done by McCall et al. (2018) anti-PXDN autoantibodies were found in 46% of the 

GP disease cohort. These patients had reduced HOBr synthesis and more severe 

inflammation in the of the vasculature. Inflammation is a key parameter in fibrogenesis and 

because in GP disease glomerulonephritis and pulmonary haemorrhaging occur, as they 

progress fibrosis would develop. Panjwani et al. (2003) report that in 80% of GP disease 

cases, patients will have abnormal chest radiography and interstitial fibrosis and the 

prognosis worsens with the detection of scarring and interstitial fibrosis.  

PXDN has been shown to cause renal, cardiac, liver and fibrosis (Péterfi et al., 2009; Liu et 

al., 2019; Sojoodi et al., 2022).  Péterfi and colleagues were the first to demonstrate that the 

protein is expressed in the endoplasmic reticulum of human pulmonary and dermal 

fibroblasts, key pro-fibrotic cells found in the connective tissue which transdifferentiate into 

myofibroblasts during wound healing and secrete matrix proteins and exert contractile forces 

on the wound area to help with its closure (Péterfi, et al., 2009). Furthermore, the study 

showed that PXDN mRNA expression increased in response to TGF-β1, a pro-fibrotic 

cytokine and it activated myofibroblasts as seen with an increase in α-SMA and in turn the 

cells secreted PXDN into the extracellular space of fibrotic kidneys of mice. Here PXDN 

colocalized with fibronectin and together these proteins enhance the stiffening of the renal 

BM (Péterfi, et al., 2009). In the heart, VPO1 expression is upregulated following myocardial 

infarction-induced fibrosis. Here PXDN induces the differentiation, migration, and 

proliferation of cardiac fibroblasts through HOCl biosynthesis which activates the Smad2/3 

and ERK1/2 pathways. The activated fibroblast in turn increases the expression of collagen I 



11 
 

and α-SMA and this promotes fibroplasia. Echocardiography analysis showed that siRNA 

knockdown of VPO1 improved cardiac function in the mouse models and they had a better 

survival rate (Liu et al., 2019).  

In liver fibrosis, PXDN is expressed and secreted into the fibrotic tissue by hepatic stellate 

cells (HSC), resident liver cells that can differentiate into myofibroblast (Puche et al., 2013; 

Sojoodi et al., 2022). PXDN is upregulated during liver fibrosis and this was detected in the 

liver biopsies of patients with cirrhosis and non-alcoholic fatty liver disease (NAFLD) and 

mouse models with carbon tetrachloride (CCl4)-induced fibrosis. The analysis of the 

deficiency of PXDN (PXDN-/-) in the CCl4-injured mice resulted in reduced collagen I 

crosslinks and fibrolysis occurred due to the increase in MMP8 and MMP13 expression thus 

reducing the density of the fibrotic deposits. Furthermore, the loss of PXDN increased the 

recruitment of M2 macrophages CD45+F4, CD206+ and CCR2+, which mitigate 

inflammation and promote normal tissue healing (Braga et al., 2015; Sojoodi et al., 2022). 

Overall, these changes improved liver function and regeneration as seen with decreased 

alanine transaminase (ALT) and aspartate transaminase (AST) (Sojoodi et al., 2022). Overall, 

these studies illustrate that PXDN mediates tissue fibrosis by remodelling the ECM, 

regulating HOBr and HOCl levels, responding to TGF-β stimulation, regulating fibroblast 

differentiation and proliferation and macrophage recruitment. Thus, targeting the PXDN may 

ameliorate fibrosis.  

1.1.6 The regulation of PXDN expression  

The regulation of PXDN expression involves epigenetic mechanisms, transcriptional and 

mRNA transcript regulation. DNA methylation is the most described epigenetic 

modifications that occurs on the PXDN promoter, and similar to other genes the modification 

occurs in, it downregulates the expression of the PXDN gene (Moore et al., 2013; Zhou et al., 

2022).  Interestingly folic acid, a molecule known to prevent oxidative damage in cells has 

been shown to be an inducer of PXDN methylation. This was illustrated in human umbilical 

vein endothelial cells (HUVECs) treated with ox-LDL and here folic acid stimulated the 

expression of DNA methyltransferases (DNMTs), and this resulted in the hypermethylation 

of the PXDN promoter thus preventing the accumulation of LDL in arteries which cause 

atherosclerosis. In the absence of folic acid however PXDN levels increased and 

atherosclerosis developed (Cui et al., 2018). This illustrates that PXDN expression can be 

regulated by the methylation of its promoter.  



12 
 

 Transcriptional regulation is another key mechanism that regulates the expression of the 

PXDN gene and so far, two upstream regulators have been identified: Snai1 and nuclear 

factor erythroid 2–related factor 2 (Nrf2). The Snai1 TF is responsible for triggering EMT 

and it primarily does this by inhibiting the expression of E-cadherin and this allows cells to 

become mesenchymal (Nieto, 2002). EMT mainly occurs through the alteration of the ECM 

and since PXDN is responsible for its formation and reinforcement it is one the key genes 

whose expression is altered to enable EMT processes to occur. Under Snai1 regulation, 

PXDN expression has been shown to become downregulated and this facilitates the 

proliferation and migration of cells (Sitole and Mavri-Damelin, 2018). As previously 

described, PXDN responds to changes in ROS levels, and in a study by Hanmer and Mavri-

Damelin (2018) they showed that Nrf2 activates its expression.  This finding is very fitting as 

Nrf2 is a master regulator of the antioxidant response and is activated in response to 

increased levels of ROS to protect cells from oxidative damage (Itoh et al., 1997).  

The expression of PXDN can also be modified by targeting its mRNA for degradation. Briem 

et al. (2019) identified miR-203a as the miRNA mediating this process. Here they showed 

that the 3′-untranslated region (3′-UTR) of the PXDN promoter contains three miR-203a 

binding sites and miR-203a repressed its expression in the mesenchymal breast epithelial cell 

line D492M leading to their decreased proliferation. The expression of the PXDN protein can 

also be altered post-translationally. PXDNL, a PXDN homolog protein which is only 

expressed in the heart and lacks any peroxidase catalytic activity has been shown to alter 

PXDN expression. PXDNL forms a complex with PXDN and this inhibits the activity of 

PXDN (Péterfi and Geiszt, 2014). To further understand the role of PXDN in fibrosis, in this 

study we focused on the transcriptional regulation of PXDN under the early growth response 

1 (EGR1) TF. 

1.2 Tissue fibrosis 

1.2.1 Pathogenesis  

Fibrosis is the excessive deposition of ECM proteins in the connective tissue of organs (Krieg 

et al., 2007). The disease may develop due to physical trauma, infections, chemical irritants, 

and autoimmune reactions (Wynn, 2008). Fibrosis can also be an end-stage of chronic 

diseases such as hypertrophic cardiomyopathy, chronic kidney disease (CKD), NAFLD and 

non-alcoholic steatohepatitis (NASH) and most recently coronavirus disease (COVID-19) has 

also been implicated (Brunt, 2004; Cho, 2010; Ho et al., 2010; Ali and Ghonimy, 2021; 

Heyens et al., 2021). These factors cause repetitive tissue injury which activates abnormal 

https://www.who.int/emergencies/diseases/novel-coronavirus-2019


13 
 

wound healing thereby leading to fibrosis. Wound healing occurs through four phases starting 

with haemostasis, followed by inflammation, then proliferation and ending with the 

remodelling phase (Figure 1.4). In haemostasis, haemorrhaging is stopped via 

vasoconstriction as platelets and fibrins are activated and form a clot that causes blood 

coagulation (Armstrong and Golan, 2011; Palta et al., 2014). In the inflammatory phase, 

macrophages, leucocytes and neutrophils are recruited and they secrete antimicrobial peptides 

and proinflammatory cytokines such as tumour necrosis α (TNF-α), interferon-gamma (IFN-

γ) and interleukins (IL), and these trigger inflammation, which clears invading microbes and 

damaged cells (Borthwick et al., 2013).  Once the inflammatory phase is completed, activated 

immune cells undergo apoptosis, but in fibroplasia, this does not happen resulting in 

uncontrolled inflammation (Lee and Kalluri, 2010).   

In the proliferative phase, the matrix is repaired and the damaged cells are replaced with new 

cells. TGF-β has been shown to be a strong driver of this phase (Meng et al., 2016). 

Fibroblasts are activated to transdifferentiate into myofibroblasts, which secrete matrix 

proteins such as collagen into the extracellular space (Darby et al., 2016). To enhance matrix 

repair, fibrocytes from the bone marrow and pericytes are also recruited to wound and secrete 

more proteins (Grieb et al., 2011; Thomas et al., 2017). Matrix crosslinking enzymes such as 

PXDN, LOX and transglutaminase 2 (TG2) are upregulated to facilitate the formation of the 

preliminary ECM granulation tissue so that cells can attach to form new tissue, but in 

fibroplasia, these enzymes excessively crosslink the matrix and it becomes stiff (Péterfi et al., 

2009; Cai et al., 2017; Tatsukawa and Hitomi, 2021). Growth factors such as fibroblast 

growth factor (FGF), PDGF, EGF, VEGF and depending on the tissue type other site-specific 

ones such as HGF and keratinocyte growth factor (KGF) are expressed to stimulate cell 

growth and proliferation (Werner et al., 2007; Demidova-Rice et al., 2012; Li et al., 2013). 

The resident cells of the affected tissue proliferate and migrate into the wound and adhere to 

the BM and reepithelialisation occurs followed by angiogenesis, which restores blood 

perfusion (Li et al., 2003; Ben Amar and Wu, 2014).  

In the remodelling phase, the wound is closed and this is facilitated by the contraction of 

myofibroblasts, which express high levels of α- SMA (Shin and Minn, 2004; Shinde et al., 

2017). The expression of MMPs is also upregulated and these degrade the excess amounts of 

ECM proteins secreted into the wound (Caley et al., 2015). Apoptosis is then initiated to 

eliminate myofibroblasts along with any unviable cells (Guerin et al., 2021). In fibrosis 

however, matrix proteolysis is reduced as TIMP are upregulated and myofibroblasts evade 



14 
 

apoptosis and senescence and as a result, the affected tissue thickens and scars (Arpino et al., 

2015; Hinz and Lagares., 2020). In the lungs, the clinical manifestation of fibrosis includes 

decreased total lung capacity (TLC), forced expiratory volume (FEV) and severe coughing 

and thromboembolisms and these cause dyspnoea (Nakamura and Suda, 2015). In the kidneys 

nephropathy occurs resulting in a decreased glomerular filtration rate, increased intratubular 

pressure and proteinuria (Kuusniemi et al., 2005; Kaissling et al., 2013). Liver fibrosis 

increases the portal hypertension, reduces clotting factor synthesis, there’s a build-up of bile 

and patients may develop jaundice and ascites (Kobelska-Dubiel et al., 2014). Fibrosis, 

therefore, alters the architecture of the ECM and this disrupts the normal physiological 

functioning of tissues and if left untreated it can cause organ failure and death.  

 

Figure 1.3: The phases of wound healing. A) Platelet and fibrin clots are formed to 

coagulate blood. B) The immune system is activated to prevent infections and clear damaged 

cells. C) Fibroblasts are recruited and they secrete proteins that repair the ECM which will 

anchor new cells and new vasculature is formed. D) The injured tissue is remodelled so that it 

closes and forms a scar (Adapted from Tracy et al., 2016).  

1.2.2 Type II EMT as a fibrosis pathway 

EMT is a process in which epithelial cells undergo a series of mechanochemical 

transformations to attain a motile mesenchymal phenotype (Kalluri and Weinberg, 2009). 

EMT involves the ECM as it is the main structural scaffolding anchoring epithelial cells 

(Morrissey and Sherwood, 2015). In EMT, the ECM adhesion complexes including adheren 

junctions, tight junctions and desmosomes are degraded and this results in the loss of cell-cell 

and cell-ECM attachments and their apicobasal polarity and cells become increasingly motile 

(Lamouille et al., 2014). During EMT, genes such as N-cadherin, vimentin, fibronectin and 

MMPs are upregulated and they increase the motility of cells and this is accompanied by the 



15 
 

downregulation of genes such as E-cadherin which facilitate the anchorage of cells to the 

ECM (Nisticò et al., 2012; Liu et al., 2015; Li et al., 2017; Loh et al., 2019). The changes in 

the expression levels of these gene can be regulated by EMT-inducing factors such as TGF-

β1 and TFs such as Snai1, Slug, twist-related protein 1 (Twist1) and EGR1 and they promote 

the acquisition of a mesenchymal phenotype (Willis and Borok, 2007; Medici et al., 2008; 

Wang et al., 2016; Wang et al., 2023). 

There are three types of EMT, type I is associated with embryo development and 

organogenesis, type II is associated with wound healing and fibrosis and type III EMT is 

involved in cell proliferation and causes cancer (Kalluri and Weinberg, 2009). Of interest in 

this project is type II EMT as it contributes to the development and progression of fibrosis. 

This EMT subtype mainly involves fibroblasts, cells that are predominantly found in 

connective tissues and responsible for the secretion of matrix proteins such as collages, 

fibronectin, elastin, and laminins (Kendall and Feghali-Bostwick, 2014). Following tissue 

injury fibroblasts cells are activated by signalling molecules such as TGF-β to undergo EMT 

to form myofibroblasts and in addition EMT can be induced on adipose cells, monocytes, and 

epithelial and endothelial cells to produce more myofibroblasts (Marconi et al., 2021). Once 

formed, myofibroblasts can migrate to the injured tissue and facilitate tissue repair by 

secreting matrix proteins (Darby et al., 2016). Myofibroblasts are also reported to generate 

contractile force in the injured sites and surrounding area, and this helps with the closure of 

the wound (Shin and Minn, 2004). During normal healing, myofibroblasts stop secreting 

matrix proteins once re-epithelialisation has occurred but with persistent tissue injury their 

activity is sustained in the proliferative phase of wound healing and in addition, they evade 

apoptosis and this results in tissue fibrosis (Hinz and Lagares, 2020).  

1.2.3 TGF-β1 as a driver of fibrosis 

TGF-β1 is a cytokine that stimulates cell growth, proliferation, differentiation, migration, and 

apoptosis (Zhang et al., 2017). It belongs to the TGF-β family which is comprised of TGF-

β1, TGF-β2 and TGF-β3. These three proteins have highly homologous structures, but their 

genes are found on different chromosomes with TGF-β1 on 19q13.1, TGF-β2 on 1q41 and 

TGF-β3 is on loci 14q24 (Fujii et al., 1986; Barton et al., 1988; Ten Dijke et al., 1988). The 

TGF-β proteins interact with the same cell surface receptors and activate similar pathways, 

but TGF-β1 is the most abundant and characterised isomer and hence the term TGF-β tends 

to be associated with it (Kubiczkova et al., 2012). TGF-β proteins are synthesized as inactive 

precursors associated with the latency-associated protein (LAP) and these form the small 



16 
 

latent complex (SLC) (Dubois et al., 2001). The SLC associates with latent TGF-β binding 

protein (LTBP) to form a large latent complex (LLC) and it is secreted into the extracellular 

space. Once the appropriate stimuli are received by the cell, such as wound healing, TGF-β is 

released from the LLC and can bind to its protein kinase receptors (Walton et al., 2010; 

Poniatowski et al., 2015). The active TGF-β ligand then binds to its TβRI and TβRII 

receptors and they form a heterotetramer, which phosphorylates intracellular proteins (Huang 

and Chen, 2012). Two TGF-β pathways can be activated by the receptors, the conical and the 

nonconical pathways. In the conical pathway, SMAD proteins are phosphorylated leading to 

a signalling cascade that eventually activates downstream TFs which then activate the 

expression of TGF-β target genes (Zi et al., 2012). In the non-conical pathway multiple 

pathways can be activated by the cytokine namely, MAPK (JNK/ERK/p38), 

PI3K/ATK/mTOR, RhoA/Rock/Cofllin, TREF/NF-κB, and Ras/RAF/ERK (Zhang, 2017).  

During persistent tissue injury, TGF-β1 acts as a key mediator of fibroproliferation. The 

levels of the cytokine are upregulated as macrophages, leucocytes, fibroblasts, epithelial and 

endothelial cells continuously secrete it and in addition, its receptors are upregulated making 

cells more responsive to its effects (Nakerakanti and Trojanowska., 2012; Meng et al., 2016). 

TGF-β1 aberrantly increases the expression of TNF-α and IL-4, IL-5, IL-13, and IL-21 

(Richter et al., 2015). Inflammation is also exasperated by the upregulation of ROS releasing 

proteins such as NOX4 and this promotes the development of fibrosis (Cucoranu et al., 

2005). The pro-fibrotic roles of TGF-β1 are predominantly directed to fibroblasts as they 

secrete ECM proteins which augment fibrosis. TGF-β1 activates type II EMT by increasing 

the expression of N-cadherin, Snai1 and α-SMA and this allows fibroblasts to differentiate 

into myofibroblasts, which can migrate into the injured fibrotic sites (Willis and Borok, 

2007). TGF-β1 also increases the numbers of myofibroblasts by aberrantly activating cyclin-

dependent kinases (CDKs) to facilitate their progression through the cell cycle and these 

increased numbers cause physiological scarring (Yamamoto et al., 2020). TGF-β1 also 

increases the survival of fibroblasts via upregulating the expression of anti-apoptotic genes 

such as BCL2, BCL-XL and X-linked inhibitor of apoptosis protein (XIAP) (Hinz and 

Lagares, 2020).  

As myofibroblasts persist in the ECM, they secrete excess amounts of proteins such as 

collagens, fibronectin, laminins, glycoproteins, and proteoglycans and the matrix thickens 

and scars (Klingberg et al., 2013). Matrix thickening is further exasperated as TGF-β1 

suppresses the catalytic activity of matrix proteases such as MMPs and plasminogen activator 



17 
 

via activating their inhibitors TIMPs and plasminogen activator inhibitor-1 (PAI-1) and 

consequently, proteins accumulate in the ECM leading to tissue fibrosis (Samarakoon et al., 

2008; Arpino et al., 2015). To stabilize the remodelled matrix which contains excess proteins, 

TGF-β upregulates the expression of matrix crosslinking enzymes such as LOX and PXDN 

and the matrix stiffens resulting in the loss of tissue elasticity (Péterfi et al., 2009; Lu et al., 

2019). TGF-β1 also creates a fibrotic microenvironment that sustains fibroproliferation 

through upregulating the expression of growth factors such as VEGF, PDGF, CTGF, and 

EGF. These promote angiogenesis and the growth and proliferation of myofibroblasts and 

organ-specific cells, and these increase the deposition of proteins into the surrounding ECM 

and therefore fibrosis worsens (Ihn, 2002; Chen et al., 2006; Pakyari et al., 2013).  

1.2.4 Pharmacological treatments of fibrosis 

Fibrosis is a progressive, life-threatening disease that remodels the connective tissue of 

organs thus interfering with their normal physiological functioning (Wynn, 2008). Various 

drugs have been approved for the treatment of the disease whilst many others are undergoing 

pre-clinical and clinical trials. Infections and pro-inflammatory cytokines are major activators 

of an exaggerated immune response and targeting them has been shown to improve 

fibroproliferation. In cystic fibrosis, inhaled antibiotics such as tobramycin, aztreonam lysine 

and levofloxacin are used (Taccetti et al., 2021). Anti-fibrotic drugs also target pro-

inflammatory cytokines and in a three-year study conducted by Brown et al. (2011) 

conducted on patients with arthrofibrosis the inhibition of IL-1 with anakinra had reduced 

patient’s knee pain and swelling and significantly improved their range of motion. TNF-α 

expression can be inhibited with infliximab, a monoclonal antibody that is used to treat 

patients with Crohn's disease (Poggioli et al., 2007). When the drug was used on Crohn's 

disease patients with fibrotic strictures, the intestinal biopsies from patients who had 

responded to infliximab had decreased depositions of collagen and fibronectin and thinner 

mucosal linings compared to their non-responder counterparts (de Bruyn, et al., 2018).  

TGF-β1 plays a central role in the development of fibrosis and various drugs have been used 

to moderate its effects. Disterine (P144) functions by binding to the TGF-β1 receptors TβRI 

and TβRII and inhibits the phosphorylation of Smad2/3 and when the drug was administered 

to rabbits with radiation-induced fibrosis (RIF) the treated group had decreased muscle 

fibrosis, alopecia, and collagen deposits (Cruz-Morande et al., 2022). Pirfenidone also targets 

TGF-β1 expression and in human lung fibroblasts, it was shown to cause a decrease in α-

SMA expression and collagen I synthesis (Conte et al., 2014). Rapamycin is another anti-



18 
 

fibrotic drug and it blocks the TGF-β1 downstream gene mammalian target of rapamycin 

(mTOR) and has been used successfully in the treatment of kidney fibrosis and it was also 

shown to reduce the proliferation, metabolic rate, and collagen I synthesis of primary 

fibroblasts from biopsies of patients with laryngotracheal stenosis (Chen et al., 2012; Namba 

et al., 2015). The profibrotic effects of TGF-β1 can also be mediated indirectly through 

targeting downstream TFs. In an idiopathic pulmonary fibrosis (IPF) study, Jayachandran et 

al. (2009) demonstrated that targeting Snai1/2 with siRNA in alveolar epithelial type II 

(ATII) cells treated with TGF-β1 decreased their migration and the expression of the EMT 

markers E-cadherin, occludin (OCCL) with a parallel increase in vimentin and α-SMA.   

Tissue fibrosis develops due to the altered synthesis and degradation of ECM proteins. 

Reduced matrix turnover occurs due to the upregulated expression of TIMP proteins which 

inhibit MMP proteases (Benyon and Arthur, 2001). When HSCs were subjected to TIMP1-

siRNA, researchers found that this reduced the proliferation of the cells and overall liver 

stiffness (Fowell et al., 2011). In fibrogenesis, cells express high levels of B-cell lymphoma-2 

(BCL2) proteins and this helps them evade apoptosis (Drakopanagiotakis et al., 2008). 

Navitoclax (ABT-263) has been used to block the activity of BCL-2 protein and in a mouse 

model of dermal fibrosis this decreased myofibroblast survival (Lagares et al., 2017). 

Similarly, in mice with radiation-induced pulmonary fibrosis, navitoclax activated the 

clearance of senescent pneumocytes and this reversed fibrosis in the animals (Pan et al., 

2017).  Growth factors are major mitogens for fibroproliferation and are also targeted in anti-

fibrotic therapies. Nintedanib competitively binds to the tyrosine kinase receptors of FGF, 

PDGF and VEGF and blocks their phosphorylation and thus fibroblast proliferation and 

migration and angiogenesis decreases (Wollin et al., 2015). Excessive matrix crosslinking is 

another key feature of fibrosis which results in a stiff matrix. The livers of CCl4 injured rats 

treated with the LOX inhibitor, β-aminopropionitrile have been shown to have reduced 

stiffness and myofibroblast activation (Georges et al., 2007). It is evident that targeting key 

signalling molecules in the fibrotic process can deliver positive outcomes and as such, it is 

important to identify more contributing genes to expand treatment options.   

1.3 EGR1 

1.3.1 Classification  

EGR1 is a zinc-finger (ZF) TF that regulates cell growth, differentiation, and apoptosis 

(Sukhatme et al., 1988). The protein was discovered independently by different researchers 

and therefore it has several names including nerve growth factor-induced A 



19 
 

(NGFI-A) (Milbrandt, 1987), Krox24 (Lemaire et al., 1988) and Zif268 (Christy and 

Nathans, 1989), but EGR1 (Sukhatme et al., 1988) is the most commonly used term. EGR1 

belongs to the immediate early gene (IEG) protein family that also includes EGR1, 2,3 and 4, 

the genes of which are separately encoded for on chromosomes 5q31, 10q31, 8p21 and 2p13, 

respectively (O'Donovan et al., 1999; Go et al., 2019). The EGR proteins share homologous 

domains, most notably the ZF DNA binding domain (DBD) and thus recognise similar 

transcription factor binding site (TFBS) sequences in target genes. The EGR isoforms do 

however perform slightly different cellular functions; EGR1 is involved in cell growth and 

differentiation; EGR2 in peripheral nerve myelination and immunity; EGR3 activates T cells 

and regulates the sympathetic nervous system, and EGR4 in involved with central nervous 

system function (Beckmann and Wilce, 1997).  

1.3.2 Structure  

EGR1 is a 57 kDa protein but based on its post-translational modifications this size can vary 

between 75-100 kDa (Sukhatme et al., 1988; Cheng et al., 2008). The protein is a monomer 

composed of three Cys2His2 (C2H2) type ZFs (Figure 1.3). Each ZF is made of two 

antiparallel β-sheets on the amino side and an α-helix and on the carboxy end. The two 

cysteine residues are situated on the β-sheets whilst the histidines are on the α-helix and 

altogether these tetrahedrally stabilize the central zinc iron (Zn2+) that is necessary for DNA 

binding (Cao et al., 1990; Pavletich and Pabo, 1991). The DBD of EGR1 is conserved within 

its protein family and all constituting TFs recognise and bind to the same 9 bp GC-rich site 

with the consensus sequence 5'-GCG(T/G)GGGCG-3' termed the EGR binding site (EBS) 

which is located in the promoter of their target genes (Christy and Nathans, 1989). Here, each 

ZF binds to a trinucleotide within the EBS sequence (Figure 1.3). The α-helices make contact 

with the major groove of DNA via hydrogen bonds and the β-sheets stabilize the interaction 

and allow the protein to transactivate genes (Pavletich and Pabo, 1991). The ZFs bind in an 

antiparallel manner on the EBS with ZF3 being on the 5' end, followed by ZF2 and then ZF3 

(Figure 1.3) (Zandarashvili et al., 2012). The other functional domains of EGR1 include 

strong and weak activation domains on either terminals and a repressor domain, and these 

allow the EGR1 to act as both a negative and positive gene regulator. Completing the 

structure of the EGR1 protein is a bipartite nuclear localization signal (NLS) which is directly 



20 
 

situated before the DBD and it is responsible for directing the protein to the nucleus once the 

appropriated stimulus is received by the cell (Gashler et al., 1993). 

 

Figure 1.4: The structure of the EGR1 and its corresponding DNA binding site. A) 

EGR1 is composed of three zinc fingers (ZF1, ZF2 and Z3), which have an α-helix and two 

β-sheets. The α-helices fit into the major groove of the DNA and interact with the target DNA 

sequences and the β-sheets hold the protein in place during binding. B)  The EGR binding 

site (EBS) is a 9 bp GC-rich sequence and the ZFs bind in an antiparallel direction starting 

with ZF3 followed by ZF2 and ZF1 (Adapted from Zandarashvili et al., 2012). 

1.3.3 EGR1 expression  

EGR1 is conserved in a host of different animal species such as zebrafish, mouse, chickens, 

cows, and humans (Close et al., 2002; Sayasith et al., 2006; Guo et al., 2014; Løtvedt, et al., 

2017). The protein is expressed in various cells such as fibroblasts, mast cells, adipocytes, 

chondrocytes, and in endothelial and epithelial cells (Schwachtgen et al., 1998. Li et al., 

2006; Bhattacharyya et al., 2008; Rockel et al., 2009; Zhang et al., 2013). The expression of 

EGR1 is induced by a broad range of biochemical and physiological stimuli such as growth 

factors, cytokines, hormones, oxidative stress, hypoxia, and tissue injury (Christy and 

Nathans, 1989; Gashler and Sukhatme., 1995; Tremblay and Drouin, 1999; Sperandio et al., 

2009; Bhattacharyya et al., 2011; Qin et al., 2022). These cues activate the expression of 

EGR1 via the mitogen-activated protein kinase (MAPK) signalling pathway which activates 

the sequential phosphorylation between protein kinases, and this activates the expression of 



21 
 

target genes so they deliver the appropriate cellular responses (Keshet and Seger, 2010; 

more). There are three distinct MAPK pathways for EGR1 expression, and these depend on 

the stimulus received, if the stimulus is biochemical the extracellular signal-regulated kinase 

(ERK) pathway is activated and if it is extra or intracellular stress, then the JNK or p58 

pathway is activated (Lim et al., 1998; Guha et al., 2001).  

1.3.4 The transactivation of genes by EGR1 

The transactivation of genes by EGR1 starts with its activation via phosphorylation. This is 

done by activation of both the protein kinase C (PKC) and tyrosine kinase pathways and 

these increase the transactivation activity of EGR1 (Huang et al., 1998). Importantly, the 

activation of EGR1 is sustained by the downregulation of protein phosphatase inhibitors 1 

and 2A and these maintain the phosphorylation modifications on the residues of EGR1 so that 

it can be able to transactive target genes (Cao et al., 1992). Once activated, EGR1 undergoes 

nuclear translocation and here the importin protein 7 (Imp7) associates with the NLS of 

EGR1 and helps it move through the nuclear pores (Chen et al., 2011). Once in the nucleus, 

EGR1 then binds as a monomer to the EBS and regulates the expression of genes (Christy 

and Nathans, 1989).  EGR1 regulates the expression of a multitude of genes involved in cell 

growth, proliferation, and apoptosis. These include cell cycle genes such as thymidine kinase 

(Molnar et al., 1994) and cyclin D proteins 1,2 and 3 (Wei et al., 2017).  Growth factors are 

also some of the EGR1-regulated genes and include the epidermal growth factor (EGF) 

(Arora et al., 2008), platelet-derived growth factor (PDGF) (Khachigian et al., 1997) and 

vascular endothelial growth factor (VEGF) (Shimoyamada et al., 2010). ECM proteins are 

also some of the most prominent EGR1 target genes and include the various types of 

collagens such as COL1A1, COL1A2 COL3A1, COL5A1, COL6A1 and COL14A1 (Chen et 

al., 2006; Havis and Duprez, 2020), fibronectin (Liu et al., 2000) and MMP9 (Shin et al., 

2010).  EGR1 also regulates the apoptotic p53 gene (Krones-Herzig et al., 2005). 

Interestingly, EGR1 can also regulate the activity of other TFs such as forkhead box protein 

C2 (FOXC2) (Zhang et al., 2013). As an IEG, the half-life of EGR1 is very short therefore 

once it has performed its task, it is quickly targeted for ubiquitination and sumoylation by 

SUMO1 and Ubc9 ubiquitin and this results in its proteasome-mediated degradation 

(Manente et al., 2011). EGR1 has also been shown to downregulate its own expression by 

recruiting its repressors NGFI-A-binding proteins 1 and 2 (NAB1 and NAB2) (Kumbrink et 

al., 2005; Bhattacharyya et al., 2009).  

https://en.wikipedia.org/wiki/SUMO_protein


22 
 

1.3.5 The pathophysiological roles EGR1  

EGR1 is involved in cell growth and proliferation, and this makes it a key contributor to the 

development of various diseases, most notably cancer and fibrosis. EGR1 is overexpressed in 

human prostate cancers due to the loss of its co-repressor NAB2 (Abdulkadir et al., 2001). 

The overexpression of EGR1 is further increased by the activity cyclin D1 and it then causes 

continuous growth of prostate cells (Xiao et al., 2005). In prostate cancer, EGR1 also 

activates the androgen receptor (AR) and this sensitizes cells to testosterone thus promoting 

tumorigenesis (Yang et al., 2003). EGR1 also acts as an oncogene that promotes metastasis 

and invasion, both key hallmarks of cancer, through the downregulation of E-cadherin, which 

is responsible for cell-cell adhesion in ECM adherens junctions (Pećina-Šlaus, 2003). 

Grotegut et al. (2006) demonstrated that in lung cancer cell lines, hepatocyte growth factor 

(HGF) activated EGR1 expression via the MAPK pathway and as well as Snai1, a regulator 

of EMT. They found that the Snai1 promoter contained four EBSs, and once activated by 

EGR1, Snai1 then reduced the expression of E-cadherin leading cells to become metastatic 

and highly invasive. Similarly, Cheng et al. (2013) showed that under the stimulation of EGF, 

EGR1 was upregulated and increased Snai2 (Slug) levels, which then reduced E-cadherin 

expression resulting in increased motility and invasion of ovarian cancer cells. EGR1 can act 

as a tumour suppressor and this is mediated via the transactivation of p53, which induces 

replicative senescence and activates apoptosis (Krones-Herzig et al., 2003).  

Fibrosis is characterized by an increased deposition of fibrous proteins and EGR1 has been 

shown to contribute to this under the TGF-β1 stimulation; EGR1 is rapidly induced in skin 

fibroblasts and binds to the COL1A2 promoter leading to increased collagen 1A2 secretion 

(Chen et al., 2006). A similar pattern has been reported in rheumatoid arthritis synovial 

fibroblasts where EGR1 upregulates the expression of type I and II collagen (Alexander et 

al., 2002). EGR1 also activates the expression of tissue inhibitor of metalloproteinases 

(TIMP) and this reduces matrix turnover thus promoting fibrosis (Aicher et al., 2003). In an 

animal-based study done by Wu et al. (2009) they used bleomycin-induced scleroderma 

mouse to investigate the expression patterns of EGR1 and the resulting phenotype. Here they 

found that EGR-null mice had reduced inflammation and fibrosis, but the wound healing was 

compromised whilst the EGR1-overexpressing counterparts had increased collagen levels and 

wound healing abilities. This study, therefore, illustrates that EGR1 has roles in wound 

healing and acts as a fibrotic factor. In myocardial fibrosis, the inhibition of EGR1 expression 

reverses fibrotic features in mouse models. When miR-150-5p is expressed, it targets the 



23 
 

EGR1 transcript reducing its levels and followed by the reduced expression of collagen I and 

III, key proteins that increase matrix stiffness (Shen et al., 2019).   

1.4 Study rationale 

The cellular functions of PXDN are not fully understood however, studies done on its 

transcriptional regulation are bridging this gap as illustrated in the studies done by Sitole and 

Mavri-Damelin (2018) and Hanmer and Mavri-Damelin (2018). Herein they contributed to 

the elucidation of the functions of PXDN by linking it to Snai1 and Nrf2 and we now know 

that PXDN functions in EMT and antioxidant responses (Sitole and Mavri-Damelin 2018; 

Hanmer and Mavri-Damelin, 2018). Our project will employ a similar approach by 

investigating whether EGR1 regulates the expression of PXDN. By establishing a link 

between the two proteins we can establish roles for PXDN in cell growth, differentiation, 

proliferation, and apoptosis which are key processes that EGR1 is known to regulate. The 

investigation of the relationship between EGR1 and PXDN will additionally provide insights 

into their role in fibrosis as they are both overexpressed in the pathology and have been 

shown to respond to the TGF-β1 which promotes the development and progression of the 

disease. From this PXDN may be used as a novel prognostic and therapeutic marker for 

fibrotic diseases. 

1.5 Aims and objectives 

 This research aimed to determine whether EGR1 regulates PXDN. The following objectives 

were performed. 

1. Detect and quantify the protein expression of EGR1 and PXDN using western blot. 

2. Identify the cellular localization of the expression of EGR1 and PXDN with 

immunofluorescence microscopy. 

3. Identify whether EGR1 interacts with the PXDN promoter using chromatin 

immunoprecipitation (ChIP). 

4. Determine if EGR1 activates or represses PXDN gene expression using the luciferase 

reporter assay.  

 

  



24 
 

2. Materials and methods 

2.1 Reagents  

Gibco® cell culture reagents were purchased from ThermoFisher Scientific (Waltham, 

Massachusetts, United States) and NEST® plasticware (Wuxi, Jiangsu, China)  was used. 

Chemical reagents were purchased from Sigma-Merck (St. Louis, Missouri, United States). 

The names of other manufacturers will be listed next to the respective product. The recipes of 

regents that were prepared are detailed in Appendix A and the catalogue numbers are listed in 

Appendix B.   

2.2 Cell culture 

Human embryonic kidney 293 cells (HEK293) were a gift from Dr Clement Penny 

(Oncology Research lab, University of the Witwatersrand, Johannesburg). The cells were 

cultured in 100 mm round plates and maintained in Dulbecco's Modified Eagle 

Medium/Nutrient Mixture F-12 (DMEM/F-12) (1:1) supplemented with 10% Fetal Bovine 

Serum (FBS) and 1% of 10,000 U/ml penicillin-streptomycin and kept at 37 ℃, in a 5% 

carbon dioxide (CO2) humidified incubator. Cells were sub-cultured when approximately 

80% confluent. The media was discarded, and the cells were washed three times with cold 1X 

phosphate-buffered saline (PBS). Following this, 0.25% trypsin-EDTA and 1X PBS (1:3) 

were added to the plate to catalyse the dissociation of cell-cell and cell-surface adhesion 

molecules and the cells were incubated for 3 minutes at 37 ℃ to facilitate the enzymatic 

dissociation reaction. The trypsinization was deactivated with 2 ml fresh media and cells 

were further detached by gently pipetting up and down to obtain single cells. From the cell 

suspension 200 µl was resuspended into a new plate containing 8 ml fresh media and the cells 

were cultured for between 48-72 hours or until confluent and used for experiments. For 

extended storage cells were cryopreserved in glycerol freezing media and stored at -80 ℃. 

Once resuscitated, cells were sub-cultured twice before use in experiments.  

2.2.1 Cell counts 

Cell counts were performed for the immunofluorescence and the luciferase reporter assay 

experiments which need a specified number of cells to be plated. Counting was done using 

the 0.0025 mm2 Neubauer haemocytometer (Paul Marienfeld, Lauda-Königshofen, Baden-

Württemberg, Germany). The cells were washed and enzymatically dissociated from the plate 

as detailed in section 2.2. The cells were mixed with 0.2% trypan blue dye (1:1) then loaded 

on the haemocytometer. Cells were viewed on the Zeiss ID-02 Inverted Microscope 

(Oberkochen, Baden-Württemberg, Germany) and counting was done on the inner 5X5 grids 

https://www.google.com/search?sxsrf=AJOqlzVg2NxrGbXl0RoSTOBm0gTYO1xY6A:1679061668027&q=Waltham&si=AEcPFx6l3RvH8SFlhHZyn7jIc6m2bU9vmoFvFAMQv2WWSYjXN8Aa11LoAMedq3c7biwJo4P_6AD0U2hy4zRorV6vC-gW4Y9EqRuA4Nn7eUDwjX4rI3fCwfzePc9Lg4IMDEjOWCK9Cix_kSSmrLP9Ix6LBMcviEjHTUc0EJa23n1Nf0dWXBNijOzJQ-DRhmyYc0jPKC4bKlF3&sa=X&ved=2ahUKEwjF_JySkOP9AhW1if0HHQA1DGcQmxMoAXoECG4QAw
https://www.google.com/search?sxsrf=AJOqlzVg2NxrGbXl0RoSTOBm0gTYO1xY6A:1679061668027&q=Waltham&si=AEcPFx6l3RvH8SFlhHZyn7jIc6m2bU9vmoFvFAMQv2WWSYjXN8Aa11LoAMedq3c7biwJo4P_6AD0U2hy4zRorV6vC-gW4Y9EqRuA4Nn7eUDwjX4rI3fCwfzePc9Lg4IMDEjOWCK9Cix_kSSmrLP9Ix6LBMcviEjHTUc0EJa23n1Nf0dWXBNijOzJQ-DRhmyYc0jPKC4bKlF3&sa=X&ved=2ahUKEwjF_JySkOP9AhW1if0HHQA1DGcQmxMoAXoECG4QAw
https://www.google.com/search?sxsrf=AJOqlzVEDB5Ny92m_9ES5dQcYu-sFJ_dOQ:1679061947814&q=St.+Louis&si=AEcPFx6l3RvH8SFlhHZyn7jIc6m2bU9vmoFvFAMQv2WWSYjXNzlvIQIitRZwtDDYqDUnJVS2NnYoS6VamNNNcNbifLPT_lXRlY92s-mB8F8JYYdtGXt1xGcz8wRNIkKH8CnbzBTNoyp406a1XOGaIyvNWo9gK7jKGtYPl4MBo1fBu3OC7jFyiiFfE3X8Y7Re6xc33bHvZQrH&sa=X&ved=2ahUKEwjG5tGXkeP9AhUlhv0HHYPNCrYQmxMoAXoECF0QAw
https://www.google.com/search?sxsrf=APwXEdcZy-Q4AcSXNxbbvtIPj0CbRlhryQ:1687515148872&q=Baden-W%C3%BCrttemberg&stick=H4sIAAAAAAAAAONgVuLQz9U3yMg2KXzEaMwt8PLHPWEprUlrTl5jVOHiCs7IL3fNK8ksqRQS42KDsnikuLjgmngWsQo5Jaak5umGH95TVFKSmpuUWpQOABnu2VxXAAAA&sa=X&ved=2ahUKEwiFpIHok9n_AhWcgv0HHQDjAsoQzIcDKAB6BAgVEAE
https://www.google.com/search?sxsrf=APwXEdcZy-Q4AcSXNxbbvtIPj0CbRlhryQ:1687515148872&q=Baden-W%C3%BCrttemberg&stick=H4sIAAAAAAAAAONgVuLQz9U3yMg2KXzEaMwt8PLHPWEprUlrTl5jVOHiCs7IL3fNK8ksqRQS42KDsnikuLjgmngWsQo5Jaak5umGH95TVFKSmpuUWpQOABnu2VxXAAAA&sa=X&ved=2ahUKEwiFpIHok9n_AhWcgv0HHQDjAsoQzIcDKAB6BAgVEAE


25 
 

in both the upper and lower chambers. For immunofluorescence, 1𝑥105cells were seeded in a 

6-well plate containing sterile 22X22 mm coverslips and for the uciferase assay 1𝑥104 

cells/well were seeded in a black 96-well plate with a clear bottom.  

2.2.3 Treatments 

When the cells were around 80% confluent, they were serum starved for 12 hours and then 

treated with TGF-β1. For western blot 5 ng/ml was used for immunofluorescence and ChIP 

10 ng/ml was used. The cells were treated for 6, 12 and 24 hours. Untreated cells were 

included to compare the differences that the cells displayed with and without the treatment. 

2.3 Western blot 

Western blot is a quantitative technique used to detect protein expression. The data obtained 

from western blotting is especially useful in comparing if protein expression levels change 

across multiple samples when cells are exposed to specific conditions or variables. We used 

the technique to determine if HEK293 cells endogenously express our proteins of interest 

(EGR1 and PXDN) and whether this expression level would change in response to TFG-β1 

treatment. In addition to obtaining qualitative data, blotting would help us pick a time point to 

use downstream in the ChIP assay when EGR1 is highly expressed to increase its binding 

chances to EBSs in the chromatin.   

2.3.1. Protein extraction 

Cells were washed three times with cold 1X PBS and scraped from the culturing dish. A 1 ml 

suspension of cells in 1X PBS was transferred into a clean microcentrifuge tube. Whole 

protein was extracted from cells by pelleting them through centrifugation at 11 000 rpm, at 

4 ℃ for 10 minutes then resuspending in 1X radioimmunoprecipitation assay (1X RIPA) 

containing 10 mM phenylmethylsulfonyl fluoride (PMSF) and 1mM leupeptin. The cells 

were lysed by vortexing the tubes and then spinning on the RM-Multi 1 Rotator Mixer 

(Starlab, Neuer Höltigbaum, Hamburg, Germany) for 1 hour at 4 ℃. The lysate was then 

centrifuged at 12 500 rpm, for 5 minutes at 4 ℃ to pellet the cell debris. The supernatant was 

then collected, and this was aliquoted into PCR tubes and stored at -80 ℃. Some was set 

aside for quantification using the Bramhall assay.  

2.3.2 Bramhall assay 

The Bramhall assay was performed to determine the concentration of the extracted protein. 

This assay is used for proteins extracted with high SDS-containing lysis buffers which are 

incompatible with other protein quantification assays such as the Bradford assay (Bramhall et 



26 
 

al., 1969; Bradford, 1976). A Whatman™ No.1 filter paper was incubated in distilled water 

for 20 minutes, then in 95% ethanol, 100% ethanol and finally in 100% acetone for 5 minutes 

each with gentle rocking at 15 rpm. The paper was dried under a fume hood and onto it, 

1,3,6,12,16 and 20 µl of 1 mg/ml of bovine serum albumin (BSA) (Glentham Life Sciences, 

Leafield, Corsham, United Kingdom) weres blotted along with 2 µl of the extracted protein 

supernatant. Following this, the paper was again air dried, and the proteins were fixed to the 

filter paper by incubating in 7.5% trichloroacetic acid (TCA) for 45 minutes. The paper was 

then stained in Coomassie brilliant blue G250 for 1 hour. The excess coomassie was removed 

by incubating in the paper in the destain solution for 1 hour. The stained protein blots were 

individually cut from the filter paper and placed in 5 ml elution buffer and incubated for 2 

hours in the dark to elute the proteins.  

The tubes containing the eluted protein were mixed and 160 µl was pipetted in triplicates 

onto a 96-well plate. The absorbance was read at 595 nm using a Multiskan GO Microplate 

Spectrophotometer (ThermoFisher Scientific) and the elution buffer was used as a blank. 

Measurements obtained from the microplate reader software were exported to Microsoft 

Excel (Office 365) and calculations for the protein concentration were performed. The BSA 

concentration (µg/µl) and corresponding absorbance values were used to plot a standard 

curve. The equation of the line of best fit (y= mx+ c) was used to determine the concentration 

(µg/µl) of the extracted protein samples using their measured absorbance values. This 

concentration was then used to find the volume of protein to load on the polyacrylamide gel.  

2.3.3 Discontinuous SDS-PAGE 

Proteins were resolved on a discontinuous sodium dodecyl sulfate–polyacrylamide gel 

electrophoresis (SDS-PAGE) to separate the protein extracts by size. EGR1 is 80 kDa (Cao et 

al., 1990) and PXDN is 165 kDa (Cheng et al., 2008). The resolving gel for EGR1 was 

prepared to 12% and for PXDN an 8% gel was made. The differences in the resolving gel 

percentages are based on the sizes of the target protein as lower molecular weight proteins 

resolve better on higher percentage gels and higher molecular weight proteins resolve better 

on low percentage gels. In addition to the gel percentages, the running voltage and protein 

transfer conditions used were based on the size of each protein. These details, together with 

the antibodies used to probe for the proteins of interest are summarized in Table 2.1.  

 

 



27 
 

Table 2.1: SDS-PAGE percentages, antibody dilutions and transfer conditions for 

EGR1, PXDN and β-actin   

 EGR1 PXDN β-actin  

SDS-PAGE 

percentage  

12% 8% 12% 

Protein loading 

amount  

Initial amount- 40 µg 

 

Optimized amount- 20 

µg 

Initial amount- 150 µg then 

100 µg 

 

Optimized amount- 80 µg 

20 µg 

SDS-PAGE 

running voltage 

150 V 180 V 150 V 

Primary 

antibody  

EGR1 rabbit 

monoclonal antibody  

(Cell Signaling 

Technology: #4154) 

PXDN mouse monoclonal 

antibody  

(Santa Cruz Biotechnology: 

sc-293408) 

β-actin rabbit 

polyclonal antibody 

(Cell Signaling 

Technology: #4967) 

Primary 

antibody 

dilution 

1:1000 Initial dilution- 1:1000 

Optimized dilution 1:2000 

1:2000 

Secondary 

antibody  

Goat anti-rabbit  

HRP antibody 

(Cell Signaling 

Technology: #7074) 

 

Horse anti-mouse IgG HRP 

(Cell Signaling 

Technology: #7076) 

 

Goat anti-rabbit  

HRP antibody 

(Cell Signaling 

Technology: #7074) 

 

Secondary 

antibody 

dilution 

1:2000 

 

Initial dilution- 1:2000 

Optimized dilution- 1:1500 

1:4000 

 

Transfer time 2 hours  

 

3 hours  2 hours  

Tween-20 

percentage in 

the 1X TBST 

wash buffer 

0.1% 0.2% 0.1% 

 

The resolving gels (pH 8.8) (Table 2.2) were prepared first and poured into the 1 mm gap 

between the short glass plate and spacer glass plate and a 0.1% SDS overlay was poured on 

top to ensure an even gel interface. The resolving gels were then left for about 20 minutes to 

polymerize, and the overlay was removed. Afterwards, the stacking gels (pH 6.8) (Table 2.2) 

were prepared and poured on top of the resolving gel and a comb was inserted to create wells 

in which to load the protein samples.  

 

 

 



28 
 

Table 2.2: Discontinuous SDS-PAGE recipes 

 Resolving gel (10 ml) Stacking gel (5 ml) 

Reagent 8% 12% 5% 

dH2O  4.6 ml 3.3 ml 3.4 ml 

29:1 Acrylamide/ bisacrylamide 2.7 ml 4.0 ml 830 µl 

1 M Tris-HCl pH 6.8 - - 630 µl 

1.5 M Tris-HCl pH 8.8 2.5 ml 2.5 ml - 

10% SDS 100 µl 100 µl 50 µl 

10% APS 100 µl 100 µl 50 µl 

TEMED 6 µl 4 µl 4 µl 

 

Once the gels had set, the extracted protein was thawed on ice and then mixed with a 4X 

protein loading buffer and the samples were linearized by boiling for 5 minutes and then 

loaded onto the gel. The PageRuler™ Plus Prestained Protein Ladder, 10-250 kDa 

(ThermoFisher Scientific) was used as the molecular weight marker. To control for equal 

protein loading between the samples on the EGR1 and PXDN gels, the same protein samples 

were used to run a parallel β-actin gel. The bands obtained from this gel would indicate 

whether proteins were loaded equivalently across the gel. The gels were electrophoresed in a 

Mini-PROTEAN® Tetra Cell tank (Bio-Rad, Hercules, California, United States) containing 

running buffer  until the dye front fell off from the gel. Using the protein ladder as a guide, 

the gel was cut across the region where the protein of interest would have migrated. These gel 

fragments were prepared for the electrophoretic transfer of the protein bands. The remaining 

gel portions were stained in coomassie overnight with constant shaking at 100 rpm then 

destained the following day for 1 hour to check if there was protein on the gel and that it was 

properly separated into different sizes.  

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