The Roles of JAB1 and NOTCH1 in the Development of Cardiovascular Disease

Karountzos, Anastasios (2019) The Roles of JAB1 and NOTCH1 in the Development of Cardiovascular Disease. PhD thesis, University of Lincoln.

Documents
The Roles of JAB1 and NOTCH1 in the Development of Cardiovascular Disease
PhD Thesis
[img]
[Download]
[img]
Preview
PDF
KAROUNTZOS, ANASTASIOS - BIOMEDICAL SCIENCES - JUNE 2019.pdf - Whole Document

5MB
Item Type:Thesis (PhD)
Item Status:Live Archive

Abstract

Background
Pulmonary arterial hypertension (PAH) and Adams-Oliver syndrome (AOS) are
rare vascular disorders, characterised by severe late-onset and developmental
cardiac abnormalities respectively. Both of them are clinically associated in a
proportion of cases. PAH is a progressive condition that is clinically characterized
by sustained elevation in mean pulmonary artery pressure, through vascular
remodelling with luminal obliteration of small vessels and increased vascular
resistance, while AOS is characterized by congenital limb defects and scalp cutis
aplasia. In a proportion of AOS-cases, notable cardiac development is also
apparent. PAH may be hereditary (HPAH), idiopathic (IPAH) or associated with
other conditions, for example AOS. Most of HPAH and 25% of IPAH cases are
associated with heterozygous mutations of a TGF-β superfamily member
transmembrane receptor type-II, known as BMPR2. The majority of mutations
suggest haploinsufficiency as the molecular mechanism of disease. In addition,
monoclonal proliferation and uncontrolled growth in pulmonary artery endothelial
(PAEC) and smooth muscle (PASMC) cells result in perturbation and
muscularization of the arterial tone. This study aims to identify the cause of a
further loss of BMPR-II expression in previous immunohistochemical studies of
patient’s lung sections, that cannot be explained by haploinsufficiency. The
defective BMP/TGF-β signalling in PAH will be addressed as well. This study also
aims to identify novel genetic determinants of AOS, along with the hitherto
unresolved underlying molecular defects, for the majority of affected subjects.
Methods and Results
Protein-protein interaction experimentation revealed a BMPR-II novel interacting
partner, namely JAB1 protein, which through association with the kinase domain
of the receptor, functions as the first identified proteasomal-associated inhibitor
of BMPR-II. In addition, the linked experimental data of this study are indicative
of BMPR-II degradation via the proteasomal pathway in the normal circulation. In
addition, transient transfection studies demonstrated that JAB1 down-regulates
BMPR-II in a dose-dependent manner. It is also confirmed that JAB1 is over-expressed during PAH progression, and proliferation studies revealed that JAB1
promotes uncontrolled cell proliferation and survival. Furthermore, functional
studies supported the hypothesis that JAB1 appears to be a key regulator of BMP
and TFG-β signalling pathways in PASMC cells, regulating differentially all stages
of the pathways’ cascades in SMAD-dependant and SMAD-independent
manners. Apart its up-stream action of promoting the proteolytic degradation of
BMPR-II, RT-qPCR experimentation and further functional studies proved that it
also acts down-stream, by suppressing ID1 transcription factor or by promoting
ID expression via up-regulation of SMAD-dependant and SMAD-independent
signalling pathways, via activation of TAK1 and p38 MAPK mitogenic cascades,
thus enhancing the pro-proliferative state of healthy and/or BMPR2 mutated
PASMC cells. Knock-down of JAB1 in healthy and BMPR2 mutant PASMC cells
resulted in control of their proliferation, an outcome which is indicative of the
essential role of JAB1 in positively regulating the cell proliferation. The anti-proliferative effect of BMP4-ligand stimulation on hyper-proliferating BMPR2
mutant PASMC cells was also established, highlighting the suppressive
regulatory role of BMP signalling on cell proliferation and differentiation.
Regarding AOS disease, NOTCH1 mutations were found as the primary cause
of AOS, in a proportion of cases of a cohort study (17%) with further
cardiovascular complications. Novel identification of 10 mutations [3 frameshifts,
6 missense (EGF-like domain) and 1 nonsense] of the NOTCH1 gene in AOS patients, along with a significant reduction of NOTCH1 expression in leukocyte-derived RNA from subjects, suggested that NOTCH1 plays a key role in the AOS
pathogenesis. Transient transfections of mutagenized NOTCH1 missense
constructs also revealed significant reduction in gene expression. Likewise,
assessment of NOTCH1 target genes HEY1 and HES1 expression via RT-qPCR
studies, verified the dysregulation of the NOTCH1 canonical signalling pathway
in AOS-patients. Lastly, functional studies demonstrated the cross talk of BMP
and NOTCH1 signalling as a synergistic effect in AOS pathology, underlined by
forms of non-canonical signalling, potentiating the suppressive BMP cell
signalling effect in AOS-cases of dysfunctional NOTCH1 signalling and further
cardiovascular defects.
Conclusions
These findings manage to provide further insight into JAB1 mediated BMPR-II
down-regulation and degradation via the proteasomal pathway, and
determination of JAB1 dysregulation and role in the aggressive mitogenic
proliferation potential, through reduction of SMAD signalling and consequently
p38 MAPK and TAK1 activation, observed in PAH cells. This study highlights the
significance of this molecule in the vasculature and sets it as a potential novel
target in PAH therapy, and for drugs against PASMC cells in pro-proliferating
state harbouring or not BMPR2 PAH-associated mutations. Finally, it is
established that haploinsufficiency of the NOTCH1 receptor is a primary cause of
AOS, while solidifies NOTCH1 as an important genetic factor in AOS with
associated cardiovascular complications. Further X-ray crystallography studies
of the identified NOTCH1 missense mutations of the EGF-like domain will also
elucidate the extend of the structural perturbation of the receptor and subsequent
impairment of the signalling cascade.

Keywords:cardiovascular disease, PAH, AOS, qPCR
Subjects:C Biological Sciences > C420 Human Genetics
C Biological Sciences > C431 Medical Genetics
Divisions:College of Science > School of Life Sciences
ID Code:48490
Deposited On:08 Mar 2022 15:38

Repository Staff Only: item control page