Monash University
Browse

Restricted Access

Reason: Access restricted by the author. A copy can be requested for private research and study by contacting your institution's library service. This copy cannot be republished

ADAM involvement in angiotensin II-mediated transactivation and cardiomyocyte hypertrophy

thesis
posted on 2017-01-16, 23:24 authored by Bourne, Allison Margaret
Prolonged pathological cardiac hypertrophy is a major risk factor for death. In the heart, angiotensin II (AngII) can cause cell growth via angiotensin type 1 receptor (AT1R)- mediated transactivation of epidermal growth factor receptors (EGFRs). This process is thought to involve intracellular stimulation of matrix metalloproteases and/or A Disintegrin And Metalloproteases (ADAMs) to release EGF-like ligands, which in turn activate EGFRs and stimulate downstream growth signalling in a molecular pathway now termed the Triple Membrane Passing Signalling paradigm (TMPS). While ADAM family members 10, 12 and 17 have been implicated in regulating the TMPS pathways, the extent to which they mediate cardiomyocyte hypertrophy has not been studied in detail. To investigate the role(s) of these ADAMs in AngII-mediated cardiomyocyte hypertrophy, I designed, created and utilised shRNAmiRs, both as plasmids, and as adenoviruses that specially knockdown the expression of their target ADAMs. By applying these unique tools, I was able to show that AngII-mediated induction of hypertrophic marker genes atrial natriuretic peptide (ANP), CyclinD, myosin light chain 2v and α skeletal actin (αSkAct) is mediated (at least in part) through the TMPS pathway – ADAM17 appears to be uniquely involved in inducing AngII-mediated ANP expression, whereas ADAM12 appears to regulate αSkAct induction. As adenoviruses, these shRNAmiRs produced targeted reduction of endogenous ADAM10 and 17 mRNA in neonatal rat ventricular myocytes, but that reduction (even in combination) was insufficient to attenuate AngII-induced cardiomyocyte hypertrophy. Taken together, these data argue against a prominent role for one specific ADAM, and instead suggests that TMPS regulated cardiomyocyte hypertrophy may involve the activation of multiple ADAMs that collectively induce cardiomyocyte hypertrophy, potentially through the regulation of different hypertrophic genes. Another key area of transactivation research that is yet to be resolved is the possibility for transactivation to occur between cells (in trans) with ADAMs cleaving ligands on nearby cells as has been shown for ADAM10-mediated ephrin shedding. To investigate this possibility, I created a model transactivation system using BaF/3 cells to enable the manipulation of key transactivation components. However, despite using a series of retroviral constructs to create cells that expressed the AT1R, EGFR, Gαq/11 and ADAMs 10, 12 and 17, I couldn’t recapitulate AngII-mediated transactivation in these cells, either via the TMPS pathway or via alternative intracellular mechanisms. Presumably some key intracellular signalling molecules involved in transactivation remained absent. Likewise, investigations into the possibility that EGF receptor transactivation involves cross-talk between myocytes and fibroblasts were unresolved by studies performed here, despite evidence that fibroblasts may provide the AT1R for activation of this pathway. In summary, despite creation of selective RNA interfering constructs against ADAM10, 12 and 17, and the establishment of a model transactivation cell line, my data indicates that the TMPS pathway is more complicated than originally conceived – multiple ADAMs, numerous EGF ligands and EGFRs, combined with likely contributions from several cardiac cell types, offers significant challenges. The reagents and approaches outlined herein provide a platform for future studies aimed at unravelling the contribution and mechanism of EGF receptor transactivation in health and disease.

History

Principal supervisor

Walter Thomas

Year of Award

2010

Department, School or Centre

Biomedical Sciences (Monash Biomedicine Discovery Institute)

Additional Institution or Organisation

Biochemistry and Molecular Biology

Campus location

Australia

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Medicine Nursing and Health Sciences

Usage metrics

    Faculty of Medicine, Nursing and Health Sciences Theses

    Categories

    No categories selected

    Exports

    RefWorks
    BibTeX
    Ref. manager
    Endnote
    DataCite
    NLM
    DC