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Type: Theses
Title: Function of latent transforming growth factor-β binding protein-2 (LTBP-2) in elastinogenesis and modulation of growth factor storage, expression and activity in normal and fibrotic tissues
Author: Mohamed Sideek, Mohamed Arshad
Issue Date: 2016
School/Discipline: School of Medicine
Abstract: LTBP-2 is tightly associated with fibrillin microfibrils and elastic fibres in a range of tissues mainly in the lung, heart, skeletal muscle, placenta, liver and the aorta. LTBP-2 belongs to the fibrillin-LTBP superfamily of extracellular matrix proteins. Unlike other LTBPs, LTBP-2 does not covalently bind TGF-beta and its molecular function remains unclear. LTBP-2 complexes with fibulin-5, an elastin-chaperone protein critical for normal elastic fibre assembly, and it has been suggested that LTBP-2 may preferentially direct fibulin-5-elastin globules onto fibrillin-1 (rather than fibrillin-2) microfibrils during elastinogenesis. However, we have now shown that LTBP-2 inhibits rather than enhances the interaction of tropoelastin with fibulin-5 in vitro. In addition LTBP-2 inhibited elastic fibre assembly in ear cartilage chondrocyte cultures largely at the stage of elastin deposition onto the fibrillin microfibril scaffold. In parallel experiments, LTBP-2 was shown to significantly inhibit the binding of heparin to tropoelastin suggesting LTBP-2 may compete with tropoelastin for binding to certain cell surface HSPGs and contribute to controlling the release of elastin microassemblies from the cell surface. Confocal microscopy showed strong co-distribution of LTBP-2 with fibulin-5 and fibrillin-1 and partial co-distribution with HSPGs, perlecan and syndecan-4 in fibroblast matrix Thus it is evident that LTBP-2 is a negative modulator of elastinogenesis and that LTBP-2 levels may regulate the rate and extent of elastinogenesis in some tissues. A recent study has linked LTBP-2 gene mutations to recessive form of Weill-Marchesani syndrome which is characterised by short stature, thick fibrotic skin and ectopia lentis. Since fibrillin-1 mutations can also cause this syndrome it is now clear that LTBP-2 is linked to fibrillin biology, growth factor regulation and fibrosis. To investigate growth factor binding to LTBP-2, our laboratory screened a number of cytokines involved in the pathogenesis of fibrotic disorders and identified a very strong specific interaction of FGF-2. The activity was confined to a central region of the LTBP-2 consisting of 6 EGF-like repeats, suggesting a single binding sequence. The finding presented in this thesis found that 5-fold molar excess LTBP-2 can completely block FGF-2 stimulation of fibroblast proliferation via its receptor. In addition increased levels and extensive co-localisation of LTBP-2 and FGF-2 were observed and quantitated in human hypertrophic scars and keloids. Furthermore, qPCR confirmed consistent elevation of LTBP-2 and FGF-2 expression in samples of these fibrotic tissues. The results support the concept that increased LTBP-2 expression in fibrotic disorders may increase FGF-2 binding and reduce FGF-2 activity, inhibiting normal repair processes. Previously we have shown that LTBP-2 competes with LTBP-1 for binding to fibrillin in vitro, suggesting that LTBP-2 may modulate TGF-β storage and activation. In experiments designed to measure displacement of TGF-β complexes from fibrillin microfibrils, our laboratory discovered addition of LTBP-2, or a small bioactive fragment LTBP-2C F3 to MSU 1.1 skin fibroblasts resulted in a large increase in TGF-β levels in culture medium. However the increase in TGF-β the medium was cycloheximide sensitive indicating elevated cellular expression and secretion of TGF-β rather than release of matrix-stored TGF-β. Exogenous LTBP-2 or fragment F3 significantly increased levels of latent TGF-β in the medium after 9h peaking at 15h. The signalling mechanism appears to involve the PI3K/Akt and p38 MAPK pathways, as incubation of cells with LTBP-2 (10μg/ml) elevated Akt 1/2/3 Ser473 and P38 D-8 phosphorylation and inhibition of each pathway completely blocked the synthesis of TGF-β. Investigation of the cell surface receptor for the bioactive fragment of LTBP-2 was less informative. Inhibitory antibody to β1 integrins did not affect the TGF-β upregulation but it was partially inhibited by an antibody to the integrin αVβ3 receptor, suggesting it may be involved in LTBP-2-cell interaction(s) resulting in elevated TGF-β expression. In conclusion, these findings are consistent with LTBP-2 having novel regulatory functions in elastinogenesis, growth factor modulation and fibrosis which may lead to novel therapy development for fibrotic diseases and tissue repair.
Advisor: Gibson, Mark Anthony
Cowin, Allison June
Kopecki, Zlatko
Dissertation Note: Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Medicine, 2016.
Keywords: LTBP-2
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at:
DOI: 10.4225/55/58ae64a06e70a
Appears in Collections:Research Theses

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