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

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.