A Central Bioactive Region of LTBP-2 Stimulates the Expression of TGF-ß1 in Fibroblasts via Akt and p38 Signalling Pathways.

Latent transforming growth factor-ß-1 binding protein-2 (LTBP-2) belongs to the LTBP-fibrillin superfamily of extracellular proteins. Unlike other LTBPs, LTBP-2 does not covalently bind transforming growth factor-ß1 (TGF-ß1) but appears to be implicated in the regulation of TGF-ß1 bioactivity, although the mechanisms are largely unknown. In experiments originally designed to study the displacement of latent TGF-ß1 complexes from matrix storage, we found that the addition of exogenous LTBP-2 to cultured human MSU-1.1 fibroblasts caused an increase in TGF-ß1 levels in the medium. However, the TGF-ß1 increase was due to an upregulation of TGF-ß1 expression and secretion rather than a displacement of matrix-stored TGF-ß1. The secreted TGF-ß1 was mainly in an inactive form, and its concentration peaked around 15 h after addition of LTBP-2. Using a series of recombinant LTBP-2 fragments, the bioactivity was identified to a small region of LTBP-2 consisting of an 8-Cys motif flanked by four epidermal growth factor (EGF)-like repeats. The LTBP-2 stimulation of TGF-ß expression involved the phosphorylation of both Akt and p38 mitogen-activated protein kinase (MAPK) signalling proteins, and specific inactivation of each protein individually blocked TGF-ß1 increase. The search for the cell surface receptor mediating this LTBP-2 activity proved inconclusive. Inhibitory antibodies to integrins ß1 and αVß5 showed no reduction of LTBP-2 stimulation of TGF-ß1. However, TGF-ß1 upregulation was partially inhibited by anti-αVß3 integrin antibodies, suggestive of a direct or indirect role for this integrin. Overall, the study indicates that LTBP-2 can directly upregulate cellular TGF-ß1 expression and secretion by interaction with cells via a short central bioactive region. This may be significant in connective tissue disorders involving aberrant TGF-ß1 signalling.

Co-localization of LTBP-2 with FGF-2 in fibrotic human keloid and hypertrophic scar.

We have recently shown that Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) has a single high-affinity binding site for fibroblast growth factor-2 (FGF-2) and that LTBP-2 blocks FGF-2 induced cell proliferation. Both proteins showed strong co-localisation within keloid skin from a single patient. In the current study, using confocal microscopy, we have investigated the distribution of the two proteins in normal and fibrotic skin samples including normal scar tissue, hypertrophic scars and keloids from multiple patients. Consistently, little staining for either protein was detected in normal adult skin and normal scar samples but extensive co-localisation of the two proteins was observed in multiple examples of hypertrophic scars and keloids. LTBP-2 and FGF-2 were co-localised to fine fibrous elements within the extracellular matrix identified as elastic fibres by immunostaining with anti-fibrillin-1 and anti-elastin antibodies. Furthermore, qPCR analysis of RNA samples from multiple patients confirmed dramatically increased expression of LTBP-2 and FGF-2, similar TGF-beta 1, in hypertrophic scar compared to normal skin and scar tissue. Overall the results suggest that elevated LTBP-2 may bind and sequester FGF-2 on elastic fibres in fibrotic tissues and modulate FGF-2's influence on the repair and healing processes.

LTBP-2 Has a Single High-Affinity Binding Site for FGF-2 and Blocks FGF-2-Induced Cell Proliferation.

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) belongs to the fibrillin-LTBP superfamily of extracellular matrix proteins. LTBPs and fibrillins are involved in the sequestration and storage of latent growth factors, particularly transforming growth factor ß (TGF-ß), in tissues. Unlike other LTBPs, LTBP-2 does not covalently bind TGF-ß, and its molecular functions remain unclear. We are screening LTBP-2 for binding to other growth factors and have found very strong saturable binding to fibroblast growth factor-2 (FGF-2) (Kd = 1.1 nM). Using a series of recombinant LTBP-2 fragments a single binding site for FGF-2 was identified in a central region of LTBP-2 consisting of six tandem epidermal growth factor-like (EGF-like) motifs (EGFs 9-14). This region was also shown to contain a heparin/heparan sulphate-binding site. FGF-2 stimulation of fibroblast proliferation was completely negated by the addition of 5-fold molar excess of LTBP-2 to the assay. Confocal microscopy showed strong co-localisation of LTBP-2 and FGF-2 in fibrotic keloid tissue suggesting that the two proteins may interact in vivo. Overall the study indicates that LTBP-2 is a potent inhibitor of FGF-2 that may influence FGF-2 bioactivity during wound repair particularly in fibrotic tissues.

LTBP-2 competes with tropoelastin for binding to fibulin-5 and heparin, and is a negative modulator of elastinogenesis.

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a protein of ill-defined function associated with elastic fibers during elastinogenesis. Although LTBP-2 binds fibrillin-1, fibulin-5, and heparin/heparan sulfate, molecules critical for normal elastic fiber assembly, it does not interact directly with elastin or its precursor, tropoelastin. We investigated the modulating effect of LTBP-2 on two key interactions of tropoelastin during elastinogenesis a) with fibulin-5 and b) with heparan sulfate (using heparin). Firstly, using solid phase assays we showed that LTBP-2 bound fibulin-5 (Kd=26.47±5.68 nM) with an affinity similar to that of the tropoelastin-fibulin-5 interaction (Kd=24.66±5.64 nM). Then using a competitive binding assay we showed that LTBP-2 inhibited the tropoelastin-fibulin-5 interaction in a dose dependent manner with almost complete inhibition obtained with 5-fold molar excess of LTBP-2. Interestingly, a fragment of LTBP-2 containing the fibulin-5 binding sequence only partially inhibited the tropoelasin-fibulin-5 interaction suggesting that LTBP-2 was directly blocking only the C-terminal tropoelastin binding site on fibulin-5 and indirectly blocking tropoelastin binding to the N-terminal region. In parallel experiments heparin was shown to have minor inhibitory effects on fibulin-5 interactions with tropoelastin and LTBP-2. However, LTBP-2 was shown to significantly inhibit the binding of heparin to tropoelastin with 50% inhibition achieved with 10 fold molar excess of LTBP-2. Confocal microscopy of fibroblast matrix showed strong co-distribution of LTBP-2 with fibulin-5 and fibrillin-1 and partial co-distribution with heparan sulfate proteoglycans, perlecan and syndecan-4. Also addition of exogenous LTBP-2 to ear cartilage chondrocyte cultures blocked elastinogenesis in a concentration-dependent manner. Overall the results indicate that LTBP-2 may have a negative regulatory role during elastic fiber assembly, perhaps in displacing elastin microassemblies from complexes with fibulin-5 and/or cell surface heparan sulfate proteoglycans.