Fibulins and LTBP4 Interact with Syndecans to Regulate Elastogenesis
H. Hakami1*; N. Dinesh1*, V. Nelea1; R. Mahalingam1; N. Lamarche-Vane1; S. Ricard-Blum2; D.P. Reinhardt1*
Affiliation of the authors: 1. UU直播, Montreal, QC H3A 0C7, Canada 2. Universit茅 de Lyon 1, UMR 5246, Villeurbanne, France.*co-senior authors
Introduction: Elastogenesis is a hierarchical process which confers elasticity to the periodontal ligament, the oral mucosa, and other tissues, requiring several extracellular matrix proteins including fibulin-4 (FBLN4), fibulin-5 (FBLN5) and latent TGF-尾 binding protein-4 (LTBP4). It is known that both FBLN4, FBLN5 and LTBP4 interact with cells, but their cell receptors and the respective molecular mechanisms in elastogenesis remain unknown.
Objective: Elucidate the role of the interaction of fibulin-4, fibulin-5 and LTBP-4 with syndecans in elastogenesis.
Methodology: Elastogenic dermal skin fibroblasts (NSF) and vascular smooth muscle cells (SMC) were employed to analyze the interaction of cells with FBLN4 and LTBP4. Baseline expression levels of FBLN4, FBLN5, and LTBP4 were analyzed by immunofluorescence. Global knockdown of syndecans (SDC1-4) via siRNA abolished interaction of NSF with both proteins. To evaluate the functional importance of these interactions, pharmacological inhibition of myosin II, focal adhesion kinase (FAK), RhoA and ERK signalling was used to analyze elastic fibre assembly upon cell interaction with FBLN4 and LTBP4. Surface plasmon resonance experiment was used to access the binding affinity of FBLN5 with syndecans.
Results: NSF and SMC bound strongly to both FBLN4 and LTBP4. FBLN4 exclusively interacted as multimers, and two novel cell interaction epitopes on FBLN4 located in cbEGF2-3 and the C-terminal domain were identified. A new cell interaction site on the LTBP4 N-terminal half was mapped. Cell binding to FBLN4 and LTBP4 was absent in the presence of heparin and significantly reduced upon heparinase treatment, suggesting heparan sulfate proteoglycans as the cell surface receptors for this interaction. Specific knockdown of SDC-2 and SDC-3 impaired interaction of cells with
FBLN4, whereas only SDC-3 knockdown abolished the interaction with LTBP4. SPR data suggest that FBLN5 binds strongly to SDC-4. The enhanced elastic fiber assembly promoted by FBLN4 and LTBP4 was mediated through an increase in cell contraction via upregulation of focal adhesion kinase (FAK), RhoA and ERK signalling. This was significantly compromised upon knockdown of SDC-2 and/or -3.
Conclusions: These results demonstrate that FBLN4 and LTBP4 cell interactions via SDC-2 and/or SDC-3 promote elastogenesis by enhancing focal adhesion formation and cell contractility through Erk1/2 and RhoA activation. Similarly, new data supporting the binding of FBLN5 to SDC-4 also suggests that this interaction seems to be involved in the formation of elastic fibers.
Key words: Fibulins, LTBPS, Syndecans, Elastogenesis