Molecular Analysis of Fibulin-5 Function during De Novo Synthesis of Elastic Fibers

Abstract

Elastic fibers contribute to the structural support of tissues and to the regulation of cellular behavior. Mice deficient for the fibulin-5 gene (fbln5 ؊/؊ ) were used to further elucidate the molecular mechanism of elastic fiber assembly. Major elastic fiber components were present in the skin of fbln5 ؊/؊ mice despite a dramatic reduction of mature elastic fibers. We found that fibulin-5 preferentially bound the monomeric form of elastin through N-terminal and C-terminal elastin-binding regions and to a preexisting matrix scaffold through calcium-binding epidermal growth factor (EGF)-like (CB-EGF) domains. We further showed that adenovirusmediated gene transfer of fbln5 was sufficient to regenerate elastic fibers and increase elastic fiber-cell connections in vivo. A mutant fibulin-5 lacking the first 28 amino acids of the first CB-EGF domain, however, was unable to rescue elastic fiber defects. Fibulin-5 thus serves as an adaptor molecule between monomeric elastin and the matrix scaffold to aid in elastic fiber assembly. These results also support the potential use of fibulin-5 as a therapeutic agent for the treatment of elastinopathies.

Loss of fibulin-5 binding to β1 integrins inhibits tumor growth by increasing the level of ROS

“…For immunohistochemistry on fixed tissues, tissues were deparaffinized in xylene, followed by a graded series of ethanol exchanges, and rehydrated in PBS containing 0.2% Tween (PBST). The primary antibodies used were: rabbit anti-phosphorylated histone H3 (phospho-H3) (Upstate Biotechnology, Inc., Lake Placid, NY); rat anti-mouse endothelial cell marker, MECA-32 (Hallmann et al, 1995) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA); rabbit anti-mouse smooth muscle actin (SMA) (RB-9010-P, LabVision, Fremont, CA); goat anti-8-hydroxydeoxyguanosine (8-OHdG) (AB5830, Chemicon, Billerica, MA); rabbit anti-gH2AX (NB100-2280, Novus Biologicals, Littleton, CO); and rabbit anti-mouse Fbln5 (BSYN1923) (Zheng et al, 2007). All antibodies were used at a concentration of 5-10 mg/ml.…”

“…The organization of, and the interaction between, these components within the tumor microenvironment are crucial determinants of tumor survival. Interactions between cells and the ECM stimulate signaling pathways that regulate cellular activities such as survival, proliferation and migration -processes that are crucial to tumor expansion (Albig and Schiemann, 2005;Li et al, 2007). Matricellular proteins, such as fibulin-5 (Fbln5), associate with the matrix and regulate the crosstalk between cells and their surrounding ECM.…”

“…Fbln5 is a member of the fibulin family of proteins and consists of six calcium-binding epidermal growth factor-like repeats, one of which contains an integrin-binding RGD motif and a COOHterminal fibulin-type module (Timpl et al, 2003;Zheng et al, 2007). Fbln5 is crucial for proper assembly of elastic fibers as it tethers tropoelastin to microfibrils where it is crosslinked by lysyl oxidase to form mature elastic fibers (Liu et al, 2004;Zheng et al, 2007).…”

“…Fbln5 is crucial for proper assembly of elastic fibers as it tethers tropoelastin to microfibrils where it is crosslinked by lysyl oxidase to form mature elastic fibers (Liu et al, 2004;Zheng et al, 2007). Mice that are deficient in Fbln5 have fragmented and disorganized elastic fibers leading to loose skin, vascular anomalies and alveolar defects (Yanagisawa et al, 2002).…”

Fibulin-2 Is Dispensable for Mouse Development and Elastic Fiber Formation

“…The protein is approximately 180 kDa in size and forms covalently linked dimers (22). The other four fibulins range between 50 and 100 kDa in size and are present mainly as monomers (8), though dimers of fibulin-5 have recently been described (31).…”