Analysis of dermal elastic fibers in the absence of fibulin-5 reveals potential roles for fibulin-5 in elastic fiber assembly

Abstract

Fibulin-5 is a 66 kDa modular, extracellular matrix protein that localizes to elastic fibers. Although in vitro protein-protein binding studies have shown that fibulin-5 binds many proteins involved in elastic fiber formation, the specific role of fibulin-5 in elastogenesis remains unclear. To provide a more detailed analysis of elastic fiber assembly in the absence of fibulin-5, the dermis of wild-type and fibulin-5 gene knockout (Fbln5 −/− ) mice was examined with electron microscopy (EM). Although light microscopy showed apparently normal elastic fibers near the hair follicles and the absence of elastic fibers in the intervening dermis of the Fbln5 −/− mouse, EM revealed the presence of aberrantly assembled elastic fibers in both locales. Instead of the elastin being incorporated into the microfibrillar scaffold, the elastin appeared as globules juxtaposed to the microfibrils. Desmosine analysis showed significantly lower levels of mature cross-linked elastin in the the Fbln5 −/− dermis, however, gene expression levels for tropoelastin and fibrillin-1, the major elastic fiber components, were unaffected. Based on these results, the nature of tropoelastin cross-linking was investigated using domain specific antibodies to lysyl oxidase like-1 (LOXL-1). Immunolocalization with an antibody to the N-terminal pro-peptide, which is cleaved to generate the active enzyme, revealed abundant staining in the Fbln5 −/− dermis and no staining in the wild-type dermis. Overall, these results suggest two previously unrecognized functions for fibulin-5 in elastogenesis; first, to limit the extent of aggregation of tropoelastin monomers and/or coacervates and aid in the incorporation of elastin into the microfibril bundles, and second, to potentially assist in the activation of LOXL-1.

Expression of Versican V3 by Arterial Smooth Muscle Cells Alters Tumor Growth Factor β (TGFβ)-, Epidermal Growth Factor (EGF)-, and Nuclear Factor κB (NFκB)-dependent Signaling Pathways, Creating a Microenvironment That Resists Monocyte Adhesion

“…Consistent with our previous studies (19,28), V3 expression by ASMCs significantly increased mRNA levels of tropoelastin as well as fibulin 5, a component of microfibrils critical in elastic fiber formation (Fig. 1, A and B) (22)(23)(24). Fibulin 1 and fibrillin 1 mRNA levels did not differ between V3-expressing ASMCs and control ASMCs (data not shown).…”

“…Previously, we have demonstrated that expression of the V3 isoform of versican in ASMCs generates an ECM enriched in elastic fibers and that this ECM had reduced capacity to bind monocytes (4,18,19,28). We now show a causal relationship between changes in elastin accumulation and monocyte adhesion such that knocking down tropoelastin or fibulin 5, a component of microfibrils critical for elastic fiber formation (22)(23)(24), partially reverses the inhibition of monocyte adhesion caused by V3 expression. in control and V3-expressing ASMCs cultured for 7 days was detected at mRNA (A) and protein (B) levels using real-time PCR and Western blotting as described under "Materials and Methods."…”

“…TGF␤ is one of the key cytokines that up-regulates tropoelastin by affecting tropoelastin promoter activity and by enhancing tropoelastin transcript stability (29 -32). Our finding that fibulin 5, a key component for proper elastic fiber formation (22)(23)(24), is also up-regulated by V3 through TGF␤ signaling in these ASMCs suggests that V3 expression may also be affecting microfibrillar protein accumulation and influencing the role of fibulin 5 in scaffold formation during elastic fiber deposition (28).…”

“…Tropoelastin and Fibulin 5 Knockdown-To determine whether monocyte adhesion is dependent on elastic fiber accumulation in ECM deposited by ASMCs, tropoelastin, a soluble monomeric subunit of elastic fiber, or fibulin 5, a scaffold protein of microfibrils critical in elastic fiber formation (22)(23)(24), was knocked down using lentiviral shRNA (Santa Cruz Biotechnology, Inc.). Briefly, control or V3-expressing ASMCs were infected with lentiviral particles containing tropoelastin shRNA sequences (CUG-CAUCCAAAGCUGCUAA, CCACUAUCAACCUGGUUCA, and CACAUGCAGUACUGUAAUC) or fibulin 5 (CCUAUUC-CUGUACGUGUAA, GGUCUUGCCAAGAUGUGAA, and CUCUAUCUCUUGCUGCAUU) at a multiplicity of infection of 0.5.…”

Fibulin-4 E57K Knock-in Mice Recapitulate Cutaneous, Vascular and Skeletal Defects of Recessive Cutis Laxa 1B with both Elastic Fiber and Collagen Fibril Abnormalities

“…However, the elastic fibers in the mutant skin are notably shorter, particularly in the dermis between hair follicles. Electron microscopic analyses indicate that ultrastructural abnormalities of the elastic fibers in Fbln4 E57K/E57K mice resemble those previously seen in the fibulin-5 null mouse skin (33). These include an elastin core consisting of small globules with low electron density, disconnection between small elastin aggregates and microfibrils, and an excess of microfibrils without an elastin core.…”