Fibulin-4 deposition requires EMILIN-1 in the extracellular matrix of osteoblasts

Abstract

Tissue microenvironments formed by extracellular matrix networks play an important role in regulating tissue structure and function. Extracellular microfibrillar networks composed of fibrillins and their associated ligands such as LTBPs, fibulins, and EMILINs are of particular interest in this regard since they provide a specialized cellular microenvironment guiding proper morphology and functional behavior of specialized cell types. To understand how cellular microenvironments composed of intricate microfibrillar networks influence cell fate decisions in a contextual manner, more information about the spatiotemporal localization, deposition, and function of their components is required. By employing confocal immunofluorescence and electron microscopy we investigated the localization and extracellular matrix deposition of EMILIN-1 and -2 in tissues of the skeletal system such as cartilage and bone as well as in in vitro cultures of osteoblasts. We found that upon RNAi mediated depletion of EMILIN-1 in primary calvarial osteoblasts and MC3T3-E1 cells only fibulin-4 matrix deposition was lost while other fibulin family members or LTBPs remained unaffected. Immunoprecipitation and ELISA-style binding assays confirmed a direct interaction between EMILIN-1 and fibulin-4. Our data suggest a new function for EMILIN-1 which implies the guidance of linear fibulin-4 matrix deposition and thereby fibulin-4 fiber formation.

Extracellular Interactions between Fibulins and Transforming Growth Factor (TGF)-β in Physiological and Pathological Conditions

“…Fibulin-4 is also expressed in articular chondrocytes and cultured chondrocyte cells [ 91 ]. In primary osteoblast cell culture, proper fibulin-4 fibril formation in the ECM requires the presence of EMILIN (Elastin-Microfibril-Interface-Located-proteIN)-1, a pro-TGF-β processing protein, for modulating collagen homeostasis [ 92 ]. Mutations of fibulin-4 cause an autosomal recessive form of cutis laxa syndrome with aortic aneurysms, arterial tortuosity and stenosis, and minor skin involvement, a different phenotype seen in other forms of cutis laxa either by elastin or fibulin-5 mutation in which skin involvement is the most prominent clinical manifestation [ 93 , 94 ].…”

EMILIN proteins are novel extracellular constituents of the dentin-pulp complex

“…SDS PAGE and immunoblotting was performed by using polyclonal antibodies raised against EMILIN-1, -2, and -3. In vitro binding assays were performed as previously described 30 . Briefly, cells were transfected with plasmids for EMILIN-1, -2, and -3 with a C-terminal HA tag.…”

“…Little is known about the localization and functional interactions of regulators of growth factor activity in the dental pulp ECM. We recently identified EMILINs (Elastin-Microfibril-Interface-Located-proteINs) as novel constituents of skeletal tissues, where they are required for the ECM incorporation of the LOX-binding protein fibulin-4 in skeletal tissues 29 , 30 . EMILINs constitute a family that exert unique functions in the regulation of connective tissue growth factors.…”

The fibrillin microfibril/elastic fibre network: A critical extracellular supramolecular scaffold to balance skin homoeostasis

“…[7] EMILIN-1 may also serve as a modulator in this process as it also interacts with fibulin-4 and fibulin-5, and is targeted to FMF. [43,56,57] Fibulin-4 ablation efficiently inhibits elastogenesis in all elastic tissues including skin leading to perinatal death. [58] Fibulin-5 null mice have a severely disorganized elastic fibre system leading to loose skin (cutis laxa) but survive well into adulthood.…”

“…[61] In vivo data suggest that LTBP-4S interactions with fibulin-5 as well as LTB4-L interaction with fibulin-4 help to orchestrate the elastogenesis process. [60][61][62]64] EMILIN-1 interactions with fibulin-5, tropoelastin and fibulin-4 are also important for the linear deposition of tropoelastin [56,57] fibronectin of around nine postnatal days. [64] Together, these data clearly indicate a functional link between Ltbp-4L and fibulin-4 as a crucial molecular requirement for survival and elastogenesis.…”