Microfibril-associated MAGP-2 Stimulates Elastic Fiber Assembly

Raphael Lemaire,Julie Bayle,Robert P Mecham,Robert Lafyatis

doi:10.1074/jbc.m609692200

Abstract

Elastic fibers are complex structures composed of a tropoelastin inner core and microfibril outer mantle guiding tropoelastin deposition. Microfibrillar proteins mainly include fibrillins and microfibril-associated glycoproteins (MAGPs). MAGP-2 exhibits developmental expression peaking at elastic fiber onset, suggesting that MAGP-2 mediates elastic fiber assembly. To determine whether MAGP-2 regulates elastic fiber assembly, we used an in vitro model featuring doxycycline-regulated cells conditionally overexpressing exogenous MAGP-2 and constitutively expressing enhanced green fluorescent protein-tagged tropoelastin. Analysis by immunofluorescent staining showed that MAGP-2 overexpression dramatically increased elastic fibers levels, independently of extracellular levels of soluble tropoelastin, indicating that MAGP-2 stimulates elastic fiber assembly. This was associated with increased levels of matrix-associated MAGP-2. Electron microscopy showed that MAGP-2 specifically associates with microfibrils and that elastin globules primarily colocalize with MAGP-2-associated microfibrils, suggesting that microfibril-associated MAGP-2 facilitates elastic fiber assembly. MAGP-2 overexpression did not change levels of matrix-associated fibrillin-1, MAGP-1, fibulin-2, fibulin-5, or emilin-1, suggesting that microfibrils and other elastic fiber-associated proteins known to regulate elastogenesis do not mediate MAGP-2-induced elastic fiber assembly. Moreover, mutation analysis showed that MAGP-2 does not stimulate elastic fiber assembly through its RGD motif, suggesting that integrin receptor binding does not mediate MAGP-2-induced elastic fiber assembly. Because MAGP-2 interacts with Jagged-1 that controls cell-matrix interaction and cell motility, two key factors in elastic fiber macroassembly, microfibril-associated MAGP-2 may stimulate elastic fiber macroassembly by targeting the release of elastin globules from the cell membrane onto developing elastic fibers.

Highlights

Lung, skin, and all other dynamic tissues and have been essential requirements in the evolution of multicellular organisms
microfibril-associated glycoproteins (MAGPs)-2 Does Not Stimulate Elastic Fiber Assembly by Altering Matrix Assembly of Fibrillin-1—To investigate whether MAGP-2 stimulates elastic fiber assembly by altering matrix assembly of fibrillins, we looked at the effect of MAGP-2 overexpression on matrix-associated fibrillin-1 in our dox-regulated MG-TE cells
MAGP-2 Does Not Stimulate Elastic Fiber Assembly by Altering Matrix-associated Levels of MAGP-1, Fibulin-2, Fibulin-5, or Emilin-1—To further understand the molecular basis for MAGP-2-induced elastic fiber assembly, we investigated whether elastic fiber components other than fibrillin-1 might mediate the stimulating effect of MAGP-2 on elastic fibers, including major structural components of microfibrils MAGP-1, as well as microfibril-elastin interface molecules fibulin-2, fibulin-5, and emilin-1 implicated in elastogenesis [3,4,5]

Summary

Introduction

Lung, skin, and all other dynamic tissues and have been essential requirements in the evolution of multicellular organisms. In this study, using cell lines conditionally overexpressing MAGP-2 and constitutively expressing EGFP2-tagged tropoelastin, we show that microfibril-associated MAGP-2 stimulates elastic fiber assembly independently of its RGD motif and without affecting microfibril assembly or matrix-associated levels of elastic fiber associated molecules, fibulin-2, fibulin-5, and emilin-1.

Results

Conclusion