DANCE/fibulin-5 promotes elastic fiber formation in a tropoelastin isoform-dependent manner.
OBJECTIVE
To investigate a function of fibulin-5 in the elastic fiber formation, we studied the molecular interactions among elastin, fibrillin-1, and fibulin-5 in the extracellular space and the maturation of tropoelastin using retinal pigment epithelial cells (ARPE-19).


METHODS
Bacterial recombinant tropoelastin (rTE) was added to ARPE-19 cells overexpressing V5-tagged fibulin-5 (ARPE-Fibulin-5). These elastic fibers were evaluated by immunofluorescence staining, the quantitative analysis of cross-linked amino acids, and semi-quantitative analysis of matrix-associated tropoelastin.


RESULTS
Immunoprecipitation assays revealed that fibulin-5 is able to separately interact with tropoelastin or fibrillin-1 in the culture medium. Moreover, immunofluorescent staining showed that elastin, fibrillin-1, and fibulin-5 co-localize in the extracellular matrix. Desmosine levels were significantly increased in ARPE-Fibulin-5 relative to untransfected cells in spite of equal deposition of tropoelastin by enzyme-linked immunosorbent assay. The addition of a tropoelastin isoform, which lacked the peptide encoded by exon 26A (Delta26A) and could bind to fibulin-5 strongly, led to a larger increase in cross-linking amino acids compared to tropoelastin containing the exon 26A peptide sequence.


CONCLUSIONS
These data provide new insights into the initial steps of elastic fiber assembly and identify fibulin-5 and tropoelastin isoforms as potential targets for the regeneration of elastic fibers in vivo.
Nonaka
Risa
R
Department of Clinical Chemistry, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Tokyo, Japan.
Onoue
Satoshi
S
Wachi
Hiroshi
H
Sato
Fumiaki
F
Urban
Zsolt
Z
Starcher
Barry C
BC
Seyama
Yoshiyuki
Y
D000818:Animals; D002417:Cattle; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000071838:Fibrillin-1; D000071837:Fibrillins; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D020894:Microfibrils / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014333:Tropoelastin / Q000235:genetics / Q000378:metabolism*; D054392:Adipokines
D016326:Extracellular Matrix Proteins; D000071838:Fibrillin-1; D000071837:Fibrillins; D008840:Microfilament Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014333:Tropoelastin; C492373:FBLN5 protein, human; C000605722:FBN1 protein, human; D054392:Adipokines