Deposition of tropoelastin into the extracellular matrix requires a competent elastic fiber scaffold but not live cells

Kozel, Beth A.; Ciliberto, Christopher; Mecham, Robert P.

doi:10.1016/j.matbio.2004.02.004

Cited by 72 publications

(66 citation statements)

References 46 publications

(44 reference statements)

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“…We also noticed that LOXL-V5 colocalized with elastin globules (Fig. 4J) that have been shown to coalesce to form fibers (21)(22)(23). In general, the immunofluorescent staining pattern obtained with LOXL-V5 was similar to what was observed with LOX-V5 and endogenous LOX and LOXL proteins in our RFL-6 model.…”

Section: The Putative Loxl Pro-region Is Required For the Associationmentioning

confidence: 80%

“…LOX and LOXL expression occurred throughout the culture period, whereas maturation was highest around 3 days of confluency (Fig. 1, A and B) at a time when RFL-6 cells start to produce and assemble extracellular matrix components (21). A band slightly smaller than 200 kDa was detectable after 8 days of confluency and most likely resulted from LOXL polymerization.…”

Section: Endogenous Lox and Loxl Are Targeted To Extracellular Fibers Ofmentioning

confidence: 94%

“…The enzymes were also detected on fibrous extracellular matrix (Fig. 2, A and B) at a time when RFL-6 produced elastic fibers (21). The specificity of the LOXL 204 -393 antibody was established by showing immunoreactivity with an appropriately sized band in 6 M urea extracts of wild type but not LOXL knock-out adult mouse heart and lung (Fig.…”

Section: Endogenous Lox and Loxl Are Targeted To Extracellular Fibers Ofmentioning

confidence: 94%

“…RFL-6 cells are derived from fetal rat lung mesenchyme. They produce LOX (20) and deposit an elaborate fibrillar matrix containing fibrillin-1, fibrillin-2, tropoelastin (21), and other ECM proteins. To characterize the time course of endogenous LOX and LOXL expression, endogenous proteins were extracted from the cell layer 2 days before and 1, 3, 5, and 8 days after confluency.…”

Section: Endogenous Lox and Loxl Are Targeted To Extracellular Fibers Ofmentioning

confidence: 99%

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The Pro-regions of Lysyl Oxidase and Lysyl Oxidase-like 1 Are Required for Deposition onto Elastic Fibers

Thomassin

Werneck

Broekelmann

et al. 2005

Journal of Biological Chemistry

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127

108

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These studies were undertaken to determine how lysyl oxidase (LOX) and lysyl oxidase like-1 (LOXL) enzymes are targeted to their substrates in the extracellular matrix. Full-length LOX/LOXL and constructs containing just the pro-regions of each enzyme localized to elastic fibers when expressed in cultured cells. However, the LOXL catalytic domain without the pro-region was secreted into the medium but did not associate with matrix. Ligand blot and mammalian two-hybrid assays confirmed an interaction between tropoelastin and the pro-regions of both LOX and LOXL. Immunofluorescence studies localized both enzymes to elastin at the earliest stages of elastic fiber assembly. Our results showed that the proregions of LOX and LOXL play a significant role in directing the deposition of both enzymes onto elastic fibers by mediating interactions with tropoelastin. These findings confirmed that an important element of substrate recognition lies in the pro-domain region of the molecule and that the pro-form of the enzyme is what initially interacts with the matrix substrate. These results have raised the interesting possibility that sequence differences between the prodomain of LOX and LOXL account for some of the functional differences observed for the two enzymes.Production of a mature and functional elastic fiber is a complex process that is only partially understood. Monomers of elastin (tropoelastin) are cross-linked in the extracellular space by one or more members of the lysyl oxidase (LO) 3 gene family to form an elastin polymer, which is the functional form of the mature protein. Fibrillin-containing microfibrils are thought to play an important role in the assembly process by serving as a scaffold for aligning cross-linking domains within tropoelastin. Recently, several other proteins, such as members of the fibulin and emilin families, have been suggested to play a role in elastic fiber formation, although their exact function has not yet been determined (1).LOs are extracellular copper-requiring enzymes that catalyze the cross-linking of collagen and elastin through oxidative deamination of lysine or hydroxylysine side chains. The resultant allysine residues can then spontaneously condense with vicinal peptidyl aldehydes or with ⑀-amino groups of peptidyl lysines to generate covalent cross-linkages.There are five members of the LO family: lysyl oxidase (LOX) and lysyl oxidase-like 1-4 (LOXL 1-4) (reviewed in Ref.2). The C-terminal region of all of the LO family members contains the elements required for catalytic activity (the copper binding site, tyrosyl and lysyl residues that contribute to the carbonyl cofactor, and 10 cysteine residues), and the high sequence homology in this region suggests that all family members share a common enzymatic mechanism. The N-terminal regions, in contrast, show the greatest variability in size and sequence.The genes for LOX and LOXL have a similar exon structure consisting of seven exons, five of which (exons 2-6) are of similar size and encode proteins with 76% amino acid iden...

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Section: The Putative Loxl Pro-region Is Required For the Associationmentioning

confidence: 80%

Section: Endogenous Lox and Loxl Are Targeted To Extracellular Fibers Ofmentioning

confidence: 94%

Section: Endogenous Lox and Loxl Are Targeted To Extracellular Fibers Ofmentioning

confidence: 94%

Section: Endogenous Lox and Loxl Are Targeted To Extracellular Fibers Ofmentioning

confidence: 99%

See 2 more Smart Citations

The Pro-regions of Lysyl Oxidase and Lysyl Oxidase-like 1 Are Required for Deposition onto Elastic Fibers

Thomassin

Werneck

Broekelmann

et al. 2005

Journal of Biological Chemistry

Self Cite

127

108

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show abstract

“…The first is that the pre-deposition of a scaffold of fibrillin microfibrils is promoted by close-range, longterm cell interaction with substratum-bound HA4; previous studies have shown soluble tropoelastin precursors to preferentially deposit on such scaffolds, even in the absence of lysyl oxidase-mediated crosslinking, to form a robust structural matrix [27]. A second possibility is that an anionic substratum, as represented by surface-tethered HA4, coacervates tropoelastin molecules and facilitates more efficient lysyl oxidase mediated stabilization via desmosine crosslinking, to form a densely crosslinked elastin matrix.…”

Section: Discussionmentioning

confidence: 99%

Impact of delivery mode of hyaluronan oligomers on elastogenic responses of adult vascular smooth muscle cells

2007

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Our prior studies demonstrated that exogenous supplements of pure hyaluronan (HA) tetramers (HA4) dramatically upregulate elastin matrix synthesis by adult vascular smooth muscle cells (SMCs). Some studies suggest that exogenous HA likely only transiently contacts and signals cells, and may elicit different cell responses when presented on a substrate (e.g., scaffold surface). To clarify such differences, we used a carbodiimide-based chemistry to tether HA4 onto glass, and compared elastin matrix synthesis by SMCs cultured on these substrates, with those cultured with equivalent amounts of exogenous HA4. Tethered HA4-layers were first characterized for homogeneity, topography, and hydrolytic stability using SEM, XPS, AFM, and FACE. In general, mode of HA4 presentation did not influence its impact on SMC proliferation, or cell synthesis of tropoelastin and matrix elastin, relative to non-HA controls; however, surface-tethered HA4 stimulated SMCs to generate significantly greater amounts of elastin-stabilizing desmosine crosslinks, which partially accounts for the greater resistance to enzymatic breakdown of elastin derived from these cultures. Elastin derived from both sets of cultures contained peptide masses that correspond to the predominant peptides present in rat aortic elastin. SEM and TEM showed that HA4 stimulated fibrillin-mediated elastin matrix deposition, and organization into fibrils. Surfaceimmobilized HA4 was particularly conducive to organization of elastin into aggregating fibrils, and their networking to form closely-woven sheets of elastin fibers, as seen in cardiovascular tissues. The results suggest that incorporation of elastogenic HA4 mers onto cell culture substrates or scaffolds is a better approach than exogenous supplementation for in vitro or in vivo regeneration of architecturally and compositionally faithful-, and more stable mimics of native vascular elastin matrices.

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Anatomy and Organization of Human Skin

McGrath

Eady

Pope³

2004

Rook's Textbook of Dermatology

151

117

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Deposition of tropoelastin into the extracellular matrix requires a competent elastic fiber scaffold but not live cells

Cited by 72 publications

References 46 publications

The Pro-regions of Lysyl Oxidase and Lysyl Oxidase-like 1 Are Required for Deposition onto Elastic Fibers

The Pro-regions of Lysyl Oxidase and Lysyl Oxidase-like 1 Are Required for Deposition onto Elastic Fibers

Impact of delivery mode of hyaluronan oligomers on elastogenic responses of adult vascular smooth muscle cells

Anatomy and Organization of Human Skin

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