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Oligomerization-dependent regulation of motility and morphogenesis by the collagen XVIII NC1/endostatin domain.

Kuo CJ, LaMontagne KR, Garcia-Cardeña G, Ackley BD, Kalman D, Park S, Christofferson R, Kamihara J, Ding YH, Lo KM, Gillies S, Folkman J, Mulligan RC, Javaherian K - J. Cell Biol. (2001)

Bottom Line: Here, the c18 NC1 domain functioned as a motility-inducing factor regulating the extracellular matrix (ECM)-dependent morphogenesis of endothelial and other cell types.This motogenic activity required ES domain oligomerization, was dependent on rac, cdc42, and mitogen-activated protein kinase, and exhibited functional distinction from the archetypal motogenic scatter factors hepatocyte growth factor and macrophage stimulatory protein.These data indicate that the collagen XVIII NC1 region encodes a motogen strictly requiring ES domain oligomerization and suggest a previously unsuspected mechanism for ECM regulation of motility and morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Children's Hospital, Harvard Medical School, Boston. Massachusetts 02115, USA. cjkuo@stanford.edu

ABSTRACT
Collagen XVIII (c18) is a triple helical endothelial/epithelial basement membrane protein whose noncollagenous (NC)1 region trimerizes a COOH-terminal endostatin (ES) domain conserved in vertebrates, Caenorhabditis elegans and Drosophila. Here, the c18 NC1 domain functioned as a motility-inducing factor regulating the extracellular matrix (ECM)-dependent morphogenesis of endothelial and other cell types. This motogenic activity required ES domain oligomerization, was dependent on rac, cdc42, and mitogen-activated protein kinase, and exhibited functional distinction from the archetypal motogenic scatter factors hepatocyte growth factor and macrophage stimulatory protein. The motility-inducing and mitogen-activated protein kinase-stimulating activities of c18 NC1 were blocked by its physiologic cleavage product ES monomer, consistent with a proteolysis-dependent negative feedback mechanism. These data indicate that the collagen XVIII NC1 region encodes a motogen strictly requiring ES domain oligomerization and suggest a previously unsuspected mechanism for ECM regulation of motility and morphogenesis.

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Inhibition of in vitro endothelial tube assembly by collagen XVIII NC1. (A) Topology of collagen XVIII. Signal peptide, triple helical collagenous repeats, NC1 domain, and a protease-sensitive “hinge” region interposed within the trimerization and ES motifs are indicated. Dotted line indicates alternative splice which occurs in nonhepatic tissues and removes the frizzled homology domain. (B) Production of recombinant c18 NC1. Human c18 NC1 purified from supernatant of stably transfected 293T cells was analyzed by SDS-PAGE with or without EGS cross-linking. Oligomeric forms corresponding to NC1 trimer at ∼114 kD (38 × 3) and 76-kD species from incomplete cross-linking (38 × 2) are indicated. (C) Recombinant c18 NC1 inhibits in vitro endothelial tube formation. HUVECs were plated on Matrigel in the absence (untreated) or presence of murine or human c18 NC1 at 50 nM and photographed under phase–contrast after 16 h (magnification ×40). A complete lack of tubular structures and a dispersed cell phenotype are apparent in the NC1-treated wells. (D) Dose-dependent inhibition of endothelial tube formation by recombinant c18 NC1. Indicated amounts of human or mouse c18 NC1 were added at the time of HUVEC seeding onto Matrigel-covered wells. Tubular structures were quantitated by manual counting of low power fields after 16 h and percent inhibition was expressed using untreated wells as 100%.
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Figure 1: Inhibition of in vitro endothelial tube assembly by collagen XVIII NC1. (A) Topology of collagen XVIII. Signal peptide, triple helical collagenous repeats, NC1 domain, and a protease-sensitive “hinge” region interposed within the trimerization and ES motifs are indicated. Dotted line indicates alternative splice which occurs in nonhepatic tissues and removes the frizzled homology domain. (B) Production of recombinant c18 NC1. Human c18 NC1 purified from supernatant of stably transfected 293T cells was analyzed by SDS-PAGE with or without EGS cross-linking. Oligomeric forms corresponding to NC1 trimer at ∼114 kD (38 × 3) and 76-kD species from incomplete cross-linking (38 × 2) are indicated. (C) Recombinant c18 NC1 inhibits in vitro endothelial tube formation. HUVECs were plated on Matrigel in the absence (untreated) or presence of murine or human c18 NC1 at 50 nM and photographed under phase–contrast after 16 h (magnification ×40). A complete lack of tubular structures and a dispersed cell phenotype are apparent in the NC1-treated wells. (D) Dose-dependent inhibition of endothelial tube formation by recombinant c18 NC1. Indicated amounts of human or mouse c18 NC1 were added at the time of HUVEC seeding onto Matrigel-covered wells. Tubular structures were quantitated by manual counting of low power fields after 16 h and percent inhibition was expressed using untreated wells as 100%.

Mentions: Collagen XVIII (c18) is a ubiquitous component of endothelial and epithelial basement membranes (Muragaki et al. 1995; Musso et al. 1998; Saarela et al. 1998) and has been identified recently as the genetic lesion in Knobloch syndrome, an autosomal recessive condition characterized by scalp and/or neural tube closure deficits and retinal degeneration (Sertie et al. 2000). In addition to triple helical repeats, the c18 gene contains an alternative exon encoding a domain homologous to the Drosophila tissue polarity gene frizzled (see Fig. 1 A) (Rehn et al. 1998; Saarela et al. 1998). The 38-kD COOH-terminal extent of c18 is nontriple helical and is designated noncollagenous (NC)1 domain. Within NC1, a nontriple helical trimerization domain redundantly trimerizes the globular COOH-terminal 20-kD ES domains (see Fig. 1 A) (Sasaki et al. 1998). The NC1 domain represents a predominant tissue form of c18 in numerous tissues, including liver and lung, and is particularly amenable to biochemical study, since it is easily produced as soluble recombinant protein (Sasaki et al. 1998) as opposed to full-length collagen XVIII.


Oligomerization-dependent regulation of motility and morphogenesis by the collagen XVIII NC1/endostatin domain.

Kuo CJ, LaMontagne KR, Garcia-Cardeña G, Ackley BD, Kalman D, Park S, Christofferson R, Kamihara J, Ding YH, Lo KM, Gillies S, Folkman J, Mulligan RC, Javaherian K - J. Cell Biol. (2001)

Inhibition of in vitro endothelial tube assembly by collagen XVIII NC1. (A) Topology of collagen XVIII. Signal peptide, triple helical collagenous repeats, NC1 domain, and a protease-sensitive “hinge” region interposed within the trimerization and ES motifs are indicated. Dotted line indicates alternative splice which occurs in nonhepatic tissues and removes the frizzled homology domain. (B) Production of recombinant c18 NC1. Human c18 NC1 purified from supernatant of stably transfected 293T cells was analyzed by SDS-PAGE with or without EGS cross-linking. Oligomeric forms corresponding to NC1 trimer at ∼114 kD (38 × 3) and 76-kD species from incomplete cross-linking (38 × 2) are indicated. (C) Recombinant c18 NC1 inhibits in vitro endothelial tube formation. HUVECs were plated on Matrigel in the absence (untreated) or presence of murine or human c18 NC1 at 50 nM and photographed under phase–contrast after 16 h (magnification ×40). A complete lack of tubular structures and a dispersed cell phenotype are apparent in the NC1-treated wells. (D) Dose-dependent inhibition of endothelial tube formation by recombinant c18 NC1. Indicated amounts of human or mouse c18 NC1 were added at the time of HUVEC seeding onto Matrigel-covered wells. Tubular structures were quantitated by manual counting of low power fields after 16 h and percent inhibition was expressed using untreated wells as 100%.
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Related In: Results  -  Collection

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Figure 1: Inhibition of in vitro endothelial tube assembly by collagen XVIII NC1. (A) Topology of collagen XVIII. Signal peptide, triple helical collagenous repeats, NC1 domain, and a protease-sensitive “hinge” region interposed within the trimerization and ES motifs are indicated. Dotted line indicates alternative splice which occurs in nonhepatic tissues and removes the frizzled homology domain. (B) Production of recombinant c18 NC1. Human c18 NC1 purified from supernatant of stably transfected 293T cells was analyzed by SDS-PAGE with or without EGS cross-linking. Oligomeric forms corresponding to NC1 trimer at ∼114 kD (38 × 3) and 76-kD species from incomplete cross-linking (38 × 2) are indicated. (C) Recombinant c18 NC1 inhibits in vitro endothelial tube formation. HUVECs were plated on Matrigel in the absence (untreated) or presence of murine or human c18 NC1 at 50 nM and photographed under phase–contrast after 16 h (magnification ×40). A complete lack of tubular structures and a dispersed cell phenotype are apparent in the NC1-treated wells. (D) Dose-dependent inhibition of endothelial tube formation by recombinant c18 NC1. Indicated amounts of human or mouse c18 NC1 were added at the time of HUVEC seeding onto Matrigel-covered wells. Tubular structures were quantitated by manual counting of low power fields after 16 h and percent inhibition was expressed using untreated wells as 100%.
Mentions: Collagen XVIII (c18) is a ubiquitous component of endothelial and epithelial basement membranes (Muragaki et al. 1995; Musso et al. 1998; Saarela et al. 1998) and has been identified recently as the genetic lesion in Knobloch syndrome, an autosomal recessive condition characterized by scalp and/or neural tube closure deficits and retinal degeneration (Sertie et al. 2000). In addition to triple helical repeats, the c18 gene contains an alternative exon encoding a domain homologous to the Drosophila tissue polarity gene frizzled (see Fig. 1 A) (Rehn et al. 1998; Saarela et al. 1998). The 38-kD COOH-terminal extent of c18 is nontriple helical and is designated noncollagenous (NC)1 domain. Within NC1, a nontriple helical trimerization domain redundantly trimerizes the globular COOH-terminal 20-kD ES domains (see Fig. 1 A) (Sasaki et al. 1998). The NC1 domain represents a predominant tissue form of c18 in numerous tissues, including liver and lung, and is particularly amenable to biochemical study, since it is easily produced as soluble recombinant protein (Sasaki et al. 1998) as opposed to full-length collagen XVIII.

Bottom Line: Here, the c18 NC1 domain functioned as a motility-inducing factor regulating the extracellular matrix (ECM)-dependent morphogenesis of endothelial and other cell types.This motogenic activity required ES domain oligomerization, was dependent on rac, cdc42, and mitogen-activated protein kinase, and exhibited functional distinction from the archetypal motogenic scatter factors hepatocyte growth factor and macrophage stimulatory protein.These data indicate that the collagen XVIII NC1 region encodes a motogen strictly requiring ES domain oligomerization and suggest a previously unsuspected mechanism for ECM regulation of motility and morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Children's Hospital, Harvard Medical School, Boston. Massachusetts 02115, USA. cjkuo@stanford.edu

ABSTRACT
Collagen XVIII (c18) is a triple helical endothelial/epithelial basement membrane protein whose noncollagenous (NC)1 region trimerizes a COOH-terminal endostatin (ES) domain conserved in vertebrates, Caenorhabditis elegans and Drosophila. Here, the c18 NC1 domain functioned as a motility-inducing factor regulating the extracellular matrix (ECM)-dependent morphogenesis of endothelial and other cell types. This motogenic activity required ES domain oligomerization, was dependent on rac, cdc42, and mitogen-activated protein kinase, and exhibited functional distinction from the archetypal motogenic scatter factors hepatocyte growth factor and macrophage stimulatory protein. The motility-inducing and mitogen-activated protein kinase-stimulating activities of c18 NC1 were blocked by its physiologic cleavage product ES monomer, consistent with a proteolysis-dependent negative feedback mechanism. These data indicate that the collagen XVIII NC1 region encodes a motogen strictly requiring ES domain oligomerization and suggest a previously unsuspected mechanism for ECM regulation of motility and morphogenesis.

Show MeSH
Related in: MedlinePlus