Limits...
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.

Show MeSH

Related in: MedlinePlus

ES domain oligomerization is necessary and sufficient for c18-induced motility. (A) Schematic of synthesis of ES domain monomer and dimer. Cleavage of an Fc-ES domain fusion produces free ES monomer. In contrast, cleavage of Fc-ES(Q7→C7) produces an ES domain dimer because of disulfide bond formation between adjacent C7 residues. (B) Nonreducing and reducing SDS-PAGE of purified recombinant human ES monomer and dimer. Migration shift from 40 to 20 kD is noted for ES dimer but not ES monomer upon disulfide reduction with DTT. (C) ES dimer but not ES monomer stimulates migration of HUVECs from pre-formed tubules on Matrigel. HUVECs were plated on Matrigel and allowed to form tubes for 16 h, followed by stimulation with ES monomer (3,000 nM) or ES dimer (50 nM) for the indicated times. Representative fields were photographed under phase–contrast (magnification ×200). (D) Dose response to recombinant human ES dimer. HUVECs were plated on Matrigel in the presence of hES dimer, and after 16 h tubular structures were quantitated by manual counting of central fields and percentage of maximal tube formation was calculated. (E) Induction of motility by Fc-ES fusions which dimerize the ES domain. Preformed HUVEC tubules were treated with Fc-mES(c18) (40 nM), Fc (3,000 nM), or mES(c18) (3,000 nM) and photographed after 30 h (magnification ×40). (F) Summary of motogenic activity of monomeric, dimeric, and trimeric ES domain derivatives.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199214&req=5

Figure 3: ES domain oligomerization is necessary and sufficient for c18-induced motility. (A) Schematic of synthesis of ES domain monomer and dimer. Cleavage of an Fc-ES domain fusion produces free ES monomer. In contrast, cleavage of Fc-ES(Q7→C7) produces an ES domain dimer because of disulfide bond formation between adjacent C7 residues. (B) Nonreducing and reducing SDS-PAGE of purified recombinant human ES monomer and dimer. Migration shift from 40 to 20 kD is noted for ES dimer but not ES monomer upon disulfide reduction with DTT. (C) ES dimer but not ES monomer stimulates migration of HUVECs from pre-formed tubules on Matrigel. HUVECs were plated on Matrigel and allowed to form tubes for 16 h, followed by stimulation with ES monomer (3,000 nM) or ES dimer (50 nM) for the indicated times. Representative fields were photographed under phase–contrast (magnification ×200). (D) Dose response to recombinant human ES dimer. HUVECs were plated on Matrigel in the presence of hES dimer, and after 16 h tubular structures were quantitated by manual counting of central fields and percentage of maximal tube formation was calculated. (E) Induction of motility by Fc-ES fusions which dimerize the ES domain. Preformed HUVEC tubules were treated with Fc-mES(c18) (40 nM), Fc (3,000 nM), or mES(c18) (3,000 nM) and photographed after 30 h (magnification ×40). (F) Summary of motogenic activity of monomeric, dimeric, and trimeric ES domain derivatives.

Mentions: HUVECs were plated into Matrigel-coated 2-well slide chambers as above, and tubes were allowed to form for 16 h. Human ES dimer (50 nM) was added at the start of videomicroscopy (see Fig. 3 C; video available at http://www.jcb.org/cgi/content/full/152/6/1233/DC1 The total video length is 12 h and was captured in a total of 360 frames at 30 frames per hour. Images were captured on a phase–contrast microscope at 200× magnification, saved to disk, and converted to avi format using Image Pro Plus software. The video was converted to Quicktime® 4 format using Vid4Win 2 QT software (shareware).


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)

ES domain oligomerization is necessary and sufficient for c18-induced motility. (A) Schematic of synthesis of ES domain monomer and dimer. Cleavage of an Fc-ES domain fusion produces free ES monomer. In contrast, cleavage of Fc-ES(Q7→C7) produces an ES domain dimer because of disulfide bond formation between adjacent C7 residues. (B) Nonreducing and reducing SDS-PAGE of purified recombinant human ES monomer and dimer. Migration shift from 40 to 20 kD is noted for ES dimer but not ES monomer upon disulfide reduction with DTT. (C) ES dimer but not ES monomer stimulates migration of HUVECs from pre-formed tubules on Matrigel. HUVECs were plated on Matrigel and allowed to form tubes for 16 h, followed by stimulation with ES monomer (3,000 nM) or ES dimer (50 nM) for the indicated times. Representative fields were photographed under phase–contrast (magnification ×200). (D) Dose response to recombinant human ES dimer. HUVECs were plated on Matrigel in the presence of hES dimer, and after 16 h tubular structures were quantitated by manual counting of central fields and percentage of maximal tube formation was calculated. (E) Induction of motility by Fc-ES fusions which dimerize the ES domain. Preformed HUVEC tubules were treated with Fc-mES(c18) (40 nM), Fc (3,000 nM), or mES(c18) (3,000 nM) and photographed after 30 h (magnification ×40). (F) Summary of motogenic activity of monomeric, dimeric, and trimeric ES domain derivatives.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199214&req=5

Figure 3: ES domain oligomerization is necessary and sufficient for c18-induced motility. (A) Schematic of synthesis of ES domain monomer and dimer. Cleavage of an Fc-ES domain fusion produces free ES monomer. In contrast, cleavage of Fc-ES(Q7→C7) produces an ES domain dimer because of disulfide bond formation between adjacent C7 residues. (B) Nonreducing and reducing SDS-PAGE of purified recombinant human ES monomer and dimer. Migration shift from 40 to 20 kD is noted for ES dimer but not ES monomer upon disulfide reduction with DTT. (C) ES dimer but not ES monomer stimulates migration of HUVECs from pre-formed tubules on Matrigel. HUVECs were plated on Matrigel and allowed to form tubes for 16 h, followed by stimulation with ES monomer (3,000 nM) or ES dimer (50 nM) for the indicated times. Representative fields were photographed under phase–contrast (magnification ×200). (D) Dose response to recombinant human ES dimer. HUVECs were plated on Matrigel in the presence of hES dimer, and after 16 h tubular structures were quantitated by manual counting of central fields and percentage of maximal tube formation was calculated. (E) Induction of motility by Fc-ES fusions which dimerize the ES domain. Preformed HUVEC tubules were treated with Fc-mES(c18) (40 nM), Fc (3,000 nM), or mES(c18) (3,000 nM) and photographed after 30 h (magnification ×40). (F) Summary of motogenic activity of monomeric, dimeric, and trimeric ES domain derivatives.
Mentions: HUVECs were plated into Matrigel-coated 2-well slide chambers as above, and tubes were allowed to form for 16 h. Human ES dimer (50 nM) was added at the start of videomicroscopy (see Fig. 3 C; video available at http://www.jcb.org/cgi/content/full/152/6/1233/DC1 The total video length is 12 h and was captured in a total of 360 frames at 30 frames per hour. Images were captured on a phase–contrast microscope at 200× magnification, saved to disk, and converted to avi format using Image Pro Plus software. The video was converted to Quicktime® 4 format using Vid4Win 2 QT software (shareware).

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