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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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Blockade of the MAPK pathway antagonizes c18-induced motility. (A) Stimulation of MAPK phosphorylation by c18 NC1 and ES dimer. HUVECs on Matrigel in the presence or absence of ES monomer (3,000 nM), ES dimer (50 nM), or c18 NC1 (50 nM), and harvested and analyzed by Western blotting using anti–phospho-MAPK antisera after 24 h. (B) Time kinetics of MAPK activation by ES dimer. HUVECs on Matrigel for 16 h were stimulated with ES dimer (50 nM) or ES monomer (3,000 nM) for the indicated times, followed by harvest and Western blotting with anti–phospho-MAPK antisera. Stimulation was measured by densitometry and represents the average of three independent experiments. Similar results were observed for NC1. (C and D) Inhibition of ES dimer–induced motility by the MEK inhibitor PD98056 but not the p38 inhibitor SB203580. HUVECs were seeded on Matrigel for 16 h followed by 60-min preincubation with or without PD98056 or SB203580 (50 μM), followed by treatment with hES dimer (50 nM) for 12 h, and then harvest for anti–phospho-MAPK Western blot (C) or phase–contrast microscopy (D) (magnification ×40).
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Figure 7: Blockade of the MAPK pathway antagonizes c18-induced motility. (A) Stimulation of MAPK phosphorylation by c18 NC1 and ES dimer. HUVECs on Matrigel in the presence or absence of ES monomer (3,000 nM), ES dimer (50 nM), or c18 NC1 (50 nM), and harvested and analyzed by Western blotting using anti–phospho-MAPK antisera after 24 h. (B) Time kinetics of MAPK activation by ES dimer. HUVECs on Matrigel for 16 h were stimulated with ES dimer (50 nM) or ES monomer (3,000 nM) for the indicated times, followed by harvest and Western blotting with anti–phospho-MAPK antisera. Stimulation was measured by densitometry and represents the average of three independent experiments. Similar results were observed for NC1. (C and D) Inhibition of ES dimer–induced motility by the MEK inhibitor PD98056 but not the p38 inhibitor SB203580. HUVECs were seeded on Matrigel for 16 h followed by 60-min preincubation with or without PD98056 or SB203580 (50 μM), followed by treatment with hES dimer (50 nM) for 12 h, and then harvest for anti–phospho-MAPK Western blot (C) or phase–contrast microscopy (D) (magnification ×40).

Mentions: The MAPK pathway regulates numerous aspects of cell growth, differentiation, and responses to extracellular stimuli (Schaeffer and Weber 1999) and is required for HGF-stimulated motility (Potempa and Ridley 1998). On ECM substrata, only c18 NC1 and ES dimer, and not ES monomer, activated MAPK. MAPK induction was first detectable within 90 min, paralleling the onset of cellular motility, and persisted for >24 h, in contrast to monomer or untreated cultures in which basal MAPK activity declined to undetectable levels at that time (Fig. 7A and Fig. B). These persistent activation kinetics are reminiscent of signaling via the DDR collagen receptor kinases, which require 18 h of exposure for maximum induction of phosphotyrosine (Vogel 1999), and HGF, which also stimulates prolonged (>16 h) MAPK activation (Potempa and Ridley 1998). Stimulation of tyrosine phosphorylation in HUVECs on Matrigel by c18 derivatives also requires ES domain oligomerization (LaMontagne, K.R., and C.J. Kuo, unpublished observations).


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)

Blockade of the MAPK pathway antagonizes c18-induced motility. (A) Stimulation of MAPK phosphorylation by c18 NC1 and ES dimer. HUVECs on Matrigel in the presence or absence of ES monomer (3,000 nM), ES dimer (50 nM), or c18 NC1 (50 nM), and harvested and analyzed by Western blotting using anti–phospho-MAPK antisera after 24 h. (B) Time kinetics of MAPK activation by ES dimer. HUVECs on Matrigel for 16 h were stimulated with ES dimer (50 nM) or ES monomer (3,000 nM) for the indicated times, followed by harvest and Western blotting with anti–phospho-MAPK antisera. Stimulation was measured by densitometry and represents the average of three independent experiments. Similar results were observed for NC1. (C and D) Inhibition of ES dimer–induced motility by the MEK inhibitor PD98056 but not the p38 inhibitor SB203580. HUVECs were seeded on Matrigel for 16 h followed by 60-min preincubation with or without PD98056 or SB203580 (50 μM), followed by treatment with hES dimer (50 nM) for 12 h, and then harvest for anti–phospho-MAPK Western blot (C) or phase–contrast microscopy (D) (magnification ×40).
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Figure 7: Blockade of the MAPK pathway antagonizes c18-induced motility. (A) Stimulation of MAPK phosphorylation by c18 NC1 and ES dimer. HUVECs on Matrigel in the presence or absence of ES monomer (3,000 nM), ES dimer (50 nM), or c18 NC1 (50 nM), and harvested and analyzed by Western blotting using anti–phospho-MAPK antisera after 24 h. (B) Time kinetics of MAPK activation by ES dimer. HUVECs on Matrigel for 16 h were stimulated with ES dimer (50 nM) or ES monomer (3,000 nM) for the indicated times, followed by harvest and Western blotting with anti–phospho-MAPK antisera. Stimulation was measured by densitometry and represents the average of three independent experiments. Similar results were observed for NC1. (C and D) Inhibition of ES dimer–induced motility by the MEK inhibitor PD98056 but not the p38 inhibitor SB203580. HUVECs were seeded on Matrigel for 16 h followed by 60-min preincubation with or without PD98056 or SB203580 (50 μM), followed by treatment with hES dimer (50 nM) for 12 h, and then harvest for anti–phospho-MAPK Western blot (C) or phase–contrast microscopy (D) (magnification ×40).
Mentions: The MAPK pathway regulates numerous aspects of cell growth, differentiation, and responses to extracellular stimuli (Schaeffer and Weber 1999) and is required for HGF-stimulated motility (Potempa and Ridley 1998). On ECM substrata, only c18 NC1 and ES dimer, and not ES monomer, activated MAPK. MAPK induction was first detectable within 90 min, paralleling the onset of cellular motility, and persisted for >24 h, in contrast to monomer or untreated cultures in which basal MAPK activity declined to undetectable levels at that time (Fig. 7A and Fig. B). These persistent activation kinetics are reminiscent of signaling via the DDR collagen receptor kinases, which require 18 h of exposure for maximum induction of phosphotyrosine (Vogel 1999), and HGF, which also stimulates prolonged (>16 h) MAPK activation (Potempa and Ridley 1998). Stimulation of tyrosine phosphorylation in HUVECs on Matrigel by c18 derivatives also requires ES domain oligomerization (LaMontagne, K.R., and C.J. Kuo, unpublished observations).

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