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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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Autoregulatory inhibition of c18 NC1 by the cleavage product ES monomer. (A) ES monomer inhibits motility induced by c18 NC1 or hES dimer. Left panels, HUVEC tubules on Matrigel for 12 h were treated with ES dimer (50 nM) or NC1/ES trimer (75 nM) for 24 h after 30-min preincubation with human ES monomer (3,000 nM) as appropriate, followed by phase–contrast microscopy (magnification ×40). B, PC12 cells were plated on Matrigel in the presence of ES dimer (50 nM) or NC1/ES trimer (75 nM) for 20 h with or without 30-min preincubation with human ES monomer (3,000 nM) as appropriate, followed by phase–contrast microscopy (magnification ×200). Similar results were obtained with treatment with factors after 12–16-h culture on Matrigel. (C) Dose-dependent reversal of c18 NC1 inhibition of HUVEC tube formation by ES monomer. Increasing concentrations of hES monomer were added to freshly seeded HUVECs on Matrigel, followed after 30 min by addition of c18 NC1 (50 or 200 nM) and quantitation of tube formation after 16 h. Higher concentrations of ES monomer were required to reverse inhibition by 200 nM as opposed to 50 nM NC1. (D) Stimulation of MAPK by c18 NC1 or ES dimer is inhibited by ES monomer. HUVEC tubules on Matrigel for 16 h were preincubated with 3,000 nM hES monomer or PBS for 30 min as appropriate, followed by stimulation with hNC1 or hES dimer (50 nM) for 24 h and Western blotting with anti–phospho- MAPK antibody.
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Figure 9: Autoregulatory inhibition of c18 NC1 by the cleavage product ES monomer. (A) ES monomer inhibits motility induced by c18 NC1 or hES dimer. Left panels, HUVEC tubules on Matrigel for 12 h were treated with ES dimer (50 nM) or NC1/ES trimer (75 nM) for 24 h after 30-min preincubation with human ES monomer (3,000 nM) as appropriate, followed by phase–contrast microscopy (magnification ×40). B, PC12 cells were plated on Matrigel in the presence of ES dimer (50 nM) or NC1/ES trimer (75 nM) for 20 h with or without 30-min preincubation with human ES monomer (3,000 nM) as appropriate, followed by phase–contrast microscopy (magnification ×200). Similar results were obtained with treatment with factors after 12–16-h culture on Matrigel. (C) Dose-dependent reversal of c18 NC1 inhibition of HUVEC tube formation by ES monomer. Increasing concentrations of hES monomer were added to freshly seeded HUVECs on Matrigel, followed after 30 min by addition of c18 NC1 (50 or 200 nM) and quantitation of tube formation after 16 h. Higher concentrations of ES monomer were required to reverse inhibition by 200 nM as opposed to 50 nM NC1. (D) Stimulation of MAPK by c18 NC1 or ES dimer is inhibited by ES monomer. HUVEC tubules on Matrigel for 16 h were preincubated with 3,000 nM hES monomer or PBS for 30 min as appropriate, followed by stimulation with hNC1 or hES dimer (50 nM) for 24 h and Western blotting with anti–phospho- MAPK antibody.

Mentions: Collagen XVIII undergoes proteolytic processing in vivo by cathepsin-L and elastase-like proteases (Wen et al. 1999; Felbor et al. 2000) to generate both NC1 trimers and ES domain monomers (Sasaki et al. 1998; Wen et al. 1999). Because of the clear oligomerization dependence of c18 motogenic activity, we investigated whether the physiologic cleavage product ES monomer might inhibit motility induced by the NC1 ES trimer. Although treatment of HUVECs or PC12 cells with NC1 or ES dimer resulted in characteristic cell motility, pretreatment with 40-fold molar excess of ES monomer before ES oligomer treatment strongly inhibited motogenic activity (Fig. 9, A–C). Identical inhibition of motility was also observed with human ES monomer produced in yeast or murine ES monomer produced in baculovirus (data not shown). In parallel, ES monomer pretreatment also abrogated c18 NC1- and ES dimer–induced MAPK activation (Fig. 9 D) and tyrosine phosphorylation (LaMontagne, K.R., and C.J. Kuo, unpublished observations). These data suggest the possibility of negative autoregulation of endogenous c18 NC1 activity mediated by in vivo proteolytic release of monomeric ES domains and reiterate the requirement for ES domain oligomerization.


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)

Autoregulatory inhibition of c18 NC1 by the cleavage product ES monomer. (A) ES monomer inhibits motility induced by c18 NC1 or hES dimer. Left panels, HUVEC tubules on Matrigel for 12 h were treated with ES dimer (50 nM) or NC1/ES trimer (75 nM) for 24 h after 30-min preincubation with human ES monomer (3,000 nM) as appropriate, followed by phase–contrast microscopy (magnification ×40). B, PC12 cells were plated on Matrigel in the presence of ES dimer (50 nM) or NC1/ES trimer (75 nM) for 20 h with or without 30-min preincubation with human ES monomer (3,000 nM) as appropriate, followed by phase–contrast microscopy (magnification ×200). Similar results were obtained with treatment with factors after 12–16-h culture on Matrigel. (C) Dose-dependent reversal of c18 NC1 inhibition of HUVEC tube formation by ES monomer. Increasing concentrations of hES monomer were added to freshly seeded HUVECs on Matrigel, followed after 30 min by addition of c18 NC1 (50 or 200 nM) and quantitation of tube formation after 16 h. Higher concentrations of ES monomer were required to reverse inhibition by 200 nM as opposed to 50 nM NC1. (D) Stimulation of MAPK by c18 NC1 or ES dimer is inhibited by ES monomer. HUVEC tubules on Matrigel for 16 h were preincubated with 3,000 nM hES monomer or PBS for 30 min as appropriate, followed by stimulation with hNC1 or hES dimer (50 nM) for 24 h and Western blotting with anti–phospho- MAPK antibody.
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Figure 9: Autoregulatory inhibition of c18 NC1 by the cleavage product ES monomer. (A) ES monomer inhibits motility induced by c18 NC1 or hES dimer. Left panels, HUVEC tubules on Matrigel for 12 h were treated with ES dimer (50 nM) or NC1/ES trimer (75 nM) for 24 h after 30-min preincubation with human ES monomer (3,000 nM) as appropriate, followed by phase–contrast microscopy (magnification ×40). B, PC12 cells were plated on Matrigel in the presence of ES dimer (50 nM) or NC1/ES trimer (75 nM) for 20 h with or without 30-min preincubation with human ES monomer (3,000 nM) as appropriate, followed by phase–contrast microscopy (magnification ×200). Similar results were obtained with treatment with factors after 12–16-h culture on Matrigel. (C) Dose-dependent reversal of c18 NC1 inhibition of HUVEC tube formation by ES monomer. Increasing concentrations of hES monomer were added to freshly seeded HUVECs on Matrigel, followed after 30 min by addition of c18 NC1 (50 or 200 nM) and quantitation of tube formation after 16 h. Higher concentrations of ES monomer were required to reverse inhibition by 200 nM as opposed to 50 nM NC1. (D) Stimulation of MAPK by c18 NC1 or ES dimer is inhibited by ES monomer. HUVEC tubules on Matrigel for 16 h were preincubated with 3,000 nM hES monomer or PBS for 30 min as appropriate, followed by stimulation with hNC1 or hES dimer (50 nM) for 24 h and Western blotting with anti–phospho- MAPK antibody.
Mentions: Collagen XVIII undergoes proteolytic processing in vivo by cathepsin-L and elastase-like proteases (Wen et al. 1999; Felbor et al. 2000) to generate both NC1 trimers and ES domain monomers (Sasaki et al. 1998; Wen et al. 1999). Because of the clear oligomerization dependence of c18 motogenic activity, we investigated whether the physiologic cleavage product ES monomer might inhibit motility induced by the NC1 ES trimer. Although treatment of HUVECs or PC12 cells with NC1 or ES dimer resulted in characteristic cell motility, pretreatment with 40-fold molar excess of ES monomer before ES oligomer treatment strongly inhibited motogenic activity (Fig. 9, A–C). Identical inhibition of motility was also observed with human ES monomer produced in yeast or murine ES monomer produced in baculovirus (data not shown). In parallel, ES monomer pretreatment also abrogated c18 NC1- and ES dimer–induced MAPK activation (Fig. 9 D) and tyrosine phosphorylation (LaMontagne, K.R., and C.J. Kuo, unpublished observations). These data suggest the possibility of negative autoregulation of endogenous c18 NC1 activity mediated by in vivo proteolytic release of monomeric ES domains and reiterate the requirement for ES domain oligomerization.

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