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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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Motogenic activity of c18 NC1 requires rac and cdc42. (A) Inhibition of c18-stimulated motility by C. difficile toxin B. HUVECs on Matrigel for 12 h were preincubated with or without C. difficile toxin B (50 ng/ml) for 4 h and treated with hES monomer (3,000 nM), hES dimer (50 nM), or c18 NC1 (50 nM) for 16 h, then photographed under phase–contrast (magnification ×200). Trypan blue staining of these cultures revealed <5% toxicity from the toxin treatment. Cell-rounding characteristic of rho inhibition is present in +toxin panels. (B) Inhibition of c18 NC1-stimulated motility by adenovirus-encoded DN alleles of rac and cdc42. Top, HUVEC tubules previously infected with adenovirus encoding racDN, rhoDN, or cdc42DN were treated with hES dimer (75 nM) and photographed (magnification ×200). Strong inhibition of motility by racDN and cdc42DN is present. RhoDN was ineffective at blocking scatter, although cell-rounding characteristic of rho inhibition was observed. Bottom, Parallel cultures were treated identically and processed for immunofluorescence using anti-myc epitope tag (rac, cdc42) or anti-rho (rhoDN and dimer alone), revealing >95% infection with racDN, rhoDN, and cdc42DN.
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Figure 8: Motogenic activity of c18 NC1 requires rac and cdc42. (A) Inhibition of c18-stimulated motility by C. difficile toxin B. HUVECs on Matrigel for 12 h were preincubated with or without C. difficile toxin B (50 ng/ml) for 4 h and treated with hES monomer (3,000 nM), hES dimer (50 nM), or c18 NC1 (50 nM) for 16 h, then photographed under phase–contrast (magnification ×200). Trypan blue staining of these cultures revealed <5% toxicity from the toxin treatment. Cell-rounding characteristic of rho inhibition is present in +toxin panels. (B) Inhibition of c18 NC1-stimulated motility by adenovirus-encoded DN alleles of rac and cdc42. Top, HUVEC tubules previously infected with adenovirus encoding racDN, rhoDN, or cdc42DN were treated with hES dimer (75 nM) and photographed (magnification ×200). Strong inhibition of motility by racDN and cdc42DN is present. RhoDN was ineffective at blocking scatter, although cell-rounding characteristic of rho inhibition was observed. Bottom, Parallel cultures were treated identically and processed for immunofluorescence using anti-myc epitope tag (rac, cdc42) or anti-rho (rhoDN and dimer alone), revealing >95% infection with racDN, rhoDN, and cdc42DN.

Mentions: During NC1- and ES dimer-induced motility, rapidly demarginating cells appeared as early as 1–2 h with prominent lamellipodia, filopodia, and stress fibers (Fig. 2 A; see also videomicroscopy at Fig. 3 C and http://www.jcb.org./cgi/content/full/152/6/1233/DC1). Given the regulation of these structures by rho-family G proteins (Hall 1998), we used C. difficile toxin B which glucosylates and inhibits rho-family GTPases. Preincubation of preformed HUVEC tubules with C. difficile toxin B produced complete blockade of ES dimer– and NC1-induced scatter (Fig. 8 A), with trypan blue staining confirming >95% cell viability (data not shown). To evaluate the potential role of individual rho-family GTPase family members, preformed HUVEC tubules were infected with adenoviruses encoding DN alleles of rac, rho, and cdc42 (Kalman et al. 1999). Strong inhibition of ES dimer–induced motility was observed with infection with racDN and cdc42DN viruses (Fig. 8 B). In contrast, no blockade was obtained with a rhoDN adenovirus, although cell rounding characteristic of stress fiber loss was observed (Fig. 8 B). Immunofluorescence revealed >95% infection of endothelial cells (Fig. 8 B, bottom). These results suggest that rac and cdc42 mediate the motile response to ES oligomers, and parallel findings that HGF-induced scatter is inhibited by racDN but not rhoDN (Ridley et al. 1995).


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)

Motogenic activity of c18 NC1 requires rac and cdc42. (A) Inhibition of c18-stimulated motility by C. difficile toxin B. HUVECs on Matrigel for 12 h were preincubated with or without C. difficile toxin B (50 ng/ml) for 4 h and treated with hES monomer (3,000 nM), hES dimer (50 nM), or c18 NC1 (50 nM) for 16 h, then photographed under phase–contrast (magnification ×200). Trypan blue staining of these cultures revealed <5% toxicity from the toxin treatment. Cell-rounding characteristic of rho inhibition is present in +toxin panels. (B) Inhibition of c18 NC1-stimulated motility by adenovirus-encoded DN alleles of rac and cdc42. Top, HUVEC tubules previously infected with adenovirus encoding racDN, rhoDN, or cdc42DN were treated with hES dimer (75 nM) and photographed (magnification ×200). Strong inhibition of motility by racDN and cdc42DN is present. RhoDN was ineffective at blocking scatter, although cell-rounding characteristic of rho inhibition was observed. Bottom, Parallel cultures were treated identically and processed for immunofluorescence using anti-myc epitope tag (rac, cdc42) or anti-rho (rhoDN and dimer alone), revealing >95% infection with racDN, rhoDN, and cdc42DN.
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Figure 8: Motogenic activity of c18 NC1 requires rac and cdc42. (A) Inhibition of c18-stimulated motility by C. difficile toxin B. HUVECs on Matrigel for 12 h were preincubated with or without C. difficile toxin B (50 ng/ml) for 4 h and treated with hES monomer (3,000 nM), hES dimer (50 nM), or c18 NC1 (50 nM) for 16 h, then photographed under phase–contrast (magnification ×200). Trypan blue staining of these cultures revealed <5% toxicity from the toxin treatment. Cell-rounding characteristic of rho inhibition is present in +toxin panels. (B) Inhibition of c18 NC1-stimulated motility by adenovirus-encoded DN alleles of rac and cdc42. Top, HUVEC tubules previously infected with adenovirus encoding racDN, rhoDN, or cdc42DN were treated with hES dimer (75 nM) and photographed (magnification ×200). Strong inhibition of motility by racDN and cdc42DN is present. RhoDN was ineffective at blocking scatter, although cell-rounding characteristic of rho inhibition was observed. Bottom, Parallel cultures were treated identically and processed for immunofluorescence using anti-myc epitope tag (rac, cdc42) or anti-rho (rhoDN and dimer alone), revealing >95% infection with racDN, rhoDN, and cdc42DN.
Mentions: During NC1- and ES dimer-induced motility, rapidly demarginating cells appeared as early as 1–2 h with prominent lamellipodia, filopodia, and stress fibers (Fig. 2 A; see also videomicroscopy at Fig. 3 C and http://www.jcb.org./cgi/content/full/152/6/1233/DC1). Given the regulation of these structures by rho-family G proteins (Hall 1998), we used C. difficile toxin B which glucosylates and inhibits rho-family GTPases. Preincubation of preformed HUVEC tubules with C. difficile toxin B produced complete blockade of ES dimer– and NC1-induced scatter (Fig. 8 A), with trypan blue staining confirming >95% cell viability (data not shown). To evaluate the potential role of individual rho-family GTPase family members, preformed HUVEC tubules were infected with adenoviruses encoding DN alleles of rac, rho, and cdc42 (Kalman et al. 1999). Strong inhibition of ES dimer–induced motility was observed with infection with racDN and cdc42DN viruses (Fig. 8 B). In contrast, no blockade was obtained with a rhoDN adenovirus, although cell rounding characteristic of stress fiber loss was observed (Fig. 8 B). Immunofluorescence revealed >95% infection of endothelial cells (Fig. 8 B, bottom). These results suggest that rac and cdc42 mediate the motile response to ES oligomers, and parallel findings that HGF-induced scatter is inhibited by racDN but not rhoDN (Ridley et al. 1995).

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