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.
The extracellular matrix (ECM) consists of a protein and carbohydrate substratum comprising the intercellular space not occupied by cell–cell adhesive complexes. Far from a passive support, the ECM has been linked to multiple activities, including structural or adhesive support, growth factor sequestration, and regulation of cell growth, differentiation, and morphogenesis. Proteinaceous ECM components include fibronectin, laminins, vitronectin, and collagens, many of which bind to various αβ integrins with transduction of intracellular signals (Giancotti and Ruoslahti 1999; Streuli 1999).
The collagens comprise a major category of intrinsic ECM proteins, encompassing ∼20 family members united by triple helical structure and Gly-Xaa-Yaa repeats. Many collagen functions have been deduced from mutant phenotypes in humans or homozygous knockout mice, including osteogenesis imperfecta, Alport syndrome, Ehlers-Danlos syndrome, and several chondrodysplasias (Prockop and Kivirikko 1995). On the other hand, collagens are ligands for transmembrane signaling proteins such as the α1β1, α2β1, and α10β1 integrins and discoidin domain receptor (DDR) transmembrane tyrosine kinases which activate downstream responses such as tyrosine phosphorylation and activation of focal adhesion kinase and phospholipase Cγ (Vogel 1999).
The multiplexin subclass of collagens, represented by collagens XV and XVIII (c15, c18), are characterized by multiple interrupted triple helical regions and a unique COOH-terminal endostatin (ES) domain (discussed below) (Oh et al. 1994; Rehn and Pihlajaniemi 1994). Collagen XV/XVIII exhibits striking evolutionary conservation in vertebrates, Caenorhabditis elegans and Drosophila, with sequencing of the C. elegans and Drosophila genomes revealing the presence of only type IV and type XV/XVIII verterbrate collagens, as described in this issue by Ackley et al. 2001 and in recent reviews (Hutter et al. 2000; Hynes and Zhao 2000). The C. elegans and Drosophila homologues more closely resemble collagen XVIII than collagen XV, based on isoform structure and motifs in a putative heparin-binding domain (Sasaki et al. 2000; Ackley et al. 2001).
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.
The ES domain within c18 NC1 has been reported to inhibit endothelial proliferation and migration, induce endothelial apoptosis, and inhibit tumor growth (O'Reilly et al. 1997; Dhanabal et al. 1999a,Dhanabal et al. 1999b; Yamaguchi et al. 1999; Dixelius et al. 2000). In vertebrates, the ES domain is only represented in collagen XVIII and the related collagen XV (Muragaki et al. 1994; Sasaki et al. 1998) and conserved (45% amino acid identity) in C. elegans and Drosophila collagen XVIII homologues. The “hinge” region between the trimerization domain and the ES domain (Fig. 1 A) is cleaved by elastase and/or cathepsin L to release free ES from c18 (Sasaki et al. 1998; Wen et al. 1999; Felbor et al. 2000). It is relevant to the present studies that physiologic proteolysis in the hinge region converts ES from an obligately trimerized form into a monomeric form, as was initially purified (O'Reilly et al. 1997).
In the current work, we describe that the collagen XVIII NC1 domain regulates ECM-dependent motility and morphogenesis of both endothelial and nonendothelial cells in a manner strictly requiring ES domain oligomerization and the activities of rac, cdc42, and the mitogen-activated protein (MAP) kinase (MAPK) pathway. We additionally demonstrate that the motogenic and morphogenic properties of the c18 NC1 trimer are distinct from and in fact antagonized by its physiologic cleavage product ES monomer, and that the c18 NC1 trimer is inhibited by ES monomer in a potential negative autoregulatory loop. Ackley et al. 2001 in this issue describe mechanistically similar findings in which the C. elegans collagen XVIII homologue CLE-1 promotes neuronal motility through its trimerization-competent NC1 domain, whereas CLE-1 ES monomers behave as dominant negative (DN) alleles which inhibit motility induced by wild-type CLE-1. These data implicate domains of the evolutionarily conserved type XVIII collagen in the regulation of cell motility and establish a previously unsuspected mechanism for ECM regulation of motility and morphogenesis.
Materials and Methods
Expression of Recombinant Proteins
Human Fc-human ES proteins, containing the 184–amino acid COOH-terminal ES domain from HSHRDF through SFMTASK, were purified from conditioned medium of stable myeloma transfectants and human ES monomer produced by subsequent enterokinase (EK) cleavage as described previously (Ding et al. 1998; Lo et al. 1998). Disulfide-bonded human ES dimer was produced similarly except that the expression vector pdCs-Fc(D4K)-huEndo was mutated Q7→C7 to produce pdCs-Fc(D4K)-huEndo(C7).
The human collagen XVIII NC1 was produced by PCR with primers 5′-GATC GGCC CAGCC GGCC cat cat cac cat cac cat gac gat gac gat aag tca ggg cag gtg agg ctc tgg gct aca cgc cag g-3′ and 5′-GATC GGATCC cta ctt gga ggc agt cat gaa gc-3′ using human collagen XVIII cDNA (a gift from B. Olsen, Harvard Medical School, Boston, MA) as template amplifying SGQvRL through the COOH terminus. Murine collagen XVIII NC1 was produced identically using the primers 5′-GATC GGCC CAGCC GGCC cat cat cac cat cac cat gac gat gac gat aag GCT GGG cag gtg agg atc tgg gcc aca tac c-3′ and 5′-GATC GGATCC cta ttt gga gaa aga ggt cat gaa gc-3′ from the template mc3b (a gift from B. Olsen), amplifying AGQVRI through the COOH terminus. Murine collagen XV NC1 was amplified from mc15 cDNA (a gift from T. Pihlajaniemi, University of Oulu, Oulu, Finland) with 5′-GATC GGCC CAGCC GGCC CAT CAT CAC CAT CAC CAT GAC GAT GAC GAT AAG gtc aca ggc ctc agc gac atg ggt gac atg cta cag-3′ and reverse primer 5′-GATC GGATCC TCA CTT CCT AGT GTC TGT CAT GAA AC-3′, amplifying LSDM through the COOH terminus. The PCR products hNC1(c18), mNC1(c18), or mNC1(c15) were cloned SfiI-BamHI into pSecTag2A (Invitrogen) in frame with the IgK signal peptide, transfected into 293T cells by calcium phosphate, and conditioned medium-purified over Ni-agarose using the NH2-terminal 6× His tag embedded by PCR.
Two versions of the ES domain of murine collagen 15 were amplified from mc15 cDNA (a gift from T. Pihlajaniemi) by PCR using the forward primers 5′-GATC C CCG GGT TCA GGG GAT GAC GAT GAC GAT AAG TTA AGT GCC AAC TAT GAG AGG CCT G-3′ or 5′-GATC C CCG GGT TCA GGG GAT GAC GAT GAC GAT AAG CCT GTT CTG CAC CTG GTT GCT CTG-3′ and the reverse primer 5′-GATC CTC GAG TCA CTT CCT AGT GTC TGT CAT GAA AC-3′, amplifying LSANYER or PVLHLVA through the COOH terminus. The resultant fragments were ligated XmaI-XhoI into the expression vector pdCs (Lexigen) in frame with a 5′ coding region for murine IgG2α Fc, and Fc-ES(c15) was purified from conditioned medium of stable myeloma transfectants using protein A–Sepharose. No differences were observed using either of the two Fc-ES(c15) recombinant proteins.
SDS-PAGE Analysis of Purified Recombinant Human c18 Fragments
Human ES monomer from Fc-ES EK cleavage or human ES dimer from EK cleavage of Fc-ES(Q7→C7) (analyzed by SDS-PAGE at 5 μg/lane under nonreducing [without DTT] or reducing [with DTT] conditions). The doublet observed with hES monomer represents a four–amino acid NH2-terminal cleavage. Human NC1 ES trimer (40 μg) with or without ethylene glycol bis-succinimidyl succinate (EGS) cross-linking (see Materials and Methods) was analyzed by SDS-PAGE.
Cell Culture and Lines
Human umbilical vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells were purchased from Clonetics and grown in EBM containing EGM-2-MV supplements (vascular endothelial growth factor [VEGF], hepatocyte growth factor [HGF], and hydrocortisone). 293 cells and MDCK cells were obtained from American Type Culture Collection and grown in DME with 10% FCS and l-glutamine. PC12 cells were a gift from M. Greenberg (Children's Hospital, Boston, MA) and grown in DME with 10% horse serum, 5% FCS, penicillin streptomycin, and l-glutamine on collagen I–coated dishes.
Matrigel Tube Formation Assay
Matrigel, an ECM preparation from the Engelbreth-Holm-Swarm tumor, was purchased from Collaborative/BD PharMingen. Multiwell dishes (24-well) were coated with 250 μl Matrigel at 4°C and incubated at 37°C for 20 min followed by seeding with HUVECs, human dermal microvascular endothelial cells, or other cell types in 1 ml complete medium at 50,000 cells/ml. Recombinant ES monomer, dimer, NC1, or Fc-ES fusion proteins were added either at plating or after 16 h of tube formation. Molecular masses used for calculation of molarity were as follows: ES monomer (20 kD), ES dimer (40 kD = 20 × 2), NC1(c18) trimer (114 kD = 38 × 3), and NC1(c15) trimer (93 kD = 31 × 3). Cells were photographed under phase–contrast and images were imported into Adobe Photoshop® or Canvas 5. Where relevant, cells were preincubated with ES monomer (30 min) or Clostridium difficile toxin B (50 ng/ml, 4 h) before addition of hNC1 or ES dimer. HGF (R&D Systems) was used at 50 ng/ml. Tubes were quantitated by manual counting in triplicate of low power fields (40×). Phalloidin-FITC staining for actin was performed after paraformaldehyde fixation using confocal microscopy.
Assay for MAPK Activation
HUVECs on Matrigel for 16 h were preincubated for 60 min with or without PD98056 or SB203580 (50 μM) (Calbiochem), followed by treatment with hES monomer (3,000 nM), hES dimer (50 nM), or c18 NC1 (50 nM). Cells were washed twice in Hepes-buffered saline and lysed with SDS-PAGE sample buffer with β-mercaptoethanol. Lysates were analyzed by SDS-PAGE and Western blotting using anti–phospho-MAPK or extracellular signal–regulated kinase (ERK)2 antisera (New England Biolabs, Inc.). Fold activation was calculated by densitometry of anti–phospho-MAPK Western blots, assigning activity of unstimulated extracts as onefold. No significant differences were observed between untreated and monomer-treated cells.
Adenoviral Infection of Endothelial Tubules
Recombinant adenoviruses encoding a chimeric tetracycline repressor/VP16 transactivator domain (Ad–tTA) or encoding DN alleles of rac or rho under the control of a minimal cytomegalovirus promoter driven by seven tetracycline operator sites have been described previously (Kalman et al. 1999). The construction of Ad-cdc42DN will be described elsewhere. HUVECs were infected at the time of plating on Matrigel in complete medium with Ad-tTA at a multiplicity of infection of 1:1,000. After 12 h, tubule formation was identical to uninfected cells (data not shown), and tubules were infected as appropriate with Ad-racDN, Ad-rhoDN, or Ad-cdc42DN for 4 h at a multiplicity of infection of 1:2,000. Subsequently, cells were treated with hES dimer (75 nM) for 24 h and photographed under phase–contrast. All incubations were in the absence of tetracycline to allow for maximal transactivation of the Ad-DN constructs. Inhibition of motility was not observed with either Ad-tTA or Ad-DN viruses alone (data not shown).
Online Supplemental Material
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).
Inhibition of Endothelial Tube Formation by the Collagen XVIII NC1 Domain
To evaluate potential functions of collagen XVIII, the soluble c18 NC1 domain was produced from conditioned medium of transfected 293T cells and migrated at 38 kD upon SDS-PAGE (Fig. 1 B, left lane) in agreement with published results (Sasaki et al. 1998). Consistent with trimerization, this recombinant protein produced an ∼114-kD species (38 × 3) upon EGS cross-linking (Fig. 1 B, right lane), confirming previous reports documenting an NH2-terminal trimerization domain, migration as a trimer upon gel filtration, and trimeric appearance upon electron microscopy (Sasaki et al. 1998).
We explored the behavior of c18 NC1 in an in vitro morphogenesis assay during which endothelial cells plated on ECM preparations, such as Matrigel, spontaneously aggregate and assemble into densely multicellular capillary-like tubular structures (Folkman and Haudenschild 1980; Grant et al. 1991). Although when used with endothelial cells, this assay recapitulates numerous aspects of angiogenesis, including cellular migration, differentiation, and metalloproteinase activation (Grant et al. 1991), nonendothelial cells such as fibroblasts can undergo similar morphologic changes, and thus this assay may best reflect ECM-induced changes in cell adhesion and morphogenesis. Human or murine c18 NC1 treatment at the time of HUVEC plating on Matrigel strongly inhibited assembly into tubular structures, with cells remaining dispersed and exhibiting a morphology resembling cells on plastic rather than aggregating into characteristic capillary-like tubes (Fig. 1 C). Quantitation of tubular structures by light microscopy revealed a dose-dependent effect with an IC50 of ∼10 nM (Fig. 1 D). Stimulation of proliferation was not detected by BrdU staining or [3H]thymidine incorporation during this time interval, nor was stimulation of cell death detected after recovery of cells from Matrigel and trypan blue staining (data not shown). Identical results were obtained with primary cultures of human microvascular, umbilical artery, or pulmonary artery endothelial cells, and morphological effects were not observed on tissue culture plastic or on gels of purified matrix proteins such as collagen I or fibrin (data not shown).
Induction of Cellular Motility by the Collagen XVIII NC1 Domain
The dispersed phenotype of HUVECs treated with c18 NC1 prompted investigation of the effects of this protein on preestablished endothelial tubules. Strikingly, when applied to HUVEC tubules, recombinant c18 NC1 stimulated active cell motility with migration of cells away from the structures easily observable by 2 h of treatment (Fig. 2 A, top). Phase–contrast microscopy revealed abundant structures resembling filopodia and lamellipodia, which were confirmed upon confocal microscopy for phalloidin-FITC reactivity (Fig. 2 A, bottom) and by videomicroscopy (see below). This cellular migration was more pronounced with time, with cells becoming progressively more dispersed and acquiring a “scattered” phenotype (Fig. 2 B). Preformed tubules were increasingly refractory to dispersion after 16–24 h and motogenic activity was not observed against HUVECs plated on plastic (data not shown).
ES Domain Oligomerization Is Necessary and Sufficient for Motogenic Activity
To localize the motogenic region of c18 NC1, a series of derivative proteins were constructed. The potential involvement of the ES domain comprising the COOH-terminal 184 amino acids of NC1 (Sasaki et al. 1998) was examined with an Fc-ES domain fusion protein (Fc-ES) containing an NH2-terminal human IgG1 Fc fragment, an interposed EK cleavage site, and a COOH-terminal ES domain (Fig. 3 A). EK cleavage of Fc-ES allowed large scale production of ES domains (Fig. 3 A) whose composition and monomeric nature were confirmed by denaturing and nondenaturing SDS-PAGE (Fig. 3 B, left), and by gel filtration and mass spectrometry (data not shown). Human and murine ES monomers thus produced were completely inactive at eliciting motility at 3,000 nM, the highest concentration tested (Fig. 3 C, top), as were murine and human ES monomers produced in baculovirus or yeast (data not shown).
Since the ES domain is physiologically trimerized in the context of collagen XVIII and NC1 (Sasaki et al. 1998) (Fig. 1 A), we evaluated whether c18 motogenic activity required ES domain oligomerization by constructing an artificial ES domain dimer. The ES crystal structure predicts a dimer in which glutamine-7 residues in adjacent subunits are in close proximity (Ding et al. 1998). Fusion of heterologous proteins to an antibody Fc fragment produces obligate dimerization via disulfide bonds in the Fc region (Lo et al. 1998). Mutation of ES glutamine-7 residue to cysteine in the context of Fc-ES fortuitously produced a novel intermolecular disulfide bond between adjacent ES molecules (Fig. 3 A, bottom). EK cleavage of this mutant Fc-ES(Q→C) then liberated free ES dimer migrating at 40 and 20 kD under nonreducing and reducing conditions, respectively (Fig. 3A and Fig. B, right). This ES domain dimer potently reproduced the inhibitory activity of the Fc-ES fusion or NC1 with half-maximal inhibition at ∼10 nM (Fig. 3C and Fig. D). Videomicroscopy of endothelial tubules treated with ES dimer confirmed rapid (<2 h) stimulation of motility, formation of filopodia and lamellipodia, and virtual absence of cell division (see video at http://www.jcb.org/cgi/content/full/152/6/1233/DC1), paralleling findings with c18 NC1 (Fig. 1 and Fig. 2). The activity of the ES dimer (10 nM) versus the inactivity of the ES monomer (>3,000 nM, the highest concentration tested) indicated that the active region of c18 NC1 localized to the ES domain and that oligomerization of the ES domain was both necessary and sufficient for motogenic activity. Further consistent with a requirement for oligomerization, the Fc-ES (wt) protein, in which the ES domain is obligately dimerized, potently stimulated motility from preformed HUVEC tubules, whereas either Fc or ES derived from EK cleavage of Fc-ES was ineffective (Fig. 3 E). Identical stimulation of motility was observed with Fc-ES(Q→C) (data not shown). The activity of c18 derivatives in which the ES domain is oligomerized, versus the inactivity of ES monomers is summarized (Fig. 3 F).
Oligomerized Collagen XV ES Domains Do Not Exhibit Motogenic Activity
Collagen XVIII and collagen XV define the multiplexin subclass of collagens, characterized by multiple interruptions in the triple helical region and a globular COOH-terminal ES domain. The NC1 domains of both c18 and c15 trimerize (Sasaki et al. 2000), and the cognate ES domains exhibit 60% amino acid identity (Muragaki et al. 1994; Oh et al. 1994; Rehn and Pihlajaniemi 1994). Despite these similarities, the c18 and c15 NC1 domains differ strikingly in their abilities to associate with heparin and zinc, in affinity for matrix components exhibited by monomers versus trimers, and a shorter “hinge” region in the NC1 domain in c15 (Sasaki et al. 2000). We produced recombinant c15 NC1 domains from the conditioned medium of transfected 293T cells but observed complete lack of inhibition of HUVEC tube formation on Matrigel at 100 nM, the highest concentration tested (Fig. 4). Similarly, dimeric Fc fusion proteins containing Fc at the NH2 terminus and the collagen XV ES domain at the COOH terminus (Fc-ES[c15]) neither scattered endothelial tubules (Fig. 4) nor antagonized the effects of Fc-ES(c18) at 30-fold molar excess (data not shown). Under these conditions, the motogenic properties of ES domain oligomers are thus highly specific for c18 and not c15, in accordance with divergent structural and functional properties (Muragaki et al. 1994; Oh et al. 1994; Rehn and Pihlajaniemi 1994; Sasaki et al. 2000).
Collagen XVIII NC1 Motogenic Activity Is Not Specific for Endothelial Cells
The growth-inhibitory, migration-inhibitory, or apoptosis-stimulatory activities of the monomeric ES domain have been reported to be selective for endothelial cells and not other cell types (O'Reilly et al. 1997; Dhanabal et al. 1999a,Dhanabal et al. 1999b). On the other hand, the precursor molecule c18 is not only expressed in vascular basement membranes, but also in most epithelial membranes such as liver, lung, and kidney, paralleling collagen IV (Muragaki et al. 1995; Musso et al. 1998; Saarela et al. 1998). Furthermore, the clinical spectrum of Knobloch syndrome has implicated collagen XVIII in diverse functions including neural tube and/or scalp closure and maintenance of retinal structure (Sertie et al. 2000), whereas cle-1 mutations in C. elegans produce neuronal migration deficits (Ackley et al. 2001).
The ability of c18 NC1 or ES dimer to induce motility of nonendothelial cell types was evaluated using PC12 pheochromocytoma cells or 293T embryonic kidney cells plated on Matrigel. Under these plating conditions, these cell lines aggregated into multicellular collections (Fig. 5). However, culture with ES dimer or c18 NC1 produced a dispersed phenotype resembling plastic-plated cells rather than forming multicellular aggregates, with ES monomer completely inactive (Fig. 5). We have also observed similar results with NIH 3T3 fibroblasts and Lewis lung carcinoma (data not shown). The trimeric c18 NC1 domain can thus modulate the matrix-regulated motility and morphogenesis of numerous cell types and possesses considerably broader tissue tropism than described for monomeric c18 ES domains (O'Reilly et al. 1997; Dhanabal et al. 1999a,Dhanabal et al. 1999b; Yamaguchi et al. 1999).
Functional Distinction between the c18 and HGF/MSP Motogens
The motile response to c18 NC1 resembled the activity of the motogens HGF/scatter factor and macrophage stimulatory protein (MSP) (Gherardi and Stoker 1991) which regulate motility, morphogenesis, proliferation, and apoptosis. HGF and MSP have been termed “scatter factors” because of their ability to induce motility and disaggregation of cells from acini formed on tissue culture plastic (Gherardi and Stoker 1991). The motogenic activity of HGF correlates with in vivo augmentation of the metastatic potential of tumorigenic lines (Jeffers et al. 1996a,Jeffers et al. 1996b) and with essential roles in the developmental migration of muscle and neural crest precursors (Tamagnone and Comoglio 1997). Additionally, HGF possesses morphogenic activities which promote angiogenesis and tubulogenesis both in vivo and in vitro (Grant et al. 1993; Tamagnone and Comoglio 1997).
Given the ability of HGF and c18 NC1 to induce motility and disaggregation of cells, effects on HUVEC tube formation were compared. In contrast to c18 NC1 and ES dimers, HGF did not promote motility of HUVECs (Fig. 6, top), despite abundant expression of the HGF receptor c-met on HUVECs (Bussolino et al. 1992). On the other hand, HGF potently disaggregated and scattered MDCK (Fig. 6, bottom) and HepG2 cells (data not shown) on plastic, whereas NC1 and ES dimers were inactive under these conditions (Fig. 6, bottom). MSP was also unable to disaggregate HUVEC tubules on Matrigel (data not shown). Notably, HGF stimulates tube formation in collagen matrices (Bussolino et al. 1992; Grant et al. 1993; Tamagnone and Comoglio 1997), as opposed to the antitubulogenic effects of c18 NC1 described here for endothelial cells. The collagen XVIII NC1 domain therefore defines a novel class of motogenic factor which strictly requires the presence of ECM and is both structurally and functionally distinct from HGF/MSP.
Collagen XVIII NC1 Requires MAPK Activation for Induction of Motility
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).
Preincubation of HUVEC tubules with the MAPK kinase (MEK) inhibitor PD98056 strongly inhibited the ability of ES dimer to elicit cellular motility and MAPK activation in parallel (Fig. 7c and Fig. d), indicating that the MAPK pathway is essential for the c18 motile response. In contrast, inhibition was not observed with SB203580, which inhibits the stress-activated p38 MAPK, indicating a selective requirement for the mitogenic MAPK pathway represented by MEK and MAPK, and not the stress response MAPK pathway represented by p38 and MKK3/4 (Fig. 7c and Fig. d).
Inhibition of ES Oligomer-mediated Motility by Blockade of rac and cdc42
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).
Autoregulatory Inhibition of c18 NC1-stimulated Motility by ES Monomer
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.
An Evolutionarily Conserved ECM Collagen as Motogen and Morphogen
The ECM regulates cellular morphogenesis by serving as a passive structural support, a repository for growth factors, and a ligand for cellular receptors such as integrins and DDR tyrosine kinases (Giancotti and Ruoslahti 1999; Vogel 1999). The collagen XV/XVIII family has been particularly interesting because of a COOH-terminal ES domain not present in other collagens, and strong evolutionary conservation, being one of only two verterbrate basement membrane collagens conserved in C. elegans and Drosophila (Hutter et al. 2000; Hynes and Zhao 2000). Despite this ancestral presence in invertebrate species, the physiologic functions of the XV/XVIII collagens have remained undefined.
In this work, we describe that the ES region of collagen XVIII functions as an effector domain regulating ECM-dependent motility and morphogenesis of numerous cell types. The composite nature of c18 in both vertebrates and C. elegans, with NH2-terminal frizzled/wingless receptor (vertebrate) or unc-40/netrin receptor (C. elegans) domains, and COOH-terminal ES/motogen domains, could certainly facilitate sophisticated and modular control of cell migration by ECM during development and morphogenesis (Muragaki et al. 1995; Rehn et al. 1998; Ackley et al. 2001). The inactivity of the homologous c15 ES domain in dimeric and trimeric forms suggests a highly specific function for c18 (Fig. 4) and is consistent with structural differences between c15 and c18 NC1 domains (Sasaki et al. 2000), although certainly other contexts may be discovered in which c15 NC1 exhibits motogenic activity.
Motility Induced by Collagen XVIII NC1 Requires ES Domain Oligomerization
The c18 motogenic activity localizes to and absolutely requires oligomerization of the ES domain based on several lines of evidence. The COOH-terminal ES domain is obligately trimerized in c18 via intrinsically triple helical collagen structure and a redundant trimerization motif within NC1 (Muragaki et al. 1994; Rehn and Pihlajaniemi 1994; Sasaki et al. 1998). The motogenic activity of the NC1 domain, representing an ES trimer, is reproduced by ES dimers in the form of either Fc-ES or the ES(Q7→C7) dimer. In turn, the activity of the ES(Q7→C7) dimer (IC50 10 nM) contrasts starkly with the inactivity of monomeric ES domains which differ by essentially a single disulfide bond (inactive at 3,000 nM, the highest concentration tested). The stimulation of motility by the pure ES domain dimer but not ES monomers clearly demonstrates that ES oligomerization is both necessary and sufficient for motogenic activity (Fig. 3). This oligomerization requirement is further corroborated by the ability of NC1 and ES dimers, but not ES monomers, to activate MAPK and tyrosine phosphorylation on ECM (Fig. 7 and Fig. 9; LaMontagne, K.R. and C.J. Kuo, unpublished observations).
Theoretically, oligomerization could facilitate c18 action upon specific cellular and/or matrix receptors. On the one hand, ES domain oligomerization could promote cross-linking of transmembrane signaling molecules, such as integrins, by analogy to growth factor receptors. On the other hand, NC1 ES trimers bind with 100–200-fold higher affinity than ES monomers to ECM components such as laminin/nidogen, perlecan, and sulfatides (Sasaki et al. 1998) and could regulate the association of matrix components with cellular receptors. We have observed ES domain binding to both membrane and matrix components, as has been described for FGF, VEGF, HGF, and Wingless (Yayon et al. 1991; Park et al. 1993; Reichsman et al. 1996; Binari et al. 1997; Chirgadze et al. 1999) and are actively investigating these two potential sites of action.
Comparison and Contrast between the c18 and HGF/MSP Motogens
As described here, collagen XVIII represents a novel motogen both structurally and functionally distinct from the archetypal HGF/MSP scatter factor class. The triple helical structure of collagen XVIII, with its three associated globular ES domains, little resembles the kringle structures of the plasminogen-like HGF and MSP proteins (Tamagnone and Comoglio 1997). HGF induces motile scatter responses on plastic within 12 h but promotes tube formation and branching morphogenesis in collagen gels over a 4–6-d period (Tamagnone and Comoglio 1997). In contrast, c18 NC1 does not promote disaggregation on plastic but induces rapid motility and disassembly on Matrigel as rapidly as 2 h, conditions in which HGF and MSP are inactive. Despite obvious differences in structure and context of activity, the motile responses induced by c18 and HGF exhibit similar downstream effector requirements for MAPK, rac, and cdc42 but not rho (Ridley et al. 1995; Potempa and Ridley 1998; Fig. 7 and Fig. 8). The distinct activities of c18 and HGF suggest the potential to provide regulatory complexity during developmental morphogenesis and tissue remodeling.
Inhibition of ECM-induced Morphogenesis by the Collagen XVIII NC1 Domain
Culture of endothelial and nonendothelial cells on rich ECM preparations such as Matrigel is associated with pronounced alterations in cellular morphogenesis and adhesion. In the case of endothelial cells, these morphologic changes resemble the formation of vascular “tubes” with recapitulation of angiogenic processes including branching, migration, and differentiation (Folkman and Haudenschild 1980; Grant et al. 1991). On the other hand, the significance of tube-like structures and aggregates formed by nonendothelial cell types such as fibroblasts and epithelial cells on ECM preparations is less clear. As described here, the c18 NC1 domain potently reverts ECM-induced morphologic changes of both endothelial and nonendothelial cell types, with cells assuming a morphology resembling cells plated on plastic. It thus seems likely that the c18 NC1 domain represents a previously unsuspected inhibitor of ECM-induced morphogenesis. Additionally, the long-described aggregation and morphogenetic responses of cells to complex ECM preparations such as Matrigel could reflect a property which is not endothelial specific but rather represents a highly conserved, c18 NC1-sensitive response to ECM component(s) exhibited by diverse endothelial and nonendothelial cell types. These ECM components likely do not include collagen I or fibrin in isolation, since we have not observed effects of c18 NC1 against cells cultured on gels composed of these purified proteins (data not shown).
Negative Autoregulation by the Cleavage Product ES Monomer
The monomeric ES domain of c18 has been reported to be an antiangiogenic agent inhibiting endothelial cell proliferation and migration and inducing endothelial apoptosis in systems not using the ECM (O'Reilly et al. 1997; Dhanabal et al. 1999a,Dhanabal et al. 1999b; Yamaguchi et al. 1999; Dixelius et al. 2000). Here, we describe that the trimeric precursor c18 NC1 exhibits motility-inducing and morphogenic activities which are distinct from and in fact antagonized by the monomeric ES domain. Indeed, although c18 NC1 promotes motility, scatter, and MAPK induction, these activities are not exhibited by and are actually inhibited by ES monomer on ECM substrata (Fig. 9). This antagonistic relationship is potentially consistent with our inability to detect responses attributed to ES monomer such as inhibition of proliferation or apoptosis induction after c18 NC1 treatment (O'Reilly et al. 1997; Dhanabal et al. 1999a,Dhanabal et al. 1999b; Yamaguchi et al. 1999; Dixelius et al. 2000) (although these reports did not utilize ECM substrata) and reports in which ES monomers actually enhance rather than inhibit endothelial tube formation (Dixelius et al. 2000). Additionally, we have not observed significant antiangiogenic activity of c18 NC1 or ES dimers in corneal micropocket assays, in contrast to reports with ES monomers (Dixelius et al. 2000). It has been reported that both ES monomers and NC1 inhibit HUVEC migration in VEGF-based transwell chambers (although these assays did not employ matrix substrata, required endothelial cells of defined passage number, and required preincubation with factor [Yamaguchi et al. 1999]). Notably, an opposing relationship between ES monomer and trimeric c18 has been suggested based on purely structural considerations (Schuppan et al. 1998).
Both full-length c18 and fragments corresponding to NC1 and ES exist in both tissues and serum (Standker et al. 1997; Sasaki et al. 1998; John et al. 1999; Wen et al. 1999). Given the description of processing proteases which cleave monomeric ES from trimeric c18/NC1 (Wen et al. 1999; Felbor et al. 2000), the in vivo proteolytic cleavage of ES could constitute an autoregulatory feedback loop which terminates and actively opposes the activity of c18.
Evolutionarily Conserved Oligomerization-dependent Motility Regulation by the Collagen XVIII NC1/ES Domain
Among the twenty-odd vertebrate collagen genes, only collagen IV and collagen XV/XVIII are conserved within the C. elegans and Drosophila genomes (Prockop and Kivirikko 1995; Hutter et al. 2000), consistent with an evolutionarily conserved function of c18. Ackley et al. 2001 in this issue characterize the C. elegans collagen XVIII homologue cle-1 which exhibits numerous structural similarities with vertebrate collagen XVIII, including conserved intron/exon boundaries, a triple helical collagenous domain, trimeric properties of the NC1 domain, and 45% amino acid identity in the ES domain. Notably, neuronal migration and axonal pathfinding deficits in cle-1 mutants lacking the COOH-terminal ES domain are rescued only by trimeric CLE-1 NC1 and not by monomeric CLE-1 ES domains. Similarly, neuronal motility stimulated by wild-type trimeric CLE-1 is dominantly inhibited by overexpression of the physiologic cleavage product ES monomer but not by trimeric CLE-1 NC1, consistent with oligomerization dependence and negative autoregulation. The extensive structural and mechanistic parallels between C. elegans cle-1 and vertebrate c18 suggest that collagen XVIII homologues throughout evolution regulate cell motility in a manner both dependent on ES domain oligomerization and negatively autoregulated by ES monomers. This convergence of function in organisms as diverse as vertebrates and C. elegans should provide a mechanistic foundation for elaborating the roles of c18 in motility-dependent processes such as angiogenesis, neurogenesis, branching morphogenesis, and metastasis in both vertebrate and invertebrate species.
We thank C. Chartier, S. Artandi, H.-P. Biemann, J.F. Kuo, G. Bergers, W. Wen, and D. Wiley for helpful discussions and John McCoy and the Genetics Institute for providing EK.
C.J. Kuo was supported by a Howard Hughes Medical Institute Physician-Scientist Fellowship. K.R. LaMontagne was supported by the Garrett B. Smith Foundation and a Leukemia and Lymphoma Society Career Development award. D. Kalman was supported by a grant from the National Cancer Institute (R35CA44338) to J. Michael Bishop. R. Christofferson was supported by the Swedish Children's Cancer Fund and Her Royal Highness Crown Princess Louisa's Fund for Child Care. Y.-H. Ding was supported by the Howard Hughes Medical Institute and a National Institutes of Health grant to D. Wiley. This work was also supported by the Howard Hughes Medical Institute and the National Institutes of Health.
The online version of this article contains supplemental material.
Abbreviations used in this paper: DDR, discoidin domain receptor; DN, dominant negative; ECM, extracellular matrix; EGS, ethylene glycol bis-succinimidyl succinate; EK, enterokinase; ERK, extracellular signal–regulated kinase; ES, endostatin; HGF, hepatocyte growth factor; HUVEC, human umbilical vein endothelial cell; MAP, mitogen-activated protein; MAPK, MAP kinase; MEK, MAPK kinase; MSP, macrophage stimulatory protein; NC, noncollagenous; VEGF, vascular endothelial growth factor.