Vol. 9, 355-365, May 1998 Cell Growth & Differentiation 355

Differential Effects of Hepatocyte Growth Factor Isoforms onEpithelial and Endothelial Tubulogenesis1

Roberto Montesano,2 Jesus V. Soriano,Katherine M. Malinda, M. Lourdes Ponce,Anna Bafico, Hynda K. Klelnman, Donald P. Bottaro,and Stuart A. Aaronson

Department of Morphology, university Medical Center, Geneva 4,Switze,land [R. M., J. v. S.]; Laboratory of Developmental Biology,Natlonal Institute of Dental Research [K M. M., M. L P., H. K. KJ, andLaboratory of Cellular and Molecular Biology, National Cancer Institute[D. P. B.], NIH, Bethesda, Maryland 20892; and Derald H. RuttenbergCancer Center, Mount 5in� Medical Center, New York. New York10029[kB., S.A.A.]

Abstract

Hepatocyte growth factor (HGF)/scatter factor (SF) is apleiotropic cytokine that acts as a mitogen, motogen,and morphogen for a variety of cell types. HGF/NKIand HGFINK2 are two naturally occurring truncatedvariants of HGF/SF, which extend from the NH2terminus through the first and second kringle domain,respectively. Although these variants have beenreported to have agonistic or antagonistic activityrelative to HGF/SF in assays of cell proliferation andmotility, their potential morphogenic actMty has notbeen investigated. To address this issue, we assessedthe ability of HGF/NKI and HGFINK2 to induce tube

formation by (a) McF-IOA mammary epithelial cellsgrown within collagen gels and (b) human umbilicalvein endothelial (HUVE) cells grown on Matrigel. Wefound that HGF/NKI stimulated tubulogenesis by both

MCF-IOA and HUVE cells, whereas HGF/NK2 did notstimulate tubulogenesis, but efficiently antagonized themorphogenic effect of full-length HGF/SF. HGFINKIand HGF/NK2 also had agonistic and antagonisticeffects, respectively, on MCF-IOA cell proliferation andHUVE cell migration. These results demonstrate thatHGF/NKI, which only consists of the NH2-terminalhairpin and first kringle domain, Is sufficient to activatethe intracellular signaling pathways required to inducemorphogenic responses in epithellal and endothelialcells. In contrast, HGFINK2, which differs from HGF/NK1 by the presence of the second krlngle domain, isdevoid of intrinsic activity but opposes the effects ofHGF/SF. The differential properties of the two HGF/SF

Received 1/26/98; accepted 3/18/98.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 u.s.c. Section 1734 solely to mdi-cate this fact.1 ThIs study was supported by 5wlss National Science Foundation Grant31-43364.95 (to A. M.) and by Grant IRol-cA 71672-O1A1 (to S. A. A.).2 To whom requests for reprints should be addressed, at Department ofMorphology. CMU, 1 rue MicheI-Servet, CH-1211 Geneva 4, 5witzerland.Phone: 41221702 52 73; Fax: 4122/347 33 34; E-mail: roberto.montesanoOniedecine.unige.ch.

isoforms provide a basis for the design of more potentHGF/SF agonists and antagonists.

Introduction

HGF3/SF is a multifunctional cytokine, secreted primarily bycells of mesenchymal origin, that acts as a paracrine medi-ator of proliferation (1), motility (2), and morphogenesis (3).Originally identified as a hepatocyte-specific mitogen (4-7),HGF/SF was subsequently demonstrated to promote theproliferation and migration of a broad spectrum of cell types(reviewed in Refs. 8-13). An additional, unique property ofHGF/SF is its morphogenic activity, i.e., the ability to transmitinformation that determines the spatial arrangement of epi-thelial and endothelial cells. Thus, HGF/SF induces formationof branching tubules by kidney and mammary gland epithe-hal cells grown in collagen gels (3, 14-17), promotes tubu-

logenesis in organocultures of kidney and mammary glandexplants (18-20), and stimulates endothelial cell organizationinto capillary-like tubes in vitro as well as angiogenesis in vivo

(21-23). These properties are likely to account for the impor-tant role of HGF/SF in organogenesis and tissue regeneration(12, 24-28).

The mitogenic, motogenic, and morphogenic responses toHGF/SF are mediated through the tyrosine kinase receptorc-Met (29-35). The mature form of c-Met consists of a Mr-50,000 extracellular a chain covalently linked to a Mr-140,000 �3 chain, which has an extracellular domain in-volved in HGF/SF binding, a transmembrane domain, and a

cytoplasmic tyrosine kinase domain. Ugand binding induces

kinase activation and autophosphorylation of several tyro-sine residues located in the (3 chain. A two-tyrosine motiflocated in the COOH-terminal tail then acts as a docking sitefor multiple SH2 domain-containing adaptor or transducerproteins (for recent reviews, see Refs. 13 and 36).

Structurally, HGF/SF has similarities to plasminogen andserine proteases involved in coagulation and fibrinolysis.

HGF/SF is secreted as a M, �90,000 inactive monomer,which is proteolytically processed to generate a functionaldisulfide-linked heterodimer consisting of a M, �60,000heavy chain and a M� �30,000 light chain. The heavy chaincontains an NH2-terminal hairpin loop (N) and four consec-utive kringle domains (Ki , K2, K3, and K4), which have acharacteristic folding pattern defined by three internal disul-fide bonds. The light chain has the structure of a serineprotease, but it lacks proteolytic activity, because two ofthree residues required for catalysis by serine proteases arenot conserved (37, 38). Binding of HGF/SF to the c-Met

3 The abbreviations used are: HGF, hepatocyte growth factor SF, scatterfactor, MDCK, Madin-Darby canine kidney; HUVE, human umbilical veinendothelial; CHO, Chinese hamster ovary; ECGS, endothelial cell growthsupplement.

356 Differential Morphogenic Activity of HGF Isoforms

receptor appears to be mediated primarily by the N, Ki , andpossibly K2 domains (39-45).

Two naturally occurring truncated variants of HGF/SF havebeen identified, which are encoded by alternative transcriptsand extend from the NH2 terminus through either the first orthe second kringle domain (40, 43, 46, 47). These smallerisoforms, designated HGF/NK1 and HGF/NK2, respectively,retain high-affinity binding to c-Met and have been variouslydescribed as having agonistic or antagonistic activity relativeto HGF/SF, depending on the assay used and the target cell(40, 42-44, 47-52). Thus, Chan et a!. (40) reported that inhuman mammary epithelial cells, HGF/NK2 has no growth-promoting activity but can block the mitogenic response tofull-length HGF/SF. The lack of mitogenic activity of HGF/NK2 was confirmed in rat hepatocytes by Hartmann et a!.(43), who found, however, that HGF/NK2 is able to stimulatescattering of MDCK cells, albeit at much higher concentra-tions than HGF/SF. The shorter variant HGF/NK1 was shown

to inhibit HGF/SF-induced DNA synthesis in rat hepatocytes(44), but to possess mitogenic activity for human mammaryepithelial cells and scattering activity for MDCK cells withnearly the potency of HGF/SF (47, 52). Finally, recent studieshave provided evidence that the agonistic/antagonistic ac-tivities of HGF/NK1 and HGF/NK2 in receptor binding andproliferation assays are dependent on HGF/SF isoform inter-

actions with hepann-like glycosaminoglycans (50, 51).Although the studies described above examined the ef-

facts of HGF/SF isoforms on baseline or HGF/SF-stimulated

cell proliferation and migration, it has not been establishedwhether HGF/NK1 and HGF/NK2 have agonistic or antago-nistic activity in experimental models that assay for inductionof epithelial and endothelial morphogenesis. To address thisissue, we have assessed the potential effects of full-lengthHGF/SF, HGF/NK1 , and HGF/NK2 on tube formation byMCF-1 OA mammary epithelial cells grown in collagen gelsand by HUVE cells grown on Matngel. We demonstrate thatHGF/NK1 stimulates tubulogenesis by these epithelial andendothelial cells, whereas HGF/NK2 is devoid of intrinsicactivity but antagonizes the morphogenic effect of HGF/SF.

ResultsEffect of HGF/SF Isoforms on Branching Tubulogenesisof MCF-IOA Mammary Epithelial OeIIs. MCF-1 OA cells area human breast cell line originated from the spontaneousimmortalization of nonmalignant mammary epithelial cells(53). They do not exhibit anchorage-independent growth, arenot tumongenic in nude mice, and possess many propertiescharacteristic of normal mammary epithelial cells (53, 54).When grown in three-dimensional collagen gels under con-trol conditions for 5-8 days, MCF-1OA mammary epithelialcells gave rise to irregularly shaped cystic structures pro-vided with very short outgrowths (Fig. 1A). In contrast, ad-dition of HGF/SF to the cultures for the same time periodresulted in the formation of highly branched structures,

which consisted of a central epithelial cell-lined cavity ex-tending into radially disposed outgrowths (Fig. 1B). The tu-bular nature of the outgrowths was confirmed by examina-tion of sections in light and electron microscopy (data notshown). A detailed quantitative analysis demonstrated that

HGF/SF induces a dose-dependent increase in the followingparameters: (a) mean number of outgrowths per colony (Fig.2�4), (b) mean total outgrowth length per colony (Fig. 2B), and(c) mean number of outgrowth branch points per colony (Fig.2C).

To elucidate structural characteristics of HGF/SF impor-tant for its morphogenic activity, we tested two naturallyoccurring truncated forms of HGF/SF for their ability to in-duce tubulogenesis by MCF-1 OA cells. HGF/NKI , whichcontains the a chain NH2-terminal hairpin loop plus the firstkringle domain (44, 47), stimulated formation of branchedtubular outgrowths in cultures of MCF-1 OA cells in a mannersimilar to that of HGF/SF (Fig. 1C). A quantitative analysisdemonstrated that at relatively low concenfrations, HGFINK1was slightly less potent than full-length HGF/SF in stimulat-ing branching tubulogenesis. Thus, 300 �M HGF/NK1 was

approximately as effective in inducing outgrowth formationas 100 �M HGF/SF. Interestingly, however, whereas the ef-fect of HGF/SF reached a plateau between 0.3 n� and 3 nM,higher concentrations of HGFINKI induced an increasinglystronger morphogenic response, which �i�3O-�nM actuallyexceeded that elicited by maximally effective concentrationsof HGF/SF (Fig. 2). In sharp contrast with the observedagonistic activity of HGFINK1 , HGF/NK2 (which contains theNH2-terminal hairpin loop and the first two kringle domains;Ref. 40) did not modify the morphogenic properties of MCF-1OA cells, even when added at concentrations as high as 100nM (Figs. 1D and 2).

HGF/NK2 was reported to act as a competitive inhibitor of

HGF/SF-induced mitogenesis in mammary epithelial cells(40). To determine whether HGF/NK2 would block the mor-phogenic response of MCF-1 OA cells to full-length HGF/SF,

increasing concentrations of HGFINK2 were coadded to col-lagen gel cultures with a fixed amount of HGF/SF (300 pM).

As shown in Fig. 3, the HGF/SF-induced increase in out-growth number, length, and branching was almost com-pletely suppressed by a 100-fold molar excess of HGF/NK2;depending on the parameter measured, 50-85% inhibitionof branching tubulogenesis was observed with 3 nt�i HGF/NK2 (10-fold molar excess).

The Effects of HGF/SF Isoforms on MCF-IOA Cells AreNot Modulated by Heparin. It has recently been reportedthat in cells deficient in hepann-like cell surface glycosami-noglycans, heparin potentiates the mitogenic activity of

HGF/SF isoforms by inducing ligand oligomerization (50, 51).In light of these findings, we examined whether coaddition ofheparin would modulate the effect of HGF/NKI or HGF/NK2on branching tubulogenesis of MCF-1 OA cells. Qualitativeobservations failed to show detectable differences in the

response of MCF-1OA cells to HGF/NK1 added alone ortogether with heparmn. In addition, combined treatment withHGF/NK2 and heparmn did not modify the morphology ofMCF-1 OA colonies in collagen gels, as is observed followingincubation with HGF/NK2 alone (results not shown).

Because the morphogenic and mitogenic responses toHGF/SF isoforms may be differentially regulated, and be-cause the branching morphogenesis assay may be less san-

sitive than cell proliferation assays, we next assessed thepotential effect of heparin on HGF/SF isoform-induced pro-

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Cell Growth & Differentiation 357

Fig. 1. Differential effect of HGF/SF isoforms on branching morphogenesis of MCF-1OA cells. MCF-1OA cells suspended in three-dimensional collagengels were incubated for 6 days in the presence or absence of HGF/SF isoforms and photographed using phase contrast optics. A, MCF-1OA cells grownunder control conditions have formed irregularly shaped cystic structures provided with very short outgrowths. B, in the presence of 300 �M HGF/SF, thecells have formed branching tubular outgrowths that extend out into the surrounding collagen gel from a central cavity. C, addition of the truncated variant

HGF/NK1 (1 nM) to the cultures has resulted in the formation of branching colonies that are morphologically indistinguishable from those induced byfull-length HGF/SF. 0, unlike HGF/NK1, HGF/NK2 (100 nM) had no effect on the morphogenic phenotype of MCF-1OA cells.

liferation of MCF-1OA cells in conventional monolayer cul-

ture. Full-length HGF/SF induced a dose-dependent in-

crease in MCF-1OA cell number, with a maximal effect at 3

nM. At HGF/SF concentrations lower than 3 nM, coaddition ofheparin (5 j�g/ml) slightly decreased ligand-induced cell pro-

liferation (Fig. 4A). HGF/NK1 stimulated MCF-1OA cell pro-

liferation to an extent similar to that of HGF/SF, but the

maximal effect was observed at 10 nM. No significant mod-

ulation of the growth response was observed following the

simultaneous addition of heparin (Fig. 4B). In contrast to

HGF/SF and HGF/NK1 , HGF/NK2, either added alone or incombination with heparin, was devoid of mitogenic activity

on MCF-1 OA cells (Fig. 4C). Taken together, these results

indicate that neither the morphogenic nor the mitogenic ef-

fects of HGF/SF isoforms on MCF-1 OA cells are substantially

altered by coaddition of heparin.

Effect of HGF/SF Isoforms on HUVE Cell Tubulogen-esis and Migration. Two widely used in vitro models of

capillary tube morphogenesis are the collagen gel invasion

assay (55, 56) and the Matrigel assay (57, 58). Because in theformer experimental system HGF/SF is devoid of activity

(59), we assessed the potential effect of HGF/SF isoforms on

the formation of tubular structures by HUVE cells grown on

Matrigel, a laminin-rich extracellular matrix (60). The effects

of HGF/SF, HGF/NK1 , and HGF/NK2 on the ability of HUVE

cells to undergo tubulogenesis in vitro are shown in Fig. 5.

HGF/SF (Fig. 5B) enhanced tube formation relative to un-treated control levels (Fig. 5A). HGF/NK1 (Fig. SC) also pro-

moted tubulogenesis, although less potently than HGF/SF.

The combination of HGF/NK1 and HGF/SF (Fig. SE) was not

readily distinguishable from the effects of HGF/NK1 alone.

HGF/NK2 (Fig. SD) failed to stimulate tube formation at any

concentration tested and visibly blocked the effects of

HGF/SF (Fig. SE). These data demonstrate that HGF/NK1

stimulates HUVE tubulogenesis, whereas HGF/NK2 has no

effect and can inhibit HGF/SF-induced tubule formation.

Because HUVE cell organization into branching and anas-

tomosing tubes requires cell motility, we next assessed the

effect of HGF/SF isoforms on cell migration in Boyden cham-

bers. Migration was significantly stimulated 7-fold over back-

ground in the presence of 100 ng/ml HGF/SF (Fig. 6). HGF/

NK1 showed less activity, with a 4-fold enhancement of

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duced the levels of migration observed with HGF/SF (Fig. 7).HGF/NK2 had the greatest inhibitory effect, reducing migra-tion as much as 4-fold when only 25 ng/ml was incubatedwith 50 ng/ml of HGF/SF (Fig. 7). HGF/NK1 (25 ng/mI) addedto HGF/SF (50 ng/ml) had less of an effect, reducing migra-tion approximately 2-fold (Fig. 7). Combinations of both iso-forms with HGF/SF (25:25:25 ng/ml) reduced migrationabout 3-fold (Fig. 7).

358 Differential Morphogenic Activity of HGF Isoforms

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Cell Growth & Differentiation 359

Taken together, these results suggest that HGF/NK2 doesnot stimulate HUVE cell migration or tubulogenesis and caneffectively block these activities Induced by HGF/SF. In con-trast, HGF/NKI displays obvious agonist activity in theseassays and, because of its intrinsic actMty, is a less effective

HGF/SF antagonist than HGF/NK2.Comparison of c-Met Tyrosine Phosphorylation in Re-

sponse to Different HGFISF Isoforms. We sought to de-

termine how the biological responses to various HGF/SF

isoforms observed with MCF-IOA and HUVE cells correlatedwith the ability of these ligands to stimulate c-Met tyrosinephosphorylation. Using ligand concentrations correspondingto those used in the bioassays described above, it waspossible to demonstrate in MCF-1OA cells a dose-dependentincrease in tyrosine phosphorylation of the M� 145,000 c-Met

13subunit (61) with both HGF/SF and HGF/NK1 . Whereas

HGF/SF was somewhat more active than HGFINK1 at 1 n�,HGF/NK1 appeared to be at least as potent at 1 0 n�. Instriking contrast, HGF/NK2 induced only a barely detectableincrease in c-Met tyrosine phosphorylation at either 10 or100 flM (Fig. 8). These results correlated well with the mor-phogenic and proliferative activities of the respective ligandson MCF-1OA cells. When we compared the effects of 1 �g/mlheparin on receptor tyrosine phosphorylation in response toeach ligand, there was no observable increase (data notshown). These findings also correlated well with the results ofthe proliferation assays (see Fig. 4). Similar results wereobtained in HUVE cells, in which HGF/SF and HGF/NK1 (300nM) stimulated c-Met autophosphorylation, whereas equimo-lar concentrations of HGF/NK2 did not (Fig. 9). These find-ings correlated well with the results of the HUVE cell tubu-logenesis assay on Matngel.

Discussion

In addition to stimulating the proliferation and motility of abroad spectrum of cell types, HGF/SF and structurally re-lated proteins have the unique ability to elicit morphogenicresponses in epithelial and endothelial cells (3, 16, 21 , 62-64). To define structural features of the HGF/SF moleculeimportant for its morphogenic activity, we assessed the abil-�ity of truncated HGF/SF variants to induce formation ofbranching tubes by MCF-1 OA mammary epithelial cells andHUVE cells. We report here that two naturally occurringHGF/SF isoforms, HGF/NK1 and HGF/NK2, have markedlydifferent effects on these target cells. HGF/NK1 , which only

comprises the NH2-terminal hairpin and the first kringle do-main of the a chain (44, 47), exhibits morphogenic activitycomparable to that of full-length HGF/SF. In contrast, thelonger isoform HGF/NK2, which extends from the NH2 tar-minus through the second krlngle domain (40), is devoid ofintrinsic activity but efficiently antagonizes the morphogeniceffect of HGF/SF. Finally, because cellular responses to

HGF/SF isoforms have recently been shown to be modulatedby heparin-like glycosaminoglycans, we assessed the bio-logical activities of HGFINK1 and HGF/NK2 in the presenceor absence of exogenous hepann and found that their mor-phogenic and mitogenic effects on MCF-1 OA cells are notostensibly altered by heparin coaddition.

Our study demonstrates that HGF/NK1 behaves as astrong agonist in the MCF-1 OA branching tubulogenesis as-say. When the activity of HGFINK1 is compared to that ofHGF/SF in the 0.1-1 n�i range, it appears that the truncated

isoform is less potent than the full-length protein. Interest-ingly, however, higher concentrations (3-30 nM) of HGF/NKIinduce a robust morphogenic response, the magnitude ofwhich actually exceeds that elicited by maximally effective

concentrations of HGF/SF (see Fig. 2). HGF/NK1 also stim-ulated migration and tube formation by HUVE cells grown on

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esis. HUVE cells were plated onMatrigel-coated 48-well platesand incubated in medium thatcontained 5% bovine calf serumand heparin [negative control�4)], HGF/SF (B), HGF/NK1 (C),or HGFINK2 (0), each at 50 ng/ml. The possible antagonist ef-fects of the HGF/SF isoformswere assessed by incubatingcells with HGF/SF and HGF/NK1combined (E) or HGF/SF andHGF/NK2 combined (F), at con-D centrations of 50 ng/ml each.Magnification, x5.

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3&1 Differential Morphogenic Activity of HGF Isoforms

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Matrigel, although not to the same extent as full-length HGF/

SF. The findings that HGF/NK1 induces formation of branch-

ing tubules by epithelial and endothelial cells correlate well

with the recently reported ability of this isoform to elicit

cellular responses, such as proliferation, migration (47, 51,

52), and production of matrix metalloproteinases (65), which

are important components of morphogenic processes.

Unlike HGF/NK1 , HGF/NK2 was consistently devoid of

morphogenic activity on both MCF-1OA and HUVE cells.

These results are therefore in agreement with previous stud-

es showing that HGF/NK2 is unable to duplicate the stimu-

lating effect of HGF/SF on cell proliferation (40, 42, 43, 48,

52), expression of urokinase and collagenase 1 (49, 65), and

angiogenesis in vivo (48). It is noteworthy, however, thatdespite the lack of mitogenic activity on rat hepatocytes,

HGF/NK2 was reported to induce scattering of canine MDCK

cells (43). Because full-length HGF/SF does not induce de-

tectable scattering of MCF-1 OA cells, we examined the po-

tential motogenic effect of our own HGF/NK2 preparation on

MDCK cells. In partial agreement with the findings of Hart-

mann et a!. (43), we found that high concentrations (1 00 nM)

of HGF/NK2 induce early events of the scattering response,

i.e., increased cell spreading and enlargement of epithelial

colonies, without causing obvious cell dissociation.4

In accordance with previous studies showing that HGF/

NK2 inhibits HGF/SF-induced mitogenesis (40, 52) and col-

lagenase 1 synthesis (65), we found that this truncated iso-

form acts as an antagonist of HGF/SF-induced branching

tubulogenesis in cultures of MCF-1 OA and HUVE cells. Thisantagonistic activity can be explained by the ability of HGF/

NK2 to competitively inhibit HGF/SF binding to c-Met (40).

If the one-kringle HGF/SF variant (HGF/NK1) is sufficient to

elicit the characteristic morphogenic and mitogenic effects of

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Fig. 6. Effects of HGF/SF isoforms on HUVE cell migration. Ordinate,mean number of cells migrating across collagen-coated microporouspolycarbonate membranes. Abscissa, concentration (in ng/m� of eachHGF/SF isoform. HUVE cell migration was stimulated 7-fold in the pres-ence of 100 ng/ml HGF/SF and 4-fold in the presence of 1 00 ng/mlHGF/NK1 , relative to the negative control (C). HGF/NK2 did not signifi-cantly stimulate HUVE cell migration. ECGS was used as a positivecontrol. Data are means; bars, SE. *, statistically significant differencesfrom the negative control (P < 0.01 , Welch’s t test).

Fig. 7. Effects of HGF isoforms in combination on HUVE cell migration.The antagonist properties of HGF/NK1 and HGF/NK2 on HGF/SF-stimu-lated HUVE cell migration was tested using various isoform combinations.Abscissa, migration expressed as a mean percentage of that observed forHGF/SF alone. �, the effects of HGF/SF, HGF/NK1 , and HGF/NK2 (50ng/m� alone. The antagonist effects of HGF/NK1 and HGF/NK2 on HGF/SF-stimulated HUVE cell migration are shown at two different HGF/SFconcentrations (25 and 50 ng/ml), as indicated at left. Concentrations ofadded HGF/NK1 or HGF/NK2 are indicated in ng/ml to the immediate leftof each bar. + NK1,2, both HGF/NK1 and HGF/NK2 at the indicatedconcentrations were simultaneously added to HGF/SF-stimulated HUVEcells. Data are means; bars, SE. All of the combined treatments (0 and �)were statistically different from HGF/SF treatment alone (P < 0.009,Welch’s t test).

Cell Growth & Differentiation 361

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the full-length protein, why then is the HGF/NK2 variantdevoid of intrinsic agonistic activity on MCF-1 OA and HUVEcells? Because the essential difference between HGF/NK1and HGF/NK2 is the presence of the second knngle domain(K2) in the latter, the strikingly different biological activities of

the two HGF/SF isoforms presumably result from changes inligand-receptor interaction attributable to the K2 domain(65). Given that we found that HGFINK2 does not signifi-cantly stimulate c-Met autophosphorylation in MCF-1 OA andHUVE cells, the distinct effects of HGF/NK1 and HGF/NK2 inour assays may be due to the inability of HGF/NK2 to effi-ciently activate the c-Met receptor. In the case of other cells

in which c-Met has been shown to become phosphorylated

in response to HGF/NK2 (43, 49, 52), it may be hypothesizedthat HGF/NK1 and HGF/NK2 induce qualitatively differentpatterns of tyrosine phosphorylation in the catalytic domainand/or COOH terminus of c-Met, resulting in differential re-cruitment of specific signal transducers that would eventually

influence the biological response (47, 49).HGF/SF is a heparin-binding polypeptide, and recent stud-

ies have suggested that interaction with heparin-like glyco-

saminoglycans has a major impact on the biological effects

of HGF/SF isoforms. Thus, Schwall et a!. (50) reported thatHGF/NK1 , which is a HGF/SF antagonist in rat hepatocyteproliferation assays, acts as a partial agonist in mink lungcells, and that hepann can convert HGF/NK1 from an antag-onist to a partial agonist in hepatocytes. Heparin also con-ferred agonistic activity onto HGF/NK1 and HGF/NK2 inc-Met-transfected BaF3 cells, which lack cell surface pro-teoglycans. On the basis of these findings, Schwall et a!. (50)proposed that interaction with endogenous heparin-like gly-cosaminoglycans on the cell surface promotes dimerizationof HGF/SF isoforms, thereby turning them from antagoniststo agonists. These conclusions are supported by the study ofSakata et a!. (51), who demonstrate that in CHO cells defi-cient in cell surface glycosaminoglycans, exogenouslyadded hepann restores HGF/NK1 binding to c-Met, induces

NK1 50

NK2 50

HGF 50

+ NK1 25

+ NK1 50

IIGF +NK225(25 nglml)

+NK250

+NKI,225

+ NK1 25HGF

(SOnglmI) +NK225+NK1,250

ligand oligomerization, and increases ligand-dependent c-

Met phosphorylation and c-fos expression. In contrast, in

wild-type CHO cells, which express abundant cell surfaceglycosaminoglycans, HGF/NK1 has intrinsic agonistic activ-ity, which is only modestly affected by the addition of exog-

enous heparin (51). In our own experiments on MCF-1OAcells, HGF/NK1 was endowed with conspicuous morpho-genic and mitogenic activity in the absence of exogenouslyadded heparin, and heparin coadministration did not furtherenhance the cellular response to HGF/NK1 . This situation isanalogous to that described in cell surface glycosaminogly-can-rich cell types, such as mink lung cells (50) and wild-typeCHO cells (51). In agreement with the conclusion of theseinvestigators, we tentatively interpret the lack of effect of

heparin in our system as reflecting an activating interactionbetween HGF/NK1 and endogenous heparin-like glycosami-noglycans on the surface of MCF-1OA cells.

Our results differ from those of Schwall et a!. (50) in that inthe presence or absence of added heparin, HGF/NK1 actedas an agonist with nearly the same potency as HGF/SF, andHGF/NK2 consistently lacked agonist activity. It is possiblethat the specific heparin-like glycosaminoglycan composi-tion on MCF-1 OA and HUVE cell surface is sufficient to

facilitate the agonist activity of HGF/NK1 , but not sufficient to

confer agonist activity to HGF/NK2. In support of this, wehave found that whereas HGF/NK2 alone only inducesspreading of MDCK cell colonies, heparin coaddition resultsin overt cell scattering, suggesting that in that system, hep-

arm potentiates the otherwise weak motogenic activity ofHGF/NK2. Coaddition of HGF/NK2 and heparin also inducesthe formation of thin processes from MDCK cysts in collagengels, whereas HGF/NK2 alone does not.4

HGF/SF HGFINK1 HGF/NK2

0 0.1 1 10 1 10100 1 10100 nM

220-

97.4 - I66- �

IP: a-met

blot:

In conclusion, although the nature of the cellular response

to HGF/NK1 and HGF/NK2 may vary in different cell typesdepending on cell surface glycosaminoglycan composition,our studies clearly establish that under identical experimentalconditions, the two truncated variants of HGF/SF exhibitmarkedly different biological properties. HGF/NK1 is capableof stimulating branching morphogenesis and proliferation ofMCF-1 OA mammary epithelial cells as well as tubulogenesisand migration of HUVE cells. In striking contrast, HGF/NK2 isdevoid of intrinsic morphogenic, motogenic, and mitogenicactivity and acts as an antagonist of HGF/SF. Additionalstudies will be aimed at elucidating the molecular basis forthe differential properties of these HGF/SF isoforms. This, inturn, may lead to the development of more potent and se-

lective agonists/antagonists for the therapeutic modulation

of HGF/SF-dependent physiological or pathological pro-

cesses.

362 Differential Morphogenic Activity of HGF Isoforms

Fig. 8. Tyrosine phosphorylation of the c-Met receptor by HGF/SF iso-forms in MCF-1OA cells. Serum-starved MCF-1OA cells were treated withvarying concentrations of HGF/SF, HGF/NK1, or HGF/NK2 for 10 mm at37#{176}C.Total cell lysates (2 mg) were immunoprecipitated with anti-c-Metantibody, resolved by 7.5% SDS-PAGE, and immunoblotted with anantiphosphotyrosine antibody. Arrow, tyrosine-phosphorylated c-Met (3subunit.

Materials and MethodsProcaryotic Expression of Truncated HGF/SF Isoforms. The trun-

cated HGF/SF isoforms HGF/NK1 and HGF/NK2 were expressed in E. coilas described previously (52). Briefly, insoluble HGF/SF proteins were

recovered by harvesting bacterial inclusion bodies and extracted with 8 Mguanidine-HCI. Solubilized proteins were fractionated by gel filtration alsoin the presence of guanidine-HCI. Purified proteins (fully denatured and

reduced) were folded into biologically active species using an equilibriumdialysis scheme that included urea to prevent protein aggregation duringthe removal of the guanidine-HCI and a glutathione-based oxido-shufflingsystem to promote the formation of disulfide linkages from the reducedprotein by thioldisulfide exchange reactions. After dialysis, the proteins

were concentrated by ultrafiltration and fractionated by gel filtration. HGF/NK1 and HGF/NK2 produced in this way displayed all ofthe structural andbiological properties of eukaryotically expressed proteins (52).

Cell Culture. MCF-1OA cells (53) were obtained from Dr. D. Salomon(National Cancer Institute, Bethesda, MD) and maintained in a 1 :1 mixture

of high-glucose DMEM and Ham’s F12 medium (DMEM/F12; Life Tech-nologies, Inc., Basel, Switzerland) supplemented with 5% horse serum(Amimed AG, Muttenz, Switzerland), 5 �g/ml insulin (Sigma Chemical Co.,St. Louis, MO), 1 �i dexamethasone (Sigma), 10 ng/ml basic fibroblastgrowth factor (a generous gift of Dr. P. Sarmientos, Farmitalia Carlo Erba,

a-met a-pY

Fig. 9. Tyrosine phosphorylation of the c-Met receptor by HGF/SF iso-forms in HUVE cells. HUVE cells were treated with an equimolar concen-tration (300 nM) of HGF/SF, HGF/NK1 , or HGF/NK2 for 10 mm at 37#{176}Candlysed with nonionic detergent-containing buffer on ice. After high-speedcentrifugation, the supematant was immunoprecipitated with anti-c-Metantiserum, subjected to SDS-PAGE, transferred to polyvinylidene difiuo-ride membranes, blotted with either anti-c-Met or antiphosphotyrosineantiserum, and then detected by chemiluminescence.

Milan, Italy), penicillin (100 units/mI), and streptomycin (110 �g/m�. HUVEcells were isolated from freshly delivered umbilical cords (66), grown as

reported previously (67), and used between passages 4 and 6.MCF-1OA Branching Morphogenesis Assay. MCF-1OA cells were

harvested using trypsin-EDTA, centrifuged, and suspended in three-di-mensional collagen gels as described (68, 69). In brief, 8 volumes of rat tail

tendon collagen solution (approximately 1 .5 mg/me were mixed with 1volume of concentrated MEM (Life Technologies) and 1 volume of sodium

bicarbonate (1 1 .76 mg/mI) in a sterile flask kept on ice to prevent prema-ture collagen gelation. MCF-1OA cells were suspended at 1 x 1 o� cells/mIin the cold collagen mixture, and 0.4-mI aliquots of cell suspension were

dispensed into 16-mm tissue culture wells (Nunc; Kamstrup, Roskilde,Denmark). After a 10-mm incubation at 37#{176}Cto allow collagen gelation,the cultures were incubated in DMEM/F12 containing 5% horse serum, 5pg/mi insulin, and 1 pi�i dexamethasone, with or without various concen-trations of HGF/SF, HGF/NK1 , or HGF/NK2. Medium and treatments wererenewed after 3 days, and the cultures were fixed in situ after an additional3 days with 2.5% glutaraldehyde in 100 m� sodium cacodylate buffer.

For quantification of branching morphogenesis, 10 randomly selected

colonies per experimental condition were photographed using the x 10

objective of a Nikon Diaphot TMD inverted photomicroscope, in each ofthree separate experiments (i.e., a total of 30 colonies per experimental

condition). On positive prints, the “central portion” of each colony wasdefined by drawing an ovoidal line that joined the points of the colony

perimeter closest to its geometrical center. When the colonies had anelongated tubular shape, the central portion was defined by a circle having

a diameter equal to the diameter of the tube. The regions of each colonythat were not comprised within the central portion were considered as

“outgrowths” when their longitudinal (radial) axis was equal or greater thantheir width. To determine total outgrowth length per colony, straight lines

were drawn along the longitudinal axis of each stem outgrowth and of itsbranches, and the additive length of these lines was measured with the aidof a Qmet 500 image analyzer (Leica Cambridge Ltd., Cambridge, United

Kingdom).MCF-IOA Cell Proliferation Assay. MCF-1OA cells were seeded in

16-mm wells at 2 x iO� cells/well in serum-free DMEM/F12 mediumsupplemented with 5 �g/ml insulin, 1 p.t�i dexamethasone, and 10 ng/mlbasic fibroblast growth factor. On the following day, the medium wasaspirated and replaced with fresh medium with or without the indicated

treatment. Media and treatments were renewed after 3 days, and the cellswere harvested by trypsinization after an additional 3 days and countedwith a hemocytometer. All data are from triplicate determinations in eachof at least three separate experiments (i.e., at least nine determinations foreach experimental condition).

HUVE CellTubulogenesis Assay. Assays were performed on 48-wellplates that had been coated with 200 �l of Matrigel (60) per well. Cells

Cell Growth & Differentiation 363

from confluent HUVE cuitures were detached with 0.05% trypsin (LifeTechnologies, Inc., Gaithersburg, MD), 0.53 m�.i EDTA in HBSS. Cells wereplated at a density of 24,000 per well in 250 �l of serum-reduced HUVE

cell medium containing 5% bovine calf serum, 200 �g/ml ECGS (Collab-orative Biomedical Products; Becton Dickinson), and 5 units/mI hepann.HGF/SF, HGF/NK1 , and HGF/NK2 were tested alone or in combination at

concentrations of 25 and 50 ng/ml as indicated. Tubulogenesis occurredduring overnight incubation at 37#{176}C.Nt-Quick solutions 1 and 2 (Baxter

Healthcare, McGaw Park, IL) were used to fix and stain cells. Each assay

was done in triplicate, and each experiment was repeated at least threetimes.

HUVE Cell Migration Assay. Migration assays were conducted in a48-well microchemotaxis chamber (Neuro Probe, Inc., Cabin John, MD).Polyvinyl pyrrolidone-free polycarbonate membranes with 12-pin pores

(Neuro Probe) were coated with a 0.1 mg/mI solution of collagen IV(Trevigen, Gaithersburg, MD) in distilled H20 and air dried. HUVE cells

were harvested using Versene (Life Technologies) and resuspended in

RPMI 1640 containing 0.1 % BSA. The bottom chamber was loaded with

RPMI 1640 containing 0.1% BSA and a mixture of HGF/NK1, HGF/NK2,and/or HGF/SF. ECGS (200 g�g/m� was added to several wells as apositive control. Medium alone was used as the negative control. Cells(50,000 perwell) were added to the upper chamber and incubated at 37#{176}Cin 5% CO2 for 4 h, and the fliters were then fixed and stained usingDiff-Quik. The cells that migrated through the filter were quantitated bycounting cells in the center of each well at x 10 magnification using anOlympus CK2 microscope. Each condition was assayed in triplicate wells,and each experiment was repeated at least three times. In Stat softwarewas used to calculate statistical significance using the Welch’s t test.

c-Met Receptor Activation. Confluent cultures of MCF-1OA andHUVE cells were serum starved for 18-20 h and exposed to varying

concentrations of HGF/SF, HGF/NK1 , or HGF/NK2 for 10 mm at 37#{176}C.

Cuitures were then rinsed in PBS containing 1 m� sodium vanadate andsolubilized in lysing buffer (0.01 M phosphate buffer, 1 % Triton, 0.5%

sodium deoxycholate, 0.1 % SDS, 0.1 M NaCI, 5 pi� EGTA, 1 m� sodium

vanadate, 10 �i sodium PP�, and 2 m� phenylmethylsulfonyl fluoride).Total cell lysates (2 mg of protein) were immunoprecipitated with anti-c-Met antibody bound to agarose beads (Santa Cruz Biotechnology). Im-munoprecipitates were resolved by 7.5% SDS-PAGE, subjected to im-munoblothng with antiphosphotyrosine monoclonal antibody (Upstate

Biotechnologyinc., Lake Placid, NV), and detected eitherwith 1251-Iabeled

protein A (Amersham Corp.; MCF-1OA cells) or by enhanced chemilumi-nescence (performed as per the manufacturer’s instructions; Amersham;

HUVE cells).

Acknowledgments

We thank Dr. L Orci forcontinuing support; M. Eissler, J. Rial-Robert, andJ-P. Gerber for skillful technical assistance; and F. Hellal for typing the

manuscript.

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