2897Development 121, 2897-2908 (1995)Printed in Great Britain © The Company of Biologists Limited 1995

HGF/SF: a potent cytokine for mammary growth, morphogenesis and

development

Birunthi Niranjan1, Lakjaya Buluwela2, Jeffrey Yant1, Nina Perusinghe1, Amanda Atherton1, Deborah Phippard1, Trevor Dale1, Barry Gusterson1 and Tahereh Kamalati1

1Division of Cell Biology and Experimental Pathology, Institute of Cancer Research, 15 Cotswald Road, Sutton, Surrey, SM2 5NG,UK2Department of Biochemistry and Medical Oncology, Charing Cross and Westminster Medical School, Fulham Palace Road,London W6 8RF, UK

The mammary gland is a renewing tissue in which mor-phogenetic processes and differentiation occur cyclicallyduring the menstrual cycle, pregnancy and lactation. Theseevents have been shown to be dependent upon epithelial-mesenchymal interactions. Studies of the effects of individ-ual factors, their cellular source and their target cell pop-ulations in the different developmental stages of themammary gland are greatly facilitated by the accessibilityof this organ and the application of new techniques thatallow purification of the major epithelial and stromal com-ponents of this tissue.

Here we demonstrate that HGF/SF and its cellularreceptor, c-met, are expressed and regulated temporallyduring mouse mammary development and differentiation.We show that human and mouse mammary fibroblasts

produce HGF/SF and that HGF/SF is not only mitogenicbut morphogenic and motogenic for both human andmouse mammary epithelial cells. We have found thathuman luminal and myoepithelial cells express c-met dif-ferentially and that HGF/SF has different effects on thesetwo mammary epithelial cell populations. HGF/SF ismitogenic for luminal cells but not myoepithelial cells, andmorphogenic to myoepithelial cells but not luminal cells.This is discussed in the context of the proliferative com-partments in the normal mammary gland and the potentialrole of the myoepithelial cells to act as the skeleton forductal development.

Key words: HGF/SF, c-met, luminal cell, myoepithelial cell,mammary fibroblast, growth and morphogenesis

SUMMARY

INTRODUCTION

The mammary gland is a unique organ since growth, morpho-genesis, cell diversification and full phenotypic differentiationoccurs in several stages during postnatal and adult life(Sakakura, 1987). In both rodent and human the rudimentaryducts elongate extensively and through dichotomous branchinginvade the mammary fat pad. In rodents the result of thisintensive growth and branching morphogenesis is a tree-likeductal network which fills the adipose-rich stroma (Williamsand Daniel, 1983). Although less is known about the humangland, similar stages of morphogenetic change appear to occur(Anbazhagan et al., 1991; Atherton et al., 1994a).

Mammary ductal elongation is the direct result of prolifera-tive activity in the mammary stroma and a highly specialisedregion of the duct called the ‘end bud’ (Berger and Daniel,1983; Bresciani, 1965; Russo and Russo, 1978, 1980;Anbazhagan et al., 1991; Atherton et al., 1994a). Theadvancing edge of the end buds appear to be specialised forpenetration of the surrounding fatty stroma (Silberstein andDaniel, 1982) while the posterior region of the end budprovides a supply of differentiating luminal and myoepithelialcells for elongation and subtending ducts.

During early mouse pregnancy, lateral (‘alveolar’) buds

appear. From these lateral buds, alveoli develop through aprocess of rapid growth and morphogenesis. The alveoliorganise into lobular structures in which the epithelia assumesa secretory function which will be continued during lactation(Cole, 1933; Russo and Russo, 1978, 1980). Similar changesare thought to occur in the human breast but there is very littledata available. In the adult human mammary gland, cyclicpatterns of proliferation, morphogenesis, differentiation andregression occur during the estrous cycle (Anderson et al.,1982). In both human and murine mammary gland, pregnancyand lactation results in a dramatic cyclic tissue remodelling.

Inductive interactions between epithelium and mesenchymeare essential for growth and differentiation of the mammarygland during embryogenesis, and continue to play a criticalrole in the growth and differentiation of this organ in thepostnatal animal and throughout adult life (Sakakura et al.,1979; Berger and Daniel, 1983; Daniel and Silberstein, 1987).The mammary gland is therefore a suitable system for identi-fication and characterisation of the mediators of theseprocesses. During normal human fetal breast development,there is an intimate relationship between the condensed spe-cialised stroma and the epithelial buds (Nathan et al., 1994).At birth and in the first two years after birth, there is a spe-cialisation of the breast stroma into the functionally distinct

2898 B. Niranjan and others

components, the interlobular stroma around the glandularepithelium and the intralobular stroma, which is found withinthe lobules (Atherton et al., 1992). These two populations offibroblasts, which are defined by their expression of theectopeptidase dipeptidyl peptidase IV (DPP IV), are charac-teristic of the adult breast. Specific benign and malignanttumours arise from the specialised intralobular stroma(Atherton et al., 1992) further exploring their distinct identity.

A complex range of molecular signals have already beenshown to be involved in the regulation of growth and branchingmorphogenesis of the mammary gland (Vonderhaar, 1987;Topper and Freeman, 1980; Coleman et al., 1988; Robinson etal., 1991). In an attempt to identify signalling molecules thatmediate mesenchymal-epithelial interactions, we sought toexamine the role of HGF/SF in normal mammary gland growthand differentiation. Hepatocyte Growth Factor/Scatter Factor,HGF/SF, is produced by mesenchymal cells and exerts itseffect on epithelial cells. It is therefore a paracrine mediator ofepithelial interactions. HGF/SF has been shown to bemitogenic for a broad spectrum of epithelial cells, to inducethe formation of branching tubules and to induce invasivenessand motility in a variety of epithelial cells (Gherardi andStoker, 1991). Further, recent studies suggest that HGF/SFmay play a significant role in mouse embryogenesis (Sonnen-berg et al., 1993; Schmidt et al., 1995; Uehara et al., 1995) andchick morphogenesis (Streit et al., 1995: Stern et al., 1990;Myokai et al., 1995). Although expression of HGF/SF tran-script and that of its cellular receptor the c-met proto-oncogenehave been demonstrated in a variety of tissues, the physiolog-ical role of HGF/SF and the molecular mechanism by which itappears to elicit the four distinct functions outlined above, viaa single receptor, remain to be elucidated (Gherardi and Stoker,1991).

In this study, using both mouse and human tissues, we haveevaluated the role of HGF/SF and c-met in normal breastgrowth and development. Using novel techniques developed inour laboratories, we have isolated and highly enriched thefibroblast (separated into interlobular and intralobular) andepithelial components (separated into luminal and myoepi-thelial) that constitute this tissue (Atherton et al., 1994b; Clarkeet al., 1994). Here we identify the mammary fibroblasts as thesource of HGF/SF and demonstrate for the first time theresponse of primary mouse and human mammary epithelialcells to the spectrum of HGF/SF’s activities. The workpresented here is unique in that it represents the first organ inwhich the contribution of HGF/SF to growth and morphogen-esis of individual cellular components has been functionallydemonstrated.

METHODS AND MATERIALS

Cell linesMRC 5, human embryonic lung fibroblasts (Jacobs et al., 1970) andMDCK, Madin canine kidney epithelial cells (Madin and Darby,1958) were purchased from the European Collection. SK23, a humanmelanoma cell line was a gift from Dr I Hart (ICRF, London). MRC5 fibroblasts and SK23 were maintained in Dulbecco’s modifiedEagle’s medium (DMEM) containing 10% foetal calf serum (FCS)while MDCK cells were maintained in DMEM plus 5% FCS. All cellswere grown at 37°C in a humidified 12% CO2 atmosphere.

Preparation of primary human mammary epithelial cellsHuman mammary luminal and myoepithelial cells were prepared fromnormal human breast material, obtained from reduction mammoplas-ties of individuals aged between 18 and 33 years. Primary mammaryepithelial cells were prepared as described by O’Hare et al., 1991.Briefly, mammary tissue was cut into small pieces and processed byprogressive collagenase digestion, followed by sedimentation and fil-tration to produce organoids (ductal and lobulo-alveolar fragments).The organoids were then placed in culture for a period of 7-10 days.This allows the organoids to attach, spread and grow into semi-confluent cultures, where myoepithelial cells grow out to form a basallayer on top of which colonies of luminal cells grow. At the end ofthis period, myoepithelial and luminal cell populations were separatedand highly enriched using an immunomagnetic separation techniquedescribed by Clarke et al. (1994). In this process, human luminal andmyoepithelial cells are separated by virtue of their exclusiveexpression of Epithelial Membrane Antigen (EMA), and CommonAcute Lymphoblastic Leukemia Antigen (CALLA/CD10), respec-tively (O’Hare et al., 1991).

ICR2, a rat monoclonal antibody against EMA (Imrie et al., 1990)and MAS 231P, a mouse monoclonal antibody against CALLA/CD10(SeraLab) were used in conjunction with appropriately coated MACSmicrobeads (Becton Dickinson, Cowley, Oxford, UK) to purifyhuman luminal and myoepithelial cells, respectively. The purity of theenriched populations of mammary epithelial cells generated by thisprocedure was checked in each preparation by immunocytochemistryusing cytokeratins 18 and 19 as markers for luminal cells and cyto-keratin 14 as the marker for myoepithelial cells (Taylor-Papadimitriouand Lane, 1987). This was done using LLOO2, (IgG3) a mouse mon-oclonal antibody against cytokeratin 14, LE61, (IgG1) a mouse mon-oclonal antibody against cytokeratin 18, and LP2K, (IgG2b) a mousemonoclonal antibody against cytokeratin, 19 (Taylor-Papadimitriouand Lane, 1987). The above antibodies (generous gifts from Dr B.Lane) were used in pairs (LE61/LLOO2 and LE61/LP2K) and simul-taneously visualised using appropriate subclass-specific tetramethylrhodamine isothiocynate (RITC) and fluorescein isothiocynate(FITC)-conjugated second antibodies (Southern Biotechnology, viaEuropath, Bude, UK).

In our hands, this technique is extremely efficient at generatinglarge quantities of highly enriched populations of human luminal andmyoepithelial cells, at 95-99% purity as checked by immunocyto-chemistry. Unless otherwise stated human luminal and myoepithelialcells were grown in RPMI 1640 supplemented with 10% FCS, insulin,I, (5 µg/ml), hydrocortisone, HC, (5 µg/ml) and cholera toxin, CT,(100 ng/ml).

Preparation of human mammary fibroblastsHeterogeneous populations of human mammary fibroblasts wereprepared by centrifugation (90 g, 5 minutes) of the filtrate (through53 µm sterile nylon filter) from the partially digested mammarytissue (see above). The cells were maintained in DMEM plus 10%FCS.

Preparation of human interlobular mammary fibroblastsThe supernatant from partially digested mammary tissue derived asdescribed above, was centrifuged (400 g for 5 minutes) and theresulting pellet washed and filtered sequentially through 140 µm, 53µm and 35 µm sterile nylon filters. The filtrate containing single cellswas collected and viable stromal cells plated at a density of 8000 cellsper cm2 in Ham’s F12:DMEM (1:1) containing 20% FCS. The cellswere subcultured at a split ratio of 1:3 on reaching confluence. Afterthree passages, the cultures were routinely fed with DMEM plus 10%FCS (Atherton et al., 1994b).

Preparation of human intralobular mammary fibroblastsThis was achieved as described by Atherton et al. (1994b). Fragmentsof undigested breast tissue, obtained by limited enzymatic digestion

2899HGF/SF in mammary development

(see above), were digested further for 3 hours at 37°C in collagenase(2 mg/ml in Leibovitz L-15 medium). The resulting preparation,composed principally of epithelial organoids, were pelleted, washedand filtered through a 140 µm sterile nylon filter in order to retain thelarger fragments, which are to be discarded. The filtrate was thenpassed through a 53 µm filter and the small/medium-sized organoidsthat were retained were retrieved, resuspended in L-15 and filteredthrough a 35 µm filter to remove all single cells.

Individual organoids that had visible intralobular stroma stillattached were plated in separate wells of a 24-well culture plate con-taining Ham’s F12:DMEM plus 20% FCS and left to mobilise andgrow for 7-14 days. After this period proliferating intralobular fibro-blasts could be seen at the periphery of some explants. Any wells thatcontained stromal cells not directly associated with an epithelialoutgrowth were neglected.

Fibroblasts were isolated from satisfactory preparations by brief (1-2 minute) trypsinisation and resuspended in Ham’s F12:DMEM plus20% FCS. The harvested cells were filtered through a 35 µm filter toremove the larger undigested epithelial fragments. The resultingintralobular fibroblasts from several original wells were pooled andgrown in Ham’s F12:DMEM plus 20% FCS. After three passages thecells were routinely maintained in DMEM plus 10% FCS.

Separated cultures of interlobular and intralobular fibroblastswere then further characterised by virtue of their exclusiveexpression of dipeptidyl peptidase IV (DPP IV) and differentialexpression of Neutral endopeptidase (NEP) at early passages,respectively, in order to assess the purity of the populations(Atherton et al., 1994b). Indirect immunofluorescence microscopywas employed to examine DPP IV and NEP expression using amouse monoclonal antibody to NEP (SS2/3b, Dako, 1:200) and arabbit antisera to DPP IV (a generous gift from Dr A. J. Kenny, Uni-versity of Leeds, Leeds, UK; 1:500).

Preparation of primary mouse mammary cellsPrimary mouse mammary epithelial cells and fibroblasts wereprepared as described by Imagawa et al. (1982) and Emerman andBissel (1988). Briefly, mammary gland (number four) from 10-12weeks old Parks mice were excised and the attached lymph noderemoved. Finely minced mouse mammary tissue was partiallydigested, with stirring for 1 hour at 37°C, using a digestion cocktailcomposed of 1.5 mg/ml trypsin (Gibco BRL) and 3 mg/ml collage-nase A (Boehringer Mannheim) dissolved in Ham’s F12 plus 10%FCS. For each gram of tissue, 4 ml of digestion cocktail was used.After centrifugation (3 g for 30 seconds) to allow undigestedfragments to settle, the fat was discarded while the supernatant andpellet were separated for further processing to yield mammary fibro-blasts and epithelial cells respectively.

Preparation of mouse mammary fibroblastsThe supernatant of the partially digested mammary tissue (obtainedas described above) was centrifuged (190 g for 10 minutes) and thepellet predominantly composed of single stromal cells washed andseeded in DMEM plus 10% FCS.

Preparation of primary mouse mammary epithelial cellsFragments of undigested mouse mammary tissue obtained by limitedenzymatic digestion (see above) were further digested with stirring at37°C for 30 minutes, using 2 ml digestion cocktail per gram of theoriginal tissue. The resulting epithelial organoids (ductal and lobulo-alveolar fragments) were then pelleted (55 g for 3 minutes) andwashed in DMEM plus 10% FCS. The organoids were then seededinto Ham’s F12:DMEM (1:1) supplemented with 10% FCS, I (10µg/ml), CT (10 ng/ml), transferin, T, (10 µg/ml) and Epidermalgrowth factor, EGF, (10 ng/ml) and, left for 2 hours. This allows anycontaminating fibroblasts to attach preferentially, effectivelyenriching the non-attached population for epithelial cells. The non-attached cells were then removed and reseeded in the above media

and left to grow for a period of 3-4 days after which the cells couldbe trypsinised into single epithelial cells and used for further study.Mouse mammary epithelial cells were routinely maintained in theabove media referred to as complete media.

This procedure is efficient in generating large quantities of highlyenriched populations of mouse mammary epithelial cells free of con-taminating fibroblasts. However, further purification of epithelial andfibroblast cells into discrete representative subpopulations is currentlynot possible with the mouse mammary cells since specific markersthat distinguish mouse mammary fibroblast subpopulations andmouse mammary epithelial subpopulations have not as yet been iden-tified. Hence the mouse mammary epithelial cells used in this studyare a mixture of myoepithelial and luminal cells, while the mousemammary fibroblasts used represent a population of interlobular andintralobular fibroblasts.

HGF/SFMouse recombinant HGF/SF (mrHGF/SF) and human recombinantHGF/SF (hrHGF/SF) were generous gifts from Dr E. Gherardi.

Mitogenicity assay2×104 cells were seeded in 24-well dishes in basal media and leftovernight to settle. The media were then changed to test media con-taining recombinant HGF/SF. Throughout this study 50 ng/mlmrHGF/SF was used for mouse epithelial cells while 50 ng/mlhrHGF/SF was used for human epithelial cells. At indicated timeintervals cells were trypsinised and cell numbers estimated using aCoulter counter (Coulter electronics). In all growth curves, the mediawas changed every three days.

Morhogenecity assayCells and organoids were embedded in collagen gels (Rat tail type I,Becton Dickinson) by mixing 250 µl collagen solution with 40 µl7.5% sodium hydrogen carbonate and 210 µl media (appropriate tothe cells) containing 2×104 cells or approximately 15 organoids. Thegels were then poured into 24-well dishes already containing 500 µlcollagen gel base per well, allowed to set, covered with media andleft overnight. The following day, recombinant HGF/SF was added tothe gels at 50 ng/ml and left for 5-7 days with daily feeding ofHGF/SF.

Motogenicity assay2×104 cells were seeded into 24-well dishes containing the appropri-ate media and left overnight to settle. Recombinant HGF/SF was thenadded at 50 ng/ml and the cells left for 24-48 hours after which timethe cells were photographed.

Co-culturesCo-cultures were initiated by seeding a well-mixed single cell sus-pension containing 4×105 epithelial cells together with 4×105 MRC 5in 2 ml DMEM containing 10% FCS in a 3.5 cm dish. The controlswere composed of 4×105 MRC 5 cells seeded in 2 ml DMEM plus10% FCS in a 3.5 cm dish. The co-cultures and their controls wereleft to condition their media for a period of 3 days. The conditionedmedia were centrifuged at 90 g for 5 minutes to remove cell debrisand stored at 4°C until use.

Scatter factor activity assayUsing 96-well plates, twofold serial dilutions of test samples wereincubated with 3000 MDCK cells at 37°C for 24 hours. The plateswere then fixed with 4% formaldehyde in PBS, stained with 0.2%Coomassie blue (15 minutes) followed by 1% crystal violet (60minutes) and each well assessed for scattering. The highest dilutionat which scattering could be observed was recorded as the end pointof the assay. Since the assay is performed in serial dilutions of 2, theresults are presented as log2, ie. SF activity at a dilution of 1/32 ispresented as (log2 1/32 =) −5.

2900 B. Niranjan and others

Fig. 1. Expression of HGF/SF and c-met during mouse mammarygland development. A northern blot of poly(A)+ RNA from differentstages development and differentiation was sequentially probed forHGF/SF and c-met (3 day exposure). Hybridisation of a controlGAPDH probe shows equal RNA loading in all lanes apart from 2and 10 day lactation (1 hour exposure). In these tracks, β caseindemonstrates the presence of comparable levels of RNA incomparison to other tracks (8 minute exposure).

Northern analysis of HGF/SF and c-met transcriptsCultured cell RNA extraction, northern blotting and hybridizationprocedures were performed as described previously (Boehm et al.,1988). For mouse developmental studies, total RNA was extracted bythe method of Chomczynski and Sacchi (1987) from the fourthabdominal mammary glands of female Parks mice (MRC; out-bred)as previously described (Weber-Hall et al., 1994). Northern blotanalysis was carried out on poly(A)+ RNA isolated from this material.HGF/SF and c-met probes were as follows; (i) a 2.6 kb cDNAfragment containing the complete mouse HGF coding region (M.Sharpe unpublished data), (ii) a 0.8 kb cDNA fragment encoding partof the C-terminal region of the mouse c-met gene (Chan et al., 1988),(iii) a human HGF/SF, 0.7 kb cDNA fragment covering the β chainregion of the h-HGF mRNA (Nakamura et al., 1989), (IV) a 886 bpcDNA fragment encoding parts of the cytoplasmic domain of thehuman c-met gene (Chan et al., 1987) and, (V) a 400 bp cDNAfragment derived from the 5′ end of the mouse β casein (a kind giftfrom R. Humphries). Following hybridisation filters were washed toa stringency of 2.5× SSC, 0.1% SDS at 65°C and autoradiographedfor 3 days at −70°C, using X-OMAT XAR-5 film. The relative qualityand quantity of RNA samples used in this study was determined byprobing for GAPDH mRNA using a 1.3 kb cDNA clone (Weber-Hallet al., 1994).

Analysis of HGF/SF and c-met gene expression by RT-PCR A 271 bp region of the human HGF/SF mRNA, encompassing exon1 and exon 2 sequences (Seki et al., 1991), and a 378 bp region of thehuman c-met mRNA, encompassing exons n-1, n and n+1 whichencode the transmembrane region of this receptor (Lee and Yamada,1994), were amplified using the following primers:

HGF/ SF-F: 5′-TTCTTTCACCCAGGCATCTC-3′, HGF/ SF-R: 5′-ATTAGCACATTGGTCTGCAG-3′

and

c-met-F: 5′-CCTGCTGAAATTGAACAGCGAG-3′,c-met-R: 5′-TGCACTTGTCGGCATGAACC-3′.

Control PCR reactions were carried out against human glucose-6phosphate dehydrogenase (G6 PD) mRNA using primers describedby Boehm et al. (1991) and human β-actin primers as described byLuqmani et al. (1992). The concentration of each RNA preparation tobe analysed by RT-PCR was adjusted to 1 µg/ml and the integritychecked by electrophoresis on 1.5% agarose mini gels. Randomprimed cDNA was reverse transcribed from 2 µg of total RNA in atotal reaction volume of 20 µl. PCR reactions (100 µl) were carriedout using 2 µl of cDNA template. Amplification consisted of 30cycles, each composed of a DNA denaturing step of 95°C for 1minute, a primer annealing step of 55°C for 1 minute and a DNAsynthesis step of 72°C for 1 minute. A final synthesis step of 72°Cfor 5 minutes was carried out. PCR products were analysed on 2%NuSieve / 1% HGT.

RESULTS

HGF/SF expression in mouse mammary tissueHGF/SF and c-met gene expression was followed duringmouse mammary development and differentiation. Northernblots of poly(A)+ RNA to the various stages of developmentand differentiation were sequentially probed for mouseHGF/SF, mouse c-met and GAPDH transcripts (Fig. 1). Theresults show that both HGF/SF and c-met are coordinatelyexpressed during the development and differentiation of mousemammary tissue. The cleared fat pad represents the mes-

enchymal component of this organ, devoid of mammaryepithelial cells, after removal of the rudimentary mammarygland by cauterisation (DeOme et al., 1959). Therefore, theexpression of HGF/SF detected in the mammary fat padindicates that the mesenchymal cells of the mammary glandare likely to be the source of HGF/SF expression in this organ.These data are in agreement with the existing body of evidencedemonstrating that HGF/SF expression is restricted to mes-enchymal cells (Stoker et al., 1987). The expression of c-metdetectable in the cleared fat pad is likely to be due to the endo-thelial cells (Bussolino et al., 1992).

Expression of HGF/SF and c-met was detected in virginmice as early as 6 weeks of age. By 12 weeks, the expressionof both genes was elevated and remained at this level for atleast the first 12.5 days of pregnancy. However, HGF/SF andc-met appeared to be expressed at undetectable levels at around17 days of pregnancy and throughout lactation. The presenceof undegraded polyadenalated mRNA in these tracks is demon-strated by the detection of full-length β casein and GAPDHmRNA (Gavin and McMahon, 1992; Barker et al., 1995) (Fig.1). It is now well established that milk protein mRNAs con-stitute up to 95% of the mRNA population in a lactating ratand mouse mammary gland (casein and whey acidic protein,respectively, represent 80% and 15% of the total poly(A)+RNA in the mammary gland (Richards et al., 1981, Hobbs etal., 1982)]. Therefore, it is likely that the apparent down reg-ulation of expression of HGF/SF and c-met is a consequenceof the feature of lactating mammary gland to express milkproteins preferentially over and above proteins, the expressionof which are not crucial for specific requirements of the gland

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A Fig. 2. HGF/SF activity inmammary fibroblasts. (A) HGF/SFactivity in supernatant of mouseand human mammary fibroblasts.Cells were seeded in basal mediaand left to condition their mediafor 4 days. The media was thenremoved and assayed for HGF/SFactivity using the MDCK assay.The error bars represent an averageof 4 independent values.(B) HGF/SF gene expression inpurified human breast cellpopulations. HGF/SF geneexpression was determined by RT-

PCR (30 cycles) on RNA made from purified luminal cells (2), Myoepithelial cells (3), heterogeneousfibroblast population derived from primary organoids (4), inter lobular fibroblasts from three separateindividuals (5, 7, 9) and intra lobular fibroblasts from the former three individuals (6, 8, 10). MRC 5 RNA wasused as a positive control for HGF/SF expression (1). Efficiency of cDNA synthesis from RNA isolated fromcell populations was monitored by co-amplification of G6PD sequences (150bP).

at this point in time. The relative abundance of these transcriptsis further reflected in the exposure times required for thedetection of HGF/SF, c-met, GAPDH and β casein transcripts(Fig. 1). Interestingly, levels of both HGF/SF and c-metremained high during the early stages of involution, a time atwhich this tissue undergoes extensive remodelling.

Mammary fibroblasts express HGF/SFIn order to identify the source of HGF/SF in the mammarytissue, populations of interlobular and intralobular fibroblasts(derived from the breast stroma) as well as luminal and myoep-ithelial (isolated from from organoids) were derived asdescribed in methods. Significant HGF/SF activity wasdetected in the culture supernatant of the mouse mammaryfibroblasts (Fig. 2A) but not in the culture supernatant of thehuman mammary fibroblasts. However, HGF/SF expressionwas readily detectable in the human mammary fibroblasts byPCR. Human luminal and myoepithelial cells do not expressHGF/SF (Fig. 2B). Interestingly, HGF/SF appears to be morehighly expressed in the human intralobular fibroblast popula-tion. HGF/SF activity was not detected in the culture super-natant of mouse (data not shown) or human mammary epi-

Fig. 3. HGF/SF induction of branching morphogenesis in human organohrHGF/SF at 50 ng/ml for a period of 5 days. (A) Untreated control cult

thelial cells (Fig. 9). Together, these results demonstrate thatmammary fibroblasts are the source of HGF/SF in the breast.

Morphogenic effects of HGF/SF on humanorganoidsMorphogenic effects of HGF/SF on the human breast wereexamined by treating organoids embedded in collagen gelswith HGF/SF at physiological concentrations. After 5 days inculture, in the absence of HGF/SF, the organoids appearedsomewhat spread with a conspicuous absence of morphogenicchanges (Fig. 3). In the presence of HGF/SF however, theorganoids exhibited a striking display of extensive branchingtubules. Although structures resembling end buds were notseen in these cultures, it is possible that these tubules werederived by processes similar to those involved in the ductalelongation phase of mammary development at puberty andhence may encompass growth together with morphogenesis(Fig. 3).

Morphogenic effects of HGF/SF on mammaryepithelial cellsIn order to examine the morphogenic effects of HGF/SF on

ids. Human organoids embedded in collagen gel were treated withures; (B) HGF/SF treated cultures. Scale bar = 200 µm.

2902 B. Niranjan and others

Fig. 4. HGF/SF induction of branching morphogenesis of mouse mammary epithelial cells. Dissociated and highly enriched mouse mammaryepithelial cells were embedded in collagen gels and treated with mrHGF/SF (50 ng/ml) for 5 days. (A) Control untreated cultures. (B) HGF/SFtreated cultures. (C) Cross section of HGF/SF treated culture demonstrating a well formed lumen. Scale bar in A, B = 200 µm. Scale bar in C=10 µm.

Fig. 5. Induction of branching morphogenesis in human mammary epithelial cells. (A) Control untreated cultures of human mammarymyoepithelial cells embedded in collagen gel. (B) Human mammary myoepithelial cells embedded in collagen gel and treated with hrHGF/SF(50 ng/ml) for 5 days, demonstrating branching morphogenesis. (C) Control untreated cultures of human mammary luminal cells embedded incollagen gel. (D) Human mammary luminal cells embedded in collagen gel and treated with hrHGF/SF (50 ng/ml) media for 5 days. Scale barfor A, B = 200 µm. Scale bar for C, D = 100 µm.

2903HGF/SF in mammary development

Fig. 6. Motogenic effect of HGF/SF on human mammary epithelial cells. (A) Control untreated cultures of human mammary myoepithelialcells. (B) Human mammary myoepithelial cells treated with hrHGF/SF (50 ng/ml) for 48 hours. (C) Control untreated cultures of humanmammary luminal cells. (D) Human mammary luminal cells treated with hrHGF/SF (50 ng/ml) for 48 hours. Scale bar for A, B = 200 µm.Scale bar for C, D = 200 µm.

mammary epithelial cells, human luminal and myoepithelialwere embedded in collagen gels. Mouse mammary epithelialcells were used as a pooled epithelial population, free fromfibroblasts. Mouse mammary epithelial cells when grownembedded in collagen gels and treated with physiologicallevels of HGF/SF produced extensive tubules with well-formed hollow lumina (Fig. 4).

Interestingly, human mammary luminal and myoepithelialcells appeared to respond differently to HGF/SF in that myoep-ithelial cells form extended branching tubules (Fig. 5A,B)whilst luminal cells were unresponsive to the morphogeniceffects of HGF/SF (Fig. 5C,D). Further, luminal cells did notappear to proliferate when embedded in collagen gels in thepresence of HGF/SF.

Motogenic effects of HGF/SF on mammary epitheliaTreatment of human luminal and myoepithelial cells withHGF/SF when grown on plastic produced classical scatteringof both human epithelial cell populations accompanied by achange in morphology of the epithelial cells to a more fibrob-lastic or polar appearance (Fig. 6). Hence both myoepithelialand luminal cells are responsive to motility effects ofHGF/SF.

Mitogenic effect of HGF/SF on mammary epithelialcellsIn order to examine the mitogenic effect of HGF/SF onmammary epithelial cells, human mammary epithelial cells(luminal and myoepithelial cells) and mixed mouse mammaryepithelial cells were treated with recombinant HGF/SF andtheir response examined using growth curves.

The results illustrate that HGF/SF produce a fourfoldincrease in growth of mouse mammary epithelial cells, whengrown in basal media [DMEM + 10% FCS] (Fig. 7A). HGF/SFproduced a further twofold increase in cell numbers in thepresence of a combination of other established growthpromoting factors (I, EGF, CT and T). This media (DMEM +10% FCS, I, EGF, CT and T) referred to as complete media,was used for basic maintenance of these cells. It is interestingto note that the addition of just HGF/SF to basal media, in theabsence of other growth-promoting agents, was sufficient toachieve the same growth rate as that achieved in completemedia which contained four different additives (Fig. 7A).Moreover, HGF/SF in conjunction with the above additivesimproved the growth of mouse mammary epithelial cellstenfold.

HGF/SF is also a potent mitogen for human luminal cells,

2904 B. Niranjan and others

Fig. 7. Mitogenic effects ofHGF/SF on mammary epithelialcells. (A) Mouse mammaryepithelial cells grown in DMEMplus 10% FCS (s); DMEM plus10% FCS and mrHGF/SF (d);DMEM:F12 plus 10% FCS, I,CT, T and EGF (,); DMEM:F12plus 10% FCS, I, CT, T, EGF andmrHGF/SF (.). (B) Humanmammary luminal cells grown inRPMI plus 1% FCS, I, CT andHC (m); RPMI plus 1% FCS, I,CT, HC and hrHGF/SF (n);RPMI plus 10% FCS, I, CT andHC (s); RPMI plus 10% FCS, I,CT, HC and hrHGF/SF (d). (C) Human mammary

myoepithelial cells grown in RPMI plus 1% FCS, I, CT and HC (s); RPMI plus 1% FCS, I, CT, HC and hrHGF/SF (d); RPMI plus 10% FCS,I, CT and HC (h); RPMI plus 10% FCS, I, CT, HC and hrHGF/SF (j).

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improving the growth rate of these cells fivefold over cellsgrown in RPMI plus 10% FCS supplemented with I, HC andCT (Fig. 7B). HGF/SF improves the growth of luminal cellsby ninefold when grown in RPMI plus 1% FCS supplementedwith I, HC and CT (Fig. 7B). However, HGF/SF appears tohave no growth stimulatory effects on myoepithelial cells (Fig.7C). It should be noted that, in RPMI supplemented with I, HCand CT, myoepithelial cells grow significantly better in thepresence of 1% FCS rather than 10% FCS. We furtherexamined the response of human myoepithelial cells toHGF/SF in a variety of growth media and so far have notobserved any mitogenic effects of HGF/SF upon myoepi-thelial cells (data not shown).

We have demonstrated that both human myoepithelial andluminal cells express c-met. Human luminal cells appear to express relatively higher levels of c-met than myoepithe-lial cells (Fig. 8A). Further, c-met expression in humanmammary fibroblasts was undetectable by RT-PCR analysis(Fig. 8C).

Co-culture of mammary epithelial cells with MRC 5fibroblastsIn our earlier studies of regulation of HGF/SF expression, we

Fig. 8. c-met gene expression in purified human luminal andmyoepithelial cells. 25 mg total RNA from purified luminal andmyoepithelial cells was probed by northern blot for c-met geneexpression (A). The position of full-length c-met transcript islabelled. This figure shows that c-met gene expression is higher inluminal cells than myoepithelial cells (4 day exposure). Equivalenceof RNA in each track is demonstrated by hybridisation to humanGAPDH probe (B) (1 day exposure). RT-PCR analysis of humanmammary fibroblast and epithelial cells is shown in C. Primers thatflank sequences encoding the transmembrane region of the human c-met receptor were used to examine c-met expression in humanluminal (lane 2), myoepithelial (lane 3) and fibroblast (lane 4) cells.A single c-met-specific PCR product (378 bp) is seen in luminal andmyoepithelial cells as well as the control melanoma cell line SK23(lane 1). No PCR product could be seen with mammary fibroblastcDNA (lane 4). Control b-actin PCR reactions for the same samplesare shown in lanes 5-8.

have shown that epithelial cells can inhibit HGF/SF expressionin HGF/SF-producing fibroblasts (i.e. MRC 5) when the twocell types are co-cultured (Kamalati et al., 1992). The capacityto inhibit HGF/SF expression in MRC 5 fibroblasts is a featureexclusive to epithelial cells, since cells of mesenchymal originfail to inhibit HGF/SF activity in co-culture with MRC 5 cells(Kamalati et al, 1992). In order to examine the capacity ofhuman myoepithelial and luminal cells to inhibit HGF/SFexpression by MRC 5 cells, we co-cultured these cells withMRC 5 fibroblasts. The results (Fig. 9) show that luminal andmyoepithelial cells do not express HGF/SF activity, inagreement with the lack of HGF/SF transcript in these cells

2905HGF/SF in mammary development

HG

F/S

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Fig. 9. Inhibition of HGF/SF activity released by MRC 5 cells in co-culture with human mammary epithelial cells. HGF/SF activity inco-cultures of MRC 5 cells with human mammary myoepithelial andluminal cells ( ) using MDCK as a positive control (h) in comparison to MRC 5 control cultures (j). The error bars representthe standard deviation of six readings.

shown above (Fig. 2). However, in co-culture with MRC 5fibroblasts, human luminal cells appear to be extremelyefficient at inhibiting HGF/SF expression while myoepithelialcells failed to inhibit HGF/SF expression by MRC 5 cells.

DISCUSSION

The importance of cellular interactions in the control of cellmotility and morphogenic events during development is nowwell established (Grobstein, 1954; Kratochwill, 1972, 1983).Interactions between epithelial and mesenchymal cellsinfluence epithelial proliferation, differentiation and morpho-genesis (Grobstein, 1967; Saxen et al., 1976; Saxen, 1977).Mesenchymal and epithelial cells use soluble growth factors tomediate local effects through binding to their cellularreceptors. Since HGF/SF is a multifunctional paracrine effectorof epithelial cells, we aimed to evaluate the role of HGF/SFand its cellular receptor, c-met, in the mammary gland. Usingboth mouse and human models, we have demonstrated thatHGF/SF may have an important role in mammary growth anddifferentiation since it is a potent mitogen for mammary epi-thelial cells and can induce motility and branching morpho-genesis in these cells.

HGF/SF and c-met expression in mouse mammarytissue.Our data demonstrate that HGF/SF and its receptor areexpressed during mouse mammary gland development anddifferentiation. This expression appears to be coordinatelyregulated through development, pregnancy, lactation and invo-lution suggesting a role for HGF/SF in growth, morphogen-

esis, differentiation and tissue remodelling. This proposed roleis supported by the in vitro findings, which show that HGF/SFis mitogenic, motogenic and morphogenic for mammaryepithelial cells. Hence, HGF/SF is likely to play an importantrole in the mammary gland at times of cellular interactions andtissue remodelling.

Our data show that mammary fibroblasts are the source ofHGF/SF in human and mouse mammary gland. Specifically,in the human mammary gland, the intralobular fibroblastsrather than interlobular fibroblasts appear to be the subpopula-tion responsible for HGF/SF expression. In contrast, c-met isexpressed by both mouse and human mammary epithelial cellpopulations. Interestingly, the c-met transcript appears moreabundant in human luminal cells relative to myoepithelial cells.

Mesenchymal-epithelial interactions in themammary glandThe retention of inductive activity in adult mammary stromaand the maintenance of the ability of adult epithelium torespond to inductive mesenchymal influences provide sugges-tive evidence that stromal-epithelial interactions are active inthe postnatal gland (Hoshino, 1964; Daniel et al., 1968;Sakakura et al., 1979; Cunha et al., 1992). Direct evidence forthis is provided by studies of postnatal mouse mammary ductalelongation, which occur at puberty (Daniel and Silberstein,1987). In this context, our data on the morphogenic effects ofHGF/SF upon human organoids suggest that the branchingmorphogenesis observed in our studies may be obtained byprocesses similar to those that are involved in the ductalelongation phase of mammary development; i.e. rapid growthaccompanied by cellular movement and morphogenesis of theepithelial component of the gland. Hence, HGF/SF may be acandidate molecule for a naturally occurring factor that triggerssignal transduction pathways leading to the activation of theseprocesses.

Role of HGF/SF in mammary gland growth anddevelopmentOur studies on the effects of HGF/SF upon mammary epi-thelial cells show that both human and mouse mammaryepithelial cells are highly responsive to the full spectrum ofHGF/SF activities. We have found HGF/SF to be a potentmitogen for mouse mammary epithelial cells causing up totenfold increase in cell growth as determined by increased cellnumbers. Previous studies of the mitogenic role of HGF/SFhave been limited to the measurements of DNA synthesis andnot absolute changes in cell number. HGF/SF is also a potentmorphogen for these cells, inducing extensive tubularbranching and well-formed lumina.

Interestingly, HGF/SF appears to effect human mammaryepithelial cells differentially, depending on the epithelial celltype. Although HGF/SF was found to be a potent mitogen forhuman luminal cells, as determined by a ninefold increase intheir cell numbers, it appeared to have no measurablemitogenic activity on myoepithelial cells. Further, we demon-strate that HGF/SF elicits a morphological response in thehuman myoepithelial cells, inducing the formation of extensivebranching tubules. Under identical experimental conditions,human luminal cells do not appear to respond to the mor-phogenic capacity of HGF/SF. Finally, HGF/SF exerts amotogenic effect on both human luminal and myoepithelial

2906 B. Niranjan and others

cells, causing classical scattering of these cells accompaniedby a change in the morphological appearance of the cells. Themitogenic response of the luminal cells to HGF/SF comparedwith myoepithelial cells is consistent with our earlier in vivoobservations. Luminal cells have been shown to have a higher[3H]thymidine labelling index in vivo whilst myoepithelialcells have very low labelling (Joshi et al., 1986). In relation tothe contrasting morphogenic responses in these two popula-tions and their motility on addition of HGF/SF, it could be pos-tulated that the myoepithelial cells form the ductal skeletalnetwork down which the luminal cells migrate. In the rodent,the cap cells of the end buds give rise to the outer sheath ofmyoepithelial cells and it is thus hypothesized that HGF/SFmay be responsible for inducing the ductal elongation, mor-phogenic and motility responses mediated through c-metexpression on the differentiated myoepithelial cell populations;whilst the effect on the luminal cells is mitogenic andmotogenic, requirements to populate the lumen. It is in thiscontext that the current evidance on the clonal analysis ofluminal and myoepithelial cells indicates that they are inde-pendent self-renewing populations (O’Hare et al, 1991;Gusterson et al., 1995)

HGF/SF has been previously shown to stimulate DNAsynthesis in transformed human mammary epithelial cells(Rubin et al., 1991). Also, fibroblasts derived from a humanbreast carcinoma have been shown to express HGF/SF tran-script but not protein (Seslar et al., 1993). The data presentedhere are the first reporting the effects of HGF/SF on primarynormal mammary epithelial cells and expression of HGF/SF inprimary normal human mammary fibroblasts.

The differential response of human luminal and myoepithe-lial cells to HGF/SF as a mitogen and morphogen, respectively,is particularly interesting since these two epithelial cell popu-lations express c-met differentially. This hence begs thequestion whether the response to different capacities ofHGF/SF by these two epithelial cell populations is governedby the receptor density or receptor subtype (given that c-metgenerates a number of transcripts) displayed on the cell surface.Identification of the number of functional receptors expressedby these cells may help elucidate this observation, since it isnow well documented that receptor density can effect theextent to which downstream signalling pathways are engagedor activated (Hempstead et al., 1992; Heasley and Johnson,1992; Traverse et al., 1994; Dikic et al., 1994).

The apparent differential response displayed by these cellscould be used as a model system to investigate the intracellu-lar signalling pathways that are involved in executingmitogenic, motogenic and morphogenic responses elicited byone ligand, apparently via one receptor. There now exists acompelling body of evidence demonstrating that individualfactors are expressed differentially throughout mammary glanddevelopment and distributed within the gland in uniquepatterns, suggesting distinct intrinsic roles for the individualfactors based on their temporal and spatial localisation as wellas their receptor density on target cells (Robinson et al., 1991;Coleman et al., 1988).

Mammary luminal and myoepithelial cells are derived fromthe basal layer of the foetal periderm (Gusterson et al., 1994).However, myoepithelial cells express smooth muscle α-actin(O’Hare et al., 1991), a feature of smooth muscle cells. In thiscontext, the inability of myoepithelial cells to inhibit HGF/SF

expression in MRC 5 fibroblasts is yet another feature distin-guishing myoepithelial cells from luminal cells and furtherdemonstrates some aspects of the non-epithelial characteristicsof these cells.

Myoepithelial cells form the outermost monolayer ofmammary ducts, with processes extending laterally along ducts(Daams et al., 1987). The functional significance of myoep-ithelial cells in secretory mammary tissue, where they causemilk ejection, is clear. However, in virgin animals, this con-tractile tissue does not appear to have an immediately obviousfunction. In this context, the differential response to HGF/SFdemonstrated by luminal and myoepithelial cells is interesting,since it suggests that myoepithelial cells may have animportant role in branching morphogenesis of the gland andhence functionally contribute to the continuously renewingarchitecture of the gland.

Evidently, other factors must be involved in the regulationof proliferation of myoepithelial cells (Coleman et al., 1988).Indeed it is rapidly becoming clear that the explosion of growthand morphogenetic events at puberty are the result of highlyregulated and precisely timed interactions between a variety ofsystemic hormones and peptide growth factors (Coleman etal.1988; Williams and Daniel, 1983; Silberstein et al., 1990).Recently, the HGF/SF gene promoter has been shown tocontain two estrogen-responsive elements (Liu et al., 1994).Using RT-PCR we could not detect estrogen receptors in thecultured human mammary fibroblast and epithelial cells.Further, using 17-β estradiol, we were unable to induceHGF/SF expression in human mammary epithelial cells or toenhance the expression of HGF/SF in human mammary fibro-blasts (data not shown).

Here we demonstrate that HGF/SF is produced by mammaryfibroblasts and can elicit mitogenic, motogenic and mor-phogenic responses in breast epithelial cells. HGF/SF musttherefore be considered a strong candidate for a naturallyoccurring mutlifunctional mammary tissue cytokine.

The authors are indebted to Dr E. Gherardi for generous gifts ofrecombinant mouse and human HGF/SF. The authors wish to thankMs C. Clarke and Dr M. O’ Hare for their advise on the separationof mammary epithelial cells, Dr S. Ali for his help and advise onestrogen receptor studies, Dr M.Crompton for his continued supportand the Cancer Research Campaign for supporting this work. L. B.would also like to thank the MRC for additional support.

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(Accepted 9 June 1995)