JOURNAL OF CELLULAR PHYSIOLOGY 149:110-116 (1991)
Human Keratinocyte Growth-Promoting Activity on the Surface of Fibroblasts -
PETER C. YAEGER, CHARLES D. STILES, AND BARRETT J. ROLLINS* Department of Cellular and Molecular Biology (P.C.Y., C.D.S.) dnd Divisiori of Medicine (B.I.R.), Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02 1 15
To proliferate in serum-containing medium, normal human keratinocytes must be co-cultured with fibroblast feeder cells. Conditioned medium from feeder cell cultures cannot substitute for the cells themselves. We tested the hypothesis that fibroblasts display a keratinocyte growth-promoting activity on their outer cell surface. The results of our investigation showed that ( I 1 glutaraldehyde-fixed fibroblast feeder cells promote keratinocyte growth, (2) the growth-promoting effect requires contact between fixed fibroblasts and keratinocytes, and ( 3 )feeder activity is highly enriched within the plasma membrane fraction of fibroblasts. We conclude that at least part of the fibroblast ”feeder” activity involves a keratinocyte growth-promoting factor which i s bound to the outer surface of fibroblast plasma membranes.
The first successful culture system for the propagation of normal keratinocytes required co-culture with fibroblasts (Rheinwald and Green, 1975). In this system, live fibroblasts, mitotically inactivated with y-irradiation or mitomycin-C, served as a “feeder layer” for murine or human keratinocytes (reviewed by Rheinwald, 1980). Growth medium has since been developed that supports keratinocyte growth in the absence of heterologous feeder cells (Peehl and Ham, 1980). However, the keratinocyte feeder function of fibroblast cells in culture is of intrinsic biological interest. It may reflect the in vivo relationship between proliferating keratinocyte stem cells and fibroblasts in the underlying dermis. In addition, the ability of fibroblasts to support tumor formation by epithelial cells in athymic nude mice suggests a possible role for fibroblast-epithelial cell interaction in neoplasia (Camps et al., 1990). The molecular basis of fibroblast feeder activity is poorly understood. No single hypothetical mechanism can fully account for the dramatic mitogenic activity which normal fibroblasts exert on keratinocyte cells in culture. For example, one could entertain the notion that fibroblasts secrete a growth factor for keratinocytes. The recent purification of keratinocyte growth factor (KGF) from medium conditioned by fibroblasts is consistent with such a classical paracrine mechanism (Rubin et al., 1989). However, culture medium conditioned by fibroblasts displays little or no keratinocyte growth-promoting activity (Rheinwald and Green, 1975).Another possibility is that fibroblasts inactivate a keratinocyte growth-inhibiting agent in serum such as transforming growth factor-p (TGF-p). Again, however, fibroblast conditioned medium does not substitute for living feeder cell activity, suggesting that detoxification cannot completely explain the feeder cell phenomenon. In his early studies on the feeder layer re0 1991 WILEY-LISS, INC
quirements of normal keratinocytes Rheinwald (1980) noted that when fibroblasts are distributed in a nonuniform way in culture dishes, keratinocyte growth is restricted to areas populated by feeder cells. This observation argues strongly that neither secretion of a growth factor nor inactivation of a growth inhibitor can account for the feeder effect. A qualitatively different explanation for the keratinocyte feeder effect can be found within the expanding inventory of growth factors which can function in a membrane-bound state. Biologically active, membrane-bound derivatives of transforming growth factor-a (TGF-a),colony-stimulating factor-1 (CSF-11,and mast cell growth factor (MGF) have all been identified (Wong et al., 1989; Rettenmier et al., 1987; Anderson et al., 1990). The membrane-bound isoforms of CSF-1 andlor MGF may account for earlier observations that glutaraldehyde-fixed 3T3 cells can substitute for bone marrow stromal cells to promote proliferation and maturation of multipotential hematopoietic stem cells (Roberts et al., 1987). In experiments presented here, we tested the keratinocyte growth-promoting activity of metabolically inactive fibroblasts, which can neither secrete growthpromoting factors into the medium nor remove growthinhibiting factors from the medium or substratum. We demonstrate that glutaraldehyde-fixed fibroblasts retain feeder activity when tested in a growth medium that does not normally support keratinocyte growth in the absence of feeder cells. The feeder activity is not released in a stable soluble form from the metabolically Received January 31, 1991; accepted May 14, 1991.
*To whom reprint requests/correspondence should be addressed. Institution a t which work was performed: Dana-Farber Cancer Institute, Boston, MA 02115.
FIBROBLAST SURFACE FACTOR AND KERATINOCYTE GROWTH
inactive fibroblasts and is enriched in the plasma membrane of fibroblasts. We propose that the feeder activity is due to a factor bound to the fibroblast surface.
MATERIALS AND METHODS Cell culture The murine embryonic fibroblast cell line, Swiss 31‘3-52 (Todaro and Green, 19631, and HeLa and MDCK cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% bovine calf serum (BCS). The Ep-2 keratinocyte cell strain was derived from human fetal skin by Dr. Kristina Lindberg (Dana Farber Cancer Institute, Boston, MA) and cells were maintained through their 11th passage on 3 T 3 5 2 feeder layers as previously described (Rheinwald, 1980) in DMEMiHam’s F12 (3:l) supplemented with 5% BCS, 0.4 pg/ml hydrocortisone, 10-l’ M cholera toxin, 24 pg/ml adenine, 5 pgiml insulin, and 0.1 mgiml penicillin and streptomycin. Epidermal growth factor (Upstate Biotechnology, Inc., Lake Placid, NY) was included at 30 ngiml a t refeedings of Ep-2 cells but not a t initial platings. Cells were refed 1 day after plating and every 2nd day thereafter. To prepare feeder layers, subconfluent 3T3-52 cells were treated with 4 kgiml mitomycin-C in growth medium for 2 hours at 37”C, washed with phosphate-buffered saline (PBS), resuspended using 0.1% trypsin/0.2% EDTA, and then replated at approximately 113 confluent density.
Glutaraldehyde fixation 3T3-52 cells were fixed with glutaraldehyde either a s attached feeder layers or in suspension. Attached cells, a t approximately 1/3 confluence, were washed twice with PBS, incubated with 0.05% glutaraldehyde in PBS for 5 minutes a t 37”C, and washed 4 times with PBS and once with Ep-2 growth medium (above) prior to plating of Ep-2 cells. Suspended cells for fixation were obtained by treating adherent cells, a t approximately 80% confluence, with 0.02% EDTA. The harvested cells were washed twice in PBS, resuspended in 0.05% glutaraldehyde at lo6 cellsiml, and incubated for 5 minutes at 37°C. After three more washes in PBS, the cells were stored for up to 3 months in PBS a t 4°C before being tested for feeder activity a t approximately 2.5 x lo5 cells/ml. Both treatments rendered cells metabolically inactive as determined by their lack of lactate production (Roberts e t al., 1987). Feeder activity assays In this report, fibroblast-derived activity that promotes keratinocyte growth specifically in the growth medium described above is referred to a s “feeder activity.” 3T3-52 feeder layers were removed from stock cultures of subconfluent Ep-2 cells after incubation in 0.02% EDTA (Rheinwald, 1980). The attached epithelial colonies were then suspended with 0.1% trypsini 0.02%EDTA, washed in Ep-2 growth medium, and plated a t a density of 50 cells/mm2 in the presence or absence of supplements described in “Results.” After 1 day, epidermal growth factor (EGF) was added to a final concentration of 30 ngiml. Cells were refed every
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2 or 3 days. For assays of fixed, attached feeder cells, Ep-2 cells were plated after the attached 31’3-32 cells had been fixed with glutaraldehyde. All other supplements were co-plated with Ep-2 cells and were included in the growth medium at refeeding. Six or 7 days after plating of Ep-2 cells, epithelial colonies were washed twice with PBS and fixed and stained by one of two methods: (1)cells were fixed 20 minutes in 100% methanol and then stained for 10 minutes in 0.2% methylene blue, or (2) cells were simultaneously fixed and stained for 15 minutes in 20% methanol/0.5% crystal violet.
Quantitation of cell proliferation Quantitation of cell proliferation was achieved by counting colonies or by elution of crystal violet stain. Methylene blue-stained colonies were counted under ~ 1 0 magnification. 0 For each 35 mm tissue culture dish, the total number of colonies in 40 random fields of approximately 0.8 mm2 (total = -30 mm2) was determined. Only colonies with a t least 16 contiguous cells having a non-differentiated epithelial morphology were counted. Each number given in “Results” represents a n average determined by two investigators, using procedures that did not permit identification of the culture dishes during counting. Alternatively, using a modification of the procedures of Dealtry and Balkwill (1987), crystal violet was quantitatively eluted from stained cells by adding 250 ~133% acetic acid to each well of 24-well tissue culture plates followed by gentle agitation on a shaker for 15 minutes. From each well, 200 pl was transferred to a well of a 96-well flatbottom microtiter plate. Absorbance a t 600 nm, calibrated against 33% acetic acid, was measured on a n ELISA reader (Bio-Rad, Richmond, CA). Plasma membrane preparation, protein determinations, and enzymic assays Near-confluent 3T3-52 cells were suspended from tissue culture dishes in 0.02% EDTA. Cells were washed in PBS and resuspended in 12 ml of sonication buffer (1mM phenylmethylsulfonyl fluoride, 10 pgiml aprotinin in PBS). Cells were homogenized with three 10 second pulses from the microtip of a Sonic Dismembranator (ARTEK, Farmingdale, NY) a t 40% of maximum output. The cell homogenate (HOM) was centrifuged a t 600g for 10 minutes to yield a post-nuclear supernatent (PNS) and the PNS was centrifuged a t 100,OOOg for 30 minutes to yield a crude membrane (CM) pellet (Ramwani and Mishra, 1986).The CM was centrifuged a t 100,OOOg for 2 hours into a 40% sucrose cushion to yield a plasma membrane band (PM) approximately 0.5-1 cm below the surface and a pellet of other crude membrane components (PEL). PM and PEL fractions were washed in PBS by centrifugation a t 170,OOOg for 1 hour. Protein concentrations were determined by the Bradford method (Bio-Rad, Richmond, CA). Alkaline phosphodiesterase (EC 3.1.4.1), NADPHcytochrome-c reductase (EC 1.6.2.41, and lactate dehydrogenase (EC 1.1.1.27) activities were determined as previously described (Storrie and Madden, 1990; Sottocasa et al., 1967).
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RESULTS Glutaraldehyde-fixedfibroblasts provide feeder activity To determine whether fibroblasts could support epithelial cell growth by means other t h a n secreted products, we tested glutaraldehyde-fixed, metabolically inactive Swiss 3T3-52 fibroblasts for feeder cell activity. After experimenting with serial dilutions, we found that 0.05%glutaraldehyde was sufficient to extinguish all metabolic activity as documented by lack of lactate production (see “Materials and Methods”). The results of several experiments, summarized in Table 1, show that feeder cells fixed in 0.05% glutaraldehyde retain feeder activity. The number of epithelial colonies increased 14-fold over background when Ep-2 cells were plated onto dishes containing attached fibroblasts fixed in 0.05% glutaraldehyde. As one of several controls, we showed that glutaraldehyde-treated tissue culture dishes without feeder layers did not support growth of epithelial colonies (data not shown). Fibroblasts fixed a t higher concentrations of glutaraldehyde (0.1% to 2%) had less feeder activity (data not shown). Even the fibroblasts fixed with 0.05% glutaraldehyde exhibited less feeder activity than unfixed fibroblasts (Table 1). Nevertheless the results summarized in Table 1 indicate t h a t feeder activity is not completely dependent upon the metabolic activity of the feeder cells. Suspensions of glutaraldehyde-fixedfibroblasts display feeder activity Encouraged by these results, we asked if glutaraldehyde-fixed fibroblasts could provide feeder activity without being attached to the tissue culture dish. Feeder activity in suspended cells could provide a n assay for testing activity after cell fractionation or other treatments that preclude feeder cell attachment. Suspended 3T3-52 cells were rendered metabolically inactive in 0.05% glutaraldehyde and tested for feeder activity a s described in “Materials and Methods.” In these experiments, the Ep-2 cells attached to the tissue culture dishes while the fixed feeders remained unattached. Control feeder activity assays were performed by testing for Ep-2 growth in the presence or absence of live attached feeder cells. Figure 1 shows that epithelial cell growth in the presence of fixed suspended feeders is nearly as extensive as growth in the presence of live attached feeders, when each feeder type is included a t its optimal concentration. For fixed suspended feeders, that concentration is approximately 2.5 x lo5 cellsiml, or a total of 4 x lo5 cells in the 1.6 ml assay. For live attached feeders, 1.6 x lo5 cells were pre-plated in the tissue culture dishes prior to adding keratinocytes. In comparison to the results from the experiment above in which the feeder layers were fixed while attached to the culture dishes, more than 100 epithelial coloniesi30 mm2 were counted in dishes that included fixed suspended feeders (see Table 1). This result has been consistently observed in several independent experiments. Experiments in which fixed suspended feeders were included a t different concentrations showed that
TABLE 1. Feeder activity of glutaraldehyde-fixedand live fibroblasts Feeder cells None Fixed, attached3 Live, attached Fixed, suspended7
No. of dishes counted 132 104 35 65
Mean No. of epithelial colonies/30 mm2 1 1.3 18.8 >loo6 >loo6
IFor each dish, colonies in 40 random fields of approximately0.8 mmz were counted (see “Materials and Methods”). 2Compositeof seven independent experiments. ”Attached feeder layers were fixed in glutaraldehydeprior to plating epithelial cells (see “Materials and Methods”). 4Compositeof six independent experiments. 5Compositeof two independent experiments. 6Merging of colonies did not allow precise quantitation. 7Suspended feeder cells were fixed in glutaraldehyde and co-plated at 7.5 X 10‘ to 2.5 X lo5cells/ml with epithelial cells.
Fig. 1. Fixed suspended and live attached feeders have comparable activity. Ep-2 cells were plated onto 35 mm tissue culture dishes with no feeder layer, a live attached feeder layer pre-plated at 10’ cellsiml, or were co-plated with feeders that had been fixed in suspension with glutaraldehyde. The fixed suspended feeders, included at 2.5 x 10’ cellsiml, had been fixed 11 weeks prior to this experiment and stored at 4°C in PBS. After 7 days in culture, the live, attached feeders were selectively removed with 0.02% EDTA and Ep-2 cells in all dishes were rinsed, fixed, and stained. Similar results were obtained from duplicate dishes (not shown). Photomicrographs ( X 100) of representative epithelial colonies grown in the respective conditions are shown below each dish.
Ep-2 growth response was dependent upon the concentration of the suspended glutaraldehyde-fixed feeders with feeder activity disappearing below 2.5 x lo4 cellsiml (data not shown). Photomicrographs of representative regions of tissue culture dishes with no feeder layer, a fixed feeder layer, or a live feeder layer are shown in Figure 1.
Glutaraldehyde-fixed fibroblasts promote keratinocyte growth in a contact-dependentfashion These experiments demonstrate that a t least some of the factods) responsible for feeder activity are not
FIBROBLAST SURFACE FACTOR AND KERATINOCYTE GROWTH
actively secreted into the medium by 3T3-52 cells. However, passive leakage of feeder activity from the fixed cells might explain these results. To test this possibility, Ep-2 cells were plated in tissue culture wells beneath cell culture inserts having membranes with 0.45 pm pores. Glutaraldehyde-fixed 3T3-52 cells in suspension were added above the membrane a t a final concentration of 2.5 x lo5 cellsiml. As a positive control, fixed 3T3-52 cells were plated together with Ep-2 cells below the membrane. As a negative control, Ep-2 cells were plated below membranes in the complete absence of suspended feeders. After 6 days, the epithelial cells were fixed and stained. As shown in Figure 2, no significant feeder activity passed through the 0.45 pm pores. This result indicates the presence of a biologically active factor bound to the surface of 31‘3-52 fibroblasts that promotes the proliferation of Ep-2 keratinocytes. We noted, in addition, that the glutaraldehyde-fixed cells used in the experiments shown in Figures 1and 2 had been stored in buffered saline more than 11 weeks and then centrifuged out of the buffer before use, without any apparent loss of activity. Furthermore, growth medium conditioned by fixed suspended feeders overnight on a roller at 37°C did not exhibit feeder activity (data not shown).
Feeder activity is cell-type specific We next asked whether epithelial cell growth promotion was a general property of glutaraldehyde-fixed mammalian cells. To answer this question, we harvested MDCK cells, HeLa cells, and 31’3-52 cells using 0.1% trypsin/0.02% EDTA, then washed and fixed the cells with glutaraldehyde. The fixed suspended cells were tested for feeder activity as described above for the experiments shown in Figure 1. Figure 3 shows t h a t among this selection of cell lines, only 3T3-52 fibroblasts exhibit feeder activity. Thus, feeder activity is not a characteristic common to all mammalian cells. Feeder activity is enriched in the plasma membrane fraction of fibroblasts Our results have demonstrated that feeder activity is bound to the fixed cells, suggesting that feeder activity might be enriched in the plasma membrane fraction of 3T3-52 cells. Accordingly we removed 31‘3-52 cells from tissue culture dishes using 0.02% EDTA, then homogenized and fractionated the cells a s described in “Materials and Methods.” Each fraction was assayed for alkaline phosphodiesterase, NADPH cytochrome-c reductase, and lactate dehydrogenase (markers for plasma membrane, endoplasmic reticulum, and cytosol, respectively). Table 2 shows t h a t the plasma membrane marker was successively enriched in the crude membrane (CM) and plasma membrane (PM) fractions, and that the endoplasmic reticulum and cytosol markers were not enriched in the PM fraction. Each fraction was tested for feeder activity by plating lo4 Ep-2 cellsiwell into 24-well tissue culture plates in growth medium supplemented with the 3T3-52 fractions a t 0, 1 , 3 , 9 , or 27 pg proteiniml. Cells were re-fed with the supplemented growth medium 3 days after plating. On the 6th day, cells were fixed and stained. Figure 4 shows that feeder activity was successively
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Fig. 2. Porous membranes block feeder activity of fixed suspended feeders. Ep-2 cells were plated into a six-well tissue culture plate below membrane inserts with .45 wm pores. Epithelial cells were plated either without feeders or with fixed, suspended feeders below or above the membrane as indicated. After 6 days, the membranes were discarded, and Ep-2 cells in the wells were rinsed, fixed, and stained. Similar results were obtained from duplicate wells (not shown). In this experiment, background growth of Ep-2 cells he., in the absence of feeders) was unusually high.
Fig. 3. Lack of feeder activity in MDCK and HeLa cells. Subconfluent MDCK, Hela, and Swiss 3T3-52 cells were suspended with 0.1% trypsin/0.02’% EDTA, washed in PBS, fixed in glutaraldehyde, and co-plated a t 2.5 x lo5 cells/ml with Ep-2 cells into 35 mm tissue culture dishes. As a negative control, Ep-2 cells were plated alone. After 7 days, the cells were fixed and stained. Similar results were obtained from duplicate dishes (not shown).
enriched in the CM and PM fractions, with maximum growth response in the PM fraction at 9 pg/ml. There was no enrichment in the membrane fraction that pelleted in 40% sucrose (PEL, see “Materials and Methods”), and activity in the cytosol (CYT) fraction was reduced relative to that of the HOM. To provide a more quantitative analysis, dye was eluted from the stained Ep-2 cells in each well and quantitated by measuring absorbance a t 600 nm (Fig. 5). By interpolating a t the absorbance value of 1.0 for the PM, CM, and HOM plots, we estimate a two-fold and nine-fold enrichment of feeder activity in the plasma membrane preparation relative to the crude membrane and homogenate, respectively. As a crude comparison of the nature of the feeder activity in PM fractions to that of intact cells fixed in glutaraldehyde, we determined their heat sensitivities. For both preparations, activity was retained after
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TABLE 2. Activities of marker enzymes in cell fractions’ Enzvme NADPH cytochrome-c reductase activity Snecific Normalized
Alkaline phosphodiesterase activity Specific Normalized
Cell fraction2 Homogenate (HOM) Post-nuclear supernatent (PNS) Cytosol (CYT) Crude membrane (CM) Plasma membrane (PM) Sucrose-Delletedmembrane (PEL)
0.10 f 0.00 0.07 5 0.00 0.01
1.0
0.7 0.1 5.2 13.1 3.9
* 0.00
0.52 f 0.01 1.31 f 0.01 0.39 f 0.00
0.055 k 0.005 0.056 i 0.005 0.045 k 0.005 0.339 f 0.005 0.057 Ifr 0.004 0.057 k 0.004
1.0 1.0 0.8 6.0
1.0 1.0
Lactate dehydrogenase activity Specific Normalized 1.34 f 0.30 1.78 f 0.09 5.61 f 0.10 0.26 f 0.10 0.60 f 0.11 0.43 k 0.15
1.0 1.3 4.2 0.2 0.4 0.3
‘Specific activities: A absorbance/min/mg protein fstandard error in assays described in “Materials and Methods”; Normalized activities: activity of HOM equals 1.00. %ee “Materials and Methods.”
HCM
PNS
PM
CM
CYT
PEL
1
3.0
1 PM CM
2.0
8
PNS
@J
0
HOM
PEL
1.0
CM
27 Fig. 4. Feeder activity in fibroblast cell fractions. Swiss 3T3-JZ cells suspended from tissue culture dishes with 0.02% EDTA were fractionated and orotein determinations were oerformed as described in “Materials and Methods.” Ep-2 cells were plated into 24-well tissue culture plates at 104 cellsiwell in growth medium containing fractions at the indicated concentrations (JLgproteiniml), After days, the cells were fixed and stained in 20% methanol,o,5% crystal violet, HOM = homogenate; PNS = post-nuclear supernatent; PM = plasma membrane; CM = crude membrane; CYT = cytosol; PEL membrane pellet in 406/csucrose, Similar results were obtained from replicate wells (two replicatesof each well containing cMand P ~ fractions; L six replicates of each well containing HOM, PNS, PM, and CYT fractions).
0.0 0
10
20
30
pg protein/ml
L -
~
treating 30 minutes at 65°C and lost after 30 minutes a t 85°C (data not shown). This was not due to the generation of toxic compounds from the heat treatment since untreated fixed cells retained activity when mixed with heat-inactivated cells. This result suggests that the feeder activity of intact metabolically inactive 3T3-52 cells and that of plasma membrane fractions may involve the same factor.
DISCUSSION We have demonstrated that glutaraldehyde-fixed fibroblasts promote the growth of normal human epidermal keratinocytes in a serum-containing medium designed for co-culture with living fibroblasts. We have
Fig. 5. Quantitative comparisons of feeder activities among cell fractions. Crystal violet was eluted into 33% acetic acid from Ep-2 cells in each of the wells shown in Figure 4 and their replicates. The eluted dye was quantitated by measuring absorbance at 600 nm on an ELISA reader. The mean absorbance is plotted as a function of the concentration of each cell fraction in the growth medium, determined on the basis of protein content. All absorbance values plotted in the graph are within the linear range of absorbance values a t 600 nm for crystal violet in 33% acetate. Error bars represent the S.E.M. Abbreviations for cell fractions are as indicated in Figure 4.
also shown that no growth response was observed when a nitrocellulose filter separated the keratinocytes from the fixed feeder cells, and that feeder activity was enriched in the plasma membrane fraction of 3T3 cells. Taken together, our results provide evidence for the existence of a membrane-bound factor on the surface of fibroblasts that promotes keratinocyte growth. In a limited survey, this activity is specific for cells that display feeder activity when metabolically active (see Fig. 3). One alternative explanation for our findings is that fibroblasts secrete a keratinocyte growth-promoting factor that remains tightly bound to the extracellular matrix. We think this is unlikely because the method
FIBROBLAST SURFACE FACTOR AND KERATINOCYTE GROWTH
used to remove cells from culture dishes (0.028 EDTA in the absence of proteases) prior to glutaraldehyde fixation and plasma membrane isolation should leave extracellular matrix components attached to the culture dishes (Roberts et al., 1987; Bradley and Brown, 1990). Moreover, plasma membrane preparations washed with 1 M NaCl retained activity after centrifugation a t 100,OOOg (data not shown). Partial protease treatment (1 mgiml pronase) of plasma membrane preparations also failed to dissociate feeder activity from the plasma membrane (data not shown). Thus the factor involved is probably tightly associated with the membrane and, if i t is a protein, it is protected from proteases in its membrane-bound form. Growth factors t h a t are known to promote keratinocyte growth include EGF (Wille et al., 19841, TGF-a (Barrandon and Green, 1987), acidic and basic fibroblast growth factor (aFGF and bFGF) (Falco et al., 1988), and KGF (Rubin et al., 1989). No integral plasma membrane form of aFGF, bFGF, or KGF has been reported nor would their primary amino acid sequences predict it (Jaye et al., 1986; Abraham et al., 1986; Finch et al., 1989). However, we cannot rule out the possibility that, in our plasma membrane preparations, FGFs were bound tightly to proteoglycans and were not completely dissociated from the membrane surface by the NaCl and protease treatments discussed above. It is also possible that related genes o r alternatively spliced mRNAs, heretofore undetected, would produce membrane-associated variants of these growth factors. While TGF-CY has a predicted transmembrane region (Derynck e t al., 1984), biological activity from a n endogenous membrane-associated TGF-a has not been demonstrated. I n a n experimental system in which TGF-a is bound to the surface of CHO cells, this form of TGF-a can activate the EGF receptor of A431 cells (Wong et al., 1989).Because EGF and TGF-a bind the same receptor, and the growth medium used in our feeder activity assay, containing 30 ng/ml EGF, does not permit keratinocyte growth in the absence of fibroblast feeder cells or plasma membranes, TGF-a can not account for the feeder activity we observe in fibroblast membranes. Furthermore, TGF-a retains activity after treatment a t 100°C (De Larco and Todaro, 19781, whereas the feeder activity we observe in fibroblast plasma membranes is lost a t 85°C (see “Results”). In addition to TGF-a, two growth factors involved in the proliferation and differentiation of hematopoietic cells, CSF-1 and MGF, have transmembrane domains as predicted from their respective cloned cDNA sequences (Clark and Kamen, 1987; Anderson et al., 1990). Both growth factors have been localized to the cell surface and the growth-promoting activity of cell surface-associated MGF on mast cells has been demonstrated (Rettenmier et al., 1987; Anderson et al., 1990). The observation that glutaraldehyde-fixed Swiss 3T3 cells promote the proliferation of hematopoietic stem cells (Roberts et al., 1987) may relate to the existence of these membrane-bound growth factors. Our results point to the existence of yet another cell surface factor involved in growth regulation, specifically the promotion of keratinocyte growth by fibroblasts (although we have not yet demonstrated that this factor is a protein). The difficulties inherent in the methodology required
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for the identification of cell surface-bound growth factors raises the possibility that many such factors remain undiscovered, and their existence may emerge as a n important theme in growth factor research. The precise mechanisms involved in the response of keratinocytes to the fibroblast surface factor have yet to be identified. We found that our fibroblast plasma membrane preparations did not stimulate proliferation of keratinocytes in bioassays in which serum-free MCDB 153 growth medium (Peehl and Ham, 1980) was used (data not shown). Thus, the surface factor may promote keratinocyte growth by acting in concert with a growth factor in the serum or it may inactivate or counteract growth inhibitory signals which are likely to be present in the serum (Wille et al., 1984). Such inhibitory signals would include those that induce differentiation or those that inhibit proliferation more directly, e.g. TGF-P (Matsumoto et al., 1990). EGF and vitamin A are examples of substances that may promote keratinocyte growth by inhibiting differentiation (Marchese et al., 1990; Kopan and Fuchs, 1989). In contrast, KGF does not inhibit calcium-mediated differentiation of keratinocytes (Marchese e t al., 1990). TGF-P is synthesized by epithelial cells (Kane et al., 1990) and may inhibit their growth in autocrine fashion (Moses et al., 1989). It has been demonstrated that live fibroblasts remove and degrade TGF-(3 spontaneously attached to the surface of tissue culture dishes (Rollins et al., 1989). We have found that the plasma membranes of fractionated fibroblasts, although exhibiting feeder activity in the absence of exogenously added TGF-P, do not interfere with the inhibition of keratinocyte growth by TGF-p (data not shown).Therefore it is unlikely that fibroblast plasma membranes promote keratinocyte proliferation by inactivating or counteracting the growth inhibitory action of TGF-(3. The localization of factors to the surface of specific cell types that influence proliferation and differentiation of heterologous cells may play a n important role in the spatial control of proliferation during development and stem cell maturation. In keratinocyte proliferation and differentiation, the undifferentiated proliferating stem cells are closer to the supporting dermal layer, while the cells that have migrated away from the basal layer become differentiated and cease to proliferate (reviewed by Fuchs et al., 1988). Our conclusion that fibroblasts bear a surface factor t h a t promotes keratinocyte growth may have relevance to this physiological phenomenon. We have recently solubilized the feeder activity from plasma membrane preparations. This will facilitate further purification and characterization of the factor and permit investigation into its mode of expression and mechanism of action.
ACKNOWLEDGMENTS This work was supported by grant HD24926 from NIH to C.S. and grant CA53091 from NIH to B.R. LITERATURE CITED Abraham, J.A., Mergia, A,, Whang., J.L., Tumolo, A., Friedman, J., Hjerreld, K.A., Gospodarowicz, D., and Fiddes, J.C. (1986) Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor. Science, 233.545-548. Anderson, D.M., Lyman, S.D., Baird, A,, Wignall, J.M., Eisenman, J.,
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Rauch, C., March, C.J., Boswell, H.S., Gimpel, S.D., Cosman, D., and Williams, D.E. (1990) Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms. Cell, G3:235-243. Barrandon, Y., and Green, H. (1987) Cell migration is essential for sustained growth of keratinocyte colonies: The roles of transforming growth factor-a and epidermal growth factor. Cell, 50:1131-1137. Bradley, R.S., and Brown, A.M.C. (1990) The proto-oncogene int-1 encodes a secreted protein associated with the extracellular matrix. EMBO J., 9:1569-1575. Camps, J.L., Chang, S.-M., Hsu, T.C., Freeman, M.R., Hong, S.-J., Zhau, H.E., Von Eschenbach, A.C., and Chung, L.W.K. (1990) Fibroblast-mediated acceleration of human epithelial tumor growth in uiuo. Proc. Natl. Acad. Sci. U.S.A., 87t75-79. Clark, S.C., and Kamen, R. (1987) The human hematopoietic colonystimulating factors. Science, 236:1229-1237. Dealtry, G.B., and Balkwill, F.R. (1987) Cell growth inhibition by interferons and tumor necrosis factor. In: Lymphokines and Interferons, a Practical Approach. M.J. Clemens, A.G. Morris, and A.J.H. Gearing, eds. IRL Press, Oxford, pp. 195-220. DeLarco, J.E., and Todaro, G.J. (1978) Growth factors from murine sarcoma virus-transformed cells. Proc. Natl. Acad. Sci. U.S.A., 75:40014005. Derynck, R., Roberts, A.B., Winkler, M.E., Chen, E.Y., and Goeddel, D.V. (1984) Human transforming growth factor-a: precursor structure and expression in E. coli. Cell, 38:287-297. Falco, J.P., Taylor, W.G., Di Fiore, P.P., Weissman, B.E., and Aaronson, S.A. (1988) Interactions of growth factors and retroviral oncogenes with mitogenic signal transduction pathways of BalbiMK keratinocytes. Oncogene, 2573-578. Finch, P.W., Rubin, J.S., Miki, T., Ron, D., and Aaronson, S.A. (1989) Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth. Science, 245t752-755. Fuchs, E., Albers, K., and Kopan, R. (1988) Terminal differentiation in cultured human epidermal cells. Adv. Cell Cult., G:1-33. Jaye, M., Howk, R., Burgess, W., Ricca, G.A., Chiu, I.-M., Ravera, M.W., O’Brien, S.J., Modi, W.S., Maciag, T., and Drohan, W.N. (1986) Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosome localization. Science, 233:541-544. Kane, C.J.M., Knapp, A.M., Mansbridge, J.N., and Hanawalt, P.C. (1990) Transforming growth factor-pl localization in normal and psoriatic epidermal keratinocytes in situ. J . Cell. Physiol., 144:144150. Kopan, R., and Fuchs, E. (1989) The use of retinoic acid to probe the relation between hyperproliferation-associated keratins and cell proliferation in normal and malignant epidermal cells. J . Cell Biol., 109:295-307. Marchese, C., Rubin, J., Ron, D., Faggioni, A., Torrisi, M.R., Messina, A., Frati, L., and Aaronson, S.A. (1990)Human keratinocyte growth factor activity on proliferation and differentiation of human keratinocytes: differentiation response distinguishes KGF from EGF family. J. Cell. Physiol., 144:326-332. Matsumoto, K., Hashimoto, K., Hashiro, M., Yoshimasa, H., and
Yoshikawa, K. (19901 Modulation of growth and differentiation in normal keratinocytes by transforming growth factor-p. J . Cell. Physiol., 245.95-101. Moses, H.L., Bascom, C.C., Lyons, R.M., Sipes, N.J., and Coffey, R.J., J r . (1989) Transforming growth factors. In: The Regulation of Proliferation and Differentiation in Normal and Neoplastic Cells. E. Frei 111, ed, Academic Press, San Diego, pp. 89-102. Peehl, D.M., and Ham, R.G. (1980) Growth and differentiation of human keratinocytes without a feeder layer or conditioned medium. In Vitro, 1G:516-525. Ramwani, J., and Mishra, R.K. (1986) Purification of bovine striatal dopamine D-2 receptor by affinity chromatography. 3. Biol. Chem., 261 :8894-8898. Rettenmier, C.W., Roussel, M.F., Ashmun, R.A., Ralph, P., Price, K., and Sherr, C.J. (1987) Synthesis of membrane-bound colony-stimulating factor 1 (CSF-1) and downmodulation of CSF-1 receptors in NIH 3T3 cells transformed by cotransfection of the human CSF-1 and c-fms (CSF-1 receptor) genes. Mol. Cell. Biol., 7:237&2387. Rheinwald, J.G. (1980) Serial cultivation of normal human epidermal keratinocytes. Methods Cell Biol., 21At229-254. Rheinwald, J.G., and Green, H. (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell, G:331-344. Roberts, R.A., Spooncer, E., Parkinson, E.K., Lord, B.I., Allen, T.D., and Dexter, T.M. (1987) Metabolically inactive 3T3 cells can substitute for marrow stromal cells to promote the proliferation and development of multipotent haemopoietic stem cells. 3 . Cell. Physiol., 132.203-214. Rollins, B.J., O’Connell, T.M., Bennett, G., Burton, L.E., Stiles, C.D., and Rheinwald, J.G. (1989)Environment-dependent growth inhibition of human epidermal keratinocytes by recombinant human transforming factor-beta. J . Cell. Physiol., 139.455462. Rubin, J.S., Osada, H., Finch, P.W., Taylor, W.G., Rudikoff, S., and Aaronson, S.A. (1989) Purification and characterization of newly identified growth factor specific for epithelial cells. Proc. Natl. Acad. Sci. U.S.A., 8G:802-806. Sottocasa, G.L., Kuylenstierna, B., Ernster, L., and Bergstrand, A. (1967) An electron-transport system associated with the outer membrane of liver mitochondria. J. Cell Biol., 32:415438. Storrie, B., and Madden, E.A. (1990) Isolation of subcellular organelles. Methods Enzymol., 182:203-225. Todaro, G.J., and Green, H. (1963) Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J. Cell Biol., 17.299-313. Wille, J.J., Jr., Pittelkow, M.R., Shipley, G.D., and Scott, R.E. (19841 Integrated control of growth and differentiation of normal prokeratinocytes cultured in serum-free medium: clonal analysis, growth kinetics, and cell cycle studies. J. Cell. Physiol., 121:31-44. Wong, S.T., Wincell, L.F., McCune, B.K., Earp, H.S., Teixido, J., MassaguB, J . , Herman, B., and Lee, D.C. 11989) The TGF-u precursor expressed on the cell surface binds to the EGF receptor on adjacent cells, leading to signal transduction. Cell, 5Gt495-506.
Human Keratinocyte Growth-Promoting Activity on the Surface of Fibroblasts -
PETER C. YAEGER, CHARLES D. STILES, AND BARRETT J. ROLLINS* Department of Cellular and Molecular Biology (P.C.Y., C.D.S.) dnd Divisiori of Medicine (B.I.R.), Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02 1 15
To proliferate in serum-containing medium, normal human keratinocytes must be co-cultured with fibroblast feeder cells. Conditioned medium from feeder cell cultures cannot substitute for the cells themselves. We tested the hypothesis that fibroblasts display a keratinocyte growth-promoting activity on their outer cell surface. The results of our investigation showed that ( I 1 glutaraldehyde-fixed fibroblast feeder cells promote keratinocyte growth, (2) the growth-promoting effect requires contact between fixed fibroblasts and keratinocytes, and ( 3 )feeder activity is highly enriched within the plasma membrane fraction of fibroblasts. We conclude that at least part of the fibroblast ”feeder” activity involves a keratinocyte growth-promoting factor which i s bound to the outer surface of fibroblast plasma membranes.
The first successful culture system for the propagation of normal keratinocytes required co-culture with fibroblasts (Rheinwald and Green, 1975). In this system, live fibroblasts, mitotically inactivated with y-irradiation or mitomycin-C, served as a “feeder layer” for murine or human keratinocytes (reviewed by Rheinwald, 1980). Growth medium has since been developed that supports keratinocyte growth in the absence of heterologous feeder cells (Peehl and Ham, 1980). However, the keratinocyte feeder function of fibroblast cells in culture is of intrinsic biological interest. It may reflect the in vivo relationship between proliferating keratinocyte stem cells and fibroblasts in the underlying dermis. In addition, the ability of fibroblasts to support tumor formation by epithelial cells in athymic nude mice suggests a possible role for fibroblast-epithelial cell interaction in neoplasia (Camps et al., 1990). The molecular basis of fibroblast feeder activity is poorly understood. No single hypothetical mechanism can fully account for the dramatic mitogenic activity which normal fibroblasts exert on keratinocyte cells in culture. For example, one could entertain the notion that fibroblasts secrete a growth factor for keratinocytes. The recent purification of keratinocyte growth factor (KGF) from medium conditioned by fibroblasts is consistent with such a classical paracrine mechanism (Rubin et al., 1989). However, culture medium conditioned by fibroblasts displays little or no keratinocyte growth-promoting activity (Rheinwald and Green, 1975).Another possibility is that fibroblasts inactivate a keratinocyte growth-inhibiting agent in serum such as transforming growth factor-p (TGF-p). Again, however, fibroblast conditioned medium does not substitute for living feeder cell activity, suggesting that detoxification cannot completely explain the feeder cell phenomenon. In his early studies on the feeder layer re0 1991 WILEY-LISS, INC
quirements of normal keratinocytes Rheinwald (1980) noted that when fibroblasts are distributed in a nonuniform way in culture dishes, keratinocyte growth is restricted to areas populated by feeder cells. This observation argues strongly that neither secretion of a growth factor nor inactivation of a growth inhibitor can account for the feeder effect. A qualitatively different explanation for the keratinocyte feeder effect can be found within the expanding inventory of growth factors which can function in a membrane-bound state. Biologically active, membrane-bound derivatives of transforming growth factor-a (TGF-a),colony-stimulating factor-1 (CSF-11,and mast cell growth factor (MGF) have all been identified (Wong et al., 1989; Rettenmier et al., 1987; Anderson et al., 1990). The membrane-bound isoforms of CSF-1 andlor MGF may account for earlier observations that glutaraldehyde-fixed 3T3 cells can substitute for bone marrow stromal cells to promote proliferation and maturation of multipotential hematopoietic stem cells (Roberts et al., 1987). In experiments presented here, we tested the keratinocyte growth-promoting activity of metabolically inactive fibroblasts, which can neither secrete growthpromoting factors into the medium nor remove growthinhibiting factors from the medium or substratum. We demonstrate that glutaraldehyde-fixed fibroblasts retain feeder activity when tested in a growth medium that does not normally support keratinocyte growth in the absence of feeder cells. The feeder activity is not released in a stable soluble form from the metabolically Received January 31, 1991; accepted May 14, 1991.
*To whom reprint requests/correspondence should be addressed. Institution a t which work was performed: Dana-Farber Cancer Institute, Boston, MA 02115.
FIBROBLAST SURFACE FACTOR AND KERATINOCYTE GROWTH
inactive fibroblasts and is enriched in the plasma membrane of fibroblasts. We propose that the feeder activity is due to a factor bound to the fibroblast surface.
MATERIALS AND METHODS Cell culture The murine embryonic fibroblast cell line, Swiss 31‘3-52 (Todaro and Green, 19631, and HeLa and MDCK cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% bovine calf serum (BCS). The Ep-2 keratinocyte cell strain was derived from human fetal skin by Dr. Kristina Lindberg (Dana Farber Cancer Institute, Boston, MA) and cells were maintained through their 11th passage on 3 T 3 5 2 feeder layers as previously described (Rheinwald, 1980) in DMEMiHam’s F12 (3:l) supplemented with 5% BCS, 0.4 pg/ml hydrocortisone, 10-l’ M cholera toxin, 24 pg/ml adenine, 5 pgiml insulin, and 0.1 mgiml penicillin and streptomycin. Epidermal growth factor (Upstate Biotechnology, Inc., Lake Placid, NY) was included at 30 ngiml a t refeedings of Ep-2 cells but not a t initial platings. Cells were refed 1 day after plating and every 2nd day thereafter. To prepare feeder layers, subconfluent 3T3-52 cells were treated with 4 kgiml mitomycin-C in growth medium for 2 hours at 37”C, washed with phosphate-buffered saline (PBS), resuspended using 0.1% trypsin/0.2% EDTA, and then replated at approximately 113 confluent density.
Glutaraldehyde fixation 3T3-52 cells were fixed with glutaraldehyde either a s attached feeder layers or in suspension. Attached cells, a t approximately 1/3 confluence, were washed twice with PBS, incubated with 0.05% glutaraldehyde in PBS for 5 minutes a t 37”C, and washed 4 times with PBS and once with Ep-2 growth medium (above) prior to plating of Ep-2 cells. Suspended cells for fixation were obtained by treating adherent cells, a t approximately 80% confluence, with 0.02% EDTA. The harvested cells were washed twice in PBS, resuspended in 0.05% glutaraldehyde at lo6 cellsiml, and incubated for 5 minutes at 37°C. After three more washes in PBS, the cells were stored for up to 3 months in PBS a t 4°C before being tested for feeder activity a t approximately 2.5 x lo5 cells/ml. Both treatments rendered cells metabolically inactive as determined by their lack of lactate production (Roberts e t al., 1987). Feeder activity assays In this report, fibroblast-derived activity that promotes keratinocyte growth specifically in the growth medium described above is referred to a s “feeder activity.” 3T3-52 feeder layers were removed from stock cultures of subconfluent Ep-2 cells after incubation in 0.02% EDTA (Rheinwald, 1980). The attached epithelial colonies were then suspended with 0.1% trypsini 0.02%EDTA, washed in Ep-2 growth medium, and plated a t a density of 50 cells/mm2 in the presence or absence of supplements described in “Results.” After 1 day, epidermal growth factor (EGF) was added to a final concentration of 30 ngiml. Cells were refed every
111
2 or 3 days. For assays of fixed, attached feeder cells, Ep-2 cells were plated after the attached 31’3-32 cells had been fixed with glutaraldehyde. All other supplements were co-plated with Ep-2 cells and were included in the growth medium at refeeding. Six or 7 days after plating of Ep-2 cells, epithelial colonies were washed twice with PBS and fixed and stained by one of two methods: (1)cells were fixed 20 minutes in 100% methanol and then stained for 10 minutes in 0.2% methylene blue, or (2) cells were simultaneously fixed and stained for 15 minutes in 20% methanol/0.5% crystal violet.
Quantitation of cell proliferation Quantitation of cell proliferation was achieved by counting colonies or by elution of crystal violet stain. Methylene blue-stained colonies were counted under ~ 1 0 magnification. 0 For each 35 mm tissue culture dish, the total number of colonies in 40 random fields of approximately 0.8 mm2 (total = -30 mm2) was determined. Only colonies with a t least 16 contiguous cells having a non-differentiated epithelial morphology were counted. Each number given in “Results” represents a n average determined by two investigators, using procedures that did not permit identification of the culture dishes during counting. Alternatively, using a modification of the procedures of Dealtry and Balkwill (1987), crystal violet was quantitatively eluted from stained cells by adding 250 ~133% acetic acid to each well of 24-well tissue culture plates followed by gentle agitation on a shaker for 15 minutes. From each well, 200 pl was transferred to a well of a 96-well flatbottom microtiter plate. Absorbance a t 600 nm, calibrated against 33% acetic acid, was measured on a n ELISA reader (Bio-Rad, Richmond, CA). Plasma membrane preparation, protein determinations, and enzymic assays Near-confluent 3T3-52 cells were suspended from tissue culture dishes in 0.02% EDTA. Cells were washed in PBS and resuspended in 12 ml of sonication buffer (1mM phenylmethylsulfonyl fluoride, 10 pgiml aprotinin in PBS). Cells were homogenized with three 10 second pulses from the microtip of a Sonic Dismembranator (ARTEK, Farmingdale, NY) a t 40% of maximum output. The cell homogenate (HOM) was centrifuged a t 600g for 10 minutes to yield a post-nuclear supernatent (PNS) and the PNS was centrifuged a t 100,OOOg for 30 minutes to yield a crude membrane (CM) pellet (Ramwani and Mishra, 1986).The CM was centrifuged a t 100,OOOg for 2 hours into a 40% sucrose cushion to yield a plasma membrane band (PM) approximately 0.5-1 cm below the surface and a pellet of other crude membrane components (PEL). PM and PEL fractions were washed in PBS by centrifugation a t 170,OOOg for 1 hour. Protein concentrations were determined by the Bradford method (Bio-Rad, Richmond, CA). Alkaline phosphodiesterase (EC 3.1.4.1), NADPHcytochrome-c reductase (EC 1.6.2.41, and lactate dehydrogenase (EC 1.1.1.27) activities were determined as previously described (Storrie and Madden, 1990; Sottocasa et al., 1967).
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RESULTS Glutaraldehyde-fixedfibroblasts provide feeder activity To determine whether fibroblasts could support epithelial cell growth by means other t h a n secreted products, we tested glutaraldehyde-fixed, metabolically inactive Swiss 3T3-52 fibroblasts for feeder cell activity. After experimenting with serial dilutions, we found that 0.05%glutaraldehyde was sufficient to extinguish all metabolic activity as documented by lack of lactate production (see “Materials and Methods”). The results of several experiments, summarized in Table 1, show that feeder cells fixed in 0.05% glutaraldehyde retain feeder activity. The number of epithelial colonies increased 14-fold over background when Ep-2 cells were plated onto dishes containing attached fibroblasts fixed in 0.05% glutaraldehyde. As one of several controls, we showed that glutaraldehyde-treated tissue culture dishes without feeder layers did not support growth of epithelial colonies (data not shown). Fibroblasts fixed a t higher concentrations of glutaraldehyde (0.1% to 2%) had less feeder activity (data not shown). Even the fibroblasts fixed with 0.05% glutaraldehyde exhibited less feeder activity than unfixed fibroblasts (Table 1). Nevertheless the results summarized in Table 1 indicate t h a t feeder activity is not completely dependent upon the metabolic activity of the feeder cells. Suspensions of glutaraldehyde-fixedfibroblasts display feeder activity Encouraged by these results, we asked if glutaraldehyde-fixed fibroblasts could provide feeder activity without being attached to the tissue culture dish. Feeder activity in suspended cells could provide a n assay for testing activity after cell fractionation or other treatments that preclude feeder cell attachment. Suspended 3T3-52 cells were rendered metabolically inactive in 0.05% glutaraldehyde and tested for feeder activity a s described in “Materials and Methods.” In these experiments, the Ep-2 cells attached to the tissue culture dishes while the fixed feeders remained unattached. Control feeder activity assays were performed by testing for Ep-2 growth in the presence or absence of live attached feeder cells. Figure 1 shows that epithelial cell growth in the presence of fixed suspended feeders is nearly as extensive as growth in the presence of live attached feeders, when each feeder type is included a t its optimal concentration. For fixed suspended feeders, that concentration is approximately 2.5 x lo5 cellsiml, or a total of 4 x lo5 cells in the 1.6 ml assay. For live attached feeders, 1.6 x lo5 cells were pre-plated in the tissue culture dishes prior to adding keratinocytes. In comparison to the results from the experiment above in which the feeder layers were fixed while attached to the culture dishes, more than 100 epithelial coloniesi30 mm2 were counted in dishes that included fixed suspended feeders (see Table 1). This result has been consistently observed in several independent experiments. Experiments in which fixed suspended feeders were included a t different concentrations showed that
TABLE 1. Feeder activity of glutaraldehyde-fixedand live fibroblasts Feeder cells None Fixed, attached3 Live, attached Fixed, suspended7
No. of dishes counted 132 104 35 65
Mean No. of epithelial colonies/30 mm2 1 1.3 18.8 >loo6 >loo6
IFor each dish, colonies in 40 random fields of approximately0.8 mmz were counted (see “Materials and Methods”). 2Compositeof seven independent experiments. ”Attached feeder layers were fixed in glutaraldehydeprior to plating epithelial cells (see “Materials and Methods”). 4Compositeof six independent experiments. 5Compositeof two independent experiments. 6Merging of colonies did not allow precise quantitation. 7Suspended feeder cells were fixed in glutaraldehyde and co-plated at 7.5 X 10‘ to 2.5 X lo5cells/ml with epithelial cells.
Fig. 1. Fixed suspended and live attached feeders have comparable activity. Ep-2 cells were plated onto 35 mm tissue culture dishes with no feeder layer, a live attached feeder layer pre-plated at 10’ cellsiml, or were co-plated with feeders that had been fixed in suspension with glutaraldehyde. The fixed suspended feeders, included at 2.5 x 10’ cellsiml, had been fixed 11 weeks prior to this experiment and stored at 4°C in PBS. After 7 days in culture, the live, attached feeders were selectively removed with 0.02% EDTA and Ep-2 cells in all dishes were rinsed, fixed, and stained. Similar results were obtained from duplicate dishes (not shown). Photomicrographs ( X 100) of representative epithelial colonies grown in the respective conditions are shown below each dish.
Ep-2 growth response was dependent upon the concentration of the suspended glutaraldehyde-fixed feeders with feeder activity disappearing below 2.5 x lo4 cellsiml (data not shown). Photomicrographs of representative regions of tissue culture dishes with no feeder layer, a fixed feeder layer, or a live feeder layer are shown in Figure 1.
Glutaraldehyde-fixed fibroblasts promote keratinocyte growth in a contact-dependentfashion These experiments demonstrate that a t least some of the factods) responsible for feeder activity are not
FIBROBLAST SURFACE FACTOR AND KERATINOCYTE GROWTH
actively secreted into the medium by 3T3-52 cells. However, passive leakage of feeder activity from the fixed cells might explain these results. To test this possibility, Ep-2 cells were plated in tissue culture wells beneath cell culture inserts having membranes with 0.45 pm pores. Glutaraldehyde-fixed 3T3-52 cells in suspension were added above the membrane a t a final concentration of 2.5 x lo5 cellsiml. As a positive control, fixed 3T3-52 cells were plated together with Ep-2 cells below the membrane. As a negative control, Ep-2 cells were plated below membranes in the complete absence of suspended feeders. After 6 days, the epithelial cells were fixed and stained. As shown in Figure 2, no significant feeder activity passed through the 0.45 pm pores. This result indicates the presence of a biologically active factor bound to the surface of 31‘3-52 fibroblasts that promotes the proliferation of Ep-2 keratinocytes. We noted, in addition, that the glutaraldehyde-fixed cells used in the experiments shown in Figures 1and 2 had been stored in buffered saline more than 11 weeks and then centrifuged out of the buffer before use, without any apparent loss of activity. Furthermore, growth medium conditioned by fixed suspended feeders overnight on a roller at 37°C did not exhibit feeder activity (data not shown).
Feeder activity is cell-type specific We next asked whether epithelial cell growth promotion was a general property of glutaraldehyde-fixed mammalian cells. To answer this question, we harvested MDCK cells, HeLa cells, and 31’3-52 cells using 0.1% trypsin/0.02% EDTA, then washed and fixed the cells with glutaraldehyde. The fixed suspended cells were tested for feeder activity as described above for the experiments shown in Figure 1. Figure 3 shows t h a t among this selection of cell lines, only 3T3-52 fibroblasts exhibit feeder activity. Thus, feeder activity is not a characteristic common to all mammalian cells. Feeder activity is enriched in the plasma membrane fraction of fibroblasts Our results have demonstrated that feeder activity is bound to the fixed cells, suggesting that feeder activity might be enriched in the plasma membrane fraction of 3T3-52 cells. Accordingly we removed 31‘3-52 cells from tissue culture dishes using 0.02% EDTA, then homogenized and fractionated the cells a s described in “Materials and Methods.” Each fraction was assayed for alkaline phosphodiesterase, NADPH cytochrome-c reductase, and lactate dehydrogenase (markers for plasma membrane, endoplasmic reticulum, and cytosol, respectively). Table 2 shows t h a t the plasma membrane marker was successively enriched in the crude membrane (CM) and plasma membrane (PM) fractions, and that the endoplasmic reticulum and cytosol markers were not enriched in the PM fraction. Each fraction was tested for feeder activity by plating lo4 Ep-2 cellsiwell into 24-well tissue culture plates in growth medium supplemented with the 3T3-52 fractions a t 0, 1 , 3 , 9 , or 27 pg proteiniml. Cells were re-fed with the supplemented growth medium 3 days after plating. On the 6th day, cells were fixed and stained. Figure 4 shows that feeder activity was successively
113
Fig. 2. Porous membranes block feeder activity of fixed suspended feeders. Ep-2 cells were plated into a six-well tissue culture plate below membrane inserts with .45 wm pores. Epithelial cells were plated either without feeders or with fixed, suspended feeders below or above the membrane as indicated. After 6 days, the membranes were discarded, and Ep-2 cells in the wells were rinsed, fixed, and stained. Similar results were obtained from duplicate wells (not shown). In this experiment, background growth of Ep-2 cells he., in the absence of feeders) was unusually high.
Fig. 3. Lack of feeder activity in MDCK and HeLa cells. Subconfluent MDCK, Hela, and Swiss 3T3-52 cells were suspended with 0.1% trypsin/0.02’% EDTA, washed in PBS, fixed in glutaraldehyde, and co-plated a t 2.5 x lo5 cells/ml with Ep-2 cells into 35 mm tissue culture dishes. As a negative control, Ep-2 cells were plated alone. After 7 days, the cells were fixed and stained. Similar results were obtained from duplicate dishes (not shown).
enriched in the CM and PM fractions, with maximum growth response in the PM fraction at 9 pg/ml. There was no enrichment in the membrane fraction that pelleted in 40% sucrose (PEL, see “Materials and Methods”), and activity in the cytosol (CYT) fraction was reduced relative to that of the HOM. To provide a more quantitative analysis, dye was eluted from the stained Ep-2 cells in each well and quantitated by measuring absorbance a t 600 nm (Fig. 5). By interpolating a t the absorbance value of 1.0 for the PM, CM, and HOM plots, we estimate a two-fold and nine-fold enrichment of feeder activity in the plasma membrane preparation relative to the crude membrane and homogenate, respectively. As a crude comparison of the nature of the feeder activity in PM fractions to that of intact cells fixed in glutaraldehyde, we determined their heat sensitivities. For both preparations, activity was retained after
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YAEGER ET AL
TABLE 2. Activities of marker enzymes in cell fractions’ Enzvme NADPH cytochrome-c reductase activity Snecific Normalized
Alkaline phosphodiesterase activity Specific Normalized
Cell fraction2 Homogenate (HOM) Post-nuclear supernatent (PNS) Cytosol (CYT) Crude membrane (CM) Plasma membrane (PM) Sucrose-Delletedmembrane (PEL)
0.10 f 0.00 0.07 5 0.00 0.01
1.0
0.7 0.1 5.2 13.1 3.9
* 0.00
0.52 f 0.01 1.31 f 0.01 0.39 f 0.00
0.055 k 0.005 0.056 i 0.005 0.045 k 0.005 0.339 f 0.005 0.057 Ifr 0.004 0.057 k 0.004
1.0 1.0 0.8 6.0
1.0 1.0
Lactate dehydrogenase activity Specific Normalized 1.34 f 0.30 1.78 f 0.09 5.61 f 0.10 0.26 f 0.10 0.60 f 0.11 0.43 k 0.15
1.0 1.3 4.2 0.2 0.4 0.3
‘Specific activities: A absorbance/min/mg protein fstandard error in assays described in “Materials and Methods”; Normalized activities: activity of HOM equals 1.00. %ee “Materials and Methods.”
HCM
PNS
PM
CM
CYT
PEL
1
3.0
1 PM CM
2.0
8
PNS
@J
0
HOM
PEL
1.0
CM
27 Fig. 4. Feeder activity in fibroblast cell fractions. Swiss 3T3-JZ cells suspended from tissue culture dishes with 0.02% EDTA were fractionated and orotein determinations were oerformed as described in “Materials and Methods.” Ep-2 cells were plated into 24-well tissue culture plates at 104 cellsiwell in growth medium containing fractions at the indicated concentrations (JLgproteiniml), After days, the cells were fixed and stained in 20% methanol,o,5% crystal violet, HOM = homogenate; PNS = post-nuclear supernatent; PM = plasma membrane; CM = crude membrane; CYT = cytosol; PEL membrane pellet in 406/csucrose, Similar results were obtained from replicate wells (two replicatesof each well containing cMand P ~ fractions; L six replicates of each well containing HOM, PNS, PM, and CYT fractions).
0.0 0
10
20
30
pg protein/ml
L -
~
treating 30 minutes at 65°C and lost after 30 minutes a t 85°C (data not shown). This was not due to the generation of toxic compounds from the heat treatment since untreated fixed cells retained activity when mixed with heat-inactivated cells. This result suggests that the feeder activity of intact metabolically inactive 3T3-52 cells and that of plasma membrane fractions may involve the same factor.
DISCUSSION We have demonstrated that glutaraldehyde-fixed fibroblasts promote the growth of normal human epidermal keratinocytes in a serum-containing medium designed for co-culture with living fibroblasts. We have
Fig. 5. Quantitative comparisons of feeder activities among cell fractions. Crystal violet was eluted into 33% acetic acid from Ep-2 cells in each of the wells shown in Figure 4 and their replicates. The eluted dye was quantitated by measuring absorbance at 600 nm on an ELISA reader. The mean absorbance is plotted as a function of the concentration of each cell fraction in the growth medium, determined on the basis of protein content. All absorbance values plotted in the graph are within the linear range of absorbance values a t 600 nm for crystal violet in 33% acetate. Error bars represent the S.E.M. Abbreviations for cell fractions are as indicated in Figure 4.
also shown that no growth response was observed when a nitrocellulose filter separated the keratinocytes from the fixed feeder cells, and that feeder activity was enriched in the plasma membrane fraction of 3T3 cells. Taken together, our results provide evidence for the existence of a membrane-bound factor on the surface of fibroblasts that promotes keratinocyte growth. In a limited survey, this activity is specific for cells that display feeder activity when metabolically active (see Fig. 3). One alternative explanation for our findings is that fibroblasts secrete a keratinocyte growth-promoting factor that remains tightly bound to the extracellular matrix. We think this is unlikely because the method
FIBROBLAST SURFACE FACTOR AND KERATINOCYTE GROWTH
used to remove cells from culture dishes (0.028 EDTA in the absence of proteases) prior to glutaraldehyde fixation and plasma membrane isolation should leave extracellular matrix components attached to the culture dishes (Roberts et al., 1987; Bradley and Brown, 1990). Moreover, plasma membrane preparations washed with 1 M NaCl retained activity after centrifugation a t 100,OOOg (data not shown). Partial protease treatment (1 mgiml pronase) of plasma membrane preparations also failed to dissociate feeder activity from the plasma membrane (data not shown). Thus the factor involved is probably tightly associated with the membrane and, if i t is a protein, it is protected from proteases in its membrane-bound form. Growth factors t h a t are known to promote keratinocyte growth include EGF (Wille et al., 19841, TGF-a (Barrandon and Green, 1987), acidic and basic fibroblast growth factor (aFGF and bFGF) (Falco et al., 1988), and KGF (Rubin et al., 1989). No integral plasma membrane form of aFGF, bFGF, or KGF has been reported nor would their primary amino acid sequences predict it (Jaye et al., 1986; Abraham et al., 1986; Finch et al., 1989). However, we cannot rule out the possibility that, in our plasma membrane preparations, FGFs were bound tightly to proteoglycans and were not completely dissociated from the membrane surface by the NaCl and protease treatments discussed above. It is also possible that related genes o r alternatively spliced mRNAs, heretofore undetected, would produce membrane-associated variants of these growth factors. While TGF-CY has a predicted transmembrane region (Derynck e t al., 1984), biological activity from a n endogenous membrane-associated TGF-a has not been demonstrated. I n a n experimental system in which TGF-a is bound to the surface of CHO cells, this form of TGF-a can activate the EGF receptor of A431 cells (Wong et al., 1989).Because EGF and TGF-a bind the same receptor, and the growth medium used in our feeder activity assay, containing 30 ng/ml EGF, does not permit keratinocyte growth in the absence of fibroblast feeder cells or plasma membranes, TGF-a can not account for the feeder activity we observe in fibroblast membranes. Furthermore, TGF-a retains activity after treatment a t 100°C (De Larco and Todaro, 19781, whereas the feeder activity we observe in fibroblast plasma membranes is lost a t 85°C (see “Results”). In addition to TGF-a, two growth factors involved in the proliferation and differentiation of hematopoietic cells, CSF-1 and MGF, have transmembrane domains as predicted from their respective cloned cDNA sequences (Clark and Kamen, 1987; Anderson et al., 1990). Both growth factors have been localized to the cell surface and the growth-promoting activity of cell surface-associated MGF on mast cells has been demonstrated (Rettenmier et al., 1987; Anderson et al., 1990). The observation that glutaraldehyde-fixed Swiss 3T3 cells promote the proliferation of hematopoietic stem cells (Roberts et al., 1987) may relate to the existence of these membrane-bound growth factors. Our results point to the existence of yet another cell surface factor involved in growth regulation, specifically the promotion of keratinocyte growth by fibroblasts (although we have not yet demonstrated that this factor is a protein). The difficulties inherent in the methodology required
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for the identification of cell surface-bound growth factors raises the possibility that many such factors remain undiscovered, and their existence may emerge as a n important theme in growth factor research. The precise mechanisms involved in the response of keratinocytes to the fibroblast surface factor have yet to be identified. We found that our fibroblast plasma membrane preparations did not stimulate proliferation of keratinocytes in bioassays in which serum-free MCDB 153 growth medium (Peehl and Ham, 1980) was used (data not shown). Thus, the surface factor may promote keratinocyte growth by acting in concert with a growth factor in the serum or it may inactivate or counteract growth inhibitory signals which are likely to be present in the serum (Wille et al., 1984). Such inhibitory signals would include those that induce differentiation or those that inhibit proliferation more directly, e.g. TGF-P (Matsumoto et al., 1990). EGF and vitamin A are examples of substances that may promote keratinocyte growth by inhibiting differentiation (Marchese et al., 1990; Kopan and Fuchs, 1989). In contrast, KGF does not inhibit calcium-mediated differentiation of keratinocytes (Marchese e t al., 1990). TGF-P is synthesized by epithelial cells (Kane et al., 1990) and may inhibit their growth in autocrine fashion (Moses et al., 1989). It has been demonstrated that live fibroblasts remove and degrade TGF-(3 spontaneously attached to the surface of tissue culture dishes (Rollins et al., 1989). We have found that the plasma membranes of fractionated fibroblasts, although exhibiting feeder activity in the absence of exogenously added TGF-P, do not interfere with the inhibition of keratinocyte growth by TGF-p (data not shown).Therefore it is unlikely that fibroblast plasma membranes promote keratinocyte proliferation by inactivating or counteracting the growth inhibitory action of TGF-(3. The localization of factors to the surface of specific cell types that influence proliferation and differentiation of heterologous cells may play a n important role in the spatial control of proliferation during development and stem cell maturation. In keratinocyte proliferation and differentiation, the undifferentiated proliferating stem cells are closer to the supporting dermal layer, while the cells that have migrated away from the basal layer become differentiated and cease to proliferate (reviewed by Fuchs et al., 1988). Our conclusion that fibroblasts bear a surface factor t h a t promotes keratinocyte growth may have relevance to this physiological phenomenon. We have recently solubilized the feeder activity from plasma membrane preparations. This will facilitate further purification and characterization of the factor and permit investigation into its mode of expression and mechanism of action.
ACKNOWLEDGMENTS This work was supported by grant HD24926 from NIH to C.S. and grant CA53091 from NIH to B.R. LITERATURE CITED Abraham, J.A., Mergia, A,, Whang., J.L., Tumolo, A., Friedman, J., Hjerreld, K.A., Gospodarowicz, D., and Fiddes, J.C. (1986) Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor. Science, 233.545-548. Anderson, D.M., Lyman, S.D., Baird, A,, Wignall, J.M., Eisenman, J.,
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