J. Cell Sci. 57, 15-23 (1982)Printed in Great Britain © Company of Biologists Limited 1982

EPIDERMAL CELL MIGRATION ON COLLAGEN

AND COLLAGEN-DERIVED PEPTIDES

DONALD J.DONALDSON, GERALD N. SMITH, JR ANDANDREW H. KANGDepartments of Anatomy and Medicine, University of TennesseeCenter for the Health Sciences, and the Veterans Administration Medical Center, U.S.A.

SUMMARYNucleopore filters coated with various genetic types of collagen and certain collagen-derived

peptides were implanted under one margin of a skin wound on adult Notophthaltmis viridescens(newt) hind limbs. In contrast to their behaviour on untreated filters, epidermal cells migratedreadily and to equal degrees on human type I, newt type I, bovine type II, and bovine type IVcollagen. Denaturation had no effect on the ability of collagen to support migration and allthree cyanogen bromide peptides tested (a^IJCI^, 7 and 8) were able to support moremigration than that seen on untreated filters. Glutaraldehyde-linked collagen gels supportedmigration but bovine serum albumin gels did not.

These results show that there is no species or collagen-type specificity shown by newtepidermal cells as they migrate over collagen-coated substrates. They also demonstrate thatthe tertiary structure of the collagen molecule is unimportant in its ability to bind to newtepidermal cells, and that the 0 (̂1) chain has at least three, and probably many epidermal bindingsites. Finally, they indicate that the improved migration on collagen is not a non-specificresponse to protein on the substrate.

INTRODUCTION

The way in which epithelial cells interact with adjacent connective tissue com-ponents is of major importance in epithelial function. For example, epithelial sheetsare normally underlain by a basement membrane that contains type IV collagen(Kefalides, 1973). The structural integrity of the sheet appears to be dependent uponcertain basement membrane proteins that bind the basal epithelial cells to the type IVcollagen (Stanley et al. 1981; Terranova, Rohrbach & Martin, 1980). In addition,collagen substrates can influence the differentiation of certain types of epithelial cells(Michalopoulos & Pitot, 1975; Meier & Hay, 1975; Murray et al. 1979). Finally, whenneoplastic epithelial cells invade deeper tissues or when normal epithelial cells migrateto close a wound, the mobility of the cells is dependent upon the availability of suit-able migration-supporting macromolecules in the connective tissue compartment.

Recently (Donaldson & Dunlap, 1981), we found that untreated nucleopore nitersinserted under one edge of a fresh skin wound on adult newts were a poor substratefor epidermal cells migrating from the wound. However, if the filters were coated withgelatin, their ability to support migration increased dramatically. Using scanningelectron microscopy, we learned that as the epithelial sheet advances on the filter thecells adjacent to the leading edge extend broad, thin, fan-shaped processes onto the

16 D.J. Donaldson, 0. N. Smith and A. H. Rang

substrate in the direction of movement. These processes possess specialized attach-ments on their undersurface that bind the cell to the substrate. This ability of gelatinto convert a poor migration substrate into a good one and the fact that collagen is amajor component of most extracellular matrices stimulated the present investigationinto the response of migrating epidermal cells to various types of collagen and certaincollagen-derived peptides.

MATERIALS AND METHODS

General

Adult male newts (Notophthalmus viridescens) obtained from Connecticut Valley Biologicalwere stored at 4 °C in 1/10 strength operating solution (Rose & Rose, 1965) containing 0-14 ml/1of Wardley's Aquatonic (a commercial product commonly used for disease prevention in tropicalfish). At least a week prior to use, the animals were moved to room temperature and kept in thesame solution under natural illumination.

Wounding, filter implantation, and measurement of migration rates

Rectangular wounds with their long axis extending proximo-distally were made by removinga piece of skin from the dorsal surface of each hind limb between the knee and ankle. Woundedlimbs were amputated through the thigh and explanted into 5 ml of full-strength Holtfretersolution (HS) containing 0-05 g/1 of streptomycin in 35 mm x 10 mm plastic dishes. The clottedblood was then cleaned from each wound, the limbs were transferred to 5 ml of fresh HS and atriangular piece of nucleopore filter was then inserted, sharp end first, under the anterior woundmargin (Fig. IA). Eight hours later, limbs were fixed overnight in 10% formalin, and then themigrating cells were stained by immersing the entire limb briefly in 0 1 % crystal violet. Afterthe filter was dissected free from the limb (Fig. IB), it was placed under a compound microscopeequipped with a Leitz drawing tube. The magnified image of the filter and wound epitheliumwas drawn onto a sheet of paper, and the area occupied by a standardized width of woundepithelium (distance migrated) was determined using a polar planimeter. These values wereused to compare the effectiveness of various substrates. Experiments were designed so that onehind limb of an animal served as a corftrol, and the other as an experimental. This allowed us toanalyse the results with a paired t-test, thereby reducing the impact of animal variability. Pvalues less than 0-05 were judged significant.

Coating of filters

Nucleopore filters (o-z /im pore size, 25 mm diameter) were immersed for 15 min in 0-5 %acetic acid and were then washed briefly with distilled water. Subsequently, 0-5 ml of o-i M-acetic acid, or the same vehicle containing, in solution, one of the following genetic types ofcollagen (human I, newt I, bovine II, bovine IV) or one of three different cyanogen bromide(CNBr) peptides (a/IJCBs, CB7, or CB8) was pipetted onto the dull side of the filter, spreadevenly, and placed at 23 °C overnight to dry. In one experiment, filters were coated withglutaraldehyde-linked collagen or bovine serum albumin (BSA). A solution of type I collagenfrom chick embryo skin (8-5 mg/ml in o-i M-acetic acid) was mixed in equal amounts with 2 %glutaraldehyde, and 200 /*1 of the resulting mixture was spread evenly over a filter and allowedto polymerize overnight in a moist chamber. Other filters were coated similarly with BSA. Tomake the viscosity of the BSA solution similar to the collagen solution, 57 mg/ml of BSA wasrequired. The polymers were then treated as follows (modified from the method of Macieira-Coelho & Avrameas, 1972): 11 h in 2% glutaraldehyde, 60 h in Na borate/HCl buffer (0-02 M,pH 8-i), 20 h in borate buffer containing 0-2 M-glycine (to block free aldehydes), 48 h inphosphate-buffered saline/EDTA at 4 °C (0-007 M-phosphate (pH 74), 2 mg EDTA/1) andfinally, overnight in HS.

Epidermal migration on collagen

B

Fig. 1 A. A limb showing a nucleopore filter inserted under the anterior margin of a skinwound. Since the wound epithelium is transparent, it shows only faintly in the photo-graph; approx. x 27. B. A filter dissected free and stained with o-i % crystal violet.The wound epithelium (arrow) is now clearly seen; approx. x 27.

Collagens and CNBr peptides

Type I collagen and a,(I) CB3, CB7, and CB8 were gifts from Dr Jerome M. Seyer. Thesecollagens were extracted from either human skin or placenta and purified as described pre-viously (Seyer, Hutchenson & Kang, 1976). Bovine type II collagen, prepared from foetalbovine skeleton (Stuart et al. 1979) was a gift from Dr Michael A. Cremer. Chick type I (Dixit,Seyer & Kang, 1977) and type IV collagen from bovine anterior lens capsule (Dixit, 1978) werecontributed by Dr Saryu N. Dixit. The newt type I collagen was prepared by digestion ofnewt tails with pepsin and purified by fractional precipitation with salt (Smith & Linsenmayer,1982).

RESULTS

Preliminary experiments in which we coated filters with 125 /Jg/ml of type Icollagen showed that this would greatly improve migration compared to untreatedfilters. We then conducted a dose-response study, in which one hind limb of eachanimal received a filter implant treated with 125 /tg/ml of type I collagen (hereafterreferred to as collagen controls) and the other limb received an implant treated withless type I collagen, the exact amount depending upon the group to which it belonged.Fig. 2 shows that the dose-response curve breaks at 1-25 /tg/ml. At this concentration,

i 8 D.J. Donaldson, G. N. Smith andA.H. Kang

110H

- 100o

12-5 1-25 0-125

Colleger! concentration0012

Fig. 2. Dose-response curve showing the effect on migration of decreasing amounts oftype I collagen on the substrate. Collagen in o i M-acetic acid was air-dried onto nucleo-pore filters. A piece of collagen-coated filter was then inserted under one edge of a skinwound on limbs explanted into a small dish of Holtfreter solution. Eight hours later,when epidermal cells had moved onto the implant, the area occupied by woundepithelium (distance migrated) was determined from a standard portion of the filter.This was done using a polar planimeter on drawings of the filter made with the aid ofa Leitz drawing tube mounted on a compound microscope. For each point shown, 6-8limbs (the collagen controls) were implanted with filters treated with 125 fig/m\ ofcollagen, while the cpntralateral limbs (the experimental) received implants treatedwith the concentration given on the abscissa. After the mean distance covered bywound epithelium was determined for each group, the means for experimental limbswere converted to a percentage of the contralateral collagen controls. NS, no significantdifference between experimentals and collagen controls; * P < o-oi; • • P < o-ooi;• • • P = o-oooi.

Table 1. Effectiveness of denatured type I collagen as a migration substrate*

GroupCollagen(/tg/ml)

Number oflimbs

Distance!migrated

NativeDenaturedNativeDenatured

125125

1-251-25

77

77

532 ±4©577 ±54467 ± 36494 ±42

NS

* Experimental design was as described in the legend to Fig. 2, except that one limb of eachanimal received an implant coated with native collagen, while the contralateral limb receivedone treated with the same amount of heat-denatured collagen.

f As defined in Materials and Methods; J standard error; § NS, no significant difference.

Epidermal migration on collagen 19

migration was still as good as that on the collagen control filters. However, loweringthe amount of collagen to 0-25 Jig/ml appeared to lessen its ability to support migra-tion (although the decline was not quite significant). Migration on filters receiving0-125 /*g/ml was significantly retarded, being only 38% as good as the collagen con-trols. Migration on untreated filters was only 11 % as good as the collagen controls.Heat-denaturing a sample of type I collagen for 55 min had no effect on its ability toserve as a migration substrate when tested at 125 fig/ml or 1-25 /<g/ml (Table 1).

600-

500-

.1 400-

8

Dis

tan

200-

100-

TT

H I N I

I

H I

L j .

B II

i

H I

A

jI

BIV

700H

600-

500-

400-

a 300-5

200-

100

nh

H I N I H I H I 3 IV

NS NS

Groups

NS /»< 0-001 P < 0002Groups

Fig. 3. Comparison of various collagen types as migration substrates. Experimentaldesign was as described in the legend to Fig. 2, except that in each pair of bars, theleft member represents limbs receiving a filter treated with human type I collagen. Theright member of each pair represents the contralateral limbs, which received implantstreated with the collagen type indicated on the bar. H, human; N, newt; B, bovine;NS, no significant difference. Distance migrated is defined in Materials and Methods.A. AH bars represent limbs receiving filters treated with 1 -25 /ig/ml of collagen, B. Ineach pair of bars the right member represents filters treated with 0-125 /<g/ml ofcollagen, while all left members represent 1*25 //g/ml.

Thus the epidermal binding sites on type I collagen do not seem to be related to thetertiary structure of the molecule but rather to the covalent structure of the componentalpha chains.

We next compared several types of collagen as migration substrates. Fig. 3 A showsthe results of experiments comparing 1-25 /tg/ml of human type I (the left bar in eachpair) to that same concentration of newt type I, bovine type II, or bovine type IVcollagens. In each experiment, the test collagen was as effective as the type I reference.Fig. 3B underscores the similarity in the various collagens by showing that when the

20 D. J. Donaldson, G. N. Smith and A. H. Kang

three test collagens were reduced to one-tenth of the amount in Fig. 3 A they losteffectiveness, just as did human type I in the dose-response curve.

Finding that the various collagens all improved migration equally well, we wonderedif this was simply a non-specific protein effect. Filters coated with BSA were no betterthan untreated filters (data not shown). However, without proof that BSA actuallyremained on the filter, no conclusion was possible. Therefore we placed glutaraldehyde-linked polymers of BSA onto filters in films thick enough to be seen. Chick embryotype I collagen films were similarly produced for comparison. Of seven limbs receivingthe collagen film, five showed migration, ranging from 126 units to 506 units, with amean of 310. No migration occurred on any BSA filter. Since the BSA was applied toa filter previously coated with 1-25 /^g/ml of type II collagen, the BSA had converteda good substrate into a poor one. Thus, while the collagen polymer was inferior to air-dried collagen as a substrate, it was clearly superior to the BSA polymer.

Table 2. Effectiveness ofaY{I) cyanogen bromidepeptides as migration substrates*

Group

Native typeCB3

Native typeCB7

Native typeCB8

I

I

I

Number oflimbs

7788

00

00

643373701443

517

Distance migratedf

±82(58%)§

±37±37(63%)±49±47(70%)

P < 001

P < 0-005

N.S.Il

• Experimental design was as described in the legend to Fig. 2, except that one limb ofeach animal received an implant coated with i'25 fig/m\ of native collagen, while the contra-lateral limb received one treated with the same amount of one of the CNBr peptides.

f As defined in Materials and Methods; J standard error; § % of the native type I mean;II N.S., no significant difference.

Table 2 shows that at 1-25 fig/m\, all three of the a^I) cyanogen bromide peptidestested were somewhat inferior to native type I collagen as a substrate. Of greatersignificance than this inferiority is the fact that when each of the peptide means inTable 2 was compared to the mean (132 + 26) for 20 untreated filters from variousother experiments, the peptides were clearly superior (P values less than 0-0002 ineach case). Thus, the ax chain has at least three, and probably many, epidermal cellbinding sites.

DISCUSSION

Attachment assays have been used by several investigators to explore epithelial-collagen interactions. Thus, freshly isolated guinea pig epidermal cells (Murray et al.1979) and a cell line (PAM212) derived from mouse epidermis (Terranova et al. 1980)both show a distinct preference for type IV collagen. In contrast, adherence of freshlyprepared rat hepatocytes to plastic dishes is improved equally well by a coating of any

Epidermal migration on collagen 21

of the five collagen types (Rubin, Hook, Obrink & Timpl, 1981). Since newt epidermalcells migrated equally well over interstitial collagen (type I), cartilage collagen (typeII) and basement membrane collagen (type IV) it might appear that these cellsresemble rat hepatocytes more than they do the epidermal cells described above.Such a conclusion assumes that migration studies and adhesion assays measure thesame thing. While migration involves adhesion to the substrate, it includes cellularevents subsequent to attachment as well. Thus, it is important to know how differentlevels of cell-substrate adhesion affect mobility. The available evidence suggests thatgiven a choice, cells will preferentially choose the more adhesive of two substrates(Harris, 1973) and this choice will then lead to a decrease in mobility. For example,Gail & Boone (1972) found that normal and transformed fibroblasts seeded onto Pyrexare less motile and more adherent to the substrate than are cells on cellulose acetate.Similarly, the more negative the substrate charge (a condition tending to decrease theaffinity of normally negative cell surfaces for the substrate) the more motile cellsbecome (Sugimoto & Hagiwara, 1979). Finally, lanthanum treatment decreases themotility of glial cells and increases their adherence to plastic culture dishes (Letourneau& Wessells, 1974). (This last example, however, may have been due in part to lan-thanum effects on calcium control of the glial contractile machinery.) If we accept thisindirect relationship between mobility and adhesion as a general principle for all cells(it has been shown convincingly only for fibroblasts) we must conclude that newtepidermal cells adhere equally well to all the native collagens tested. How can thisconclusion be reconciled with adhesion assays that show a preference of epidermalcells for type IV collagen ?

Attachment assays are conducted after cells in culture, or tissue cells, are dis-aggregated by enzymes. If protein synthesis in such cells is shut off, they show a greatreduction in the ability to bind collagen (Terranova et al. 1980). These assays thereforeactually measure the ability of enzymically altered cell surfaces to regenerate attach-ment factors for collagen. Consequently, when a particular collagen preference isfound in attachment assays this may simply reflect a difference in the rate of regenera-tion of separate classes of collagen-binding proteins. By contrast, in migration studiessuch as ours, where epithelial cells are not exposed to enzymes, the interaction withcollagen may involve a wider spectrum of collagen receptors. While not designed toaddress this question, the work of Stenn, Madri & Roll (1979), in which mouse skinexplants were made into plastic dishes coated with collagen types I, III, IV and V,suggests this is the case. The authors state that after 3 days of culture, migration oncollagen was 59% greater than on uncoated dishes. No preference for any of thecollagens was mentioned. In addition, their figure 2, which shows the ability of aproline analogue to inhibit migration on the various collagens when added after 2 daysin culture, shows similar end-points at day 3 for all collagen types. Thus, in mouseepidermis a preference for type IV in attachment assays is apparently not reflected inmigration behaviour.

There has been considerable interest recently in collagen attachment factors. Fromstudies in tissue culture, two major adhesive proteins have been implicated, fibro-nectin and laminin. Fibronectin is a fibroblast cell-surface and blood-borne glyco-

22 D. J. Donaldson, G. N. Smith and A. H. Kang

protein found in many species (Yamada & Olden, 1978), including salamanders(Repesh, Furcht & Smith, 1981). In attachment assays, fibronectin improves theability of fibroblasts to bind to type I and type IV collagen, but has no effect on thebinding of freshly isolated guinea-pig epidermis to these collagens (Murray et al.1980). Since in the 0 (̂1) chains, the major fibronectin binding site is found in CB7(Kleinman, McGoodwin & Klebe, 1976), the fact that migration was similar on allthree peptides in our experiments suggests that fibronectin is not necessary in thissystem for epidermal-collagen interactions. However, because we have not actuallymeasured the affinity of newt fibronectin for the three human peptides, this conclusionmust be considered tentative.

Laminin is another cell surface glycoprotein, but is secreted primarily by epithelialcells (Foidart et al. 1980). Unlike fibronectin, it is not found in serum (Terranova et al.1980). It is a component of basement membranes where it is localized in the laminalucida between the epithelial cells and the type IV collagen making up the lamina densa(Foidart et al. 1980). The preferential adherence of epidermal cells to type IV collagen,appears to be mediated by laminin (Terranova et al. 1980). This specific affinity fortype IV collagen plus its location in basement membranes suggest that laminin is the'glue' that binds epithelial cells to their basement membranes. At this point it is notpossible to comment on the role of laminin in migrating newt epidermis, except tosay that since this system shows no preference for type IV collagen, any type IV-specific ligand must be accompanied by other collagen binding proteins as well.

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(Received 3 March 1982)