EpitheIiaIspecific Gene Expression during Differentiation ... - CiteSeerX

August 1992

Vol. 3, 471-483,

Cell Growth

& Differentiation

471

EpitheIiaIspecific Gene Expression during Differentiation of Stratified Primary Human Keratinocyte Cultures1

Jeffrey

L Wilson,

Sheila

C. Dollard,

vated

Louise T. Chow,

Department Medicine

of Biochemistry, and

Dentistry,

University

Rochester,

of Rochester,

New

York

School

of

14642

cells are used to generate

patches of autologous burns or wounds

differentiation undertaken

skin equivalent

and to investigate

of epithelia

for patients with the

growth

and

in vitro. We have

a comprehensive

study of the

morphological and molecular events that occur during culturing of human foreskin keratinocytes at the liquidair interface on a dermal equivalent consisting of a collagen matrix containing fibroblasts. Using radioadively

labeled

RNA probes

for mRNAs

and

stage-specific genes was affeded by the type of fibroblasts included in the matrix as well as by the age of the culture. The expression of these genes was not always coordinated and could not be predided from Surprisingly,

appearance

the mouse

of the stratified

fibroblasts

epithelium.

promoted

epithelia

pany

the morphological

partner

keratins

sized

in the basal

first

appear

Thus,

KHGs these

K14,

which

to differentiate are

are

ideal

molecular events associated thelial cells in culture. Several culture systems differentiation of epithelial

left

the

synthe-

Ki and

basal

layer

K1O and

Filaggrin is associated only in the granular layer. (2).

(3, 4) and is made genes

example,

(1), whereas have

family of accom-

The basic-acidic

for

keratinocytes

in cells

are committed with

differentiation.

K53 and

of histologically and cornified

of the large proteins

probes

with

for

monitoring

differentiation sustained in vitro.

by Grant

the

of epi-

growth and When coculti-

Biochemistry,

University

2550 from The council

collagen

marker

of Rochester,

School

of Medicine

and

601 Elmwood Avenue, Rochester, NY 14642. 3 The abbreviations used are: K-, keratin; KHG, keratohyalin PHF, primary human fibroblast; FBS, fetal bovine serum; DMEM, co’s minimum essential medium; cDNA, complementary DNA;

ing temperature

(Celsius).

matrix

prepared

Ki

can also

placed

at the

been developed Ref. 9). It consists

is topographically

delayed

markers such as fibronectin, but found in hyperproliferative

fibro-

skin,

are

abnormally

(15).

Fur-

not seen in normal epidermis such

expressed

(1 3). When

the matrices contained mouse 3T3 fibroblasts, other differences were detected. For example, K14 mRNA was correctly expressed in the basal layer but also persisted within all epidermal layers, albeit at lower abundance (10). Moreover, K13 was expressed in the superficial cells in culture but Submerged

not in normal or raft cultures

epidermis (16). of epithelial cells

used to generate skin equivalents wounds and to examine epithelial

lomaviruses with host cells4 (Refs. therein). However, characterization typically

restricted

ment.

to short

of marker

Furthermore,

term

genes

there

have

also

keratinocytes

blast

cell

been papil-

18-20 and references of epithelial rafts was cultures

assayed

has been

and

limited

in

in any one experino report

of side-by-

side comparisons of different raft culture conditions. Herein, we describe a systematic morphological

investigation

been

for grafting to burns or cell differentiation (see

review in Ref. 1 7). Recently, raft cultures have used to investigate interactions of epitheliotropic

natal foreskin

for Tobacco

Research Grant CA36200. should be addressed, at Department

dermis

with dermal

foreskin Research-U.S.A. and by USPHS 2 To whom requests for reprints

(6). Keratinocytes

of a porous

molecular Received 2/13/92. 1 This project was sponsored

do not occur

on de-epidermized

the resulting epinormal epidermis this system is not

the number allow cells

cells

medium-air interface (7, 8). Although thelium is morphologically similar to and expresses Ki and filaggrin markers, amenable to routine handling. An effective dermal equivalent has for ease of manipulation (reviewed in

thermore, epidermis

Introdudion

strata. Alterations in the expression keratins and other epithelium-specific

lethally

complete stratifiof native squa-

as psoriatic

consists granular,

been

C, epithelial

of serum or high Ca2 concentration, cation and differentiation characteristic

entiation

epithelial

differentiation much more closely resembling foreskin than did the homologous primary foreskin fibroblasts.

A mature squamous epithelium distinguishable basal, spinous,

have

blasts (10), mouse 3T3 fibroblasts (11, 12), or embryonic human fibroblasts (13, 14). When raised to the liquid-air interface, dispersed keratinocytes placed on top of the dermal equivalent receive moisture and nutrients through the support matrix. The keratinocytes in this “raft” culture proliferate, stratify, and differentiate with morphologies similar to natural epithelia. Growth of human foreskin keratinocytes on a collagen matrix containing human embryonic fibroblasts reconstitutes a homologous system, yet expression of the differ-

monoclonal antibodies for proteins, we found that the expression of a comprehensive set of differentiation

the histological

that

mitomycin

cultures can be in the presence

be cultured

extensive

feeders

with

(5). Although such some differentiation

mous epithelial

fibroblast

or treated

proliferate rapidly induced to undergo

Abstrad Cultured

with

irradiated

and Thomas R. Broker2

of raft cultures

of primary

and

human

supported either by primary neoor by established mouse fibropatterns of expression of a compre-

fibroblasts

lines.

The

of

Dentistry,

granule; DulbecTm, melt-

4

5. C. Dollard, J. L.

Wilson,

L. M.

Demeter,

W.

Bonnez,

R. C. Reichman,

1. R. Broker, and L. 1. Chow. Production of human papillomavirus and modulation of the infectious program in epithelial raft cultures. Genes & Dev., 6: 1131-1142, 1992.

472

Epithelial

Differentiation

hensive

set

in Raft Cultures

of differentiation

compared

to those

by mRNA

hybridization

tochemical

protein

stage-specific

observed

genes

in foreskin,

in situ

as well

localization.

We

were

as monitored

as by immunocy-

demonstrate

that

epithelial differentiation in vitro is dramatically influenced by the type of fibroblasts in the dermal equivalent and by the age of the culture.

Morphology Matrices

and Life Span of Raft Cultures

Containing

Fibroblasts

from

Different

Sources.

human foreskin keratinocytes showed orderly stratification and differentiation after 9 days at the medium-air interface (Fig. 1C). The epithelium that developed included in succession: (a) an organized layer of polygonal basal keratinocytes, several layers of cells

(b)

several containing

layers of spinous cells, (c) KHGs, and (d) flattened,

cornified cells with nuclear degeneration. were virtually identical to native foreskin

These features (Fig. 1A) except

for

KHGs

a thicker

granular

layer,

Appropriate morphological over a period of 32 days,

nucleated

cells became

but

fewer

per

in the 32-day

Using

the

same

batch

we also tested cultured

from

of

foreskin

collagen

cultures

neonatal

keratinocytes,

matrices

foreskin

prepared

(PHF

raft

The resulting cultures were morphologically the A31-supported cultures except for one difference. 18). In the

in

with

cultures).

similar to unexpected

The 9-day PHF cultures had no KHGs (Fig. 21-day cultures, KHGs were observed sporad-

ically in small and isolated areas in the uppermost stratum (Fig. 1 D). At 32 days, keratohyalin granules increased in number and were more uniformly distributed in most of the uppermost cell layer, although they remained far less abundant than in the A31 raft cultures or in foreskin (Fig. iF). Thus, although either culture could be maintained for at least 1 month, to differentiate more

the homologous slowly than the

system appeared heterologous sys-

tem. Qualitatively similar results were edly with fibroblasts and keratinocytes different donor foreskins.

obtained repeatprepared from

cell

differentiation

repeat

unit-length

clone

unique

sequences

(carboxyl

sequences) and basic cDNA

stage-specific

of profilaggrin terminus

from a comprehensive keratin cDNAs and

were

obtained

or subcloned

A

and subclones and

from

genes.

11Materials

or to neonatal autoradiographic cytoplasm

and

alignments (data not shown). Tntwere applied to histological secparaffin-embedded raft cultures

foreskin to assess mRNA expression. Most silver grains were observed over the

of the

epithelial

cells,

as would

be expected

in the cultures and temporal

relative to foreskin. differences among

were also detected. These observations detail below. Markers for Proliferating Epithelial

as sum-

mRNAs

for the

basic-acidic

differdistni-

Striking quanraft cultures

are discussed Cells. partner

in

In native keratins KS

expressed at moderate intensity only in (Fig. 3 and Fig. 4, A and B), as reported

previously (1). However, both mRNAs were expressed in moderate to strong levels in A31 or PHF raft cultures in most or all of the cell layers (Figs. 3 and 4). K6 protein expression is regarded as a marker for hyperproliferation and wound healing or for malignant transformation in vivo (11, 21). As reported previously (11, 21), we observed no K6 mRNA expression in foreskin (Fig. 5, A and B). Again, all raft cultures expressed moderate to strong

K6 RNA signals there appeared of all three expression

aged,

in most of the cells (Fig. 5). Furthermore, to be some modulation in the expression

genes with time. In the was highest at 9 days,

it became

either

reduced

PHF raft cultures, the but, as the cultures

in abundance,

more

restricted

to the lower strata, or both (compare Figs. 4 and 5, C, E, C, and Fig. 3). In contrast, transcription of these genes was strongest in the 21-day A31 raft cultures (compare Figs. 4 and 5, 0, F, H, and Fig. 3). By 32 days, the expression of K5 and K14 in both raft cultures more

approximated

that

in foreskin,

whereas

the

K6

RNA signal was still at considerable abundance. Although the failure of suprabasal cells to shut off transcription of basal cell marker genes suggested a delay in differentia-

tion,

each

cornified

of the layers

the terminal med

(see

,

interest

pressed

expressed suprabasal

Fig.

cultures 1)

nevertheless

contained

and expressed some or all of

differentiation-specific

for Terminal

involucnin,

committed entiation

raft

(see

marker

genes

exam-

below).

Markers Ki

Methods”) (Fig. 2). They did not cross-hybridize with one another in Southern blots performed at Tm15#{176}C, nor did they share significant sequence homology with other known intermediate filament genes, as tested by computer-assisted pairwise ium-Iabeled niboprobes tions of formalin-fixed,

bution titative

of

3’ untranslated

set of human acidic human involucrin (see

or strong,

thelium (Fig. 3). Other probes revealed dramatic ences in the message abundance and topographic

closely

Cellular Differentiation Revealed by in Situ Hybridization Using Riboprobes for Differentiation Stage-specific mRNAs. To evaluate cellular differentiation with molecular markers, we examined native foreskin and the various raft cultures of different ages for the transcription epithelial

moderate,

signals were detected at weak to moderate intensities only in the upper strata of raft cultures, in contrast to their appearance throughout most strata in foreskin epi-

and K14 were the basal layer

(Fig. 1, E and C). parallel

weak,

K4, K8, K13, and K18, characteristic of simple epithewere weakly to moderately expressed in foreskin

foreskin,

cell.

appearance was maintained although the strata containing

thinner

sporadic, in Fig. 3.

in the dermis or dermal negative controls. mRNA respect to the intensity of distribution across the Intensity was recorded as

epithelium (Fig. 3). The expression patterns of K8 and K18 in all raft cultures examined were similar. K4 did not exhibit any signal in raft cultures of any age, whereas Ki 3

on Collagen

When cultivated on a dermal equivalent prepared with type I rat tail collagen and mouse BALB/c 3T3 A31 fibroblasts (referred to as A31 raft cultures), primary

of

negative, manized ha,

Results

PHFs

from mRNAs. The fibroblasts equivalent served as internal expression was evaluated with signals and their topographical different strata of the epithelia.

because

Differentiation.

and profilaggnin they

are

mRNAs considered

to differentiation or achieving (2, 3, 22). Involucnin mRNA in all suprabasal

layers

weak to moderate layers (Fig. 3).

K1 mRNA expression layers of native foreskin,

was with

Expression were

of

of particular

markers

of

cells

terminal differwas weakly ex-

in foreskin.

All raft cultures

involucnin

message

in the

moderate in all suprabasal some negative cells in the uppermost strata (Fig. 6, A and B). Raft cultures supported by A31 fibroblasts showed moderate K1 mRNA expression in the upper cell layers, and expression appeared to diminish at 32 days (compare Fig. 6, D, F, and H). Surprisingly, the 9-day PHF raft culture was essentially

Cell

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fig. 1. Foreskin and rat culture morphology. Thin sections were stained with lens. A, foreskin; B, D, and F, 9-, 21-, and 32-day PHF-supported raft cultures, respectively. Bar, 50 Mm. (All photomicrographs are the same magnification.)

hematoxylin respectively;

and eosin C, F, and

prior to being photographed using C. 9-, 21-, and 32-day A31-supported

a 40X objective raft cultures,

473

474

Epithelial Differentiation

in Raft Cultures

- Protein

*

-400

Termination

Codon in mRNA

200

-200

400 3’

5’

+238

.94

K-I

(1396-1719)

K-4

(1235-1650)

K-5

(2271-2503)

+423

+8

.3

+232

AAA +361

-170

K-6

(O2-1435)

-

K-8

(657-1064)

K-13

(1487-1691)

K-14

(1061-1500)

K-iS

(1001-1346)

+69

+273 AAA

+23

-417

+72

-273

AAA

+295

-278

Involucnn

AAA

+225

-182

(1504-2078)

Profilaggrin

-

riboprobes used for in situ hybridization studies. The numbers in parentheses following each probe designation cDNA subcloned. Each line diagrams the region of the subclone with respect to the translation stop codon (*) as a generic mRNA. The nucleotide position of the subcloned fragment 5’ or 3’ of the stop codon (- or +, respectively) is indicted above each line. AAA at the 3’ end of a probe denotes the presence of a polyadenylated tail in the cDNA clone. The profilaggrin mRNA encodes a polyprotein. The probe consists of one 1-kilobase region which included a 3’ portion of one repeat followed by the linker region and the majority of the following repeat. The profilaggrin line represents the length of the probe only and is not intended to designate the location of the probe with respect to any particular repeat unit. Fig. 2. indicate

Differentiation the region

indicated

stage-specific of the original

on the numbered

scale representing

negative for Ki mRNA (Fig. 6C), with sporadic and weak signals in only a few isolated loci in the upper strata. After 21 days, K1 message was weakly expressed in

for K1 antigen

approximately one-half (Fig. 6E). By 32 days,

somewhat reduced in the 32-day cultures. In contrast, the 9-day PHF raft cultures were totally negative for K1 antigen, but weak signals were detected in some regions after 21 days and were more uniform after 32 days in the upper one-third of the cell layers (data not shown). The results of the filaggnin antibody which also recognizes profilaggnin were in agreement with the mRNA distribution. The antibody generated strong signals co-

served,

rather

of the cells in the upper strata moderate Ki message was ob-

uniformly

(Fig. 6C). The uppermost

layer

distributed

in the

of neonatal

erated strong profilaggnin mRNA the appearance of keratohyalin

human

signals

upper

strata

foreskin

gen-

coincidental

with

granules

(Fig. 7, A and B), which are reactive with anti-filaggnin antibodies (4) (see Fig. 8A). Strong expression was observed in the upper layers of 9- and 21-day A31 raft cultures. At 32 days,

the

signals

moderated

slightly,

and

fewer

layers

were positive. However, more cell layers were positive than in foreskin (compare Fig. 7, D, F, and H, with A). In contrast, when 9- and 21-day PHF cultures were probed, expression

of

profilaggnin

message

was

evident

only

in

small, isolated areas in the upper stratum (Fig. 7, C and E). At 32 days, there was weak to moderate expression

in the single cell layer just below most

areas

of the

epithelial

sheet

the cornified (Fig.

7C).

cells in The

same

cells also displayed keratohyalin granules, although they were fewer in number than in foreskin or in the A31 raft cultures (see Fig. 1). Together, these results suggested that the human fibroblast-supported cultures matured or differentiated more mouse A31 cells.

slowly

than

cultures

supported

by

incidental

with

culture

was

anti-

completely

cells.

signals epithelium

in the

devoid

observed

In the 9- and were detected and became

uppermost

layer

of

(Fig.

8C).

These

of filaggrin

antigen

(Fig.

in a few cells in the upper

culture (Fig. 8E). At 32 days, weak in most cells in the upper stratum

results

deed differentiated cultures.

confirmed

more

that

quickly

A31

than

cultures

did

the

in-

PHF

Discussion Epithelial

raft

cultures

neonatal

foreskin

matrices

containing

tamed

monoclonal

weaker of the

granules

layers in the 21-day signals were detected

immunocytochemical

using

the

8C). Signals were

established

analyses

cultures, one-half

cells in foreskin (Fig. 8A). The 9-day (Fig. 8D) and 21-day (Fig. 8F) A31-supported raft cultures also had strong granular signals in the upper cell layers, which then became weaker at 32 days (Fig. 8H). The 9-day PHF raft

To corroborate the expression of Ki and profilaggnin genes, or lack thereof, we localized these proteins by bodies. The results were consistent with the in situ hybnidization oftheir mRNAs. Foreskin was strongly positive

in all of the suprabasal

21-day A31 raft only in the upper

mouse

over a period

Each of the cultures similar

to that

were

established

keratinocytes either

primary

fibroblast

with

supported cell

foreskin lines.

fibroblasts

They

of 32 days at the liquid-air

achieved

of foreskin

and

a morphology produced

primary

on collagen were

or main-

interface.

remarkably a stratum

con-

Cell Growth

keratin F C

keratin

1

PHF

I

313

& Differentiation

475

4

NIH 3T3

A31

x

x

G S R

9

9

21

32

9

21

keratin

32

9

9

9

9

21

32

5

9

21

keratin PHF

F

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32

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x

32

j 9

9

32

9

9

S B

bars,

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zones

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Relative

intensities

9

-

Diagrammatic representation of the expression patterns of the various differentiation stage-specific probes as revealed by in situ hybridization studies. The probes used are indicated above each graph, and the epithelial layer is shown on the left: B, basal; 5, spinous; C, granular; and C, corneum. The specimens probed were foreskin (F) or raft cultures supported by primary human foreskin fibroblasts (PHF), irradiated foreskin fibroblasts (X), BALB/c 3T3 A31 fibroblasts (313 A31), irradiated NIH 313 (X), and nonirradiated NIH 3T3 cells. Diagonally cropped 3.

abundance. are shown.

9

6 3T3A31

I

G

Fig.

9

keratin

9

9

21

32

9

9

21

21

32

, ,

99

32

9

9

21

21

21

32

13

9

21

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14

9

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keratin

F

9

32

8

keratin

9

21

32

9

9

9

18

PHF

9

21

32

9

NIH 3T3

A31

313

21

32

9

9

pro filaggrin

Involucrin PHF

F

313

NIH 3T3 x

A31

x

T’

***l****NNr!

9

9

21

32

DAYS

LII neum.

After

as judged

epithelia.

1 month, by

This

duced

growth

blasts,

or both.

the

the cultures reduced

negatIve

began

thickness

thinning

might

potential

of the epithelial

The

only

clear

:

sporadIc

to deteriorate, of

be attributable morphological

the

stratified

to the re-

cells, the fibrodifference

between rafts supported by primary human fibroblasts and those grown with mouse fibroblasts was that, with PHF, keratohyalin granules and were not as abundant. tion and immunocytochemical

had a delayed appearance In contrast, in situ hybnidizastudies revealed signifi-

9

21

32

9

9

DAYS

Ei

weak

moderate

cant molecular profiles

differences

of the

strong

with

differentiation

respect

stage-specific

L::::7 to the expression genes

among

the foreskin

and the two types of raft cultures, as summanized in Fig. 3. The most dramatic results were with regard to markers

ofterminal

differentiation.

expressed

in the raft cultures

K1 and

filaggnin

supported

were

faithfully

by mouse

3T3 A31 fibroblasts for as short a time as 9 days. When cultivated in the presence of primary human foreskin fibroblasts,

K1

and

filaggnin

were

not

detected

until

3

476

Epithelial

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in raft cultures All photographs

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illumination respectively;

and foreskin. were taken with arrows D, F, and

In situ

with

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a 40x

delineating H, 9-, 21-,

objective

results

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Profilaggrin

mRNA

expression

in raft

cultures

and

foreskin.

1, an atypical

region

of profilaggrin

0

expression.

The

majority

of

the

epithelia

were

479

480

Epithelial

Differentiation

in Raft

Cultures

B

A

. .,

,.,_

:40.

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.

Cell

weeks,

and

The signals culture. Significant

type

then

expression

were

still

differences

of raft culture

was

relatively

only

between

included

weak

weak

and

after

focal.

1 month

foreskin

and

the continued

KS and K14 mRNAs as basal cells divided the suprabasal strata in vitro. Constitutive

and

either

of

moved

to

expression of KS and K14 mRNAs is characteristic ofcertain carcinoma cell lines as well as raft cultures established from them (11). K6, a marker for hyperproliferation and wound healing (21) and abnormal keratinization (23), was not expressed in the foreskin but produced strong signals in the raft cultures throughout the entire time period. Stolen et a!. (11) K14. The

also reported similar protracted expression

observations of K5, K6,

mouse

is a marker

for differentiation

(22),

was

level compared

for K6 and and K14 in in vitro are in

transcribed

to foreskin

at an

throughout

the entire period, in spite of the delay and reduction in the expression of the terminal differentiation marker genes Ki and profilaggnin in the PHF raft cultures (Fig. 3). Conversely, the genes KS and K14 normally expressed in basal cells and K6 in healing wounds or psoniasis were coexpressed in the suprabasal cells with Ki, profilaggnin, and involucnin (Fig. 3). Together, these observations suggest that there are multiple regulatory circuits modulating the expression of the various genes, and some of the regulatory machinery is uncoupled in vitro. Thus, the most useful probes for monitoring differentiation and improving raft cultures in the future should include the basal-specific keratins KS or K14, the differentiationcommitment marker Ki, and the terminal differentiation marker profilaggnin. The

ability

to

promote

terminal

differentiation

for

2 weeks

(Fig.

3; and

data

not

factor,

supported

by established

mouse

the

9 days

at the

medium-air

the ability to do so through matured much more slowly

Fig.

8.

Profilaggrin/filaggrin

human

filaggrin

by A31

mouse

A31 -supported

interface

distribution

was used as described fibroblasts.

raft cultures,

and

This

Arrowheads,

respectively.

in

raft

cultures

and

remained

growth

human

potential

factor

foreskin

of

the

produced in more

interpretation

terminal

3T3) or slowly

viable

or

were

at

a, keratinocyte

growth

fibroblasts,

the

established

human

fibroblasts

may

is consistent

differentiation,

have

less proliferation-promoting rapid epithelial differentiation. with

our

observation

expressing

K1

and

that,

irradiated achieved profilaggnin

messages and producing keratohyalin granules in the uppermost stratum. When the irradiation was sublethal (1000 rads), the 9-day culture exhibited an intermediate state of differentiation (data not shown). In summary, no raft culture described to date exhibits an expression pattern of all of the epithelial genes identical to native foreskin. Nonetheless, cultures supported by the established mouse fibroblast cell lines closely resembled foreskin. Our study supports the concept that

the latter the same

foreskin

(NIH

when primary human fibroblasts were lethally with 137Cs (5000 nads), the 9-day raft culture

cell

as detected

in Materials and Methods.” A, neonatal KHGs. C, E, and G, 9-, 21-, and 32-day

primary

decreased and factors, resulting

maintained

32 days, whereas and did not reach

collagen active.

transforming

growth

lines much more closely resembled native epithelium than cultures supported by primary human foreskin fibroblasts. The former achieved terminal differentiation within

rapidly

mouse fibroblasts may have produced on elicited a more natural and balanced response to a combination of growth factors, resulting in terminal differentiation. In contrast, the cultures with primary human fibroblasts may have contained a higher level of one on more of the growth-promoting factors relative to the differentiationstimulating factors. However, as the cultures aged, the

How-

fibroblast

divided

factor, interleukin 6, and transforming growth factor /3) (reviewed in Ref. 6). It is unlikely that the fibroblasts exerted their effect by directly altering the Ca2 gradient in the epithelium. We postulate that, when compared to

ever, at 3 weeks, the epithelium appeared disorganized and necrotic, probably because the fibroblasts were no longer metabolically active (data not shown). On the basis of these results, foreskin keratinocyte raft cultures

that

The growth and differentiation of epithelial cells are affected by Ca2 concentration and retinoic acid as well as by a number of growth factors (e.g., epidermal growth

is not

shown).

fibroblasts

fibroblasts in the least metabolically

unique to mouse 3T3 A31 cells, as similar results were also obtained by including mouse NIH 3T3 fibroblasts in the collagen (see Fig. 3). However, NIH 313 cells multiplied rapidly in the dermal equivalent and, by 3 weeks, invaded the epithelium, causing disorganization without affecting terminal differentiation of the epithelial strata (data not shown). When lethally irradiated with 137Cs to block their multiplication, they supported keratinocyte cultures

481

(3T3 A31) (see “Materials and Methods”) on not at all (irradiated NIH 3T3) in the collagen matrix all supported swift terminal differentiation. Differential viability of the fibroblasts in the collagen matrix does not appear to be the cause of the observed differences, either. We believe that the mouse and human fibroblasts remained alive throughout most of the experiments, if not for the entire duration, as they were continuously shed into the media and grew to confluence on the bottom of the culture dishes (see “Materials and Methods”). Furthermore, raft cultures prepared without fibroblasts are only two or three cells thick and become immediately flattened upon leaving the basal layer, thus showing neither spinous nor granular layers (24, 25). No terminal differentiation-specific kenatin marker K1 was observed in these cultures (25). These observations are different from ours and support our interpretation that both mouse and human

suprabasal cells suggests that keratinocytes a more proliferative mode than are cells in native foreskin, perhaps simulating wound healing. It was unexpected that the expression of one gene specific for terminal differentiation could not be used to predict that of a second gene normally coexpressed in the same stratum in vivo. For instance, involucnin, which

equal or elevated

& Differentiation

degree of differentiation by 32 days in culture. The reason for this distinction is not clear. The number of viable or metabolically active fibroblasts in the collagen matrix appeared not to be a critical determinant of kenatinocyte molecular differentiation. Cultures prepared by using one-half the usual number of primary human fibroblasts in the collagen matrix had similar expression patterns of marker genes (data not shown). In addition, the

in

presence

Growth

by

immunocytochemistry. B, hematoxylin

human foreskin; PHF-supported

raft cultures,

A monoclonal

and eosin respectively;

antibody

directed

against

stain of the 9-day raft supported 0, F, and F!, 9-, 21-, and 32-day

482

Epithelial

Differentiation

in Raft Cultures

the collagen raft culture system is an extremely useful tool for studying epithelial stratification and differentiation. By changing the type of fibroblasts within the collagen matrix, one can achieve different degrees of keratinocyte differentiation. This manipulation could be used to study the regulation of host genes and to dissect the stages of gene expression of viruses that normally depend on terminal epithelial cell differentiation for their productive life cycle. Taking advantage of this information, we have recently recapitulated the first in vitro reproductive

program

supported viral

of a papillomavirus

raft

expression

ported

by primary

Materials

culture from

system, the

human

late

using

the

while

mouse

segregating

events

in

cultures

A31-

early sup-

fibroblasts.4

and Methods

Primary Neonatal Foreskin, keratinocytes, and Fibroblasts. Human neonatal foreskins obtained from area hospitals were fixed in neutral buffered 10% formalin and embedded in paraffin. Four-zm sections were cut for histological evaluation following hematoxylin and eosin staining, for in situ hybridization with radioisotopically labeled RNA probes, or for immunocytochemical evaluation as described below. For keratinocyte cultures, two or more neonatal foreskins were combined, minced in 30 ml of 0.25% trypsin and 1 mM EDTA in Hanks’ balanced salt solution without Ca2 or Mg2’ (GIBCO). Digestion was carried out at 37#{176}C for 1 h in a BelIco 125-mI trypsinizing flask while being mixed gently with a stirbar. The supernatant containing the dispersed keratinocytes was collected, and the trypsin was

quenched

with

an equal

volume

of Ham’s

Keratinocyte

Raft Culture.

Approximately

DNA

F-12

medium supplemented with 10% FBS. Cells were pelleted and resuspended in serum-free medium (GIBCO) or keratinocyte growth med ium (Collaborative Research) and then plated on 100-mm dishes previously seeded with NIH 3T3 mouse fibroblasts (at 20-30% confluency) lethally irradiated with 5000 rads using a 137Cs source. Primary human dermal fibroblasts were recovered from neonatal foreskins in the same manner as keratinocytes and grown in DMEM with 10% FBS. Mouse BALB/c 3T3 A31 cells were received from Dr. Vince Groppi (Upjohn Co., Kalamazoo, Ml) and were propagated in DMEM supplemented with 10% bovine calf serum. Mouse NIH 3T3 cells were also grown in DMEM supplemented with 10% bovine calf serum.

Primary

5% CO2 at 37#{176}C. Media were changed every 24-48 h, as needed, for up to 32 days. Through this duration, the A31 cells did not migrate; however, a small fraction of the cells divided, but no more than two to three times. In contrast, the human fibroblasts did not divide or migrate. Because live human or mouse fibroblasts were continuously shed into the media, plates were changed when they grew to confluence at the bottom of the culture plates. Portions of the raft cultures were harvested at different times (9, 21, and 32 days), fixed in 10% buffered formalin, and embedded in paraffin. Fourzm sections were stained with hematoxylin and eosin for histological evaluation or were used for in situ hybnidization or for immunocytochemistry (see below). Plasmid Construdion and Riboprobe Synthesis. The following cDNA clones of differentiation stage-specific human genes (for a review of the keratin nomenclature, see Ref. 27) were obtained: K1, plasmid pK4S6 (28); K4, pKH4 (29); K8, pKH8 (30); K18, pKH18 (30); KS, pBRS81 (31); K13, pBR54-1 (from Dr. Richard L. Eckert5); K6, 1SP (1); Kl4, 3-SP (1); and involucnin, p1-2 (32). For each of these genes, segments consisting entirely or largely of the 3’ noncoding region, sometimes together with the unique carboxyl terminal domain, were recloned or subcloned as needed into the dual promoter transcription vector pGEM-l (Promega). The profilaggnin probe pHx-1 is equivalent to the coding region of one protein repeat unit.6 Computer analyses of DNA sequences were performed using programs from The University of Wisconsin Genetics Computer Group Software Package, Version 6.1 (33).

106

mouse BALB/c 3T3 A31 fibroblasts or primary neonatal human fibroblasts in 0.5 ml 100% FBS were suspended in a mixture of 8 ml of type IA rat tail collagen (Collabonative Research), 1 ml of lox Ham’s F-12 media without NaHCO3 and 1 ml of 2.2% NaHCO3, 0.05 N NaOH, and 200 mti 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer. Three ml of the mixture were poured into a 35-mm culture dish, allowed to gel at 37#{176}C for at least 1 h, and then fed raft culture medium, which consisted of DMEM:Ham’s F-12 (3:1) plus 10% FBS, supplemented with 0.4 ig/ml hydrocortisone, 0.1 nt’i cholera toxin, 0.5 zg/mI transfernin, 5 ng/ml epidermal growth factor, and 5 zg/ml insulin (25). Approximately 2 x l0 primary human keratinocytes were seeded onto the collagenfibroblast matrix and maintained submerged for 24-48 h until a confluent monolayer was established. The cell matrix was then raised to the medium-air interface on a stainless steel grid placed in a 60-mm culture dish, exposing the keratinocytes to an atmosphere containing

plasmids

were

linearized

with

appropriate

re-

stniction enzymes prior to synthesis of radiolabeled riboprobes by in vitro transcription, as previously descnibed (34). Approximately 60 Ci/mmol total of [3H]UTP and [3H]CTP were added to each transcription reaction. Final niboprobe preparations had a specific activity of about 3 x 10 cpm/zg. The tnitium-labeled transcripts were hydrolyzed to a mean length of 300 nucleotides, as confirmed by formaldehyde-agarose gel electrophoresis. In Situ Hybridization of mRNAs. Four-zm sections were mounted on 3-aminopropyl-tniethoxysilane-treated glass microscope slides (35). In situ hybridization was performed essentially as described (34). The shortest probe was applied at 100% saturation, and the remaining probes were normalized such that the quantity of radioactivity applied was the same for all probes regardless of their complexity (100% saturation = 0.2-0.3 ag probe/ ml

kilobase

of probe).

Antisense

probes

containing

po-

lyunidylate were prehybnidized with polyadenylate to ensure specificity of binding. High stringency washes were carried out in 0.ix standard saline citrate (0.15 M NaCl-0.015 M sodium citrate) at approximately Tm S#{176}C to eliminate nonspecific retention of probes. Tm calculations for RNA:RNA hybrids have been described (36). Slides were dipped in liquefied Kodak NTB-2 radiographic emulsion, exposed at 4#{176}C for 4 weeks, and photographically developed in Kodak D19. The sections were stained with hematoxylin and eosin and subsequently examined and photographed with an Olympus BH-2 light microscope.

S

6

R. 1. Eckert, P. Fleckman,

unpublished P. Haydock,

observations. and R. Presland,

personal

communication.

Cell Growth

lmmunocytochemistry. Tissue and raft culture sections mounted on 3-aminopropyl-tniethoxysilane-coated slides were probed with monoclonal antibodies specific for either the 67-kilodalton human K1 (34/3B4; ENZO Diagnostics,

New

York,

NY)

or

human

profilaggnin/filaggnin

(Biomedical Technologies, Stoughton, epitope accessibility, partial proteolytic sues

was

performed

with

either

trypsin

in phos-

phate-buffered saline, pH 7.2, for 10 mm at 37#{176}C (for anti-human filaggnin) or 0.4% pepsin in 0.01 N HCI for 30 mm at 37#{176}C (for 34flB4). Slides were then incubated in 3% normal goat serum prior to an overnight incubation with the primary antibody (1:100 dilution for anti-human filaggnin

or

1 :50

dilution

for

34/3B4).

Antibody

reactivity

was detected with peroxidase-conjugated goat antimouse IgG (Sigma) followed by aminoethyl carbazole development (Zymed Laboratories, South San Francisco, CA). Slides were then counterstained with hematoxylin. Acknowledgments We thank

Lucy Watson

Laboratory

of Strong

for collecting

Memorial

Hospital

foreskins

and the Surgical

for sectioning

tissues

Pathology and

keratin-

ocyte raft cultures. We also thank Drs. Dennis Roop, Werner Franke, Richard Eckert, and Elaine Fuchs for providing the human keratin and involucrin cDNA clones, and Paul Haydock, Philip Fleckman, Richard Presland, and Beverly Dale for sharing the profilaggrin cDNA clone prior to publication.

keratinocytes 1985.

16. Kopan, of terminal

epidermal 440,

MA). To increase digestion of tis-

0.1%

of human 536-539,

at the

liquid-air

interface.

& Differentiation

Exp. Cell

Res.,

483

159:

R., Traska, G., and Fuchs, differentiation: examining

cells

at various

stages

E. Retinoids as important regulators keratin expression in individual of keratinization. J. Cell Biol., 105: 427-

1987.

17. Parenteau, N. L., Nolte, C. M., Bilbo, P., Rosenberg, M., Wilkins, L. M., Johnson, E. W., Watson, S., Mason, V. S., and Bell, E. Epidermis generated in vitro: practical considerations and applications. j. Cell. Biochem., 45: 245-251, 1991. 18. Bedell, M. A., Hudson, J. B., Golub, 1. R., Turyk, M. E., Hosken, M., Wilbanks, G. D., and Laimins, L. A. Amplification of human papillomavirus genomes in vitro is dependent on epithelial differentiation. J. Virol., 65: 2254-2260,

1991.

19. Blanton, R. A., Perez-Reyes, N., Merrick, D. 1., and McDougall, j. K. Epithelial cells immortalized by human papillomaviruses have premalignant characteristics in organotypic culture. Am. J. Pathol., 138: 673-685, 1991.

20. Woodworth, C. D., Cheng, S., Simpson, S., Hamacher, L., Chow, L. 1., Broker, T. R., and DiPaolo, J. A. Recombinant retroviruses encoding human papillomavirus type 18 E6 and E7 genes stimulate proliferation and delay differentiation of human keratinocytes early after infection. Oncogene, 7: 619-626, 1992. 21 . Weiss, R. A., Eichner, R., and Sun, 1-1. Monoclonal antibody analysis of keratin expression in epidermal disease: a 48- and 56-kdalton keratin as molecular markers for hyperproliferative keratinocytes. j. Cell Biol., 98: 1397-1406,

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