Monoclonal Antibody Studies of Mammalian ... - Sun Laboratory
Monoclonal Antibody Studies of Mammalian Epithelial Keratins: A Review" TUNG-TIEN SUN,* SCHEFFER C. G. TSENG," ANDREW J.-W. HUANG,' DAVID COOPER,* ALEXANDER SCHERMER,*MARION H. LYNCH,* ROBERT WEISS,dAND RIVA EICHNER Departments of Dermatology and Pharmacology New York University School of Medicine New York, New York 10016 'Department of Ophthalmology Harvard University Medical School Boston, Massachusetts 02115 Dermatology Branch, National Cancer Institute National Institutes of Health Bethesda, Maryland 20205 Departments of Dermatology and Cell Biology and Anatomy The Johns Hopkins University School of Medicine Baltimore, Maryland 21205
INTRODUCTION Significant progress has been made during the past decade with regard to the biochemistry and immunology of keratin filaments. The results indicate that keratin represents a family of more than 17 water-insoluble, cytoskeletal proteins,"2 that keratins are restricted to epithelia and their derivatives;d that different subsets of 2- 10 keratins are expressed in different epithelia,1~2~7-12 that at least two keratin species are required for filament a~sembly,'".'~ and that keratin molecules consist of a central helical domain with two nonhelical end^.^^-^^ Despite these advances, however, it remains a puzzle as to why such a large number of keratin species have evolved to form tonofilaments, while other morphologically similar, intermediate-sized filaments are characterized by a much simpler protein composition with only one to three subunits (for reviews, see Lazaridesl' and other chapters in this volume). To address this fundamental question, we have prepared several monoclonal antikeratin antibodies2' and used them as a tool for studying keratin expression. In this 'This work was supported by grants from the National Institutes of Health (EY 04722 and AM 3451 1). T.-T. Sun is the recipient of a Monique Weill-Caulier Career Scientist Award. 307
308
ANNALS NEW YORK ACADEMY OF SCIENCES
paper, we will review our data, which indicate that all human epithelial keratins can be divided into two subfamilies according to their charges and their immunoreactivities with AEl and AE3 monoclonal antibodies. We will also discuss how the expression of specific keratins in the epidermis and several nonepidermal epithelia can vary as a function of environmental modulation and disease. Finally, we will present new data on keratins expressed in rabbit epidermis and other epithelia during embryonic development. Taken together, the data suggest the existence of a number of “rules” governing the expression of specific keratin molecules and help to formulate the concept of “keratin pairs” as defined by co-expression.
TWO KERATIN SUBFAMILIES AS DEFINED BY MONOCLONAL ANTIBODIES
Recent results from two-dimensional gel analyses have established the existence of 17- 19 human keratin species132*12s2%22. , many of them have been shown to be distinct translational prod~cts.Z’~*~ To study these keratins, we have prepared water-insoluble cytoskeletal proteins from five representative human tissues (abdominal epidermis, corneal epithelium, esophageal epithelium, cultured epidermal cells, and mesothelial cells) and analyzed the proteins by immunoblotting using our AEl and AE3 monoclonal anti-keratin antibodies (FIGUREl).22s27-29 Consistent with cDNA data from Fuchs et al.,24,30 peptide mapping data of Franke et aL,I,31and our own earlier immunoblotting the results indicate that all known human epithelial keratins can be divided into two subfamilies based on their charges and immunoreactivities with AEl and AE3 monoclonal antibodies (FIGURE2)?’,” One subfamily consists of the 65-67K (no. 1 and 2, according to Moll et a l l ) , 64K (no. 3), 59K (no. 4),58K (no. 5), 56K (no. 6), 54K (no. 7),and 52K (no. 8) keratins. These keratins have isoelectric points (PIS) greater than 6.0and share an antigenic determinant as defined by our AE3 antibody (FIGURES1 and 2).Franke and co-workers have shown that all of these proteins are structurally related according to peptide mapping and, noting their molecular weights and charge, referred to them as a subfamily of “relatively large and basic cytokeratins.”” Hybrid-selection data from Fuchs and co-workers have demonstrated that the 58K,56K,54K,and 52K keratins share sequence homologies and are related to the Type I1 wool keratins previously defined by Crewther and c ~ - w o r k e r s ? ” ~Thus ~ * ~ the ~ . ~keratins ~ described above are closely related and constitute the “basic” subfamily (Type 11). The other subfamily consists of the 56.5K (no. lo),55K (no. 12), 51K (no. 13), 50K (no. 14),50’K(no. 15), 48K (no. 16), 46K (no. 17), 45K (no. IS), and 40K (no. 19)keratins. These keratins have PI values of less than 5.7 and many of them (56.5K, 50K,50K,48K,48K,and 48K)share an AE1 determinant (FIGURES1 and 2). Data on cDNA from Fuchs et al. have shown that the 50K,46K,45K,and 40K keratins share sequence homologies and are related to Type I wool keratin^?^.^'.^^ Franke and colleagues have established by peptide mapping that the 50K,48K,and 46K keratins are structurally These keratins thus form the “acidic” subfamily (Type I), which is structurally and immunologically distinct from the basic subfamily.
TUNG et al.: RULES OF KERATIN EXPRESSION
309
“KERATIN PAIRS” AS DEFINED BY CO-EXPRESSION In order to gain insight into the possible functional meaning of these heterogeneous keratin species, we have performed a series of experiments to determine whether the expression of different keratin molecules can be correlated with specific morphological or physiological features of epithelial differentiation. Our data suggest that there exist a number of simple rules regarding the expression of individual keratin species (TABLE 1). Moreover, a close examination of these rules reveals a strikingly parallel relationship between the keratins of the two subfamilies. Thus, with the exception of the 40K keratin (of the acidic subfamily), which is wides~read,’.’~*~~ each acidic keratin is coexpressed with a particular basic keratin, forming a “keratin pair” (TABLE1). Within each pair, the two keratins have identical “size-ranks” in their respective subfamilies
HGURE 1. Immunoblot analyses of human epithelial keratins using AEl and AE3 monoclonal antibodies. (a-c) One-dimensional SDS gels (Laemmli, 12.5% acrylamide)” stained by (a) Fast green, (b) AE3, and (c) AEI. Antibody staining was performed by the peroxidase-antiperoxidase (PAP) te~hnique.’~ Samples are (1) in vivo abdominal epidermis, (2) corneal epithelium, (3) esophageal epithelium, (4) cultured foreskin epidermal cells, and ( 5 ) cultured mesothelial cells (courtesy of James Rheinwald). The molecular weights (MWs) of the four major epidermal keratins are marked to the left. Note in (b) and (c) that AE3 and AEl recognize two mutually exclusive subfamilies of keratin^.^^.'^ (d-f) Two-dimensional nonequilibrium gel analysis 28 of a mixture of keratins from the five tissues listed above. The proteins were stained by (d) Fast green, (e) AE3, and (f) AEl antibodies. All keratins are identified by MWs; for their corresponding catalogue numbers’, see FIGURE 2 and TABLE1. Note that all AE3-reactive keratins are relatively basic (on the left of the gel), while all AEl-reactive ones are acidic. B and P denote bovine serum albumin and 3-phosphoglycerate kinase’; V denotes vimentin. (From Cooper et al.2’ With permission from Laboratory Investigation.)
PI
I
acidic
\
‘45 (18) -‘40 (19)
(closed circle). This is a schematic tracing of an actual two-dimensional gel (as shown in FIGURE Id) showing that human epithelial keratins can be divided into acidic (A or Type I) and basic (B or Type 11) subfamilies. The charge heterogeneity of individual keratins is at least in part due to phosphorylation.’2.21.77 The positions of bovine serum albumin (BSA), vimentin (V), and 3-phosphoglycerate kinase (PGK) are shown for reference.’.I2 For reasons mentioned elsewhere?’ keratins no. 9 (64K) and no. 11 (56K) are not included in this scheme. (From Cooper et al.” With permission from Laboratory Investigation.)
FIGURE 2. Classification of human epithelial keratins by their charge and their immunoreactivities with AE3 (hatched circle) and AEl antibodies
basic
+o-+ PGK
c)o BSA
w 0 c
AE 1
AEl
5.1
5.3/4.9
5.1 5.1 5.7 5.2
51 (13)
50/50’ (14/ 15) 48 (16) 46 (17) 45 (18) 40 (19) 7.8 6.0 6.1
7.4
58 (5) 56 (6) 54 (7) 52 (8)
7.3
6-8 7.5
PI
59 (4)
65-67 (1,2) 64 (3)
MW X lo-’
Basic (Type 11)
AE3 AE3 AE3
AE3
AE3
AE3 AE3
Antibody
8 8 7
8
8
8 9
AMW x lo-’ @-A)
Hyperproliferative keratinocytes Simple and some stratified epithelia Simple epithelia
“Skin type” differentiation “Corneal-epithelial type” differentiation “Esophageal-epithelial type” differentiation Keratinocytes
Markers
Keratins are arranged according to their relative sizes under the acidic and basic subfamilies. Numbers in parentheses are keratin nomenclatures according to Moll er nl.’ Note the striking parallelism between the two subfamilies with respect to their relative size and tissue distribution.
AE 1
AE 1
5.3 4.9
56.5 (10) 55 (12)
Antibody
Keratin Subfamily
PI
Acidic (Type I)
Classification and Expression of Human Epithelial Keratins
MW x 10-3
TABLE 1.
312
ANNALS NEW YORK ACADEMY OF SCIENCES
and follow similar rules for expression (TABLE1). In the next three sections we will briefly review the evidence from which these rules are derived.
KERATIN EXPRESSION IN HUMAN EPIDERMIS Immunolocalitation Studies Normal human epidermis is a keratinized” epithelium that expresses four major keratins (50K, 56.5K, 58K, and 65-67K).1s20.37 Using monoclonal antibodies, we have localized these four keratins in different epidermal layers by a combination of immunohistochemical and biochemical techniques?’ The results indicate that the 50K and 58K keratins are present in all cell layers including the relatively undifferentiated basal layer, whereas the 56.5K and 65-67K keratins are associated only with the more differentiated cells above the basal layer (FIGURES 3 and 7)?0.3842In other experiments surveying the tissue distribution of keratins, we found that the “basal type” 50K and 58K keratins are present in various quantities in all stratified squamous epithelia, whereas the “suprabasal type” 56.5K and 65-67K keratins are mainly expressed by the keratinized e p i d e r m i ~ . These ~ . ~ ~ findings ~ ~ ~ support the notion that the 50K and 58K keratins (or 50K/58K pair) may be regarded as molecular markers for keratinocytes (the major cell type of all stratified squamous epithelia), and that the 56.5K and 65-67K keratins (56.5K/65-67K pair) may be regarded as markers for keratinization or “skin type” differentiation (TABLE1).
Reversible Modulation of Keratin Expression in Cultured Human Epidermal Keratinocytes In the presence of lethally irradiated 3T3 cells, human epidermal cells form stratified squamous colonies and undergo terminal differentiati0n.4~Unlike their in vivo Morphological keratinization is defined by the formation of granular cells and eosinophilic, anucleated cornified cells.
FIGURE 3. Sequential and coordinate expression of the four major epidermal keratins accompanying morphological differentiation of normal human epidermis. (A) Morphology of the epidermis, which can be divided into four layers: basal (B), spinous (S), granular (G), and cornified (C). Note the presence of hemidesmosomes (HD), desmosomes (D), tonofilaments (F), keratohyalin granules (K), membrane-coating granules (or lamellar granules; MCG), and cornified envelope (CE). (B) A schematic summary of keratin localization data showing that basal cells (B) express mainly an acidic 50K and a basic 58K keratin, while suprabasal cells ( S , G, and C ) contain, in addition, an acidic 56.5K and a basic 65-67K keratin. In normal cornified cells (not callus), most of these keratins are either completely (e.g., the 50K component), or partially (all of the other keratins) degraded, resulting in two diffuse, ill-defined, major bands (one 55-58K, another 63-653). Keratins of the living cellular layers (B, S , and G) are not crosslinked by intermolecular disulfide bonds (K-SH) and are therefore extractable with urea or SDS alone (“prekeratin” of Matoltsy’’); whereas keratins in the nonviable cornified layer are known to be crosslinked extensively by intermolecular disulfide bonds (K-S-S-K) and are therefore extractable only after reduction.21
313
TUNG et al.: RULES OF KERATIN EXPRESSION
EPIDERMAL DIFFERENTIATION A. MORPHOLOGY
I
I I I I I I I Basal + Spinous + Granular + Cornified I
B. KERATIN EXPRESSION B
S
67 6665-
G
C 63-65 55- 58
K-SH
K-SIS-K
ANNALS NEW YORK ACADEMY OF SCIENCES
3 14
counterparts, however, these cultured keratinocytes fail to form granular layers or 4)?‘’,, Kerenucleated cornified layers, and are therefore “nonkeratinized” (FIGURE atin analysis showed that these cells continue to synthesize the 50K/58K keratin pair, a marker for keratinocytes, but cease to make the 56.5K/65-67K keratin pair (FIGURE5).21t37.45 Interestingly, these latter two “keratinization markers” are coordinately expressed when the cells are induced to keratinize, both in athymic mice’ and in culture medium deprived of certain growth factors such as vitamin A, hydro4)?’-‘‘ cortisone, and epidermal growth factor (FIGURE
Keratin Expression in Human Epidermal Diseases Although cultured human epidermal cells do not synthesize the “keratinization markers” (56.5K and 65-67K keratins), they express 56K, 48K,46K, and several
Cell culture; Vit. A, HC, EGF, CAMP CT, etc.
Normal (keratinized)
Hyperproliferated (non- or para-keratinized)
FIGURE 4. A schematic drawing illustrating the morphological changes that occur when human epidermal cells are grown in tissue culture media containing various growth-stimulating substances such as vitamin A, hydrocortisone (HC), epidermal growth factor (EGF), cyclic AMP (CAMP), cholera toxin (CT), etc. The arrows indicate that this process can be reversed by transplanting the cells to an in vivo permissive envir~nrnent,’.’~ or by growing the cells in media deprived of certain growth factors, such as vitamin A.45.”
TUNC et al.: RULES OF KERATIN EXPRESSION
315
FIGURE 5. A comparison of keratins expressed by human epidermal cells in vivo versus in culture. Keratins were isolated from in vivo normal (keratinized) epidermis (lane 1, 3, and 5 ) and cultured (nonkeratinized) epidermal colonies (lanes 2, 4, and 6), separated by SDS gel electrophoresis, transferred to nitrocellulose paper, and stained with (lanes 1 and 2) Fast green, (3 and 4) AEl antibody, and ( 5 and 6) AE3 antibody. Note that the two samples share the (acidic) 50K and (basic) 58K keratins. The (acidic) 56.5K and (basic) 65-67K keratins are unique to the keratinized epidermis, while the (acidic) 48K and (basic) 56K keratins are present only in cultured cells. (From Eichner el ~ 1 , With ~ ’ permission from Journal of Cell Biology.)
other small keratins that are undetectable in normal interfollicular epidermis (FIGURE 5).2’,3’,45 We have found recently that the 48K and 56K “culture type” keratins are expressed in a wide variety of hyperproliferative epidermal diseases including psoriasis, warts, actinic keratoses, and squamous cell carcinomas (FIGURE 6):’ Furthermore, these two keratins become readily detectable when the epidermis is stimulated to undergo hyperproliferation either by the tape-stripping technique (Weiss et al., unis ~important ~~~ to note, however, published) or by placing the skin in organ c ~ l t u r e . 8It. ~ that these two keratins clearly are not epidermis specific, because they are also expressed by cultured keratinocytes derived from various stratified squamous epithelia Since the 48K including those of the esophagus, conjunctiva, cornea, and ~agina.2’’’~’~~
316
ANNALS NEW YORK ACADEMY OF SCIENCES
TUNG el al.: RULES OF KERATIN EXPRESSION
317
and 56K proteins are not detectable in normal epidermis or in nonhyperproliferative epidermal diseases such as ichthyosis vulgaris, the results suggest that this keratin pair may serve as a useful molecular marker for hyperproliferative keratinocytes
(FIGURES 5-7).47,48
KERATIN EXPRESSION IN NONEPIDERMAL EPITHELIA Normal Epithelia Tissue-distribution data have shown that (1) 56.5Kand 65-67Kkeratins are mainly associated with keratinized tissues including the epidermis and thymic Hassall’s corp u s c l e ~ . These ~~~~ two ~ ~keratins ’ are also present in vaginal and exocervical epithelia?’ whether this reflects a partially keratinized state of the epithelia remains unknown. (2)55K and 65K keratins are corneal epithelium-~pecific.’*~*’~~~~ (3) 51K and 59K keratins are mainly found in esophageal, tongue, and other internal, nonkeratinized stratified epithelia.12’’ (4)50/50‘Kand 58K keratins are found in various quantities in all stratified squamous epithelia, but not in simple epithelia.1r2r32.52*53 (5)45K and 52K keratins are the major keratins in many simple epithelia.1*z-32 Glandular, transitional, and pseudostratified columnar epithelia usually express a complex mixture of “keratinocyte type” as well as “simple epithelial-type” keratin^.^.^.^' These results are summarized schematically in FIGURE 8.
CuItu red Cells
In general, stratified squamous epithelial cells undergo major morphological and biochemical (keratin) changes when they are placed under culture conditions designed for maximum cellular growth. Thus, cultured human epidermal, corneal, and esophageal epithelial cells do not express their differentiation-specific keratins, i.e., the 56.5K/65-67K, 55/64K,and 51K/59K keratin pairs, respectively. Instead, all three cultured epithelia express the 50K/58K keratins, the 48K/56K hyperproliferation markers, a 46K keratin, and a few other small keratins normally associated with As we have shown earlier, however, these cultured cells (with simple epithelia.2~7.2’*37*so
FIGURE 6. Keratin expression in human epidermal diseases. Keratins were isolated from (1) normal epidermis, (2) ichthyosis vulgaris, (3) atopic dermatitis, (4) and ( 5 ) psoriasis, ( 6 ) keratoacanthoma, (7) actinic keratosis, (8) wart, (9) verrucous carcinoma, (10) and (1 1) squamous cell carcinomas, (12) basal cell carcinoma, and (13) cultured human epidermal cells. The proteins were separated by 12.5% SDS gel electrophoresis, transferred to nitrocellulose paper, and stained with (a) Fast green (FG), (b) AEl antibody, and (c) AE3 antibody. Note the striking similarity between the overall keratin pattern of some diseases (e.g., lanes 7-12) and that of cultured epidermal cells (lane 13). Also note the detection of an AEl-reactive (acidic) 48K keratin and an AE3-reactive (basic) 56K keratin in all hyperproliferative epidermal diseases, and their absence in normal epidermis (lane 1) or the nonhyperproliferative disease ichthyosis vulgaris (lane 2). With permission from Journal of Cell Biology.) (From Weiss ef
ANNALS NEW YORK ACADEMY OF SCIENCES
318
Normal Keratinized
58 56.5 56 52 2 v 50
____t
Hyper proliferative Non-keratinized
t
a c
4R
.-
c
0 L-
a 40 4s
Y
65-67 58 56.5 56 52
50
I
I
A
48 6
4
B
r
C
D
FIGURE 7. A schematic diagram illustrating a continuous spectrum of keratin expression by human epidermal cells. Bars represent specific keratins (identified by MW X Position A denotes the keratin pattern of normal in vivo (keratinized) epidermis; B denotes that seen in a variety of epidermal diseases with different degrees of hyperproliferation and/or altered differentiation; C denotes keratins of basal cell carcinomas; and D represents keratin pattern of human epidermal cells cultured in media designed for maximum cellular growth. Note that the 50K/ 58K keratin pair (black bars) is present throughout the spectrum, the 56.5K/65-67Kkeratin pair (dotted) is expressed mainly during keratinization, the 48K/56K keratin pair (hatched) is associated with “hyperproliferation.” The above three keratin pairs are keratinocyte specific, whereas the remaining keratins (open bars) are found as major keratins mainly in simple epithelia. This diagram emphasizes (1) the continuous spectrum of keratin expression, (2) the tremendous overlap of keratin pattern in various diseases, and (3) on a tissue level, the reciprocal expression of the “keratinization” and “hyperproliferation” markers. (From Weiss et aL4’ With permission from Journal of Cell Biology.) the possible exception of corneal cells) can rapidly regain their normal in vivo morphology and keratin patterns once the cells are provided with a permissive in vivo en~ironment.~.“’~~ In contrast, simple epithelial cells rarely undergo major changes in their keratin composition, either in culture or when they become tran~formed.‘**.~~.’~
Keratin Expression in Nonepidermal Epithelia during Vitamin A Dejciency Although the 56.5K and 65-67K keratins are normally absent from nonkeratinized esophageal, conjunctival, and corneal epithelia, this keratin pair becomes readily detectable when the tissues become keratinized as a result of vitamin A deficiency (FIGURE9).“*” This observation indicates that the 56.5K/65-67K keratins are not
TUNG et ab: RULES OF KERATIN EXPRESSION
319
epidermis specific, and therefore may be regarded as molecular markers for the program of keratinization or “skin type” differentiati~n?”~’
SEQUENTIAL EXPRESSION OF KERATINS DURING EMBRYONIC DEVELOPMENT The embryonic development of epidermis provides a unique opportunity for studying keratin expression. Morphologically, the early epidermis is a simple epithelium that later becomes stratified, and finally keratinized. Immunoblot analysis of keratins isolated from rabbit epidermis at different stages of embryonic development confirmed earlier r e p ~ r t s ~ that * - ~ showed that keratins undergo sequential changes during development. FIGURE1O(f) shows the keratin changes in rabbit epidermis as detected by immunoblotting using our AEl antibody. In this tissue, AE1 recognizes mainly three keratins (MK, 50K, and 56.5K)that appear sequentially in that order during embryonic development (FIGURE10, f). The 40K keratin is present throughout the developmental period that we have studied (12-day-old to term), but disappears from the skin after A 38K band appears in the 29-day epidermal sample (FIGURE
Stratif ied Simple
Esophagus, etc.
Cornea
Skin
-
-
6465-67
1
-
52
-1
-
-
45 -
-59 58
1111111111111111--
. . . 1 1 1 1 1 -
56.5 55
I
1111111
5051 -
320
ANNALS NEW YORK ACADEMY OF SCIENCES
TUNG et al.: RULES OF KERATIN EXPRESSION
321
10, a and f); presumably this represents a degradative product of the 40K keratin. The 50K (and a variable amount of 48K) and 56.5K keratins are first detected in significant quantities in the skin on days 22 and 23, respectively, around the time when the epithelium becomes stratified and keratinized. The 65-67K and 56.5K keratins appear concurrently, as can be detected by AE3 antibody (data not shown), or, as shown in FIGURE 11, by AE2 antibody, which specifically recognizes these two “keratinization m a r k e ~ s . ” ~These ~ ~ ’ ~data, in conjunction with the earlier data by Da1e,58.79 Banks-S~hlegel,~~ Mol,@’ and their co-workers, suggest that during embryonic development the appearance of specific keratin markers accompanies or slightly precedes morphological differentiation. Developmental studies of nonepidermal epithelia (FIGURE10, a-e) show that the AEl-reactive 40K keratin is expressed in simple epithelia (e.g., small intestinal epithelium) throughout development (FIGURE10, b). In corneal (FIGURE10, c), con10, d), and esophageal (FIGURE10, e) epithelia, the 50K keratin junctival (FIGURE first becomes detectable in 22-23-day-old embryos, shortly before these epithelia become stratified. Finally, consistent with the fact that these epithelia are not keratinized, they do not express the 56.5K (or 65-67K) keratin at any developmental stage.
RULES OF KERATIN EXPRESSION The results described above suggest that there exist a number of rules governing keratin expression. These rules and their supporting evidence are summarized below. (1) 56.5/65-67K keratins as markers of keratinization or “skin type” differentiation. Evidence in support of this notion includes that (a) these keratins are expressed 3)2023842; (b) they are during advanced stages of epidermal keratinization (FIGURE 4 and absent when the epidermis becomes nonkeratinized in cell culture (FIGURES 5)”,” and in diseases (FIGURE 6),47,48,6143 but reappear coordinately when the cells are induced to keratinize (FIGURE7)7.45.46; (c) they are also made by corneal, conjunctival, and esophageal epithelia when the tissues undergo partial keratinization as a result of vitamin A deficiency (FIGURES 8 and 9).46,57 It should be noted that in embryonic epidermis these two keratins actually appear before morphological keratinization becomes apparent, suggesting that biochemical keratinization precedes morphological change^.""^ (2) 55K/64K keratins as markers for “corneal type” epithelial differentiation. Tissue-distribution including those obtained with several new hybridoma antibodies monospecific for the 64K component,64indicate that these two keratins may be regarded as markers of normal corneal epithelial differentiation (FIGURE 8). Similar to the 56.5K/65-67K keratins of the epidermis, the corneal 55K/64K keratin pair is diminished in cultured corneal epithelial cells, suggesting that its expression is also differentiation de~endent.’.~’.~~ FIGURE 9. The expression of the 56.5K and 65-67K keratins in rabbit corneal, conjunctival, and esophageal epithelia during vitamin A deficiency. Keratins were isolated from various rabbit epithelia of normal (A+) and vitamin A-deficient (A-) rabbits, and analyzed by immunoblotting using AE1 and AE3 antibodies. Note the synthesis of the AEl-reactive 56.5K and AE3-reactive 65-67K keratins (arrows) in all three nonepidermal epithelia during vitamin A deficiency-induced keratinization. The 66K (equivalent to the 64K human keratin) band in normal rabbit cornea is biochemically and immunologically distinguishable from the 65-67K keratinization marker.64 (From Tseng et al. With permission from Journal of Cell Biology)
’’
as detected by immunoblotting using AE1 antibody. (a) 12-day whole embryo, (b) intestine, (c) corneal epithelium, (d) conjunctival epithelium, (e) esophageal epithelium, and (f) skin epidermis. Numbers denote days. Nb and Ad denote newborn and adult, respectively. Note the appearance of the 40K keratin as the only AEl-positive component in early embryos, the expression of the 50K keratin in all stratified epithelia around day 22-23 when stratification occurs, and the appearance of the 56.5K keratin in epidermis on day 23, shortly before morphological keratinization. Also note in (0 the abrupt disappearance of the 40K keratin (upper arrow) from the epidermis at birth (lower arrow denotes a “38K band,” which is presumably a degradative product of the 40K species).
FIGURE 10. The sequential expression of the 40K, 50K,and 56.5K keratins in rabbit epidermis and several other epithelia during embryonic development,
v1
3R
2 v1 aR
5
0
*
*
!a R
0
cc
v1
F
z
5
w
W
TUNG et al.: RULES OF KERATIN EXPRESSION
323
(3) 51K/59K keratins as markers for stratified squamous epithelia of internal organs including esophagus and tongue (“esophageal type” differentiation)?’ This 8).1s22These two conclusion is based primarily on tissue-distribution data (FIGURE keratins are also differentiation dependent since they are diminished in cultured esophageal epithelial cells as well as in “de-differentiated” esophageal or tongue squamous cell carcinomas.1’2z’65 (4) 50K/58K keratins as keratinocyte markers. These two keratins are present in reasonably large quantities in almost all stratified epithelia except in corneal epi-
FIGURE 11. The coordinate expression of the 56.5K and 65-67K keratin in epidermis during (rabbit) embryonic development. Keratins were extracted from rabbit embryonic epidermis at different stages of development and from postnatal animals and analyzed by immunoblotting using AE2 monoclonal antibody, which specifically recognizes the 56.5K and 65-67K “keratinization marker^."^^^'^ Note the coordinate expression of these two keratins, first detected around day 23 (cf. FIGURE10).
324
ANNALS NEW YORK ACADEMY OF SCIENCES
thelium where they are present only in minute quantities (FIGURE 8).’,12*22*32 Interestingly, these two keratins are expressed in 10-week-oldembryonic human skin, when the epidermis is still largely a simple epithelium.60This result is consistent with our earlier observation that cultured postnatal human epidermal keratinocytes continue to express the 50K/58K keratins even when the cells form a monolayer in low calcium medium, suggesting that these keratins are “permanently” expressed by epidermal cells regardless of their state of cellular stratification?2 Taken together, these results suggest that 50K/58K keratins may be regarded as markers for keratinocytes or, in the embryo, cells that have committed to become keratinocytes. The fact that most stratified epithelia continue to express the 50K/58K keratins in culture and in neoplasms makes these two keratins valuable markers for identifying cells of stratified epithelial ~rigin.’~.’~ However, in rare situations, stratified epithelia can lose their 50K/ 58K keratin markers. For example, some cultured SV40-transformed keratinocytes lose their 50K/ 58K keratins, involucrin (another keratinocyte marker),66,67as well as their ability to stratify.6’ In fact, these cells appear to have acquired the properties of a simple epithelium because they express as their major cytoskeletal components the 40K, 45K, and 52K keratins, which are characteristic of simple epithelia.’.2.49.68-70 Thus, although the presence of the 50K/58K keratins establishes the stratified epithelial nature of a cell, their absence (or the presence of simple epithelial markers) does not rule out the possibility that the tumor is originally derived from a stratified epitheli~m.~’.~’ (5) 48K/56K keratins as markers for “hyperproliferative/de-differentiated” keratinocytes. These two keratins are present in (a) a variety of hyperproliferative epi6): (b) cultured keratinocytes and carcinomas derived from dermal diseases (FIGURE 5)J.2.7,8,71 and skin, corneal, conjunctival, tongue, and esophageal epithelia (FIGURE (c) normal human trunk epidermis stimulated to hyperpr~liferate?’.~~ (6) 45K/52K keratins (and to a lesser extent the 46K/54K keratins) as markers for simple epithelia. Tissue-distribution studies showed that these keratins are most frequently found in simple epithelia.’.’’ The two cow keratins that are equivalent to the 45K and 52K human keratins have been shown to form a complex that is stable even in the presence of high concentrations of urea.‘2’80 (7) The 40K keratin is present as a major cytoskeletal component only in simple epithelia, although it can be readily detected in almost all epithelia except adult epidermis.1,2,32,36,73
A MODEL FIGURE 12 is a schematic diagram summarizing the concepts of keratin subfamilies and keratin pairs.” In this diagram, keratins of both subfamilies are arranged according to an identical molecular weight scale (note the 5K bar), except that the scale for the basic subfamily is shifted upwards by 8,OOO. The 51K/59K and 55K/64K keratin pairs are placed as branches to indicate that they, like the 56.5K/65-67K keratins, represent markers for advanced states of stratified epithelial differentiation (see rules 1, 2, and 3). This arrangement provides a striking demonstration that the relative size distribution of the keratins in the two subfamilies is highly conserved, and that keratins with identical “size ranks” in the two subfamilies form a pair, with both members following similar rules for expression. This scheme also emphasizes that (1) the larger keratins of both subfamilies are characteristic of stratified epithelia (rules 1 to 5) whereas smaller keratins are expressed mainly by simple epithelia (rules 6 and 7), and
TUNG et 01.: RULES OF KERATIN EXPRESSION
325
Skin Skin Cornea
other s.e.
\
TF
other s.e.
Keratin subfamilies FIGURE 12. A unifying model of keratin expression. In this diagram, the acidic and basic keratins are arranged vertically according to their molecular weights (see the 5K scale at lower left corner). To align the two subfamilies, the MW scale of the basic keratins is 8K lower than that used for the acidic keratins. Keratins above the horizontal line are mainly expressed by keratinocytes of stratified squamous epithelia, whereas those below the line are mainly expressed by simple epithelia. The top three pairs of keratins are markers for skin, cornea, and esophageal “types” of epithelial differentiation. S.e. denotes stratified epithelia. The expression of these top three differentiation-specific keratin pairs is frequently diminished when the epithelia become “de-differentiated” as a result of disease or growing in a tissue culture environment. This diagram , ~only ~ difference is that, based on the SDS gel shown in FIGURE is modified from Sun et ~ l . the 1, the molecular weight of the acidic “other s.e.” member was changed from 54‘K to 51K. (From Cooper et aL2’ With permission from Laboratory Investigation.)
326
ANNALS NEW YORK ACADEMY OF SCIENCES
(2) a common set of keratins (box in FIGURE 12) is expressed by stratified epithelia in culture and in some diseases. The implications of this model on keratin structure and evolution have been discussed elsewhere.”
CONCLUDING REMARKS AND PERSPECTIVE In this paper, we have reviewed our current knowledge concerning the classification and expression of specific keratin molecules. Our results indicate that all human epithelial keratins can be divided into two subfamilies, and that there exist specific “keratin pairs” as defined by co-expression. These data have allowed us to construct a model (FIGURE12 and TABLE1) that defines and emphasizes the concepts of keratin subfamilies and keratin pairs.22This model also raises several important questions. (1) What are the detailed structural relationships among different keratins within a subfamily? The answer to this question will have to come from cDNA or protein sequence analysis and should shed light on the molecular mechanisms by which the two keratin subfamilies may have evolved. ( 2 ) What is the structural significance of the 8,OOO dalton size difference between the acidic and basic keratins of each keratin pair? Two extra pieces of sequence, totaling approximately 8,000 daltons, have been localized by Steinert and co-workers to the two ends of the central alpha-helical domain of a basic keratin?’ These two pieces are missing in the corresponding acidic keratin, thus providing a possible explanation for the 8,OOO dalton size difference. The structural significance of these pieces, however, remains to be elucidated. (3) How do the acidic and basic keratins interact with each other to form filaments? Even before the existence of two keratin subfamilies became known, early in vitro reconstitution data suggested that at least two keratins are required for tonofilament f~rmation.”.’~ More recently, Franke et al. have shown that certain acidic and basic keratins can form specific complexes in the presence of high concentrations of urea.72s8o We have also found recently that the 50K acidic and 58K basic keratins of normal human epidermis can interact specifically in 8.5 M urea, forming a high molecular weight aggregate that can be isolated by gel filtration (R.Eichner et a/., unpublished observation). These results are consistent with the idea that keratins of both acidic and basic subfamilies may be required for filament formation. Whether such a requirement applies at the level of a 2 nm protofilament, 4.5 nm protofibril, or 10 nm filament,74will have to be determined. (4) At what stages of epithelial maturation are the differentiation-related keratins expressed? In stratified epithelia, the basal cells are relatively undifferentiated, whereas the suprabasal cells are in advanced stages of maturation and take on the unique properties of the individual tissues. In this regard, it is interesting that the 56.5K and 65 -67K markers for keratinization or “skin type” differentiation have actually been localized to the suprabasal layers of the epidermis (FIGURE 3)?0,3542 Whether the other two pairs, i.e., 55K/64K and 51K/59Kkeratins, are also expressed suprabasally in corneal and esophageal epithelia, respectively, remains unknown. Similarly, the 48K/ 56K keratins have not yet been localized in hyperproliferative epidermis or cultured human epidermal colonies. Since on a tissue level, the expression of these two keratins (48 /56K) appears to be inversely proportional to that of the suprabasally located keratinization markers (FIGURE 7);’ we postulate that in individual cells the expression of these two types of keratin markers (56.5K/65-67K versus 48K/56K keratins) may be mutually exclusive. This possibility will have to be tested by immunolocalization.
TUNG et al.: RULES O F KERATIN EXPRESSION
327
(5) Finally, and most important, what are the detailed, specific functions of individual keratins or keratin pairs? The fact that each keratin molecule can be associated with specific morphological and/or physiological features of various epithelia strongly implies that different keratins must play unique functional roles during in vivo epithelial differentiation. Thus, significant headway has been made in addressing our original inquiry as to why such a large number of keratin species may have evolved. To further define these roles in detailed biochemical and structural terms now becomes the principal challenge, not only for “keratinologists,” but also for investigators interested in the mechanisms of normal and abnormal epithelial differentiation.
REFERENCES B. GEIGER& R. KREPLER.1982. Cell 31: 1. MOLL,R., W. W. FRANKE,D. L. SCHILLER, 11-24. N. E. BINDER,M. A. BECKETT,J. H. SINARD,C. T. GRIFFITHS 2. Wu, Y.J., L. M. PARKER, & J. G. RHEINWALD. 1982. Cell 31: 693-703. 3. SUN,T.-T. & H. GREEN.1978. Cell 1 4 468-476. W. W., E. SCHMID,M. OSBORN & K. WEBER.1978. Proc. Natl. Acad. Sci. USA 4. FRANKE, 7 5 5034-5038. 5. SUN,T.-T., C. SHIH& H. GREEN.1979. Proc. Natl. Acad. Sci. USA 7 6 2813-2817. 6. FRANKE, W. W., B. APPELHANS, E. SCHMID, C. FREUDENSTEIN, M. OSBORN & K. WEBER. 1979. Differentiation 15: 7-25. 7. DORAN,T. I., A. VIDRICH& T.-T. SUN. 1980. Cell 22: 17-25. 8. FUCHS,E. & H. GREEN.1980. Cell 19 1033-1042. 9. GIPSON,I. K. & R. A. ANDERSON.1980. Exp. Cell Res. 128 395-406. 10. MILSTONE,L. M. & J. MCGUIRE.1981. J. Cell Biol. 8 8 312-316. 11. FRANKE,W. W., H. DENK,R. KAIT & E. SCHMID.1981. Exp. Cell Res. 131: 299-318. 12. FRANKE,W. W., D. L. SCHILLER, R. MOLL,S. WINTER,E. SCHMID,I. ENGELBRECHT, H. DENK,R. KREPLER& E. PLATZER.1981. J. Molec. Biol. 153 933-959. 13. STEINERT, P. M., W. W. IDLER& S. B. ZIMMERMAN. 1976. J. Molec. Biol. 108: 547-567. 14. LEE, L. D. & H. P. BADEN.1976. Nature 264: 377-379. 15. HANUKOGLU, I. & E. FUCHS. 1982. Cell 31: 243-252. 16. GEISLER,N. & K. WEBER.1982. EMBO J. 1: 1649-1656. 17. STEINERT, P. M., R. H. RICE,D. R. ROOP, L. T. BENES& A. C. STEVEN.1983. Nature 302 794-800. 18. WEBER,K. & N. GEISLER.1984. In The Cancer Cell. A Levine, W. Topp, G. Vande Woude & J. D. Watson, Eds.: 153-159. Cold Spring Harbor Laboratories. Cold Spring Harbor, N.Y. E. 1982. Ann. Rev. Biochem. 51: 219-250. 19. LAZARIDES, J., R. EICHNER,W. G. NELSON& T.-T. SUN.1982. J. Cell Biol. 20. WOODCOCK-MITCHELL, 95: 580-588.
21. SUN,T.-T. & H. GREEN.1978. J. Biol. Chem. 293 2053-2060. W. G. NELSON& R. A. WEISS. 22. SUN, T.-T., R. EICHNER,D. COOPER,A. SCHERMER, 1984. In The Cancer Cell. A Levine, W. Topp, G. Vande Woude & J. D. Watson, Eds.: 169-176. Cold Spring Harbor Laboratories. Cold Spring Harbor, N.Y. 23. FUCHS,E. & H. GREEN.1979. Cell 17: 573-582. & E. V. FUCHS. 1983. Molec. Cell Biol. 3 495-502. 24. KIM, K. H., J. RHEINWALD & W. W. FRANKE. 1983. EMBO J. 2: 1387-1392. 25. MAGIN,T. M., J. L. JORCANO 1980. J. Invest. Dennatol. 74: 382-388. 26. GIBBS,P. E. M. & 1. M. FREEDBERG. & T.-T. SUN. 1985. Lab. Invest. 5 2 243-256. 27. COOPER,D., A. SCHERMER & P. H. O’FARRELL.1977. Cell 12: 1133-1142. 28. OFARRELL,P. Z., H. M. GOODMAN & J. GORDON. 1979. Proc. Natl. Acad. Sci. USA 7 6 4350-4354. 29. TOWBIN,H., T. STAEHELIN 1981. Cell 27: 75-84. 30 FUCHS,E., S. M. COPPOCK,H. GREEN& D. W. CLEVELAND. D. L., W. W. FRANKE & B. GEIGER.1982. EMBO J. 1: 761-769. 31. SCHILLER,
328
ANNALS NEW YORK ACADEMY OF SCIENCES
W. G. NELSON,A. J.-W. HUANG,J. WOODCOCK32. TSENG,S. C. G., M. J. JARVINEN, MITCHELL& T.-T. SUN. 1982. Cell 3 0 361-372. I. & E. FUCHS. 1983. Cell 33: 915-924. 33. HANUKOGLU, W. G. & L. M. DOWLING.1971. Appl. Polm. Symp. 18: 1-20. 34 CREWTHER, & R. KREPLER.1982. J. Cell Biol. 95: 35. MOLL, R., W. W. FRANKE,B. VOLC-PLATZER 285-295. 1981. Cell 2 5 627-635. 36. Wu, Y.-J. & J. G. RHEINWALD. 37. FUCHS,E. & H.GREEN.1978. Cell 15 887-897. 38. VIAC,J., J. M. STAQUET,J. THIVOLET& C. GOUJON.1980. Arch. Dermatol. Res. 267: 179-188. W. G. NELSON,A. VIDRICH & J. WOODCOCK-MITCHELL. 1983. 39. SUN,T.-T., R. EICHNER, In Normal and Abnormal Epidermal Differentiation. M. Seiji & I. A. Bernstein, Eds.: 277-291. University of Tokyo Press. Tokyo. D. & C. J. SKERROW. 1983. Exp. Cell Res. 143: 27-35. 40. SKERROW, J., M. KINJO,G. FURSTENBERGER & H. WINTER.1984. Cell 37: 159-170. 41. SCHWEIZER, D., A. BOHNERT,E. HERZMANN, P. BOWDEN,P. BOUKAMP & N. E. 42. BREITKREUTZ, FUSENIG. 1984. Differentiation. (In press.) J. G. & H. GREEN.1975. Cell 6 331-343. 43. RHEINWALD, 44. LAVKER,R. M. & T.-T. SUN. 1983. J. Invest. Dermatol. 81: 121s-127s. R., P. BONITZ& T.-T. SUN. 1984. J. Cell Biol. 98: 1388-1396. 45. EICHNER, 46. FUCHS,E. & H. GREEN.1981. Cell 25: 617-625. 47. WEISS,R.A,, R. EICHNER& T.-T. SUN. 1984. J. Cell Biol. 98: 1397-1406. 1984. Br. J. Dermatol. (Suppl. 27) 111:27-37. 48. MCGUIRE,J., M. OSBER& L. LIGHTFOOT. 49. CELIS,J. E., S. J. FEY,P. M. LARSEN & A. CELIS.1984. Proc. Natl. Acad. Sci. USA 81: 3128-3132. 50. SUN,T.-T. & H. GREEN.1977. Nature 269: 489-493. P. HOLLWEG-MAJERT, G. DALLENBACH-HELL51. MOLL,R., R. LEVY,B. CZERNOBILSKY, WEG & W. W. FRANKE.1983. Lab. Invest. 49: 599-610. 52. NELSON,W. G. & T.-T. SUN. 1983. J. Cell Biol. 97: 244-251. H. SANTANA & T.-T. SUN. 1984. Cancer Res. 44. 53. NELSON,W. G., H. BATTIFORA, 1600-1603. 54. BANKS-SCHLEGEL, S. P. & H. GREEN.1980. Transplantation 2 9 308-313. W. W., D. MAYER,E. SCHMID,H. DENK& E. BORENFREUND. 55. FRANKE, 1981. Exp. Cell Res. 134 345-365. 56. DENK,H., R. KREPLER,E. LACKINGER, U. ARTLIEB& W. W. FRANKE. 1982. Lab. Invest. 46: 584-596. A.J.-W. HUANG& T.-T. SUN. 1984. J. Cell Biol. 99: 57. TSENG,S. C. G., D. HATCHELL, 2279. 58. DALE,B. A., I. B. STERN,M. RABIN& L.-Y.HUANG.1976. J. Invest. Dermatol. 6 6 230-235. 59. BANKS-SCHLEGEL, S. P. 1982. J. Cell Biol. 9 3 551-559. 60. MOLL,R., I. MOLL& W. WIEST. 1983. Differentiation 23: 170-178. M., K. FUKUYAMA, W. L.EPSTEIN& K. A. FISHER. 1978. J. Invest. Dermatol. 61. THALER, 75: 156-158. 62. BADEN,H. P., N. MCGILVRAY, C. K. CHENG,L. D. LEE & J. J. KUBILUS.1978. J. Invest. Dermatol. 7 0 294-297. 63. SKERROW, D. & I. HUNTER.1978. Biochem. Biophys. Acta 537: 474-484. 64. SCHERMER, A. & T.-T. SUN.Submitted for publication. 65. BANKS-SCHLEGEL, S. P. & C. C. HARRIS.1983. Exp. Cell Res. 146 271-280. 66. RICE,R. H. & H. GREEN.1979. Cell 18: 681-694. 67. GREEN,H.,E. FUCHS & F. WATT. 1981. Cold Spring Harbor Symp. Quant. Biol. 4 6 293-301. 68. HRONIS,T. S., M. L. STEINBERG, V. DEFENDI& T.-T. SUN. 1984. Cancer Res. 44: 5797. 69. LANE,E. B. 1982. J. Cell biol. 9 2 665-673. J., P. PURKIS,E. B. LANE,I. A. MCKAY& S . CHANG.1982. 70. TAYLOR-PAPADIMITRIOU, Cell Differ. 11: 169-180. 11. MOLL,R., R. KREPLER & W. W. FRANKE. 1983. Differentiation 23: 256-269. 7 .
TUNG et al.: RULES OF KERATIN EXPRESSION
329
M. HATZFELD & S. WINTER.1983. Proc. Natl. Acad. 72. FRANKE,W. W., D. L. SCHILLER, Sci. USA 80: 7113-7117. 73. FRANKE, W. W., S. WINTER,C. GRUND,E. SCHMID,D. L. SCHILLER & E. D. JARASCH. 1981. J. Cell Biol. 90: 116-127. 74. AEBI, U., W. E. FOWLER,P. REW & T.-T. SUN. 1983. J. Cell Biol. 97: 1131-1143. 75. LAEMMLI,U. K. 1970. Nature 227: 680-685. I. A. 1979. In Immunocytochemistry. 122-129. John Wiley & Sons. New 76. STERNBERGER, York. M. E., V. B. CULBERTSON & I. M. FREEDBERG. 1980. J. Invest. Dermatol. 77. GILMARTIN, 75: 211-216. A. G. 1964. Nature 201: 1130-1131. 78. MATOLTSY, 79. DALE,B. et al. In preparation 80. QUINLAN,R. A,, D. L. SCHILLER,M. HATZFELD,T. ACHTSTATTER,R. MOLL, J. L. JORCANO, T. M. MAGIN& W. W. FRANKE. 1985. Ann. N.Y. Acad. Sci. (This volume.) 81. STEINERT,P. M., W. W. IDLER,X.-M. ZHON, L. D. JOHNSON,D. A. D. PARRY,A. C. STEVEN& D. R. ROOP. 1985. Ann. N.Y. Acad. Sci. (This volume.)
INTRODUCTION Significant progress has been made during the past decade with regard to the biochemistry and immunology of keratin filaments. The results indicate that keratin represents a family of more than 17 water-insoluble, cytoskeletal proteins,"2 that keratins are restricted to epithelia and their derivatives;d that different subsets of 2- 10 keratins are expressed in different epithelia,1~2~7-12 that at least two keratin species are required for filament a~sembly,'".'~ and that keratin molecules consist of a central helical domain with two nonhelical end^.^^-^^ Despite these advances, however, it remains a puzzle as to why such a large number of keratin species have evolved to form tonofilaments, while other morphologically similar, intermediate-sized filaments are characterized by a much simpler protein composition with only one to three subunits (for reviews, see Lazaridesl' and other chapters in this volume). To address this fundamental question, we have prepared several monoclonal antikeratin antibodies2' and used them as a tool for studying keratin expression. In this 'This work was supported by grants from the National Institutes of Health (EY 04722 and AM 3451 1). T.-T. Sun is the recipient of a Monique Weill-Caulier Career Scientist Award. 307
308
ANNALS NEW YORK ACADEMY OF SCIENCES
paper, we will review our data, which indicate that all human epithelial keratins can be divided into two subfamilies according to their charges and their immunoreactivities with AEl and AE3 monoclonal antibodies. We will also discuss how the expression of specific keratins in the epidermis and several nonepidermal epithelia can vary as a function of environmental modulation and disease. Finally, we will present new data on keratins expressed in rabbit epidermis and other epithelia during embryonic development. Taken together, the data suggest the existence of a number of “rules” governing the expression of specific keratin molecules and help to formulate the concept of “keratin pairs” as defined by co-expression.
TWO KERATIN SUBFAMILIES AS DEFINED BY MONOCLONAL ANTIBODIES
Recent results from two-dimensional gel analyses have established the existence of 17- 19 human keratin species132*12s2%22. , many of them have been shown to be distinct translational prod~cts.Z’~*~ To study these keratins, we have prepared water-insoluble cytoskeletal proteins from five representative human tissues (abdominal epidermis, corneal epithelium, esophageal epithelium, cultured epidermal cells, and mesothelial cells) and analyzed the proteins by immunoblotting using our AEl and AE3 monoclonal anti-keratin antibodies (FIGUREl).22s27-29 Consistent with cDNA data from Fuchs et al.,24,30 peptide mapping data of Franke et aL,I,31and our own earlier immunoblotting the results indicate that all known human epithelial keratins can be divided into two subfamilies based on their charges and immunoreactivities with AEl and AE3 monoclonal antibodies (FIGURE2)?’,” One subfamily consists of the 65-67K (no. 1 and 2, according to Moll et a l l ) , 64K (no. 3), 59K (no. 4),58K (no. 5), 56K (no. 6), 54K (no. 7),and 52K (no. 8) keratins. These keratins have isoelectric points (PIS) greater than 6.0and share an antigenic determinant as defined by our AE3 antibody (FIGURES1 and 2).Franke and co-workers have shown that all of these proteins are structurally related according to peptide mapping and, noting their molecular weights and charge, referred to them as a subfamily of “relatively large and basic cytokeratins.”” Hybrid-selection data from Fuchs and co-workers have demonstrated that the 58K,56K,54K,and 52K keratins share sequence homologies and are related to the Type I1 wool keratins previously defined by Crewther and c ~ - w o r k e r s ? ” ~Thus ~ * ~ the ~ . ~keratins ~ described above are closely related and constitute the “basic” subfamily (Type 11). The other subfamily consists of the 56.5K (no. lo),55K (no. 12), 51K (no. 13), 50K (no. 14),50’K(no. 15), 48K (no. 16), 46K (no. 17), 45K (no. IS), and 40K (no. 19)keratins. These keratins have PI values of less than 5.7 and many of them (56.5K, 50K,50K,48K,48K,and 48K)share an AE1 determinant (FIGURES1 and 2). Data on cDNA from Fuchs et al. have shown that the 50K,46K,45K,and 40K keratins share sequence homologies and are related to Type I wool keratin^?^.^'.^^ Franke and colleagues have established by peptide mapping that the 50K,48K,and 46K keratins are structurally These keratins thus form the “acidic” subfamily (Type I), which is structurally and immunologically distinct from the basic subfamily.
TUNG et al.: RULES OF KERATIN EXPRESSION
309
“KERATIN PAIRS” AS DEFINED BY CO-EXPRESSION In order to gain insight into the possible functional meaning of these heterogeneous keratin species, we have performed a series of experiments to determine whether the expression of different keratin molecules can be correlated with specific morphological or physiological features of epithelial differentiation. Our data suggest that there exist a number of simple rules regarding the expression of individual keratin species (TABLE 1). Moreover, a close examination of these rules reveals a strikingly parallel relationship between the keratins of the two subfamilies. Thus, with the exception of the 40K keratin (of the acidic subfamily), which is wides~read,’.’~*~~ each acidic keratin is coexpressed with a particular basic keratin, forming a “keratin pair” (TABLE1). Within each pair, the two keratins have identical “size-ranks” in their respective subfamilies
HGURE 1. Immunoblot analyses of human epithelial keratins using AEl and AE3 monoclonal antibodies. (a-c) One-dimensional SDS gels (Laemmli, 12.5% acrylamide)” stained by (a) Fast green, (b) AE3, and (c) AEI. Antibody staining was performed by the peroxidase-antiperoxidase (PAP) te~hnique.’~ Samples are (1) in vivo abdominal epidermis, (2) corneal epithelium, (3) esophageal epithelium, (4) cultured foreskin epidermal cells, and ( 5 ) cultured mesothelial cells (courtesy of James Rheinwald). The molecular weights (MWs) of the four major epidermal keratins are marked to the left. Note in (b) and (c) that AE3 and AEl recognize two mutually exclusive subfamilies of keratin^.^^.'^ (d-f) Two-dimensional nonequilibrium gel analysis 28 of a mixture of keratins from the five tissues listed above. The proteins were stained by (d) Fast green, (e) AE3, and (f) AEl antibodies. All keratins are identified by MWs; for their corresponding catalogue numbers’, see FIGURE 2 and TABLE1. Note that all AE3-reactive keratins are relatively basic (on the left of the gel), while all AEl-reactive ones are acidic. B and P denote bovine serum albumin and 3-phosphoglycerate kinase’; V denotes vimentin. (From Cooper et al.2’ With permission from Laboratory Investigation.)
PI
I
acidic
\
‘45 (18) -‘40 (19)
(closed circle). This is a schematic tracing of an actual two-dimensional gel (as shown in FIGURE Id) showing that human epithelial keratins can be divided into acidic (A or Type I) and basic (B or Type 11) subfamilies. The charge heterogeneity of individual keratins is at least in part due to phosphorylation.’2.21.77 The positions of bovine serum albumin (BSA), vimentin (V), and 3-phosphoglycerate kinase (PGK) are shown for reference.’.I2 For reasons mentioned elsewhere?’ keratins no. 9 (64K) and no. 11 (56K) are not included in this scheme. (From Cooper et al.” With permission from Laboratory Investigation.)
FIGURE 2. Classification of human epithelial keratins by their charge and their immunoreactivities with AE3 (hatched circle) and AEl antibodies
basic
+o-+ PGK
c)o BSA
w 0 c
AE 1
AEl
5.1
5.3/4.9
5.1 5.1 5.7 5.2
51 (13)
50/50’ (14/ 15) 48 (16) 46 (17) 45 (18) 40 (19) 7.8 6.0 6.1
7.4
58 (5) 56 (6) 54 (7) 52 (8)
7.3
6-8 7.5
PI
59 (4)
65-67 (1,2) 64 (3)
MW X lo-’
Basic (Type 11)
AE3 AE3 AE3
AE3
AE3
AE3 AE3
Antibody
8 8 7
8
8
8 9
AMW x lo-’ @-A)
Hyperproliferative keratinocytes Simple and some stratified epithelia Simple epithelia
“Skin type” differentiation “Corneal-epithelial type” differentiation “Esophageal-epithelial type” differentiation Keratinocytes
Markers
Keratins are arranged according to their relative sizes under the acidic and basic subfamilies. Numbers in parentheses are keratin nomenclatures according to Moll er nl.’ Note the striking parallelism between the two subfamilies with respect to their relative size and tissue distribution.
AE 1
AE 1
5.3 4.9
56.5 (10) 55 (12)
Antibody
Keratin Subfamily
PI
Acidic (Type I)
Classification and Expression of Human Epithelial Keratins
MW x 10-3
TABLE 1.
312
ANNALS NEW YORK ACADEMY OF SCIENCES
and follow similar rules for expression (TABLE1). In the next three sections we will briefly review the evidence from which these rules are derived.
KERATIN EXPRESSION IN HUMAN EPIDERMIS Immunolocalitation Studies Normal human epidermis is a keratinized” epithelium that expresses four major keratins (50K, 56.5K, 58K, and 65-67K).1s20.37 Using monoclonal antibodies, we have localized these four keratins in different epidermal layers by a combination of immunohistochemical and biochemical techniques?’ The results indicate that the 50K and 58K keratins are present in all cell layers including the relatively undifferentiated basal layer, whereas the 56.5K and 65-67K keratins are associated only with the more differentiated cells above the basal layer (FIGURES 3 and 7)?0.3842In other experiments surveying the tissue distribution of keratins, we found that the “basal type” 50K and 58K keratins are present in various quantities in all stratified squamous epithelia, whereas the “suprabasal type” 56.5K and 65-67K keratins are mainly expressed by the keratinized e p i d e r m i ~ . These ~ . ~ ~ findings ~ ~ ~ support the notion that the 50K and 58K keratins (or 50K/58K pair) may be regarded as molecular markers for keratinocytes (the major cell type of all stratified squamous epithelia), and that the 56.5K and 65-67K keratins (56.5K/65-67K pair) may be regarded as markers for keratinization or “skin type” differentiation (TABLE1).
Reversible Modulation of Keratin Expression in Cultured Human Epidermal Keratinocytes In the presence of lethally irradiated 3T3 cells, human epidermal cells form stratified squamous colonies and undergo terminal differentiati0n.4~Unlike their in vivo Morphological keratinization is defined by the formation of granular cells and eosinophilic, anucleated cornified cells.
FIGURE 3. Sequential and coordinate expression of the four major epidermal keratins accompanying morphological differentiation of normal human epidermis. (A) Morphology of the epidermis, which can be divided into four layers: basal (B), spinous (S), granular (G), and cornified (C). Note the presence of hemidesmosomes (HD), desmosomes (D), tonofilaments (F), keratohyalin granules (K), membrane-coating granules (or lamellar granules; MCG), and cornified envelope (CE). (B) A schematic summary of keratin localization data showing that basal cells (B) express mainly an acidic 50K and a basic 58K keratin, while suprabasal cells ( S , G, and C ) contain, in addition, an acidic 56.5K and a basic 65-67K keratin. In normal cornified cells (not callus), most of these keratins are either completely (e.g., the 50K component), or partially (all of the other keratins) degraded, resulting in two diffuse, ill-defined, major bands (one 55-58K, another 63-653). Keratins of the living cellular layers (B, S , and G) are not crosslinked by intermolecular disulfide bonds (K-SH) and are therefore extractable with urea or SDS alone (“prekeratin” of Matoltsy’’); whereas keratins in the nonviable cornified layer are known to be crosslinked extensively by intermolecular disulfide bonds (K-S-S-K) and are therefore extractable only after reduction.21
313
TUNG et al.: RULES OF KERATIN EXPRESSION
EPIDERMAL DIFFERENTIATION A. MORPHOLOGY
I
I I I I I I I Basal + Spinous + Granular + Cornified I
B. KERATIN EXPRESSION B
S
67 6665-
G
C 63-65 55- 58
K-SH
K-SIS-K
ANNALS NEW YORK ACADEMY OF SCIENCES
3 14
counterparts, however, these cultured keratinocytes fail to form granular layers or 4)?‘’,, Kerenucleated cornified layers, and are therefore “nonkeratinized” (FIGURE atin analysis showed that these cells continue to synthesize the 50K/58K keratin pair, a marker for keratinocytes, but cease to make the 56.5K/65-67K keratin pair (FIGURE5).21t37.45 Interestingly, these latter two “keratinization markers” are coordinately expressed when the cells are induced to keratinize, both in athymic mice’ and in culture medium deprived of certain growth factors such as vitamin A, hydro4)?’-‘‘ cortisone, and epidermal growth factor (FIGURE
Keratin Expression in Human Epidermal Diseases Although cultured human epidermal cells do not synthesize the “keratinization markers” (56.5K and 65-67K keratins), they express 56K, 48K,46K, and several
Cell culture; Vit. A, HC, EGF, CAMP CT, etc.
Normal (keratinized)
Hyperproliferated (non- or para-keratinized)
FIGURE 4. A schematic drawing illustrating the morphological changes that occur when human epidermal cells are grown in tissue culture media containing various growth-stimulating substances such as vitamin A, hydrocortisone (HC), epidermal growth factor (EGF), cyclic AMP (CAMP), cholera toxin (CT), etc. The arrows indicate that this process can be reversed by transplanting the cells to an in vivo permissive envir~nrnent,’.’~ or by growing the cells in media deprived of certain growth factors, such as vitamin A.45.”
TUNC et al.: RULES OF KERATIN EXPRESSION
315
FIGURE 5. A comparison of keratins expressed by human epidermal cells in vivo versus in culture. Keratins were isolated from in vivo normal (keratinized) epidermis (lane 1, 3, and 5 ) and cultured (nonkeratinized) epidermal colonies (lanes 2, 4, and 6), separated by SDS gel electrophoresis, transferred to nitrocellulose paper, and stained with (lanes 1 and 2) Fast green, (3 and 4) AEl antibody, and ( 5 and 6) AE3 antibody. Note that the two samples share the (acidic) 50K and (basic) 58K keratins. The (acidic) 56.5K and (basic) 65-67K keratins are unique to the keratinized epidermis, while the (acidic) 48K and (basic) 56K keratins are present only in cultured cells. (From Eichner el ~ 1 , With ~ ’ permission from Journal of Cell Biology.)
other small keratins that are undetectable in normal interfollicular epidermis (FIGURE 5).2’,3’,45 We have found recently that the 48K and 56K “culture type” keratins are expressed in a wide variety of hyperproliferative epidermal diseases including psoriasis, warts, actinic keratoses, and squamous cell carcinomas (FIGURE 6):’ Furthermore, these two keratins become readily detectable when the epidermis is stimulated to undergo hyperproliferation either by the tape-stripping technique (Weiss et al., unis ~important ~~~ to note, however, published) or by placing the skin in organ c ~ l t u r e . 8It. ~ that these two keratins clearly are not epidermis specific, because they are also expressed by cultured keratinocytes derived from various stratified squamous epithelia Since the 48K including those of the esophagus, conjunctiva, cornea, and ~agina.2’’’~’~~
316
ANNALS NEW YORK ACADEMY OF SCIENCES
TUNG el al.: RULES OF KERATIN EXPRESSION
317
and 56K proteins are not detectable in normal epidermis or in nonhyperproliferative epidermal diseases such as ichthyosis vulgaris, the results suggest that this keratin pair may serve as a useful molecular marker for hyperproliferative keratinocytes
(FIGURES 5-7).47,48
KERATIN EXPRESSION IN NONEPIDERMAL EPITHELIA Normal Epithelia Tissue-distribution data have shown that (1) 56.5Kand 65-67Kkeratins are mainly associated with keratinized tissues including the epidermis and thymic Hassall’s corp u s c l e ~ . These ~~~~ two ~ ~keratins ’ are also present in vaginal and exocervical epithelia?’ whether this reflects a partially keratinized state of the epithelia remains unknown. (2)55K and 65K keratins are corneal epithelium-~pecific.’*~*’~~~~ (3) 51K and 59K keratins are mainly found in esophageal, tongue, and other internal, nonkeratinized stratified epithelia.12’’ (4)50/50‘Kand 58K keratins are found in various quantities in all stratified squamous epithelia, but not in simple epithelia.1r2r32.52*53 (5)45K and 52K keratins are the major keratins in many simple epithelia.1*z-32 Glandular, transitional, and pseudostratified columnar epithelia usually express a complex mixture of “keratinocyte type” as well as “simple epithelial-type” keratin^.^.^.^' These results are summarized schematically in FIGURE 8.
CuItu red Cells
In general, stratified squamous epithelial cells undergo major morphological and biochemical (keratin) changes when they are placed under culture conditions designed for maximum cellular growth. Thus, cultured human epidermal, corneal, and esophageal epithelial cells do not express their differentiation-specific keratins, i.e., the 56.5K/65-67K, 55/64K,and 51K/59K keratin pairs, respectively. Instead, all three cultured epithelia express the 50K/58K keratins, the 48K/56K hyperproliferation markers, a 46K keratin, and a few other small keratins normally associated with As we have shown earlier, however, these cultured cells (with simple epithelia.2~7.2’*37*so
FIGURE 6. Keratin expression in human epidermal diseases. Keratins were isolated from (1) normal epidermis, (2) ichthyosis vulgaris, (3) atopic dermatitis, (4) and ( 5 ) psoriasis, ( 6 ) keratoacanthoma, (7) actinic keratosis, (8) wart, (9) verrucous carcinoma, (10) and (1 1) squamous cell carcinomas, (12) basal cell carcinoma, and (13) cultured human epidermal cells. The proteins were separated by 12.5% SDS gel electrophoresis, transferred to nitrocellulose paper, and stained with (a) Fast green (FG), (b) AEl antibody, and (c) AE3 antibody. Note the striking similarity between the overall keratin pattern of some diseases (e.g., lanes 7-12) and that of cultured epidermal cells (lane 13). Also note the detection of an AEl-reactive (acidic) 48K keratin and an AE3-reactive (basic) 56K keratin in all hyperproliferative epidermal diseases, and their absence in normal epidermis (lane 1) or the nonhyperproliferative disease ichthyosis vulgaris (lane 2). With permission from Journal of Cell Biology.) (From Weiss ef
ANNALS NEW YORK ACADEMY OF SCIENCES
318
Normal Keratinized
58 56.5 56 52 2 v 50
____t
Hyper proliferative Non-keratinized
t
a c
4R
.-
c
0 L-
a 40 4s
Y
65-67 58 56.5 56 52
50
I
I
A
48 6
4
B
r
C
D
FIGURE 7. A schematic diagram illustrating a continuous spectrum of keratin expression by human epidermal cells. Bars represent specific keratins (identified by MW X Position A denotes the keratin pattern of normal in vivo (keratinized) epidermis; B denotes that seen in a variety of epidermal diseases with different degrees of hyperproliferation and/or altered differentiation; C denotes keratins of basal cell carcinomas; and D represents keratin pattern of human epidermal cells cultured in media designed for maximum cellular growth. Note that the 50K/ 58K keratin pair (black bars) is present throughout the spectrum, the 56.5K/65-67Kkeratin pair (dotted) is expressed mainly during keratinization, the 48K/56K keratin pair (hatched) is associated with “hyperproliferation.” The above three keratin pairs are keratinocyte specific, whereas the remaining keratins (open bars) are found as major keratins mainly in simple epithelia. This diagram emphasizes (1) the continuous spectrum of keratin expression, (2) the tremendous overlap of keratin pattern in various diseases, and (3) on a tissue level, the reciprocal expression of the “keratinization” and “hyperproliferation” markers. (From Weiss et aL4’ With permission from Journal of Cell Biology.) the possible exception of corneal cells) can rapidly regain their normal in vivo morphology and keratin patterns once the cells are provided with a permissive in vivo en~ironment.~.“’~~ In contrast, simple epithelial cells rarely undergo major changes in their keratin composition, either in culture or when they become tran~formed.‘**.~~.’~
Keratin Expression in Nonepidermal Epithelia during Vitamin A Dejciency Although the 56.5K and 65-67K keratins are normally absent from nonkeratinized esophageal, conjunctival, and corneal epithelia, this keratin pair becomes readily detectable when the tissues become keratinized as a result of vitamin A deficiency (FIGURE9).“*” This observation indicates that the 56.5K/65-67K keratins are not
TUNG et ab: RULES OF KERATIN EXPRESSION
319
epidermis specific, and therefore may be regarded as molecular markers for the program of keratinization or “skin type” differentiati~n?”~’
SEQUENTIAL EXPRESSION OF KERATINS DURING EMBRYONIC DEVELOPMENT The embryonic development of epidermis provides a unique opportunity for studying keratin expression. Morphologically, the early epidermis is a simple epithelium that later becomes stratified, and finally keratinized. Immunoblot analysis of keratins isolated from rabbit epidermis at different stages of embryonic development confirmed earlier r e p ~ r t s ~ that * - ~ showed that keratins undergo sequential changes during development. FIGURE1O(f) shows the keratin changes in rabbit epidermis as detected by immunoblotting using our AEl antibody. In this tissue, AE1 recognizes mainly three keratins (MK, 50K, and 56.5K)that appear sequentially in that order during embryonic development (FIGURE10, f). The 40K keratin is present throughout the developmental period that we have studied (12-day-old to term), but disappears from the skin after A 38K band appears in the 29-day epidermal sample (FIGURE
Stratif ied Simple
Esophagus, etc.
Cornea
Skin
-
-
6465-67
1
-
52
-1
-
-
45 -
-59 58
1111111111111111--
. . . 1 1 1 1 1 -
56.5 55
I
1111111
5051 -
320
ANNALS NEW YORK ACADEMY OF SCIENCES
TUNG et al.: RULES OF KERATIN EXPRESSION
321
10, a and f); presumably this represents a degradative product of the 40K keratin. The 50K (and a variable amount of 48K) and 56.5K keratins are first detected in significant quantities in the skin on days 22 and 23, respectively, around the time when the epithelium becomes stratified and keratinized. The 65-67K and 56.5K keratins appear concurrently, as can be detected by AE3 antibody (data not shown), or, as shown in FIGURE 11, by AE2 antibody, which specifically recognizes these two “keratinization m a r k e ~ s . ” ~These ~ ~ ’ ~data, in conjunction with the earlier data by Da1e,58.79 Banks-S~hlegel,~~ Mol,@’ and their co-workers, suggest that during embryonic development the appearance of specific keratin markers accompanies or slightly precedes morphological differentiation. Developmental studies of nonepidermal epithelia (FIGURE10, a-e) show that the AEl-reactive 40K keratin is expressed in simple epithelia (e.g., small intestinal epithelium) throughout development (FIGURE10, b). In corneal (FIGURE10, c), con10, d), and esophageal (FIGURE10, e) epithelia, the 50K keratin junctival (FIGURE first becomes detectable in 22-23-day-old embryos, shortly before these epithelia become stratified. Finally, consistent with the fact that these epithelia are not keratinized, they do not express the 56.5K (or 65-67K) keratin at any developmental stage.
RULES OF KERATIN EXPRESSION The results described above suggest that there exist a number of rules governing keratin expression. These rules and their supporting evidence are summarized below. (1) 56.5/65-67K keratins as markers of keratinization or “skin type” differentiation. Evidence in support of this notion includes that (a) these keratins are expressed 3)2023842; (b) they are during advanced stages of epidermal keratinization (FIGURE 4 and absent when the epidermis becomes nonkeratinized in cell culture (FIGURES 5)”,” and in diseases (FIGURE 6),47,48,6143 but reappear coordinately when the cells are induced to keratinize (FIGURE7)7.45.46; (c) they are also made by corneal, conjunctival, and esophageal epithelia when the tissues undergo partial keratinization as a result of vitamin A deficiency (FIGURES 8 and 9).46,57 It should be noted that in embryonic epidermis these two keratins actually appear before morphological keratinization becomes apparent, suggesting that biochemical keratinization precedes morphological change^.""^ (2) 55K/64K keratins as markers for “corneal type” epithelial differentiation. Tissue-distribution including those obtained with several new hybridoma antibodies monospecific for the 64K component,64indicate that these two keratins may be regarded as markers of normal corneal epithelial differentiation (FIGURE 8). Similar to the 56.5K/65-67K keratins of the epidermis, the corneal 55K/64K keratin pair is diminished in cultured corneal epithelial cells, suggesting that its expression is also differentiation de~endent.’.~’.~~ FIGURE 9. The expression of the 56.5K and 65-67K keratins in rabbit corneal, conjunctival, and esophageal epithelia during vitamin A deficiency. Keratins were isolated from various rabbit epithelia of normal (A+) and vitamin A-deficient (A-) rabbits, and analyzed by immunoblotting using AE1 and AE3 antibodies. Note the synthesis of the AEl-reactive 56.5K and AE3-reactive 65-67K keratins (arrows) in all three nonepidermal epithelia during vitamin A deficiency-induced keratinization. The 66K (equivalent to the 64K human keratin) band in normal rabbit cornea is biochemically and immunologically distinguishable from the 65-67K keratinization marker.64 (From Tseng et al. With permission from Journal of Cell Biology)
’’
as detected by immunoblotting using AE1 antibody. (a) 12-day whole embryo, (b) intestine, (c) corneal epithelium, (d) conjunctival epithelium, (e) esophageal epithelium, and (f) skin epidermis. Numbers denote days. Nb and Ad denote newborn and adult, respectively. Note the appearance of the 40K keratin as the only AEl-positive component in early embryos, the expression of the 50K keratin in all stratified epithelia around day 22-23 when stratification occurs, and the appearance of the 56.5K keratin in epidermis on day 23, shortly before morphological keratinization. Also note in (0 the abrupt disappearance of the 40K keratin (upper arrow) from the epidermis at birth (lower arrow denotes a “38K band,” which is presumably a degradative product of the 40K species).
FIGURE 10. The sequential expression of the 40K, 50K,and 56.5K keratins in rabbit epidermis and several other epithelia during embryonic development,
v1
3R
2 v1 aR
5
0
*
*
!a R
0
cc
v1
F
z
5
w
W
TUNG et al.: RULES OF KERATIN EXPRESSION
323
(3) 51K/59K keratins as markers for stratified squamous epithelia of internal organs including esophagus and tongue (“esophageal type” differentiation)?’ This 8).1s22These two conclusion is based primarily on tissue-distribution data (FIGURE keratins are also differentiation dependent since they are diminished in cultured esophageal epithelial cells as well as in “de-differentiated” esophageal or tongue squamous cell carcinomas.1’2z’65 (4) 50K/58K keratins as keratinocyte markers. These two keratins are present in reasonably large quantities in almost all stratified epithelia except in corneal epi-
FIGURE 11. The coordinate expression of the 56.5K and 65-67K keratin in epidermis during (rabbit) embryonic development. Keratins were extracted from rabbit embryonic epidermis at different stages of development and from postnatal animals and analyzed by immunoblotting using AE2 monoclonal antibody, which specifically recognizes the 56.5K and 65-67K “keratinization marker^."^^^'^ Note the coordinate expression of these two keratins, first detected around day 23 (cf. FIGURE10).
324
ANNALS NEW YORK ACADEMY OF SCIENCES
thelium where they are present only in minute quantities (FIGURE 8).’,12*22*32 Interestingly, these two keratins are expressed in 10-week-oldembryonic human skin, when the epidermis is still largely a simple epithelium.60This result is consistent with our earlier observation that cultured postnatal human epidermal keratinocytes continue to express the 50K/58K keratins even when the cells form a monolayer in low calcium medium, suggesting that these keratins are “permanently” expressed by epidermal cells regardless of their state of cellular stratification?2 Taken together, these results suggest that 50K/58K keratins may be regarded as markers for keratinocytes or, in the embryo, cells that have committed to become keratinocytes. The fact that most stratified epithelia continue to express the 50K/58K keratins in culture and in neoplasms makes these two keratins valuable markers for identifying cells of stratified epithelial ~rigin.’~.’~ However, in rare situations, stratified epithelia can lose their 50K/ 58K keratin markers. For example, some cultured SV40-transformed keratinocytes lose their 50K/ 58K keratins, involucrin (another keratinocyte marker),66,67as well as their ability to stratify.6’ In fact, these cells appear to have acquired the properties of a simple epithelium because they express as their major cytoskeletal components the 40K, 45K, and 52K keratins, which are characteristic of simple epithelia.’.2.49.68-70 Thus, although the presence of the 50K/58K keratins establishes the stratified epithelial nature of a cell, their absence (or the presence of simple epithelial markers) does not rule out the possibility that the tumor is originally derived from a stratified epitheli~m.~’.~’ (5) 48K/56K keratins as markers for “hyperproliferative/de-differentiated” keratinocytes. These two keratins are present in (a) a variety of hyperproliferative epi6): (b) cultured keratinocytes and carcinomas derived from dermal diseases (FIGURE 5)J.2.7,8,71 and skin, corneal, conjunctival, tongue, and esophageal epithelia (FIGURE (c) normal human trunk epidermis stimulated to hyperpr~liferate?’.~~ (6) 45K/52K keratins (and to a lesser extent the 46K/54K keratins) as markers for simple epithelia. Tissue-distribution studies showed that these keratins are most frequently found in simple epithelia.’.’’ The two cow keratins that are equivalent to the 45K and 52K human keratins have been shown to form a complex that is stable even in the presence of high concentrations of urea.‘2’80 (7) The 40K keratin is present as a major cytoskeletal component only in simple epithelia, although it can be readily detected in almost all epithelia except adult epidermis.1,2,32,36,73
A MODEL FIGURE 12 is a schematic diagram summarizing the concepts of keratin subfamilies and keratin pairs.” In this diagram, keratins of both subfamilies are arranged according to an identical molecular weight scale (note the 5K bar), except that the scale for the basic subfamily is shifted upwards by 8,OOO. The 51K/59K and 55K/64K keratin pairs are placed as branches to indicate that they, like the 56.5K/65-67K keratins, represent markers for advanced states of stratified epithelial differentiation (see rules 1, 2, and 3). This arrangement provides a striking demonstration that the relative size distribution of the keratins in the two subfamilies is highly conserved, and that keratins with identical “size ranks” in the two subfamilies form a pair, with both members following similar rules for expression. This scheme also emphasizes that (1) the larger keratins of both subfamilies are characteristic of stratified epithelia (rules 1 to 5) whereas smaller keratins are expressed mainly by simple epithelia (rules 6 and 7), and
TUNG et 01.: RULES OF KERATIN EXPRESSION
325
Skin Skin Cornea
other s.e.
\
TF
other s.e.
Keratin subfamilies FIGURE 12. A unifying model of keratin expression. In this diagram, the acidic and basic keratins are arranged vertically according to their molecular weights (see the 5K scale at lower left corner). To align the two subfamilies, the MW scale of the basic keratins is 8K lower than that used for the acidic keratins. Keratins above the horizontal line are mainly expressed by keratinocytes of stratified squamous epithelia, whereas those below the line are mainly expressed by simple epithelia. The top three pairs of keratins are markers for skin, cornea, and esophageal “types” of epithelial differentiation. S.e. denotes stratified epithelia. The expression of these top three differentiation-specific keratin pairs is frequently diminished when the epithelia become “de-differentiated” as a result of disease or growing in a tissue culture environment. This diagram , ~only ~ difference is that, based on the SDS gel shown in FIGURE is modified from Sun et ~ l . the 1, the molecular weight of the acidic “other s.e.” member was changed from 54‘K to 51K. (From Cooper et aL2’ With permission from Laboratory Investigation.)
326
ANNALS NEW YORK ACADEMY OF SCIENCES
(2) a common set of keratins (box in FIGURE 12) is expressed by stratified epithelia in culture and in some diseases. The implications of this model on keratin structure and evolution have been discussed elsewhere.”
CONCLUDING REMARKS AND PERSPECTIVE In this paper, we have reviewed our current knowledge concerning the classification and expression of specific keratin molecules. Our results indicate that all human epithelial keratins can be divided into two subfamilies, and that there exist specific “keratin pairs” as defined by co-expression. These data have allowed us to construct a model (FIGURE12 and TABLE1) that defines and emphasizes the concepts of keratin subfamilies and keratin pairs.22This model also raises several important questions. (1) What are the detailed structural relationships among different keratins within a subfamily? The answer to this question will have to come from cDNA or protein sequence analysis and should shed light on the molecular mechanisms by which the two keratin subfamilies may have evolved. ( 2 ) What is the structural significance of the 8,OOO dalton size difference between the acidic and basic keratins of each keratin pair? Two extra pieces of sequence, totaling approximately 8,000 daltons, have been localized by Steinert and co-workers to the two ends of the central alpha-helical domain of a basic keratin?’ These two pieces are missing in the corresponding acidic keratin, thus providing a possible explanation for the 8,OOO dalton size difference. The structural significance of these pieces, however, remains to be elucidated. (3) How do the acidic and basic keratins interact with each other to form filaments? Even before the existence of two keratin subfamilies became known, early in vitro reconstitution data suggested that at least two keratins are required for tonofilament f~rmation.”.’~ More recently, Franke et al. have shown that certain acidic and basic keratins can form specific complexes in the presence of high concentrations of urea.72s8o We have also found recently that the 50K acidic and 58K basic keratins of normal human epidermis can interact specifically in 8.5 M urea, forming a high molecular weight aggregate that can be isolated by gel filtration (R.Eichner et a/., unpublished observation). These results are consistent with the idea that keratins of both acidic and basic subfamilies may be required for filament formation. Whether such a requirement applies at the level of a 2 nm protofilament, 4.5 nm protofibril, or 10 nm filament,74will have to be determined. (4) At what stages of epithelial maturation are the differentiation-related keratins expressed? In stratified epithelia, the basal cells are relatively undifferentiated, whereas the suprabasal cells are in advanced stages of maturation and take on the unique properties of the individual tissues. In this regard, it is interesting that the 56.5K and 65 -67K markers for keratinization or “skin type” differentiation have actually been localized to the suprabasal layers of the epidermis (FIGURE 3)?0,3542 Whether the other two pairs, i.e., 55K/64K and 51K/59Kkeratins, are also expressed suprabasally in corneal and esophageal epithelia, respectively, remains unknown. Similarly, the 48K/ 56K keratins have not yet been localized in hyperproliferative epidermis or cultured human epidermal colonies. Since on a tissue level, the expression of these two keratins (48 /56K) appears to be inversely proportional to that of the suprabasally located keratinization markers (FIGURE 7);’ we postulate that in individual cells the expression of these two types of keratin markers (56.5K/65-67K versus 48K/56K keratins) may be mutually exclusive. This possibility will have to be tested by immunolocalization.
TUNG et al.: RULES O F KERATIN EXPRESSION
327
(5) Finally, and most important, what are the detailed, specific functions of individual keratins or keratin pairs? The fact that each keratin molecule can be associated with specific morphological and/or physiological features of various epithelia strongly implies that different keratins must play unique functional roles during in vivo epithelial differentiation. Thus, significant headway has been made in addressing our original inquiry as to why such a large number of keratin species may have evolved. To further define these roles in detailed biochemical and structural terms now becomes the principal challenge, not only for “keratinologists,” but also for investigators interested in the mechanisms of normal and abnormal epithelial differentiation.
REFERENCES B. GEIGER& R. KREPLER.1982. Cell 31: 1. MOLL,R., W. W. FRANKE,D. L. SCHILLER, 11-24. N. E. BINDER,M. A. BECKETT,J. H. SINARD,C. T. GRIFFITHS 2. Wu, Y.J., L. M. PARKER, & J. G. RHEINWALD. 1982. Cell 31: 693-703. 3. SUN,T.-T. & H. GREEN.1978. Cell 1 4 468-476. W. W., E. SCHMID,M. OSBORN & K. WEBER.1978. Proc. Natl. Acad. Sci. USA 4. FRANKE, 7 5 5034-5038. 5. SUN,T.-T., C. SHIH& H. GREEN.1979. Proc. Natl. Acad. Sci. USA 7 6 2813-2817. 6. FRANKE, W. W., B. APPELHANS, E. SCHMID, C. FREUDENSTEIN, M. OSBORN & K. WEBER. 1979. Differentiation 15: 7-25. 7. DORAN,T. I., A. VIDRICH& T.-T. SUN. 1980. Cell 22: 17-25. 8. FUCHS,E. & H. GREEN.1980. Cell 19 1033-1042. 9. GIPSON,I. K. & R. A. ANDERSON.1980. Exp. Cell Res. 128 395-406. 10. MILSTONE,L. M. & J. MCGUIRE.1981. J. Cell Biol. 8 8 312-316. 11. FRANKE,W. W., H. DENK,R. KAIT & E. SCHMID.1981. Exp. Cell Res. 131: 299-318. 12. FRANKE,W. W., D. L. SCHILLER, R. MOLL,S. WINTER,E. SCHMID,I. ENGELBRECHT, H. DENK,R. KREPLER& E. PLATZER.1981. J. Molec. Biol. 153 933-959. 13. STEINERT, P. M., W. W. IDLER& S. B. ZIMMERMAN. 1976. J. Molec. Biol. 108: 547-567. 14. LEE, L. D. & H. P. BADEN.1976. Nature 264: 377-379. 15. HANUKOGLU, I. & E. FUCHS. 1982. Cell 31: 243-252. 16. GEISLER,N. & K. WEBER.1982. EMBO J. 1: 1649-1656. 17. STEINERT, P. M., R. H. RICE,D. R. ROOP, L. T. BENES& A. C. STEVEN.1983. Nature 302 794-800. 18. WEBER,K. & N. GEISLER.1984. In The Cancer Cell. A Levine, W. Topp, G. Vande Woude & J. D. Watson, Eds.: 153-159. Cold Spring Harbor Laboratories. Cold Spring Harbor, N.Y. E. 1982. Ann. Rev. Biochem. 51: 219-250. 19. LAZARIDES, J., R. EICHNER,W. G. NELSON& T.-T. SUN.1982. J. Cell Biol. 20. WOODCOCK-MITCHELL, 95: 580-588.
21. SUN,T.-T. & H. GREEN.1978. J. Biol. Chem. 293 2053-2060. W. G. NELSON& R. A. WEISS. 22. SUN, T.-T., R. EICHNER,D. COOPER,A. SCHERMER, 1984. In The Cancer Cell. A Levine, W. Topp, G. Vande Woude & J. D. Watson, Eds.: 169-176. Cold Spring Harbor Laboratories. Cold Spring Harbor, N.Y. 23. FUCHS,E. & H. GREEN.1979. Cell 17: 573-582. & E. V. FUCHS. 1983. Molec. Cell Biol. 3 495-502. 24. KIM, K. H., J. RHEINWALD & W. W. FRANKE. 1983. EMBO J. 2: 1387-1392. 25. MAGIN,T. M., J. L. JORCANO 1980. J. Invest. Dennatol. 74: 382-388. 26. GIBBS,P. E. M. & 1. M. FREEDBERG. & T.-T. SUN. 1985. Lab. Invest. 5 2 243-256. 27. COOPER,D., A. SCHERMER & P. H. O’FARRELL.1977. Cell 12: 1133-1142. 28. OFARRELL,P. Z., H. M. GOODMAN & J. GORDON. 1979. Proc. Natl. Acad. Sci. USA 7 6 4350-4354. 29. TOWBIN,H., T. STAEHELIN 1981. Cell 27: 75-84. 30 FUCHS,E., S. M. COPPOCK,H. GREEN& D. W. CLEVELAND. D. L., W. W. FRANKE & B. GEIGER.1982. EMBO J. 1: 761-769. 31. SCHILLER,
328
ANNALS NEW YORK ACADEMY OF SCIENCES
W. G. NELSON,A. J.-W. HUANG,J. WOODCOCK32. TSENG,S. C. G., M. J. JARVINEN, MITCHELL& T.-T. SUN. 1982. Cell 3 0 361-372. I. & E. FUCHS. 1983. Cell 33: 915-924. 33. HANUKOGLU, W. G. & L. M. DOWLING.1971. Appl. Polm. Symp. 18: 1-20. 34 CREWTHER, & R. KREPLER.1982. J. Cell Biol. 95: 35. MOLL, R., W. W. FRANKE,B. VOLC-PLATZER 285-295. 1981. Cell 2 5 627-635. 36. Wu, Y.-J. & J. G. RHEINWALD. 37. FUCHS,E. & H.GREEN.1978. Cell 15 887-897. 38. VIAC,J., J. M. STAQUET,J. THIVOLET& C. GOUJON.1980. Arch. Dermatol. Res. 267: 179-188. W. G. NELSON,A. VIDRICH & J. WOODCOCK-MITCHELL. 1983. 39. SUN,T.-T., R. EICHNER, In Normal and Abnormal Epidermal Differentiation. M. Seiji & I. A. Bernstein, Eds.: 277-291. University of Tokyo Press. Tokyo. D. & C. J. SKERROW. 1983. Exp. Cell Res. 143: 27-35. 40. SKERROW, J., M. KINJO,G. FURSTENBERGER & H. WINTER.1984. Cell 37: 159-170. 41. SCHWEIZER, D., A. BOHNERT,E. HERZMANN, P. BOWDEN,P. BOUKAMP & N. E. 42. BREITKREUTZ, FUSENIG. 1984. Differentiation. (In press.) J. G. & H. GREEN.1975. Cell 6 331-343. 43. RHEINWALD, 44. LAVKER,R. M. & T.-T. SUN. 1983. J. Invest. Dermatol. 81: 121s-127s. R., P. BONITZ& T.-T. SUN. 1984. J. Cell Biol. 98: 1388-1396. 45. EICHNER, 46. FUCHS,E. & H. GREEN.1981. Cell 25: 617-625. 47. WEISS,R.A,, R. EICHNER& T.-T. SUN. 1984. J. Cell Biol. 98: 1397-1406. 1984. Br. J. Dermatol. (Suppl. 27) 111:27-37. 48. MCGUIRE,J., M. OSBER& L. LIGHTFOOT. 49. CELIS,J. E., S. J. FEY,P. M. LARSEN & A. CELIS.1984. Proc. Natl. Acad. Sci. USA 81: 3128-3132. 50. SUN,T.-T. & H. GREEN.1977. Nature 269: 489-493. P. HOLLWEG-MAJERT, G. DALLENBACH-HELL51. MOLL,R., R. LEVY,B. CZERNOBILSKY, WEG & W. W. FRANKE.1983. Lab. Invest. 49: 599-610. 52. NELSON,W. G. & T.-T. SUN. 1983. J. Cell Biol. 97: 244-251. H. SANTANA & T.-T. SUN. 1984. Cancer Res. 44. 53. NELSON,W. G., H. BATTIFORA, 1600-1603. 54. BANKS-SCHLEGEL, S. P. & H. GREEN.1980. Transplantation 2 9 308-313. W. W., D. MAYER,E. SCHMID,H. DENK& E. BORENFREUND. 55. FRANKE, 1981. Exp. Cell Res. 134 345-365. 56. DENK,H., R. KREPLER,E. LACKINGER, U. ARTLIEB& W. W. FRANKE. 1982. Lab. Invest. 46: 584-596. A.J.-W. HUANG& T.-T. SUN. 1984. J. Cell Biol. 99: 57. TSENG,S. C. G., D. HATCHELL, 2279. 58. DALE,B. A., I. B. STERN,M. RABIN& L.-Y.HUANG.1976. J. Invest. Dermatol. 6 6 230-235. 59. BANKS-SCHLEGEL, S. P. 1982. J. Cell Biol. 9 3 551-559. 60. MOLL,R., I. MOLL& W. WIEST. 1983. Differentiation 23: 170-178. M., K. FUKUYAMA, W. L.EPSTEIN& K. A. FISHER. 1978. J. Invest. Dermatol. 61. THALER, 75: 156-158. 62. BADEN,H. P., N. MCGILVRAY, C. K. CHENG,L. D. LEE & J. J. KUBILUS.1978. J. Invest. Dermatol. 7 0 294-297. 63. SKERROW, D. & I. HUNTER.1978. Biochem. Biophys. Acta 537: 474-484. 64. SCHERMER, A. & T.-T. SUN.Submitted for publication. 65. BANKS-SCHLEGEL, S. P. & C. C. HARRIS.1983. Exp. Cell Res. 146 271-280. 66. RICE,R. H. & H. GREEN.1979. Cell 18: 681-694. 67. GREEN,H.,E. FUCHS & F. WATT. 1981. Cold Spring Harbor Symp. Quant. Biol. 4 6 293-301. 68. HRONIS,T. S., M. L. STEINBERG, V. DEFENDI& T.-T. SUN. 1984. Cancer Res. 44: 5797. 69. LANE,E. B. 1982. J. Cell biol. 9 2 665-673. J., P. PURKIS,E. B. LANE,I. A. MCKAY& S . CHANG.1982. 70. TAYLOR-PAPADIMITRIOU, Cell Differ. 11: 169-180. 11. MOLL,R., R. KREPLER & W. W. FRANKE. 1983. Differentiation 23: 256-269. 7 .
TUNG et al.: RULES OF KERATIN EXPRESSION
329
M. HATZFELD & S. WINTER.1983. Proc. Natl. Acad. 72. FRANKE,W. W., D. L. SCHILLER, Sci. USA 80: 7113-7117. 73. FRANKE, W. W., S. WINTER,C. GRUND,E. SCHMID,D. L. SCHILLER & E. D. JARASCH. 1981. J. Cell Biol. 90: 116-127. 74. AEBI, U., W. E. FOWLER,P. REW & T.-T. SUN. 1983. J. Cell Biol. 97: 1131-1143. 75. LAEMMLI,U. K. 1970. Nature 227: 680-685. I. A. 1979. In Immunocytochemistry. 122-129. John Wiley & Sons. New 76. STERNBERGER, York. M. E., V. B. CULBERTSON & I. M. FREEDBERG. 1980. J. Invest. Dermatol. 77. GILMARTIN, 75: 211-216. A. G. 1964. Nature 201: 1130-1131. 78. MATOLTSY, 79. DALE,B. et al. In preparation 80. QUINLAN,R. A,, D. L. SCHILLER,M. HATZFELD,T. ACHTSTATTER,R. MOLL, J. L. JORCANO, T. M. MAGIN& W. W. FRANKE. 1985. Ann. N.Y. Acad. Sci. (This volume.) 81. STEINERT,P. M., W. W. IDLER,X.-M. ZHON, L. D. JOHNSON,D. A. D. PARRY,A. C. STEVEN& D. R. ROOP. 1985. Ann. N.Y. Acad. Sci. (This volume.)
