Cell, Vol. 64, 365-360,

January

25, 1991, Copyright

0 1991 by Cell Press

Mutant Keratin Expression in Transgenic Mice Causes Marked Abnormalities Resembling a Human Genetic Skin Disease Robert Vassar, Pierre A. Coulombe, Linda Degenstein, Kathryn Albers, and Elaine Fuchs Howard Hughes Medical Institute and Department of Molecular Genetics and Cell Biology The University of Chicago Chicago, Illinois 60637

Summary To explore the relationship between keratin gene mutations and genetic disease, we made transgenic mice expressing a mutant keratin in the basal layer of their stratified squamous epithelia. These mice exhibited abnormalities in epidermal architecture and often died prematurely. Blistering occurred easily, and basal cell cytolysis was evident at the light and electron microscopy levels. Keratin filament formation was markedly altered, with keratin aggregates in basal cells. In contrast, terminally differentiating cells made keratin filaments and formed a stratum corneum. Recovery of outer layer cells was attributed to down-regulation of mutant keratin expression and concomitant induction of differentiation-specific keratins as cells terminally differentiate, and the fact that these cells arose from basal cells developing at a time when keratin expression was relatively low. Collectively, the pathobiology and biochemistry of the transgenic mice and their cultured keratinocytes bore a resemblance to a group of genetic disorders known as epidermolysis bullosa simplex. Introduction As surface and lining cells, epithelial cells share in common a protective function, which is manifested by the production of an extensive cytoskeletal network of 10 nm intermediate filaments composed of keratin. The keratins are a complex family of ~30 proteins (40,000-70,000 daltons) that can be subdivided into two distinct classes, type I and type II (Fuchs et al., 1981). At least one member of each type is essential for filament formation (Franke et al., 1983) and keratins are frequently expressed as specific pairs (Sun et al., 1984). Stratified squamous epithelia including epidermis, cornea, and tongue have especially abundant keratin networks, and their mitotically active basal cells are similar in that they all express the type II keratin K5 (58 kd) and the type I keratin K14 (50 kd) (Mall et al., 1982a; Nelson and Sun, 1983). In the developing embryo, basal cells first express appreciable levels of K14 at a time when stratification takes place; thereafter, K14 mRNA and protein increase progressively until several days after birth (Jackson et al., * Present address: Center, University

Department of Kentucky,

of Pathology, Lucille I? Markey Lexington, Kentucky 40536.

Cancer

1981; Moll et al., 1982b; Dale et al., 1985; Kopan and Fuchs, 1989a). As basal cells differentiate and move into suprabasal layers, they down-regulate their synthesis of K5 and K14 (Tyner and Fuchs, 1986; Roop et al., 1987) and induce expression of new pairs of keratins in a tissuespecific fashion (Sun et al., 1984). Thus, for example, suprabasal cells of epidermis synthesize Kl (67 kd) and KlO (56.5 kd), differentiating esophageal cells make K4 (59 kd) and K13 (53 kd) (van Muijen et al., 1986) and keratinizing cornea1 cells express K3 (64 kd) and K12 (52 kd) (Schermer et al., 1986). In a number of epidermal skin diseases, K6 (56 kd) and K16 (56.5 kd) are often expressed suprabasally (Weiss et al., 1984; Stoler et al., 1988). Expression of keratins is often much greater in differentiating cells than in basal cells (Fuchs and Green, 1980; Moll et al., 1982a, 1982b; Sun et al., 1984; Roop et al., 1987). Since the discovery of keratins and other 10 nm cytoskeletal filaments, their function has remained elusive. In vivo, most if not all vertebrate cells seem to have at least a modest intermediate filament network (for review see Lazarides, 1982; Moll et al., 1982a). However, in vitro, cell lines lacking these filaments have been selected, thereby suggesting that an intermediate filament cytoskeletal network is not required for cells to divide and carry out normal metabolic and growth processes (Venetianer et al., 1983; Hedberg and Chen, 1986). Hence, it has generally been assumed that if keratins and other intermediate filaments have a purpose, it must be one manifested by the differentiated tissue as a whole, perhaps in providing architecture and cellular organization. Despite the widespread acceptance of this notion, however, direct in vivo demonstration has been difficult to obtain. Elucidating keratin function has been further hampered by the lack of known genetic diseases involving keratin mutations. The premise that a mutation in a keratin gene might give rise to a dominant alteration in cytoskeletal architecture has been tested and verified in vitro by transfecting truncated human keratin genes into cultured cells (Albers and Fuchs, 1987, 1989; Lu and Lane, 1990). In some cases, e.g., a truncated keratin missing 135 amino acid residues from the carboxyl terminus of K14 (CA135K14P), mutant protein integrated into and disrupted the endogenous keratin filament network (Albers and Fuchs, 1987). Complementary in vitro filament assembly studies on bacterially produced human K5, K14, and mutant CA135K14P showed that addition of as little as 1% of the mutant CA135K14P to the assembly mixture caused a detectable alteration in filament formation (Coulombe et al., 1990). That small amounts of mutant keratins can cause dominant phenotypic changes in epithelial sheets in culture suggests that such mutations might also cause dominant phenotypic changes in stratified squamous epithelia in vivo. Are such mutations lethal? Can proper embryonic development and tissue organization take place? Do such mutations correlate with known genetic diseases of unknown etiology? To investigate these questions, we have

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Figure 1. Genetic Map of Expression pgCA135K14PI(-6000)

Plasmid

The vector is pGEM3. The components of this plasmid are the 5’ upstream sequence of the human K14 gene (stippled box) (Marchuk et al., 1964) extending from the ATG translation initiation codon to an EcoRl site located *6 kb 5’ to the TATA box; the coding sequence of the human CA135K14P cDNA (black boxes); the first four introns of the K14 gene (open boxes); the sequence coding for the substance P tag (gray box); K14 3’ untranslated sequence extending from 21 bp 3’from the TGA stop codon to the poly(A) signal and extending -600 bp 3’from the poly(A) signal (diagonally striped box). Bg, the Bglll site used to splice together the K14 gene and cDNA segments; RI, EcoRI; K, Kpnl.

introduced into the germline of transgenic mice a hybrid gene consisting of the human K14 enhancer/promoter and a K14 gene cDNA encoding the truncated, P-tagged keratin CA135K14P Previously, we demonstrated that sequences 5’ to the functional human K14 gene were sufficient to drive stratified squamous epithelial-specific expression of a human K14 cDNA that had been tagged at the C-terminus with sequences encoding the antigenic portion of neuropeptide substance P (Vassar et al., 1989). We now show that in striking contrast to K14P-expressing transgenic mice, transgenics expressing a truncated K14P have gross abnormalities in the intra- and intercellular architecture of their stratified squamous epithelial tissues. At least some functions of the tissues appear to be compromised by this disorganization, as judged by basal cell cytolysis and subsequent blistering, and death shortly after birth.

Figure

2. Phenotype

of CAl35K14P

?ansgenic

Mice

(Top) Control newborn mouse. (Bottom) Fl generation transgenic offspring from mosaic mouse CAl35K14P-1B. Note the presence of epidermal blistering around the limbs, particularly at the upper-left forearm.

Results Construction of the Mutant Keratin Gene and Production of Transgenic Mice The expression vector construct used for most of our studies is illustrated in Figure 1. Six thousand base pairs of 5’ upstream human K14 gene sequence were used to drive expression of a P-tagged, truncated K14 protein, CA135K14P, missing 135 amino acid residues at the C-terminal end. This mutant lacks >30% of the central a-helical domain and all of the nonhelical carboxyl tail of the keratin polypeptide (Albers and Fuchs, 1987). The presence of the P tag and the absence of K14 C-terminal residues enabled use of monospecific anti-P antibodies to track expression of the transgene product, and a polyclonal antiserum to the C-terminus of K14 to track expression of endogenous mouse K14 (Vassar et al., 1989). Transgenic mice were produced using an outbred strain (CD-l) of mice as described previously (Vassar et al., 1989). Eleven mice were transgenic, as judged by polymerase chain reaction (PCR) analysis of their skin DNAs. Of these, one male mouse (CA135K14P-16, or 16 for short) was used for mating to generate transgenic offspring (CAl35K14PlB-Fl). Expression of CAl35K14P Correlates with Skin Abnormalities and Premature Death There were two pecularities characteristic of litters obtained from fertilized mouse eggs injected with the CAl35K14P gene construct and not seen with K14P transgenies. First, CAl35K14P litters had a high incidence of neonatal mortality, often occurring within 24 hr after birth. Second, dead litter mates often showed gross skin abnormalities. One of seven mice with these characteristics (2A) appeared to be without an epidermis altogether. While skins of five other mice (lA, 28,3A, 3C, and 3D) seemed relatively normal by sight, the skin was often blistered around the limbs, and it blistered further when subjected to trivial trauma, such as mild rubbing of the skin (Figure 2, compare normal control mouse, top, with transgenic mouse, bottom). Four additional positives (lB, lC, lD, and 3E) did not show these characteristics, but upon breeding, mouse CAl35K14P-lB gave rise to lo%-20% transgenic Fl offspring, and these transgenic Fl mice all exhibited the same aberrant phenotypes as the seven founder mice. Thus, mouse 1B appeared to have survived because it was mosaic. In contrast, neither a stillborn mouse nor any of the surviving litter mates exhibited these peculiar skin abnormalities.

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9 10 11 1213 1415 16 17

c.

1 2 3

Figure 3. Intermediate Filament Proteins from CAi35K14P Transgenic Founder Mice and Fl Offspring of Founder Mouse 16

Intermediate filament proteins from skin of a control mouse, ten founder transgenic mice, and one Fl offspring transgenic were extracted and resolved by electrophoresis through triplicate 6.5% SDS-polyacrylamide gels. Proteins from two gels (A and B) were subjected to immunoblot analysis using an anti-P (A) or antiK14 (B) antiserum. The other gel (C) was stained with Coomassie blue. Samples in gel (A) were from, lane 1, control newborn mouse (10 pg); lane 2, CA135K14PV1A transgenic (10 wg); lane 3, CA135K14P-28 (2.5 ug); lane 4, CA135K14P-1B (IO Kg); lane 5, CA135K14P-1C (10 99); lane 6, CA135K14P-1D (10 ug); lane 7, CA135K14P-3A (2.5 Kg); lane 6. CA135K14P-38 (10 ug); lane 9, CAl35K14P-3C (2.5 vg); lane IO, CAl35K14P-3D (2.5 99); lane 11, CAl35K14P-3E (10 ug); lane 12, CA135K14P-lB-Fl (5 pg); lanes 13-17, CAl35K14P protein purified from overexpressing bacteria (Coulombe et al., 1990) and loaded at IO, 50, 100, 250, and 500 ng, respectively. Mobilities of molecular weight standards are indicated at left. Molecular mass of CAl35K14P protein based on electrophoretic mobility was 37 kd, in agreement with its predicted size based on sequence. Samples (0.9 ug) in lanes l-12 (B) were in same order as those in (A). Lanes 13-17 (B), human K14 protein purified from bacteria, loaded at 20, 50, 100, 200, and 400 ng, respectively (Coulombe and Fuchs, 1990). Mouse K14 runs as a 52 kd protein, whereas human K14 runs as a 50 kd protein. Samples in gel (C) were, lane 1, bacterially produced CAl35K14P control (1 Hg); lane 2, control newborn back skin keratins (12 pg); lane 3, mouse 28 keratins (12 wg). Keratins are identified at right; molecular masses of standards and their mobilities are indicated at right

To investigate further the basis for mutant versus wildtype phenotypes among our 11 transgenic founders and mouse 1B’s transgenic Fl offspring, we isolated intermediate filament proteins from their epidermis. As judged by immunoblot analysis, samples from the mice with the aberrant phenotypes contained the diagnostic 37 kd CA135K14P band showing antigenic cross-reactivity with anti-P antiserum (Figure 3A, lanes 2, 3, 7-10, 12; compare with aliquots of bacterially expressed and purified CA135 K14P protein in lanes 13-17). For this particular assay, we could not obtain sufficient intermediate filament proteins from the skin of mouse 2A, which had almost no epidermis. However, subsequent anti-P staining of cultured keratinocytes from this mouse (see below) confirmed that it too expressed the CA135K14P gene. None of the proteins from surviving transgenics (lanes 4-6) nor the control (lane 1) cross-reacted with anti-P However, since the founder mouse 16 (lane 4) generated CAl35K14P-expressing transgenic offspring (lane 12) the founder 16 mouse must have been a mosaic transgenic with few expressing cells in the epidermis. Overall, a nearperfect correlation existed between the mutant phenotype and expression of the truncated keratin transgene. In contrast, >20 different founder mice expressing the wild-type equivalent, i.e., a human K14P transgene, lived to adult age, and none exhibited the abnormal skin phenotype characteristic of CAl35K14P mice (see Vassar et al., 1989; Leask et al., 1990; R. V and E. F., unpublished data). Thus, neonatal death and trauma-induced denuding of epidermal tissue appeared to be phenotypic traits of transgenic mice expressing a truncated keratin gene known to be a potent disrupter of keratin filament networks in vitro. To estimate the amount of CA135K14P produced relative to the endogenous basal counterpart, we conducted anti-K14 immunoblot analysis on a duplicate gel, this time containing various known amounts of bacterially expressed and purified human K14 protein as standards (Figure 38). Wild-type mouse and human K14 proteins have identical C-terminal sequences and both show com-

parable cross-reactivity with the C-terminal anti-K14 peptide antisera. After adjustments for molecular mass and loading differences, it was estimated that CAl35K14P levels varied substantially among these transgenics, ranging from -5% of the molar amounts of total K14 in mouse 1A skin (Figures 3A and 36, lane 2) to m30% in mouse 28 (Figures 3A and 38, lane 3) to -8O%-90% in mouse 3A and 3C skin (Figures 3A and 38, lanes 7 and 9, respectively). Densitometric scanning of a corresponding Coomassie blue-stained gel gave results consistent with these estimations (see Figure 3C, lane 3, for example of total keratins from mouse 28). From the stained gel, it was also apparent that endogenous and mutant K14, combined, represented only a small fraction of the total keratin in transgenic mouse skin. Thus, induction of the larger, differentiation-specific keratins occurred at comparable and abundant levels in both control and transgenic mice (compare lanes 2 and 3 in Figure 3C). Intra-Epithelial Blistering and Gross Disorganization of Stratified Squamous Epithelial Architecture in CAl35K14P-Expressing Transgenic Mice To investigate abnormalities in transgenic skin in greater detail, we stained tissue sections (5 urn) with hematoxylin and eosin (Figure 4). For reference, a skin section from a normal newborn mouse is shown in Figure 4A. In contrast to the control, gross disorganization occurred in some, but not all, regions of the epidermis of CA135K14P-1A mouse skin (see affected area shown in Figure 48). The alternating regions of normal and abnormal morphology suggested that this mouse was mosaic, and additional lines of evidence presented below were consistent with this notion. Mouse 28 skin showed abnormalities similar to, but more severe than, mouse 1A skin. Moreover, in this case, all areas of the epidermis appeared affected (Figure 4C). The skin from a transgenic Fl offspring of mouse 1B was also grossly blistered, although the suprabasal layers within the tissue were not as disorganized as in many of the other transgenics (Figure 4D). in all skin samples of CAl35K14P-expressing trans-

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genie mice that we examined, evidence of cellular degeneration and cytolysis was prevalent (see arrowheads in Figures 4B-4D). Cytolysis was restricted to the epidermis and often localized within the inner layers: the granular and stratum corneum layers appeared intact and less affected. Blistering occurred both intra-epidermally and at the dermal-epidermal junction. Remnants of cytolyzed basal cells were often seen attached to the dermis at the site of a blister, indicating that the separation may have occurred as a consequence of basal cell cytolysis (Figures 4C and 4D). While the morphology of transgenic epidermis was clearly altered, signs of terminal differentiation were evident, as indicated by the presence of granular cells (GR) and a stratum corneum (SC) in the outermost layers (Figures 4B-4D). These features of keratinization were consistent with the large amounts of differentiation-specific keratins seen in intermediate filament extracts from transgenic skin. Beneath the stratum corneum of affected areas, terminal differentiation was often aberrant, as judged by the presence of pearls of keratinized material and atypically large cells. To examine the relationship between mutant morphology and CAl35K14P expression, we exposed control and transgenic skin sections to antibodies specific for mutant and K14 proteins, respectively. As expected from previous studies, a transgenic mouse expressing a human K14 promoter-driven “wild-type” K14P showed uniform anti-P staining in the basal layer (Figure 4E; Vassar et al., 1989). This pattern was similar to that observed with nontransgenie mouse skin stained with an anti-K14 antiserum (Figure 4F). In contrast to these controls, transgenic mouse 28 skin showed anti-P and anti-K14 staining throughout most of the basal and inner suprabasal layers (Figures 4G and 4H, respectively). Similar patterns were also seen for the severely affected regions of most other transgenic mice, with the exception of mouse 1BFl skin, which was basal restricted and similar to control newborn skin. For all transgenic mice, anti-P was not detected in the stratum corneum, consistent with the known absence of K14 in these outermost layers (Fuchs and Green, 1980). There are multiple reasons as to why alterations in antiK14 and anti-P staining patterns might be expected in CA135K14P-expressing mouse skin. The change could reflect an alteration in K14 and CA135K14P gene expression as a consequence of putative perturbation of the cytoskeletal architecture of the cell. However, since K14 protein is known to persist in normal spinous cells even though K14 mRNA is down-regulated (Fuchs and Green,

Figure

4. Sections

of Skin and Tongue

from CAl35K14P-Expressing

1980; Roop et al., 1987), and CA135K14P was predicted to inhibit filament assembly and induce keratin aggregation (Albers and Fuchs, 1987; Coulombe et al., 1990) antigen unmasking and/or stabilization of keratins in aggregate form seemed a more likely contributor to this phenomenon. Ultrastructural analyses (see below) were consistent with this notion. In all cases, however, anti-P staining and aberrant anti-K14 staining were restricted to affected regions, providing definitive evidence that the gross abnormalities in epidermal phenotype were directly due to CA135K14P expression. Previously, we showed that the human K14 promoter was broadly active in stratified squamous epithelia of transgenic mice (Vassar et al., 1989). Since K14 is expressed in tongue, cornea, esophagus, and trachea (Mall et al., 1982a; Nagle et al., 1985) we expected abnormalities in their tissue architecture as well. A particularly striking example of this was the ventral epithelium of tongue, where K14 expression is known to be high (Figures 4l-4L). Hematoxylin and eosin-stained sections of mouse 2A tongue revealed severe disorganization of the ventral epithelium, where intra-epidermal and dermal-epidermal separations were also prevalent (Figure 4J, compare with control in Figure 41). CA135K14P was clearly expressed in this tissue, as evidenced by anti-P staining (Figure 4K), which was similar to that seen with anti-K14 (Figure 4L). The dorsal epithelium was also affected (data not shown), but blistering was less severe. Thus, while some variation was seen in the severity with which CA135K14P induced abnormalities in stratified squamous epithelial architecture, these perturbations were not restricted to epidermis. The Program of Terminal Differentiation in CAl35K14P-Expressing Transgenic Mice Is Present, but Aberrant To examine terminal differentiation in epidermis of CA135K14P-expressing mice, we stained mouse 28 skin sections with a cross-reacting antiserum for Kl. Figure 5A shows the presence of strong staining in the spinous cells of transgenic skin. Despite the gross disorganization of cells within epidermis, the stage of differentiation at which Kl was detected in transgenic skin corresponded quite well with that for normal mouse skin (Figure 58). Filaggrin, a late differentiation marker of granular cells (Dale et al., 1985) was expressed in mouse 2B (Figure 5C) in a fashion similar to control epidermis (Figure 5D). Overall, the major aberrancies in staining patterns for these differentiation-specific antisera seemed to arise from the disorganization of cells within the epidermis, rather than from ab-

Transgenic

Mice

Skin (A-H) and tongue (I-L) from four CA135K14P-expressing transgenic mice (lA, 2A, 28, and lB-Fl), one nontransgenic newborn mouse, and one control K14P-expressing transgenic mouse were fixed in Bouin’s fixative, embedded in paraffin, and sectioned (5 pm). Sections were subjected to hematoxylin and eosin (H/E) staining, or immunohistochemistry with anti-P or anti-K14 antisera. (A) Nontransgenic control, H/E; (B) mouse 1A (affected), H/E; (C) mouse 28, H/E; (D) mouse IB-Fl, H/E; (E) K14P transgenic mouse, anti-P; (F) nontransgenic mouse, anti-K14; (G) mouse 28, anti-P; (H) mouse 28, anti-Kl4. (I-L) Ventral tongue epithelium: (I) nontransgenic control, H/E; (J) mouse 2A, H/E; (K) mouse 2A, anti-P; (L) mouse 2A, anti-K14. Note that tissues from nontransgenic control, K14P transgenic. and unaffected regions of mouse 1A were indistinguishable morphologically. The bar in(L) represents 16 pm. SC, stratum corneum; GR, granular layer; SP spinous layers; BL, basal layer; ep, epidermis; dr, dermis. Arrowheads denote cytolysis; double arrows denote intra-epidermal separations.

_, i

Figure

5. Expression

of Differentiation-Specific

Markers

in CA135K14P-Expressing

Transgenic

Mouse

Epidermis

Skin from transgenics and a nontransgenic newborn was fixed and sectioned as described in the legend to Figure 4. Sections were subjected to immunostaining with anti-human stratum corneum antiserum (A and B), anti-mouse filaggrin (C and D), or anti-mouse K6 (E and F). Regions shown are mouse 28 skin (A and C); nontransgenic skin (8); unaffected region of mouse 1A skin (D); mouse IA skin showing affected (anti-KG+) (E and F) and unaffected (anti-KG-)(F) regions. Double arrows denote blister. Dotted lines indicate dermal-epidermal border. dr, dermis; hf, hair follicle. The bar in (A) represents 16 urn for (A) through (E) and 63 pm for (F).

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normalities in the biochemical program of expression per se. Recent studies have indicated that K6 and K16 expression is associated with aberrant keratinization, independent of the proliferative status of epidermis (Kopan and Fuchs, 1969b; Schermer et al., 1969; Choi and Fuchs, 1990). Since a band (46-50 kd) the size of mouse K16 was present in the intermediate filament extract from mouse 28 (Figure 3C, lane 3) and since abnormalities in differentiation were also observed, this prompted us to stain skin sections with a monospecific antiserum against mouse K6 (Roop et al., 1967). Indeed, anti-K6 staining was found in transgenic epidermis, where it was restricted to the suprabasal cells of CA135K14P-expressing regions. This was most striking in skin from mosaic mouse lA, where anti-K6 staining coincided with the mosaic pattern of disorganized epidermal morphology (Figure 5E, high magnification of an affected region; Figure 5F, low magnification showing patchwise staining). However, when coupled with the gel analysis in Figure 3C, the levels of K6 and K16 relative to Kl and KlO were judged to be low. CA135K14P-Expressing Keratinocytes Cultured from Transgenic Mice Show Mutant Keratin Filament Networks Indistinguishable from Keratinocytes Transfected In Vitro The finding that CA135K14P-expressing keratinocytes existed in newborn transgenic mice indicated that the cells were at least transiently viable and should be able to be cultivated in vitro. While we were able to obtain keratinocytes from transgenic skin, colony growth was slow and cytolysis appeared to be prevalent. In contrast to control cultures, the colonies from transgenic epidermis did not survive beyond the primary culture stage. This finding suggests a possible explanation for why we have not been able to isolate permanent lines of transfected human keratinocytesexpressing CA135K14P (K. A. and E. F., unpublished data). The ability to culture transgenic mouse keratinocytes for short periods enabled us to investigate the 10 nm filament networks of these cells. Cultures were made from mouse 2A and lB-Fl skins. Two distinct staining patterns were observed in mouse 2A cultures stained with anti-K5 and anti-P (Figure 6). One type of keratinocyte showed only anti-K5 staining and no anti-P staining, as illustrated in the representative example in the upper right of Figures 6A (anti-KS) and 6B (anti-P). These cells showed typical keratin filament networks, suggesting that they arose from nontransgenic, i.e., wild-type, cells of mosaic skin. In contrast to normal keratinocytes, transgenic cells from both 2A and lB-Fl mice costained with anti-P and anti-K5, as indicated by the other keratinocytes in Figures 6A and 6B, respectively. A representative range of additional CA135K14P-expressing keratinocytes from the full transgenic mouse lB-Fl and from mosaic mouse 2A can be seen in Figure 6C. These mutant keratin networks were similar to those seen in cultured human epidermal cells transiently transfected in vitro with an SV40 promoter-driven CA135K14P cDNA (Figure 6D, anti-P staining, and Figure 6E, anti-K5

staining) (see also Albers and Fuchs, 1967). While evidence of filamentous-like structures was sometimes present, staining was predominantly punctate or clumped. In some cells, there were a few regions of the keratin network where either anti-K5, but no anti-P, staining was observed (see example in Figures 6A and 6B), or anti-P, but no antiK5, staining was seen (see Albers and Fuchs, 1967). In general, however, most regions of each perturbed cytoskeletal network showed costaining with anti-P and antiK5. Collectively, these data indicated that the transgenic mouse keratinocytes contained levels of CA135K14P sufficient to perturb keratin filament networks, a feature that apparently led to cytolysis and restricted growth within the basal epidermal cell population. Ultrastructure of CA135K14P Transgenic Mouse Skin Reveals Cytolysis and Keratin Aggregates in the Inner Layers, but a Near-Normal Stratum Corneum To examine in greater detail the CA135K14P-mediated disruption of keratin networks and its consequences on epidermal tissue architecture, we conducted electron microscopic studies of sections of skin from transgenic mice 28, 3C, and lB-Fl (Figure 7). The three sets of examples in Figure 7A illustrate regions at the dermal-epidermal junction of transgenic skin. Basal cell cytolysis was often present and extensive, while other cell types and dermal structures including the basement membrane (BM) appeared largely unaffected. In all sections, hemidesmosome (he) attachments were numerous and interconnected basal cells to the basement membrane. On the inner surface of the plasma membrane, keratins (ke) were associated with hemidesmosomes, although they often appeared nonfilamentous, in contrast to the keratin filament bundles typically associated with normal epidermal hemidesmosomes. Large clumps of keratin also existed throughout the cytoplasm of basal cells. These aggregates contained CA135K14P as confirmed by anti-P immunolabeling (Figure 78). They also labeled with an antiserum specific for endogenous basal keratins (data not shown). The keratin in these clumps did not appear to be in filamentous form, at least within the limits of resolution afforded by ultrastructural analysis. In basal cells with very large keratin clumps (e.g., lowest frame in Figure 7A), keratin tonofilament bundles were usually rare. In other basal cells, a mixture of keratin aggregates and tonofilament bundles (kf) were sometimes seen (Figure 7C). In these cases, anti-P immunolabeling was always stronger over the aggregates than over the filaments (Figure 7C), suggesting that some segregation of wild-type and mutant K14 may have taken place in these cells. This notion was consistent with our immunofluorescence data (see Figure 6). Several interesting morphological features were evident in the suprabasal layers of transgenic mouse epidermis (Figures 7D-7F). Typical of normal spinous cells, desmosomes in transgenic spinous cells were often prevalent. This was true even when the cells were heavily damaged, as indicated in the example in Figure 7D. As seen by higher magnification (Figure 7E), transgenic mouse des-

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Figure

6. Cultured

Transgenic

Mouse

and Human

Keratinocytes

Expressing

CA135K14P

Epidermis from transgenic newborns was trypsinized, and cells were plated on a lawn of mitomycin C-treated mouse 3T3 fibroblast feeder cells. Cells were fed on low calcium-containing medium as described by Hennings et al. (1960). Epidermal cells from mice 2A and lB-Fl were fixed and costained with guinea pig anti-human K5 (A) and rabbit anti-P(B). Bound antibodies were visualized with Texas red-conjugated anti-guinea pig IgG antiserum and fluoresceinconjugated anti-rabbit IgG antiserum, respectively. Note the presence of two types of keratinocytes: one that had a normal keratin filament network and did not stain with anti-P (cell in upper right of [A] and [B]), and one that had a disrupted keratin filament network and costained with anti-P and anti-K5 (other cells in [A] and [B]; note additional examples in [C] that costained with anti-K5 and anti-P). Cultured keratinocytes from squamous cell carcinoma cell line XX-13 (D and E) (Wu and Rheinwald, 1961) were transfected with an BV40 enhancer/promoter-driven CAl35K14P cDNA (Albers and Fuchs, 1967) and processed at 6.5 hr posttransfection as above. (D) Anti-P antiserum; (E) anti-K5 antiserum. The bar in (A) represents 16 urn.

mosomes in spinous cells appeared normal in structure. However, associated tonofilament bundles were often shorter than normal (see example in Figure 7E), and sometimes keratin clumps were seen, similar to those associated with hemidesmosomes (see Figure 7A). In all of the transgenic samples that we examined, the basal layer always showed extensive cytolytic damage and keratin aggregation. In contrast, the degree of abnormal morphology in spinous cells was variable and correlated with the relative level of CA135K14P expression. Thus, while some transgenic samples showed broad

damage in the spinous layers, others appeared nearly normal. In all cases, cells in the outermost layers were much less affected than cells in the basal and inner spinous layers (Figure 7F). In many outer layer spinous cells, tonofilament bundles of keratin (kf) were numerous, and these filaments often associated with desmosomal plaques (Figure 7F). Only a few clumps of keratin (ke) were present at the cytoplasmic periphery of these cells, and this was often the only marked difference between these cells and the outer spinous layers of normal epidermis. The intercellular, lipid-like material typically seen between

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the granular and stratum corneum layers was present, and these layers were usually normal in appearance. Two factors seemed to be playing a role in generating the observed ultrastructure in newborn transgenic mouse epidermis. First, since K14 expression continues to rise in basal cells from the time at which the epidermis stratifies to several days after birth (Kopan and Fuchs, 1989a), cells that underwent a commitment to terminally differentiate in the fetus (i.e., the outermost cells) must have arisen from basal cells that produced less K14 (and CA135K14P) than those that underwent a commitment at a later time (i.e., the innermost suprabasal layers). Second, at all developmental stages after epidermal stratification, basal cells down-regulate expression of K5 and K14 and produce large quantities of differentiation-specific keratins when they undergo a commitment to terminally differentiate (Fuchs and Green, 1980; Roop et al., 1987; Kopan and Fuchs, 1989a; see also Figure 3C). This process must have led to progressively reduced proportions of CA135K14P relative to wild-type keratins in the differentiating layers. If intracellular levels of CA135K14P correlated with the potential for inhibition of keratin filament assembly, accumulation of keratin aggregates, and basal cell cytolysis, this might explain why the most fully differentiated cells often appeared the least affected, and how a near-normal stratum corneum ever developed, considering the extensive cytolysis in the basal layer of some newborn transgenies. To examine this possibility further, we measured the effects of having increasing amounts of CA135K14P present in keratin filament assembly mixtures in vitro (Figures 7G-7K). Figure 7G shows representative examples of keratin filaments assembled from wild-type K14P and K5. When CA135K14P was present at 1% (Figure 7H), 10% (Figure 71) 50% (Figure 7J), or 100% (Figure 7K) of the total K14 levels, assembly with wild-type K5 into 10 nm filaments was progressively restricted. However, even when CA135K14P constituted as much as lo%-50% of the type I keratin in the assembly reaction, some evidence of filament formation was seen, although the filaments were few in number and also abnormal. The in vitro studies confirm the notion that the restrictive effects of CA135K14P on filament formation should be most severe under conditions where the concentration of mutant relative to wild-type keratin is highest. Coupled with previous in vitro studies by Franke et al. (1983) demonstrating that Kl and KlO can coassemble with K5 and K14 into 10 nm filaments, this explains why there were usually more tonofilaments relative to aggregates in differentiating epidermal cells than in basal cells. While the CA135K14P-induced perturbation of filament formation undoubtedly contributed heavily to the various phenotypic abnormalities in the transgenic mice, it is likely that secondary factors also played a role. One of these is the accumulation of keratin aggregates in the cytoplasm of affected cells. Since the total amount of keratin in basal cells was increasing with time, and since our morphological studies suggested that basal cell cytolysis became progressively worse with time, the presence of large aggregates of keratin may have interfered with cellu-

lar function. A second factor may have been the birthing process itself, which subjected substantial trauma to the skin. CA135K14P-Expressing Transgenic Mice Exhibit a Pathobiology Resembling the Epidermolysis Bullosa Simplex Class of Human Skin Disorders The pathobiology of CA135K14P-expressing transgenic mice resembled that of a heterogeneous group of human genetic skin diseases, referred to as epidermolysis bullosa simplex, or EBS (for reviews see Kero and Niemi, 1986; Fine, 1986; Fitzpatrick et al., 1987). Similar to CA135K14P transgenic mice, blistering in EBS can occur in response to mild trauma. Intra-epidermal separations with subsequent dermal-epidermal separations often arise directly from cytolytic disintegration of basal cells. Ultrastructural examinations of skin sections of the herpetiformis (DowlingMeara) subclass of EBS in particular have revealed gross alterations in keratin filament networks, ranging from perinuclear organization of keratin filament bundles (Pearson and Spargo, 1961) to the presence of ball-like structures assumed to be keratin (Anton-Lamprecht and Schnyder, 1982; Anton-Lamprecht, 1983; Fine, 1986; Buchbinder et al., 1986). Finally, lesions in internal stratified squamous epithelial tissues, particularly oral epithelia, have been seen in the more severe forms of EBS, where the incidence of infant mortality can be high (Buchbinder et al., 1986; Fitzpatrick et al., 1987). To explore further the possible relationship between EBS and CA135K14P transgenic mice, we examined the ultrastructure of skin from a patient diagnosed as having EBS. To determine whether the previously described cytoplasmic aggregates in EBS basal cells were indeed composed of K14 and K5, immunoelectron microscopy was required. Since this necessitated mild fixation and hence reduced preservation of tissue morphology, we chose to examine asample of nonblistering (i.e., “uninvolved”) EBS skin (Figures 8A-8C). Typical of EBS skin and similar to CAl35K14P-expressing mouse skin, dermal-basement membrane junctions did not seem to be affected (Figure 8A). Although minimal, some basal cell cytolysis could be seen even in this uninvolved region (cell denoted by arrow in Figure 8A). Desmosomal plaques appeared normal, while desmosomes sometimes showed reduced or absent bundles of tonofilaments (arrowheads in Figure 8A). Keratin filament bundles (kf) were intermixed with aggregates (ke), which were similar in appearance, although not as numerous, as those seen in CA135K14P-expressing transgenic mice. That the clumps in EBS cells were truly composed of basal keratins was shown by immunoelectron microscopy using an anti-K14 (Figure 8B) or an anti-K5 antiserum (data not shown). Gold particles were specifically localized over filaments, aggregates, and desmosome-associated keratin. The similarities between EBS and CA135K14P-expressing transgenic mouse skin extended to the differentiating cells, where a marked restoration of keratin filament networks (kf) and absence of keratin aggregates were seen (Figure 8C). In addition, the desmosomes (de) in spinous

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Figure 7. Basal and Suprabasal In Vitro

Cells from

CA135K14P-Expressing

Transgenic

Mouse

Skin and Filament

Assembly

Properties

of CA135K14P

Skin from transgenic mice 28, 3C, and lB-Fi was fixed and sectioned for regular and immunoelectron microscopy (see Experimental Procedures). (A) Three representative examples showing dermal-epidermal junctions of transgenic skin. The first frame shows a relatively healthy basal cell, while the other two show cytolyzed cells. Note that in all cases, the basement membrane and dermis were largely intact. Note also that keratin associated with hemidesmosomes was in seemingly nonfilamentous form. ke, keratin aggregates; he, hemidesmosomes; BM, basement membrane; rer, rough endoplasmic reticulum; mi, mitochondrion. (B) Anti-P immunolabeling of a section of transgenic mouse 28 epidermis depicting a basal cell. Note that keratin aggregate is densely labeled with gold particles. (C) Region of cytoplasm from mouse 28 basal cell, immunolabeled with anti-P antibodies. Note presence of a large clump of anti-P-labeled keratin

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layers appeared normal and numerous, with typical tonofilament bundles often emanating from the inner membrane plaque surface. Despite many similarities between CAl35K14P-expressing transgenic mouse skin and EBS skin, some significant differences were also noted. In particular, the skins of most CAl35Kl4P transgenic mice that we have thus far examined showed a much more severe phenotype, with gross disorganization of cells within the tissue, very few basal keratin filaments, much larger keratin aggregates, and more cytolysis. While inspection of the literature suggests that some of these features may vary with each particular EBS case and also on the degree of blistering within the sample (Anton-Lamprecht, 1983; Fine, 1986) the phenotype of the CAl35Kl4P mice we have studied was still generally more exaggerated than even the more severe EBS cases. To begin to distinguish whether these differences in severity can be attributed to species-specific variations, variations in the particular K14 or K5 mutation, or lack of genetic correlation between EBS and keratin gene mutations, we made transgenic mice expressing another truncated keratin, CA50Kl4P known to cause only mild perturbations in keratin filament assembly in vitro (Coulombe et al., 1990; see also Albers and Fuchs, 1987). Interestingly, transgenic mice expressing this truncated keratin were wild type in appearance and could be bred to homozygosity (Ft. V., K. A., and E. F., unpublished data). In contrast to EBS skin or to CAl35K14P-transgenic skin, CA50Kl4P skin showed no blistering or basal cell cytolysis. In fact, the only defect that we have noted in these mice is at the ultrastructural level: in some basal cells, aggregates of keratin (ke) could be seen interspersed with tonofilament bundles (kf) (Figure 8D). While the number of aggregates were fewer than in the EBS basal cells that we examined, their structure was strikingly similar (compare brackets in Figure 8A with those in Figure 8D). Similar to EBS skin, spinous cells of CASOKl4P-expressing mice were indistinguishable from control skin and showed no keratin aggregates (data not shown). Collectively, the phenotypes of our CAl35Kl4P and CA50Kl4P transgenics seemed to flank those of EBS, with CA135Kl4P mouse skin showing a more severe phenotype and CA50K14P skin showing a less severe phenotype. Most importantly, these findings indicated that transgenic mice can cope with at least a mildly perturbed

keratin filament network with no apparent consequences, and the severity in phenotype, including disorganization of cells within an epithelium, can vary dramatically with the particular K14 truncation utilized. Thus, if there is a genetic link between EBS and K14 or K5 gene mutations, this could explain why cases range in severity from death at or shortly after birth to mild blistering that clears with age. Discussion This report describes the effects of expressing a truncated keratin, CAl35Kl4P in the stratified squamous epithelia of transgenic mice. For this work, eleven transgenie founder mice and one transgenic Fl offspring mouse harboring the truncated Kl4P construct were examined. Eight of these mice showed appreciable CAl35K14P protein expression, and they died within 24 hr after birth. Although there may have been multiple causes of lethality, a major contributor to death seemed to arise from gross damage to the epidermis during or after birth. In at least some cases, death may have been accelerated by the mothers, who seemed to quickly recognize these transgenics as being abnormal. A direct correlation existed between expression of the truncated keratin, disruption of keratin filament assembly, basal cell cytolysis, and neonatal death. In contrast, transgenic mice expressing intact Kl4P or a less severe truncation were healthy and showed no gross alterations in phenotype. Basis for the Phenotype of Newborn CA135K14P-Expressing Mice In the developing embryo, it is not until several days prior to birth, when the epidermis stratifies, that K14 expression becomes appreciable in the basal layer (Jackson et al., 1981; Kopan and Fuchs, 1989a). Thereafter, basal-specific expression of K14 continues to increase dramatically until several days after birth. The relatively late appearance of elevated CAl35Kl4P expression was evident by the seemingly normal development that occurred in the early stages of gestation. In skin, for example, processes that precede epidermal stratification, including differentiation of the embryonic basal layer into epidermis and hair follicles (Jackson et al., 1981; Dale et al., 1985; Kopan and Fuchs, 1989a), appeared to have progressed normally in these mice. Indeed, the only gross alterations in tissue development were those involving epithelial stratification, a

adjacent to a keratin filament bundle that shows little or no labeling. This is suggestive of intracellular segregation of mutant and wild-type K14. respectively. kf, keratin filaments. (D) Representative examples of seemingly normal desmosomes (arrowheads) linking suprabasal cells of mouse 3C. Often, short or no filaments were anchored to the desmosomal plaques. (E) High magnification of desmosomal plaques from suprabasal cells of mouse 3C. Note presence of associated short filament bundles. (F) Region of outermost layers of mouse 28 skin. Note the presence of extensive keratin filaments, with only a few clumps of keratin. (G-K) In vitro assembly of purified mutant and wild-type keratins from bacterial clones. As described in Experimental Procedures, keratin filaments were assembled in vitro from anion exchange-purified K5-K14 complexes involving CA135K14P and K14P in the following ratios: 0:l control (G); 1:lOO (H); I:10 (I); I:2 (J); and 1:0 (K). Samples of the reconstituted filaments were then visualized by electron microscopy. Note that while keratins were not organized into clumps as seen in vivo in (A), filament assembly was inhibited in the presence of increasing amounts of CA135K14P The bars in (A) through (C) and (E) represent 0.5 urn; the bars in (D) and (F) represent 1 vm; the bar in (G) represents 0.1 pm and applies for (G) through (K).

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Figure

8. Skin Sections

from a Patient

with EBS and a Transgenic

Mouse

Expressing

a Milder

Keratin

Mutant,

CA50K14P

(A-C) Skin from a nonblistered region of a patient with EBS was fixed and processed for electron microscopy and for immunoelectron microscopy as described in Experimental Procedures. (A) Region from basal layer. ke, keratin clumps or aggregates; kf, keratin (tono)filaments; N, nucleus; arrowheads denote keratin attached to desmosomes. The thick arrow denotes presence of cytolyzed cell despite absence of blistering. (B) Anti-K14 immunolabeling of keratin filaments and clumps from basal cell cytoplasm. Note also labeling of keratin at desmosomes (de). All K14containing structures are also labeled with anti-K5 (data not shown). (C) Spinous layer. Note absence of keratin clumps and substantial increase in concentration of tonofilament bundles (kf). (D) Basal cell layer of skin from a transgenic mouse expressing CA50K14P a mutant keratin that was less severe than CA135K14P in its ability to disrupt keratin filament assembly in transfected keratinocytes (Albers and Fuchs, 1987) or in vitro (Coulombe et al., 1990). Note that keratin aggregates (enclosed by brackets) and desmosomal-associated keratin appear very similar to the corresponding structures in (A). Spinous, granular, and stratum corneum layers of CA50K14P mouse skin were indistinguishable from those of control mouse skin (data not shown). Bars represent 1 pm.

feature that, at least in skin, is coincident with the onset of the rise in K14 expression in the basal layer (Kopan and Fuchs, 1989a). Mutant keratin expression severely impaired proper development and maintenance of stratified squamous epi-

thelial tissue function, as judged by the rapidity with which these tissues degenerated within only a few days of the onset of appreciable K14 expression. Since stratification was quite extensive, and since CA135K14P-expressing cells could be further expanded in culture, mutant keratin

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expression did not immediately lead to cell death, although it clearly shortened the lifetime of the keratinocyte. Interestingly, the degree to which basal cells were affected seemed to increase rapidly during a time when the overall levels of basal cell K14 were increasing dramatically. Although we cannot exclude the possibility that fluctuations in the ratio of CA135K14P to K14 were a contributing factor, it seems more likely that the increase in total concentration of basal cell keratin was the primary factor leading to this phenomenon. If true, the ensuing cytolysis may have stemmed from accumulation of large aggregates of mutant keratin, rather than the lack of an extensive keratin filament network per se. Nevertheless, absence of a proper keratin filament network might have contributed to disorganization of cellular organelles, e.g., lysozomes, and subsequent cytolysis, or loss of tensile strength and structural integrity, resulting in cytolysis in response to mechanical stress. Such factors might be exacerbated during the birth process. Since K5 and K14 are both expressed and necessary for keratin filament formation in basal keratinocytes, homologous recombination to eliminate at least two genes in mice might be necessary to distinguish which abnormalities were due to lack of a proper filament network and which were the cause of accumulation of keratin aggregates. Irrespective of the underlying mechanism involved, however, our results demonstrate that stratified squamous epithelial tissues in vivo are not able to maintain proper tissue architecture when the keratin filament networks of their basal cells have been extensively disrupted. Whether this will also hold true for other intermediate filament networks in other tissues is an intriguing question and awaits further investigation. CA135K14P-Expressing Transgenic Mice and Relevance to Human Disease Keratins are the major structural proteins of epidermis, and consequently it has been of long-standing interest to know whether mutations in keratins might lead to human genetic skin diseases and indeed whether any of the myriad of genetic skin diseases of unknown etiology may in fact be due to mutations in epidermal keratin genes. Recently, many of the epidermal keratin genes have been mapped to human chromosomes 17 (type I) and 12 (type II) (Rosenberg et al., 1988; Nadeau et al., 1989; Lessin et al., 1988; Popescu et al., 1989; also M. Rosenberg, M. Le Beau, and E. Fuchs, unpublished data). Interestingly, several mutant murine skin phenotypes have also mapped to the corresponding murine chromosomes harboring the epidermal keratin genes (Nadeau et al., 1989). As mapping of keratin genes and mutant mouse skin diseases becomes more refined, as possible relations between mouse and human skin diseases are established, and as chromosomal linkages to human skin diseases are identified, this approach will clearly be an important one in the quest for understanding the molecular defects involved in the etiology of human skin diseases. An alternative approach to examine whether a keratin gene mutation might lead to a genetic skin disease is to create a mutation in a keratin gene and determine whether

it can generate a dominant phenotype in vitro and in vivo. Previous in vitro studies had indicated that some keratin mutations were dominant in their ability to disrupt endogenous keratin filament networks in transfected cultured cells (Albers and Fuchs, 1987, 1989; Lu and Lane, 1990). A priori, we reasoned that if disruption of keratin filament networks were also deleterious in vivo, then natural mutations in the simple epithelial keratin genes (encoding K7, K8, K18, and K19; Lu and Lane, 1990) might give rise to embryonic lethality, since these genes are expressed in the embryonic ectoderm during the early development of skin (Jackson et al., 1981). However, keratin mutations in stratified squamous epithelia, particularly the skin, should not have gone unnoticed. Indeed, our transgenic studies have not only verified the dominant behavior of mutations such as CA135K14P in vivo, but more importantly, they have yielded new phenotypes attributable to these mutations, thereby providing strong clues as to which human genetic skin diseases might be candidates for natural mutations in K5 and K14. Because no known genetic disease has stood out as an obvious candidate for a natural keratin gene mutation, our experiments have necessarily been exploratory in nature. This said, the parallels were both striking and extensive between transgenic mouse skin expressing truncated K14 and the EBS class of human genetic skin diseases. Considering the diagnostic hallmarks of the herpetiformis (Dowling-Meara) subclass of EBS, namely a notable reduction in numbers of tonofilament bundles and concomitant presence of protein aggregates within the cytoplasm of basal keratinocytes, it seems remarkable that attempts have not been made to analyze biochemically the keratins synthesized by patients with this or other forms of EBS. To our knowledge, the only data on keratin expression in skin from any EBS class stem from immunohistochemical studies (Tidman et al., 1988). In part, intermediate filament extracts and SDS-PAGE analyses may not have been attempted because keratin patterns in diseases are often difficult to interpret due to changes in the differentiation program and natural proteolytic processmg of keratins. In addition, however, there are other unusual features of EBS, such as blistering and basal cell cytolysis, that were not thought to be likely consequences of mutant keratin expression and which have instigated investigations into possible enzymatic defects in EBS (see, for example, Savolainen et al., 1980; Sanchez et al., 1983). Thus far, our data suggest that many keratin gene mutations, e.g., CA50K14P may have no apparent phenotype, while other K5 and K14 gene mutations, e.g., CAl35K14P or somewhat less severe mutations, might be able to account for some EBS cases. However, even if K5 or K14 mutations are involved, the etiology of EBS subgroups may be complex, and there may be other mutations, perhaps in genes encoding intermediate filament-associated proteins, desmosomal proteins, or proteolytic enzymes, that might also give rise to an EBS phenotype. Hence, while more extensive analyses are required, including cloning and sequencing of K5 and K14 cDNAs from skin mRNAs of EBS patients, a number of EBS cases will need to be examined before the etiology of this class of disease is

Cell 378

fully understood. Nevertheless, while our findings are necessarily speculative at this stage, they provide evidence that the pioneering observations associating keratin filament disorganization with some EBS cases (Pearson and Spargo, 1961; Anton-Lamprecht and Schnyder, 1982; Anton-Lamprecht, 1983; Fine, 1986; Kitajima et al., 1989) may in fact be the most important clue to the genetic basis for this subclass of diseases. Interestingly, the linkage of blistering, cytolysis, and tonofilament clumping to filament-disrupting mutations in keratin genes suggests a molecular basis for several additional types of human genetic skin diseases. One such disease is bullous ichthyosiform erythroderma (epidermolytic hyperkeratosis), in which basal cells are normal, but tonofilament clumping and blistering begin at the first suprabasal layer (Anton-Lamprecht, 1983; Fitzpatrick et al., 1987). While other mutations may also give rise to such a phenotype, a mutation in one of the differentiationspecific keratin genes might be predicted to generate these abnormalities. In addition, there are several other diseases, such as Darier’s disease and familial benign pemphigus, that have been associated in part with abnormalities in keratin filament organization (Anton-Lamprecht, 1983; Fitzpatrick et al., 1987; Burge, 1989). While the phenotype of our mutant keratin-expressing mice do not correlate as well with these diseases as with EBS, it is still possible that species-specific or mutation-specific differences may account for this. As more extensive analyses are conducted, both on keratin gene mutants in transgenie mice and on the biochemistry and molecular biology of genetic disorders involving blistering and alterations in keratin filament organization, the extent to which keratin gene mutations play a role in the manifestations of these diseases should become more apparent. Experimental Procedures Plasmid Construction for the Transgene Plasmid pgCA135K14PI(-6000) was made in several steps using conventional DNA recombinant technology. Essentially,the sequences extending from the first EcoRl site at 6000 bp Sof the transcription start site of the human K14 gene to the first Bglll site shown in Figure 1 came from GKl, a genomic clone containing the human K14 gene (Marchuk et al., 1984). The sequences extending from the first Bglll site to the end of the P tag came from pJK14CA135 (Albers and Fuchs, 1987) and include the sequences encoding the truncated end of K14, followed by the P tag and a TGA stop codon. The remaining sequences to the 3’ EcoRl site include -800 bp of 3’downstream sequences from the human K14 gene (Vassar et al., 1969). Intermediate Filament Protein Extraction, Electrophoresis, and lmmunoblot Analysis Intermediate filament proteins were isolated by the procedure of Wu et al. (1982). Final pellets were resuspended in 8 M urea and 10% P-mercaptoethanol, and protein concentrations were determined by the method of Bradford (1976). Intermediate filament proteins were resolved using SDS-PAGE, and either stained with Coomassie blue or electrophoretically transferred to nitrocellulose paper for immunoblot analysis as described by Choi and Fuchs (1990). Antisera used were anti-P rabbit polyclonal (Wako Chemicals, Dallas, TX) and antiK14 rabbit polyclonal (Stoler et al., 1988). Primary antibody binding was detected with alkaline phosphate-coupled goat anti-rabbit IgG (Bio-Rad, Richmond, CA), according to the manufacturer’s specifications.

Preparation and Identification of Transgenlc Mice Mutant keratin genes were isolated from plasmid expression vectors and microinjected into male pronuclei of single cell embryos from an outbred strain (CD-l) of mice with a 19 day gestation period (Charles River Laboratories, Wilmington, MA). Isolation of embryos and microinjection methods were as described by Vassar et al. (1989). Mouse ear or tail DNAs were isolated and assayed by PCR analysis for the presence of human K14P sequences. Primers chosen were specific for human K14P sequences and gave rise to a 500 bp diagnostic fragment in all and only DNAs from mice harboring the CA135K14P gene. This band was detected by agarose gel electrophoresis. lmmunohistochemistry: Light Microscopy Level Tissues from control and transgenic mice were placed in Bouin’s fixative for 2 hr at room temperature. Following fixation, samples were dehydrated, embedded in paraffin, and sectioned (5 pm). Sections were processed for immunogold enhancement immunohistochemistry as described by the manufacturer (Janssen Life Science Products, Piscataway, NJ). Primary antibodies used for staining were a 1:200 dilution of anti-K14 (Stoler et al., 1988); a 1:200 dilution of a rabbit polyclonal antiserum against gel-purified 63 kd human stratum corneum keratin (Fuchs and Green, 1978); a 1:lOO dilution of a rat monoclonal antibody, NCl/34, against the C-terminus of neuropeptide substance P (Accurate Corp., Westbury, NY); a 1:200 dilution of a rabbit polyclonal anti-mouse filaggrin antiserum (courtesy of Beverly Dale, University of Washington, Seattle, WA); and a 1:200 dilution of a rabbit polyclonal anti-K6 antiserum (courtesy of Dennis Roop, Baylor University, Houston, TX). Immunocytochemlstry: Electron Microscopy Level For regular electron microscopy, mouse or EBS human tissues were fixed in 2% glutaraldehyde and postfixed in 0.8% osmium tetroxide, followed by embedding in LX-112 (Ladd Research Industries, Inc.. Burlington, VT). For immunoelectron studies, tissues were fixed in 4% paraformaldehyde and embedded in Lowicryl K4M according to Stromer and Bendayan (1988) with some modifications, as described by Coulombe et al. (1989). lmmunogold labeling was carried out as described by Coulombe et al. (1989), using either rabbit polyclonal anti-P antiserum (Wake Chemicals, Dallas, TX), used at a 1:200 dilution; rabbit polyclonal anti-K14 antiserum (Stoler et al., 1988), used at a 1:300 dilution; or guinea pig anti-K5 antiserum (R. Lersch and E. Fuchs, unpublished data), used at a 1:200 dilution. Gold particle-conjugated (15 nm) secondary antibodies (Amersham Corporation, Arlington Heights, IL) were used, followed by washing, drying, and staining as described (Coulombe et al., 1989). Prepared grids were examined using a Philips CM-10 electron microscope. The specificity of cytochemical labeling was assessed through repeating the above procedure using preimmune sera, rather than primary anti-keratin antisera, and repeating the above procedure, but omitting the primary antiserum incubation step. Overexpression of Keratlns in E. coli, FPLC Purification, and In Vitro Filament Assembly Bacterial clones of BL21(DE3), transformed with plasmids PET-K5, PET-K14F’, or pETCA135K14P were derived, cultivated, and induced to express keratin as described previously (Coulombe and Fuchs, 1990; Coulombe et al., 1990). Following purification, human K5 was combined with human K14P and/or CA135K14R and tetrameric complexes were isolated by FPLC anion exchange chromatography as described (Coulombe et al., 1990). In vitro filament assembly was performed according to Coulombe and Fuchs (1990). and negatively stained filaments (1% uranyl acetate) were visualized with a Philips CM-10 electron microscope. Magnification was calibrated using a #lo021 diffraction grating replica from Ernest Fulham, Inc. (NY).

We thank Grazina Traska for her expert technical assistance in culturing keratinocytes from the transgenic mice and Anton Callaway for his assistance in PCR analyses of mouse DNAs. In addition, we thank Diane Gingras (Department of Anatomy, University of Montreal, Montreal, Canada) for her help in preparing electron microscopy sections, Dr. Robert Josephs for enabling us to use his electron microscope fa-

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cility, and Gerald Grofman for printing electron micrographs. Special thanks go to Dr. Adelaide A. Hebert (University of Texas Health Science Center, Houston, TX) for providing us with EBS tissue, Dr. Dennis Roop (Baylor University, Houston, TX) for anti-mouse K6 antiserum, and Dr. Beverly Dale (University of Washington, Seattle, WA) for anti-mouse filaggrin antiserum. Finally, we thank Philip Galigafor his artful presentation of the data. This work is supported by a grant from the National Institutes of Health (AR27663). E. F is an Investigator of the Howard Hughes Medical Institute. R. V. is a graduate student supported by an NIH Predoctoral Training Grant in Molecular Biology. P. A. C. is the recipient of a Centennial Fellowship from the Medical Research Council of Canada. K. A. was a recipient of a Postdoctoral Fellowship from the Dermatology Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

September

13, 1990

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Mutant keratin expression in transgenic mice causes marked abnormalities resembling a human genetic skin disease.

To explore the relationship between keratin gene mutations and genetic disease, we made transgenic mice expressing a mutant keratin in the basal layer...
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