Classification of epidermal keratins according to their immunoreactivity, isoelectric point, and mode of expression

Human epidermal keratinocytes express under various growth conditions a total of at least nine keratins that can be divided into two subfamilies. Subfamily A comprises 40-, 46-, 48-, 50-/50'-, and 56.5-kilodalton (kd) keratins which are relatively acidic (pI less than 5.5) and, with the exception of 46-kd keratin, are recognized by AE1 monoclonal antibody. Subfamily B comprises 52-, 56-, 58-, and 65-67-kd keratins which are relatively basic (pI greater than 6) and are recognized by AE3 monoclonal antibody. Within each keratin subfamily, there is a constant member (50-/50'- and 58-kd keratins of the subfamilies A and B, respectively) that is always expressed. The other seven keratins of both subfamilies are variable members whose expression depends upon the cellular differentiated state, which is in turn modulated by the growth environment. The 56.5-kd keratin (subfamily A) and the 65-67-kd keratins (subfamily B) are coordinately expressed during keratinization. In contrast, the 40-, 46-, and 48-kd keratins (subfamily A) and the 52- and 56-kd keratins (subfamily B) are characteristic of cultured epidermal cells forming nonkeratinized colonies. These results demonstrate that human epidermal keratins can be classified according to their reactivity with monoclonal antikeratin antibodies, isoelectric point, and mode of expression. The classification of keratins into various subgroups may have important implications for the mechanisms of epidermal differentiation, the evolution of keratin heterogeneity, and the use of keratin markers for tumor diagnosis.     

Correlation of specific keratins with different types of epithelial differentiation: monoclonal antibody studies

We have prepared three monoclonal antibodies against human epidermal keratins. These antibodies were highly specific for keratins and, in combination, recognized all major epidermal keratins of several mammalian species. We have used these antibodies to study the tissue distribution of epidermis-related keratins. In various mammalian epithelia, the antibodies recognized seven classes of keratins defined by their immunological reactivity and size. The 40, 46 and 52 kilodalton (kd) keratin classes were present in almost all epithelia; the 50 kd and 58 kd keratin classes were detected in all stratified squamous epithelia, but not in any simple epithelia; and the 56 kd and 65-67 kd keratin classes were unique to keratinized epidermis. Thus the expression of specific keratin classes appeared to correlate with different types of epithelial differentiation (simple versus stratified; keratinized versus nonkeratinized).     

Changes in keratin gene expression during terminal differentiation of the keratinocyte

Cells of the inner layers of the epidermis contain small keratins (46-58K), whereas the cells of the outer layers contain large keratins (63-67K) in addition to small ones. The changes in keratin composition that take place within each cell during the course of its terminal differentiation result largely from changes in synthesis. Cultured epidermal cells resemble cells of the inner layers of the epidermis in synthesizing only small keratins. The cultured cells possess translatable mRNA only for small keratins, whereas mRNA extracted from whole epidermis can be translated into both large and small keratins. As no synthesis takes place in the outermost layer of the epidermis (stratum corneum), the keratins of this layer must be synthesized earlier, but in some cases they then become smaller: this presumably occurs by post-translational processing of the molecules during the final stages of differentiation. Stratified squamous epithelia of internal organs do not form a typical stratum corneum and do not make the large keratins characteristic of epidermis. Their keratins are also different from those of cultured keratinocytes, implying that they have embarked on an alternate route of terminal keratin synthesis.     

Regulation of terminal differentiation of cultured human keratinocytes by vitamin A

Vitamin A is known to exert an important influence on epithelial differentiation. The fetal calf serum supplement of cell-culture medium contains enough of the vitamin to affect the differentiation of cultured keratinocytes derived from epidermis and from other stratified squamous epithelia. The cellular and molecular properties of the cultures are altered when the medium is supplemented with serum from which the vitamin A has been removed by solvent extraction (delipidized serum). Cell motility is reduced, the adhesiveness of cells increases and pattern formation is prevented. In both epidermal and conjunctival keratinocytes, removal of vitamin A leads to the synthesis of a 67 kd keratin characteristic of terminally differentiating epidermis and to much reduced synthesis of the 52 kd and 40 kd keratins typical of conjunctiva. These changes, both cellular and molecular, are reversed by the addition of retinyl acetate to the medium containing delipidized serum. Cell motility and pattern formation are restored, and detachment of the most mature cells from the surface of the stratified epithelium is promoted. Synthesis of the 67 kd keratin is prevented and the synthesis of the 40 and 52 kd keratins is stimulated. The nature of the keratins synthesized is regulated by the concentration of vitamin A, and each cell type adjusts its synthesis differently at a given vitamin concentration.     

Tissue-specific expression of keratin proteins in human esophageal and epidermal epithelium and their cultured keratinocytes

In contrast to the simplified keratin content of bovine, rabbit, and rat esophageal epithelium (composed mainly of a 57 and 46 or 51 kD keratin, depending on the animal species), human esophageal epithelium contained a quantitatively different array of keratin proteins, ranging in molecular weight from 37 to 61 kD. The pattern of keratin proteins from human esophageal epithelium differed qualitatively and quantitatively from that of human epidermis. Human esophageal epithelium lacked the 63, 65, and 67 kD keratins characteristic of human epidermis, consistent with the absence of a granular layer and an anucleate stratum corneum. Moreover, human esophageal epithelium contained a distinctive 61 kD keratin protein which was either not present or present in only small amounts in human epidermis and variable amounts of a 37 kD keratin. Whereas the 56, 59, and 67 kD keratins were the most abundant keratins in human epidermis, the 52, 57, and 61 kD keratins predominated in human esophageal epithelium. During in vitro cultivation, both human epidermal and esophageal keratinocytes produce colonies which are stratified, but the morphologic appearance of these cultured epithelia differs. Only cultured human epidermal keratinocytes contain keratohyalin granules in the outermost layers and a prominent 67 kD keratin on immunoprecipitation. Otherwise the keratin contents appear similar. In conclusion, human esophageal epithelium exhibited intertissue and interspecies differences in the pattern of keratin proteins. During in vitro cultivation, human esophageal keratinocytes retained some aspects of their distinctive program of differentiation.     

Vitamin adeficiency and keratin biosynthesis in cultured hamster trachea

Summary Tracheas from vitamin A-deficient hamsters in organ culture in vitamin A-free medium developed squamous metaplasia. Addition of retinyl acetate to the medium prevented squamous metaplasia and a mucociliary epithelium was maintained. Indirect immunofluorescent staining with antikeratin antibodies AE1 and AE3 indicated positive reactions with epithelium of tracheas either cultured in vitamin A-free or retinyl acetate (RAc)-containing medium. The “stratum corneum”-like squames in metaplastic tracheas were strongly stained by AE3. Immunoprecipitation of cytoskeletal extracts from [³⁵S]methionine labeled tracheas with a multivalent keratin antiserum indicated that the concentration of keratins synthesized in tracheas cultured in vitamin A-free medium was greater than that observed in tracheas cultured in the presence of RAc. In addition, new species of keratin were expressed in tracheas cultured in RAc-free medium. Alterations in the program of keratin synthesis were clearly detectable after 1 d in vitamin A-free medium, even though squamous metaplasia was not yet obvious. Squamous tracheas were shown by immunoblot analysis to contain keratins of 50, 48, 46.5, and 45 kilodalton (kd) detected with AE1; and 58, 56, and 52 kd detected with AE3. Immunoblot analysis with monospecific antimouse keratin sera also demonstrated the presence of 60, 55, and 50 kd keratins in the metaplastic tracheas. All these various species of keratins were either absent or present in much reduced quantity in mucociliary tracheas in RAc-containing medium. Interestingly, the induction of squamous metaplasia in tracheal epithelium did not result in the expression of the 59 and 67 kd keratins which are characteristically expressed in the differentiated layers of the epidermis. Therefore, this study shows that squamous metaplasia of tracheas due to vitamin A-free cultivation is accompanied by an increase in keratin synthesis as well as by the appearance of keratin species not normally present in mucociliary tracheal epithelium.     

Keratin alterations during embryonic epidermal differentiation: a presage of adult epidermal maturation

Differentiation of the epidermis during embryonic rabbit development was found to be accompanied by dramatic changes in keratin proteins. Immunofluorescent labeling with keratin antiserum revealed that the undifferentiated epithelium of 12-d embryos was already committed to making keratin proteins. At 18 d of embryogenesis, the epithelium contained keratin proteins in the molecular weight range of 40,000-59,000. The stratification of the epithelium into two cell layers at 20 d of development coincided with the appearance of a 65-kdalton keratin. When a thick stratum corneum developed at 29 d, several additional keratins became prominent, most notably the large keratins (61- and 64-kdalton) and a 54-kdalton keratin. In addition, the 40-kdalton keratin, which had been present in earlier embryonic epidermis, disappeared. Newborn epidermis resembled that of a 29-d embryonic epidermis, with the exception of the appearance or increase in concentration of two more keratin species (46- and 50-kdalton). In vitro culturing of keratinocytes from 12- and 14-d embryonic skin demonstrated that these cells contained essentially the same keratin profiles as the undifferentiated epithelium of 18-d embryos (40-59 kdalton). Keratinocytes grown from older embryos contained increased amounts of keratin, similar to the in vivo situation, but did not synthesize the high molecular weight keratins. The changes observed during embryonic epidermal differentiation appear to be recapitulated during the sequential maturation steps of adult epidermis.     

Keratin filaments of cultured human epidermal cells. Formation of intermolecular disulfide bonds during terminal differentiation.

Human epidermal cells grown in culture synthesize abundant keratins. These keratins are similar to those of stratum corneum of human epidermal callus in their insolubility in dilute aqueous buffers, their molecular weight range of 40,000 to 60,000, their immunolgical reactivity, and their ability to assemble into 80 A tonofilaments in vitro; but there are differences in the molecular weights of some of the proteins, the number of components, and their charge heterogeneity, related at least in part to phosphorylation. About 30% of all the proteins of living cultured keratinocytes consists of keratins, compared with over 85% of stratum corneum. All the keratins of human stratum corneum were found to be cross-linked by intermolecular disulfide bonds while most keratins of the living cells were not. As the cells mature in Methocel-stabilized suspension culture, their keratins become increasingly disulfide cross-linked. When uncross-linked tonofilaments of living keratinocytes are dissolved in 8 M urea and the filaments reconstituted in vitro their keratins become disulfide cross-linked under aerobic conditions and consequently insoluble in solutions of 8 M urea or sodium dodecyl sulfate. The results indicate that the uncross-linked state of the keratins in living cells is due to the reducing intracellular environment and not to a precursor state related to the primary structure of the proteins. The disulfide cross-links stabilizing the keratin filaments must be distinguished from the epsilon-(gamma-glutamyl)lysine cross-links stabilizing the cornified cell envelope.     

Monoclonal antibody analysis of keratin expression in epidermal diseases: a 48- and 56-kdalton keratin as molecular markers for hyperproliferative keratinocytes

The polypeptide composition of epidermal keratin varies in disease. To better understand the biological meaning of these variations, we have analyzed keratins from a number of human epidermal diseases by the immunoblot technique using AE1 and AE3 monoclonal antikeratin antibodies. The results reveal a continuous spectrum of keratin expression ranging from one closely resembling the normal in vivo pattern to one almost identical to cultured epidermal keratinocytes. Specifically, a 50-kilodalton (kd) (AE1-positive) and a 58-kd (AE3-positive) keratin are present in all diseases, supporting the concept that they represent "permanent" markers for keratinocytes. A 56.5-kd (AE1) and a 65-67-kd (AE3) keratin, previously shown to be markers for keratinization, are expressed only by lesions retaining a keratinized morphology. A 48-kd (AE1) and a 56-kd (AE3) keratin are present in all hyperproliferative (para- or nonkeratinized) disorders, but not in normal abdominal epidermis or in ichthyosis vulgaris which is a nonhyperproliferative disease. These two keratins have previously been found in various nonepidermal keratinocytes undergoing hyperproliferation, suggesting that these keratins are not epidermis-specific and may represent markers for hyperproliferative keratinocytes in general. In various epidermal diseases, there is a reciprocal expression of the (keratin) markers for hyperproliferation and keratinization, supporting the mutual exclusiveness of the two cellular events. Moreover, our results indicate that, as far as keratin expression is concerned, cultured human epidermal cells resemble and thus may be regarded as a model for epidermal hyperplasia. Finally, the apparent lack of any major, disease-specific keratin changes in the epidermal disorders studied so far implies that keratin abnormalities probably represent the consequence, rather than the cause, of these diseases.     

Altered patterns of keratin synthesis in human epidermal keratinocytes transformed by SV40

Transformation of human epidermal keratinocytes by the oncogenic virus SV40 is a stage-specific process in which normal patterns of differentiation are progressively altered over time following infection. Within the context of this scheme, we examined the keratins produced by the infected cells. Immunofluorescence studies indicated that viral infection led to the formation of variant cells visibly lacking the normal keratin cytoskeleton after about 10-15 serial passages (60-90 cell generations) post infection. Analyses of variant cell formation in clonal populations grown on palladium islands revealed that the variants were derived within 2-3 cell divisions from cells containing an apparently normal keratin cytoskeleton, but that variant formation depended upon cell density. Immunoprecipitation of 35S-methionine labelled keratins from the infected keratinocytes revealed a gradual loss of the normal 46, 50, 56 and 58Kd keratin species over a period of many months after infection. The loss of the normal keratins was accompanied by the appearance of at least two species in the 48-52Kd size range not present in uninfected cells and the enhancement of a third, 40Kd, protein quite early after infection. Analysis of the altered keratin patterns on two-dimensional acrylamide gels using either isoelectric focusing (IEF) or non-equilibrium pH gradient electrophoresis (NEPHG) along the first dimension showed that the infected cells produced basic keratins which increased in relative abundance as cells became more transformed with serial passage including at least five isoelectric forms not seen in uninfected cells. Translation of poly A+ RNAs from the infected cells indicated that the altered keratin synthesis probably reflects changes in the translatable mRNA pool.     

Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies

Three monoclonal antibodies (AE1, AE2, and AE3) were prepared against human epidermal keratins and used to study keratin expression during normal epidermal differentiation. Immunofluorescence staining data suggested that the antibodies were specific for keratin-type intermediate filaments. The reactivity of these antibodies to individual human epidermal keratin polypeptides (65-67, 58, 56, and 50 kdaltons) was determined by the immunoblot technique. AE1 reacted with 56 and 50 kdalton keratins, AE2 with 65-67 and 56-kdalton keratins, and AE3 with 65-67 and 58 kdalton keratins. Thus all major epidermal keratins were recognized by at least one of the monoclonal antibodies. Moreover, common antigenic determinants were present in subsets of epidermal keratins. To correlate the expression of specific keratins with different stages of in vivo epidermal differentiation, the antibodies were used for immunohistochemical staining of frozen skin sections. AE1 reacted with epidermal basal cells, AE2 with cells above the basal layer, and AE3 with the entire epidermis. The observation that AE1 and AE2 antibodies (which recognized a common 56 kdalton keratin) stained mutually exclusive parts of the epidermis suggested that certain keratin antigens must be masked in situ. This was shown to be the case by direct analysis of keratins extracted from serial, horizontal skin sections using the immunoblot technique. The results from these immunohistochemical and biochemical approaches suggested that: (a) the 65- to 67-kdalton keratins were present only in cells above the basal layer, (b) the 58-kdalton keratin was detected throughout the entire epidermis including the basal layer, (c) the 56- kdalton keratin was absent in the basal layer and first appeared probably in the upper spinous layer, and (d) the 50-kdalton keratin was the only other major keratin detected in the basal layer and was normally eliminated during s. corneum formation. The 56 and 65-67- kdalton keratins, which are characteristic of epidermal cells undergoing terminal differentiation, may be regarded as molecular markers for keratinization.     

Immunofluorescent staining of keratin fibers in cultured cells.

Antibody prepared against a group of keratins purified from human stratum corneum was used to identify cells containing keratins by immunofluorescence. In sectioned tissue and in culture, keratinocytes of skin and other stratified squamous epithelia-whether human, rabbit of mouse-stained strongly, indicating homologous amino acid sequences in the keratins of these species. In all cases, the antibody revealed a dense cytoplasmic network of discrete fibers probably consisting of aggregated (tono-) filaments. The pattern of staining was not affected by cytochalasin B or colcemid. No keratins were detected in cultured cells of mesenchymal origin (3T3, NIL, BHK, human diploid fibroblasts) or in connective tissues, indicating that the 100 A filaments of fibroblasts are not related to the keratins. Keratinocytes at all stages of differentiation, including basal cells, stained brightly and therefore contained abundant keratins.     

Keratin protein expression during the development of conducting airway epithelium in nonhuman primates

Keratin protein expression during the development of Rhesus monkey conducting airway epithelium was investigated by both biochemical and immunohistochemical methods. Keratin proteins were extracted from tracheal and intrapulmonary airway tissues of fetal (at 80- and 140-day gestational ages), neonatal, and adult animals. Using immunoblot analyses and immunohistochemistry with various monoclonal (AE1, AE3, AE8, 6.01 and 6.11) and monospecific antibodies (anti-50/55 and anti-40 kDa), the presence of keratins 5, 6, 8, 13, 14, and 19 in adult airway epithelium were demonstrated. Except for keratin 13 (51 kDa), the remaining keratins could be immunologically detected in fetal and neonatal tissues. To further understand the nature of the synthesis of keratin 13 during development, airway epithelial cells from different ages were isolated and cultured in vitro. Cultured cells were labeled with 35S-methionine, and the patterns of keratin protein were analyzed by one- and two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Results indicated that the cultured airway cells synthesized additional keratins including 7, 15, 16, 17, and 18. However, consistent with the in vivo finding, fetal cells synthesized less or no keratin 13. These in vivo and in vitro studies strongly suggest that the synthesis of the keratin 13 in monkey conducting airway epithelium is developmentally regulated.     

The cDNA sequence of a human epidermal keratin: divergence of sequence but conservation of structure among intermediate filament proteins

We have determined the DNA sequence of a cloned cDNA that is complementary to the mRNA for the 50 kilodalton (kd) human epidermal keratin. This provides the first amino acid sequence for a cytoskeletal keratin. Comparison of this sequence with those of other keratins reveals an evolutionary relationship between the cytoskeletal and the microfibrillar keratins, but shows no homology to matrix or feather keratins. The 50 kd keratin shares 28%-30% homology with partial sequences of other intermediate filament proteins, which suggests that keratins may be the most distantly related members of this class of fibrous proteins. Our computer analyses predict that the 50 kd keratin contains two long alpha-helical domains separated by a cluster of helix-inhibitory residues in the middle of the protein. These findings indicate that despite major sequence divergence among intermediate filament proteins, they retain sequences compatible with secondary structural features that appear to be common to all of them.     

The expression of keratin genes in epidermis and cultured epidermal cells

Cultured human epidermal cells and human stratum corneum (callus) contain a number of keratins of different molecular size, but the size distribution is not the same in the two cases. To characterize these keratins in more detail, we compared them by amino acid analysis, immunological reactivity and one-dimensional peptide mapping (Cleveland et al., 1977). No differences in amino acid composition could be detected among keratins of stratum corneum differing in molecular size by as much as 50%, suggesting that some repeating structure may be present in these molecules. Examination of polypeptide fragments produced by partial enzymatic hydrolysis showed strong similarities among all the keratins of stratum corneum and of cultured epidermal cells, even extending to the keratins of rodents; but the keratins of similar size, whether of stratum corneum or cultured cells, were more closely related than keratins of different size. This conclusion was supported by studies of the immunological reactivity of the keratins.How the epidermal cell generates a family of keratins is a problem of considerable interest. The differences in size and structure between the keratins of stratum corneum and cultured epidermal cells suggest that the epidermal cell can modify the expression of its keratin genes.     

Distribution profiles of keratin proteins during rat amelogenesis

Summary We examined rat cells undergoing amelogenesis for the presence of three types of keratin proteins using a polyclonal antibody to keratin (against total keratins (TK) with molecular masses ranging from 41 to 65 kilodaltons (kd) and monoclonal antibodies keratins to KL1 and PKK1 (reactive with keratins with molecular masses of 55–57 and 41–56 kd, respectively). In normal oral epithelia from young rats, the TK, KL1, and PKK1 antibodies bound to all of the epithelial strata. The epithelial cap on the top of incisors and the dental lamina of molar teeth exhibited strong TK staining, moderate staining KL1, and little or no PKK1 staining. In developing molar enamel organs, both the outer and inner enamel epithelia, the stratum intermedium, and stellate reticulum cells were all positively stained by the TK immunoreagent. In developing incisors, TK only bound strongly to stratum-intermedium cells, and no KL1 and PKK1 staining antibodies was observed in ameloblasts or the stratum intermedium.     

Distribution profiles of keratin proteins during rat amelogenesis

We examined rat cells undergoing amelogenesis for the presence of three types of keratin proteins using a polyclonal antibody to keratin (against total keratins (TK) with molecular masses ranging from 41 to 65 kilodaltons (kd) and monoclonal antibodies keratins to KL1 and PKK1 (reactive with keratins with molecular masses of 55-57 and 41-56 kd, respectively). In normal oral epithelia from young rats, the TK, KL1, and PKK1 antibodies bound to all of the epithelial strata. The epithelial cap on the top of incisors and the dental lamina of molar teeth exhibited strong TK staining, moderate staining KL1, and little or no PKK1 staining. In developing molar enamel organs, both the outer and inner enamel epithelia, the stratum intermedium, and stellate reticulum cells were all positively stained by the TK immunoreagent. In developing incisors, TK only bound strongly to stratum-intermedium cells, and no KL1 and PKK1 staining antibodies was observed in ameloblasts or the stratum intermedium.     

Sequential expression of mRNA-encoded keratin sets in neonatal mouse epidermis: basal cells with properties of terminally differentiating cells

The keratin pattern of newborn mouse epidermis was investigated during terminal differentiation. In highly pure fractions of basal and suprabasal cells, obtained by Percoll density gradient centrifugation, we identified two sets of mRNA-encoded proteins: a basal set of 58.5, 52, and 47 kd subunits and a suprabasal set of 67 and 60 kd subunits. The large subunits of each set were alkaline to neutral, while the small subunits were acidic. Polyclonal antibodies against the suprabasal, acidic 60 kd protein and the basal, alkaline 58.5 kd protein selectively recognized their antigens in immunoblots of NEPHGE -resolved keratins and decorated the corresponding epidermal compartments in frozen sections. The antibody to the suprabasal 60 kd protein also recognized distinct cells in the basal cell layer. Quantification of this cell population revealed a 10% cell fraction, morphologically indistinguishable from the total cell population, that, in addition to expressing basal keratin proteins, was already synthesizing suprabasal keratin subunits.     

Isopeptide bond formation in epidermis

Summary Proteins which are major substrates of epidermal transglutaminases can be identified in cultured keratinocytes of human, cow, and new-born rat.Cow and human keratinocytes both contain substrate proteins which are 30000 to 50000 daltons in size but dissociable in SDS to 12000 daltons or less. In both species these proteins correspond to in vivo synthesized proteins which are probable precursors of cornified envelope. Human keratinocytes synthesize a 125000 dalton protein which is also a precursor of cornified envelope both in cells and tissue. By SDS electrophoresis two 100000 dalton substrate proteins are seen in cow keratinocyte extracts and a 23000 dalton substrate protein is seen in rat keratinocyte extracts. Minor substrates of transglutaminase are seen in human keratinocytes, and one has been isolated by preparative electrophoresis. Major structural proteins of epidermis which are in vitro substrates of epidermal transglutaminase include the keratins and the stratum corneum basic protein.     

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 of their stratified squamous epithelia. These mice exhibited abnormalities in epidermal architecture and often died prematurely. Blistering occurred easily, and basal cell cytolysis was evidence 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.