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.

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) (AEl-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 non hyperproliferative 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, diseasespecific keratin changes in the epidermal disorders studied so far implies that keratin abnormalities probably represent the consequence, rather than the cause, of these diseases.The keratins are a group of water-insoluble proteins (40-70 Kdaltons [kd])' that form 10-nm desmosome-associated tonofilaments in all epithelia (15,17,18,61,63,64). Four major keratins (50, 56.5, 58, and 65-67 kd) have been identified in normal human epidermis (7, 21, 40, 72; cf. 4, 49, 62); significant alterations in epidermal keratin composition can occur, however, as a result of disease (1,3,5,25,26,28,33,34,36,37,40,50,52,54,66,71) as well as changes in cellular growth environment (10,20,29,55,61,62 -67-kd keratins (1, 5, 8, 37, 50, 54, 66). In addition, Thaler et al. (65,66) reported that two water-insoluble proteins of -54 and 57 kd are unique to psoriatic scale, although Baden et al. (5) found no "psoriasis-specific" keratin changes . Hunter and Skerrow (26) reported that certain abnormal keratin polypeptides ranging from 55-63 kd (probably corresponding to 50-58 kd in our gel system) are present in the scales of psoriasis, atopic dermatitis, and seborrheic dermatitis, as well as in the stratum comeum oftape-stripped epidermis . Interestingly, Kubilus et al. (28) found that keratins of another disease, basal cell 13...

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 (pl <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 (pl >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, iscelectric 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 .The keratins represent a family of water-insoluble proteins (40-70-kilodalton [kd]') that form intermediate filaments (tonofilaments) in epidermal cells as well as almost all other epithelial cells (9,12,13,55,57,58). Unlike other types of intermediate filaments (vimentin, desmin, glial filament, and neurofilament) which are relatively simple in subunit composition (for review, see 31), keratin composition is highly heterogeneous and varies depending on epithelial cell type, period of embryonic development, stage of epithelial differentiation, disease state, and cellular growth environment (for references, see 23,34,37,61,65).To investigate the biological significance of keratin heterogeneity, we have produced three monoclonal antibodies (designated AE 1, AE2, and AE3) against human epidermal keratins (67). Immunofluorescence data have established that 'Abbreviations used in this paper. EGF, epidermal growth factor; HC, hydrocortisone; kd, kilodalton; NEpHG, nonequilibrium-pHgradient. these antibodies are highly specific for keratin-type intermediate filaments. Immunoblot analysis further suggested that AEI and AE3 antibodies react with two different groups of keratins and, in combination, recognize most keratin species (61,67). Using these antibodies, we have shown that the expression ofspecific keratins can be correlated with different types of epithelial differentiation (simple vs. stratified, keratinized vs. nonkeratinized; 61 ; cf. 11, 34), and that individual epide...

No abstract

Abstract. The four major keratins of normal human epidermis (molecular mass 50, 56.5, 58, and 65-67 kD) can be subdivided on the basis of charge into two subfamilies (acidic 50-kD and 56.5-kD keratins vs. relatively basic 58-kD and 65-67-kD keratins) or subdivided on the basis of co-expression into two "pairs" (50-kD/58-kD keratin pair synthesized by basal cells vs. 56.5-kD/65-67-kD keratin pair expressed in suprabasal cells). Acidic and basic subfamilies were separated by ion exchange chromatography in 8.5 M urea and tested for their ability to reassemble into 10-nm filaments in vitro. The two keratins in either subfamily did not reassemble into 10-nm filaments unless combined with members of the other subfamily, While electron microscopy of acidic and basic keratins equilibrated in 4.5 M urea showed that keratins within each subfamily formed distinct oligomeric structures, possibly representing precursors in filament assembly, chemical cross-linking followed by gel analysis revealed dimers and larger oligomers only when subfamilies were combined. In addition, among the four major keratins, the acidic 50-kD and basic 58-kD keratins showed preferential association even in 8.5 M urea, enabling us to isolate a 50-kD/58-kD keratin complex by gel filtration. This isolated 50-kD/58-kD keratin pair readily formed 10-nm filaments in vitro.These results demonstrate that in tissues containing multiple keratins, two keratins are sufficient for filament assembly, but one keratin from each subfamily is required. More importantly, these data provide the first evidence for the structural significance of specific co-expressed acidic/basic keratin pairs in the formation of epithelial 10-nm filaments.T HE intermediate-sized filaments (10-nm diameter) of epithelial cells are composed of a family of waterinsoluble proteins termed keratins (molecular mass 40-70 kD). Although ~20 different human keratin polypeptides have been identified, data from monoclonal antikeratin antibody reactivity (4,6, 11,35), nucleic acid hybridization (9, 13), peptide mapping (25), and two-dimensional gel electrophoresis (17, 32) have demonstrated that all keratins can be subdivided into two subfamilies: acidic keratins (isoelectric point < pH 5.5) and relatively basic keratins (isoelectric point > pH 6.0). Detailed analysis of the keratins in various epithelia has shown that only 2-10 keratins are expressed in any individual tissue, but at least one member of each subfamily is always represented (4,17,25,32,35,41). More recently, Franke and co-workers have demonstrated the association of specific acidic and basic keratins as "complexes" in polyacrylamide gels containing 4.0-9.5 M urea (7). In rat hepatocytes, which contain only a relatively basic 55-kD keratin (A) and an acidic 49-kD keratin (D), the complex observed in 4 M urea gels was shown to represent heterotypic tetramers which could reassemble in vitro into 10-nm filaments (22). Taken together with earlier data suggesting that at least two different keratins are required for keratin filament reco...