Cell type-specific expression of bovine keratin genes as demonstrated by the use of complementary DNA clones

Cytokeratins are a family of polypeptides that form the intermediate-sized filament characteristic of epithelial cells. The cytoskeletons of different types of epithelial cells have been reported to possess specific combinations of the members of this protein family. Therefore, we have sought to examine the correspondence between such differential protein expression and the expression of cytokeratin genes at the nucleic acid level. A library of recombinant plasmids carrying cDNA sequences synthesized from bovine epidermal mRNAs was constructed. Clones of about 10(3) base-pairs coding for all the major epidermal keratins of molecular weights of 50,000, 54,000, 59,000, 60,000 and 68,000 were identified by means of hybridization-selection, followed by one and two-dimensional gel electrophoresis of products of translation in vitro. Under stringent conditions, each of these clones hybridizes specifically with its corresponding mRNA and does not show significant cross-hybridization with mRNAs coding for the other keratins, including those belonging to the same subfamily. Using these clones in RNA blot hybridization analysis, we have studied the expression of keratin genes in diverse bovine epithelial tissues (muzzle epidermis, cornea, esophagus, bladder urothelium, liver) and cultured cell lines from kidney (MDBK) and mammary gland (BMGE + H, BMGE -H). In each case we have found a correlation between the respective keratin polypeptides and the corresponding mRNAs. Whereas mRNA coding for keratins Ia and VIb have been found only in epidermis, genes coding for other epidermal keratins are expressed also in certain non-epidermal epithelia and in cells of the BMGE + H line. In contrast, epidermal keratin mRNA sequences have not been detected in liver or bladder tissue, nor in cultured kidney cells (MDBK) or mammary gland cells of the BMGE - H line, which all express a set of cytokeratin polypeptides entirely different from those of epidermis. In all cases, only one mRNA size species has been found, suggesting that in different cell types the same mRNA species is synthesized from the same keratin gene. We conclude that the mechanisms controlling the cell type-specific synthesis of the diverse keratin genes act at a pre-translational level.     

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.     

Integration of different keratins into the same filament system after microinjection of mRNA for epidermal keratins into kidney epithelial cells

We have isolated poly (A)+ RNA, highly enriched in keratin mRNA from bovine muzzle epidermis, and injected it into epithelial cells of a different type, i.e., cultured kidney epithelial cells of the same (MDBK) or taxonomically distant (PtK2) species. Both recipient cell lines contain keratin polypeptides that are different from those present in epidermal cells. Using keratin subtype-specific antibodies in immunofluorescence and immunoelectron microscopy, we show that foreign keratin mRNAs when injected into a different type of epithelial cell can recruit polyribosomes and are translated together with the keratin mRNAs of the host cell. Foreign epidermal keratins are excluded from vimentin filaments and other structures but readily coassemble with the endogenous keratins and appear to be integrated into the meshwork of the preexisting kidney-type keratin filaments. Our observations indicate that different sets of keratin polypeptides from the same or different species can coassemble in the living cell into a common filament system. Thus we have developed a procedure that allows experimental alteration of the intermediate filament cytoskeleton within living epithelial cells.     

Sequence and expression of a human type II mesothelial keratin

Using mRNA from cultured human mesothelial cells, we constructed bacterial plasmids and lambda phage vectors that contained cDNA sequences specific for the keratins expressed in these cells. A cloned cDNA encoding keratin K7 (55 kD) was identified by positive hybrid selection. Southern Blot analysis indicated that this sequence is represented only once in the human genome, and Northern Blot analysis demonstrated that the gene encoding K7 is expressed in abundance in cultured bronchial and mesothelial cells, but only weakly in cultured epidermal cells and not at all in liver, colon, or exocervical tissue. The predicted amino acid sequence of this keratin has revealed a striking difference between this keratin and the type II keratins expressed in epidermal cells: whereas all of the epidermal type II keratins thus far sequenced have long nonhelical termini rich in glycine and serine, this mesothelial type II keratin has amino and carboxy terminal regions that are unusually short and lack the inexact repeats of glycine and serine residues.     

Tissue specificity of epithelial keratins: differential expression of mRNAs from two multigene families.

Human epithelial cells cultured from stratified and simple squamous tissues all produce keratins of 40,000 to 58,000 daltons, but within this range the number and sizes vary with different epithelial cells. We have shown that this tissue-specific variation in the keratins is not due to posttranslational modification or processing, but rather to the differential expression of a family of heterogeneous but closely related mRNAs. All of these epithelial keratin mRNAs can be further grouped into two distinct subfamilies by their ability to hybridize with either of two cloned epidermal keratin cDNAs. All of the keratin mRNAs hybridize to one or the other, but not both, of the two cloned cDNAs. However, the mRNAs within each group hybridize with varying degrees of stringency, indicating that they are of similar but not identical sequence. Both types of keratin mRNAs are always expressed in every epithelial cell line studied, suggesting that filament assembly is dependent on the presence of both types of keratins. Within each of these two groups, the slight sequence differences in each class may reflect subtle tissue-specific variations in the structural and functional requirements of the epithelial cytoskeleton.     

Translational products of mRNAs coding for non-epidermal cytokeratins

Total RNA and poly(A)+ RNA were isolated from tissues and cultured cells of various mammalian species (bovine muzzle epidermis and bladder urothelium; rat hepatoma cells; human cell lines HeLa, MCF-7 and A-431) and examined by translation in vitro using the reticulocyte lysate system. Polypeptides were separated and identified by two-dimensional electrophoresis and cytokeratins were selectively enriched from the translation assays by co-polymerization with added heterologous cytokeratins. In all three species, non-epidermal cytokeratins A, D and mol. wt. 40,000 (corresponding to numbers 8, 18 and 19 of the human cytokeratin catalog of Moll et al., 1982) were identified as translation products capable of co-polymerization with epidermal keratins. Several other basic and other acidic cytokeratins were also identified as translational products. In addition, two unidentified polypeptides (mol. wt. 52,000 and 43,000) which were minor polypeptides in cytoskeletons and translation assays were found to be specifically enriched in co-polymers with bovine epidermal keratins. The results indicate that many, perhaps all, non-epidermal cytokeratins characteristic of simple epithelia are genuine products of translation and that their diversity is not due to post-translational modification or processing. These findings, taken together with observations of in vitro translation of epidermal mRNAs, suggest that the diversity of cell type-specific expression of the different members of the cytokeratin polypeptide family is largely due to the cell type-specific synthesis of diverse mRNAs.     

Isolation of messenger RNA coding for the "fast" protein of embryonic chick feathers.

The messenger RNA coding for the "Fast" protein of embryonic chick feathers has been purified from the overwhelming relative amounts of keratin mRNA which are present in the developing feathers. The "Fast" protein mRNA represents about 4-8% of the total mRNA population of the feather. Despite differences between the size of the "Fast" proteins and the keratins the two mRNA species are very similar in molecular weight as judged by electrophoresis under denaturing conditions. However, by electrophoresis in 8 M urea gels at 55 degrees C, the "Fast" protein mRNA could be separated from keratin mRNA, presumably reflecting differences in messenger RNA secondary structure.     

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.     

Identification of two types of keratin polypeptides within the acidic cytokeratin subfamily I

Cytoskeletal filaments of the α-keratin type (cytokeratins) are a characteristic of epithelial cells. In diverse mammals (man, cow and rodents) these cytokeratins consist of a family of approximately 20 polypeptides, which may be divided into the more acidic (I) and the more basic (II) subfamilies. These two subfamilies show only limited amino acid sequence homology. In contrast, nucleic acid hybridization experiments and peptide maps have been interpreted to show that polypeptides of the same subfamily share extended sequence homology.We compare two polypeptides of the acidic cytokeratin subfamily, VIb (M_r 54,000) and VII (M_r 50,000), which are co-expressed in large amounts in bovine epidermal keratinocytes. These two epidermal keratins can be distinguished by specific antibodies and show different patterns of expression among several bovine tissues and cultured cells. In addition, they differ in the stability of their complexes with basic keratin polypeptides and in their tryptic peptide maps. The amino acid sequences deduced from the nucleotide sequences of complementary DNA clones containing the 3′ ends of the messenger RNAs for these keratins are compared with each other and with available amino acid sequences of human, murine and amphibian epidermal keratins. Bovine keratins VIb and VII share considerable sequence homology in the α-helical portion (68% residues identical) but lack significant homology in the extrahelical portion. Bovine keratin VIb shows, in its α-helical region, a pronounced sequence homology (88% identity) to the murine epidermal keratin of M_r 59,000. In addition, the non-helical carboxy-terminal regions of both proteins are glycinerich and contain a canonic sequence GGG^S_GYGG, which may be repeated several times. Moreover, their mRNAs present a highly conserved stretch of 236 nucleotides containing, in the murine sequence, the end of the coding and all of the non-coding region (81% identical nucleotides). Bovine keratin VII is considerably different from the murine M_r 59,000 keratin but is almost identical to the human cytokeratin number 14 of M_r 50,000, both in the α-helical and in the non-α-helical regions of the proteins, and the mRNAs of the human and the bovine keratins also display a high homology in their 3′ non-coding ends.The results show that in the same species keratins of the same subfamily can differ considerably, whereas equivalent keratin polypeptides of different species are readily identified by characteristic sequence homologies in the α-helical and the non-helical regions as well as in the 3′ non-coding portions of their mRNAs. Among the members of the acidic subfamily I of cytokeratin polypeptides that are co-expressed in bovine epidermis, at least two types can be distinguished by their carboxy-terminal sequences. One type is characterized by its abundance of glycine residues, a consensus GGG^S_GYGG heptapeptide sequence, which may be repeated several times, and an extended stretch of high RNA sequence homology in the 3′ non-coding part. The other type shows a predominance of serine and valine residues, a subterminal GGG^S_GYGG sequence (which has been maintained in Xenopus, cow and man) and also a high level of homology in the 3′ non-coding part of the mRNA. The data indicate that individual keratin type specificity overrides species diversity, both at the protein and the mRNA level. We discuss the evolutionary conservation and the tissue distribution of these two types of acidic keratin polypeptides as well as their possible biological functions.     

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.     

Amino acid sequence of the carboxy-terminal part of an acidic type I cytokeratin of molecular weight 51 000 from Xenopus laevis epidermis as predicted from the cDNA sequence

The DNA sequence of a clone from a cDNA library made from Xenopus laevis skin is described. This sequence represents the 3'-terminal end of an mRNA which codes for an epidermal cytokeratin polypeptide of mol. wt. 51 000 of the acidic (type I) subfamily as identified by hybridization-selection of mRNAs, followed by gel electrophoretic identification of the polypeptides synthesized by translation in vitro. The partial amino acid sequence of the amphibian cytokeratin shows strong similarity to type I cytoskeletal keratins from human (mol. wt. 50 000) and murine (mol. wt. 59 000) epidermis. In the non alpha-helical tail region the human and the non-mammalian (Xenopus) keratins are more similar to each other than to the murine protein, indicating that the former are equivalent cytokeratin polypeptides and belonging to a special subclass of type I keratin polypeptides devoid of glycine-rich regions in the carboxy-terminal portion. The evolutionary conservativity of the genes coding for cytokeratins is discussed.     

Use of monospecific antisera and cRNA probes to localize the major changes in keratin expression during normal and abnormal epidermal differentiation

We report here the isolation and characterization of three antisera, each of which is specific for a single keratin from one of the three different pairs (K1/K10, K14/K5, K16/K6) that are differentially expressed in normal human epidermis and in epidermal diseases of hyperproliferation. We have used these antisera in conjunction with monospecific cRNA probes for epidermal keratin mRNAs to investigate pathways of differentiation in human epidermis and epidermal diseases in vivo and in epidermal cells cultured from normal skin and from squamous cell carcinomas in vitro. Specifically, our results suggest that: (a) the basal-specific keratin mRNAs are down-regulated upon commitment to terminal differentiation, but their encoded proteins are stable, and can be detected throughout the spinous layers; (b) the hyperproliferation-associated keratin mRNAs are expressed at a low level throughout normal epidermis when their encoded proteins are not expressed, but are synthesized at high levels in the suprabasal layers of hyperproliferating epidermis, coincident with the induced expression of the hyperproliferation-associated keratins in these cells; and (c) concomitantly with the induction of the hyperproliferation-associated keratins in the suprabasal layers of the epidermis is the down-regulation of the expression of the terminal differentiation-specific keratins. These data have important implications for our understanding of normal epidermal differentiation and the deviations from this process in the course of epidermal diseases of hyperproliferation.     

Differential expression of keratin genes during mouse development

Suprabasal layers of the newborn mouse epidermis contain two mRNAs of 2.0 and 2.4 kb which are translated into keratins of 59 and 67 kDa, respectively. To study their expression during development, cDNA sequences corresponding to the 2.0- and the 2.4-kb mRNAs were cloned, characterized by hybridization selection assay, and used as probes to detect keratin sequences in polyadenylated RNA from Day 11, 13, 15, and 17 embryos. In RNA from Day 11 of gestation, two RNAs of 2.8 and 1.8 kb were identified. They were found to have homologies with both epidermal RNAs, suggesting that they are coding for proteins of the keratin family. These two sequences were not detected in sample of later stages. RNAs comigrating with the two epidermal keratin RNAs were identified only in Day 15 and 17 embryos indicating that their expression was induced between Day 13 and 15. Finally, the localization of the 59-kDa keratin mRNA was examined by in situ hybridization. The spinous and granulous cell layers were found to be heavily covered with grains while other regions of the tissue sections were unlabeled. All these results support the hypothesis of a sequential expression of keratins during differentiation of epidermal cells and suggest that proteins related to the keratins expressed specifically in keratinizing cells are expressed earlier during development.     

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.     

Genetic polymorphism of silk fibroin studied by two-dimensional translation pause fingerprints.

Silk fibroins from a number of B mori strains have been compared, and at least six different polypeptide size phenotypes, which show apparent molecular weights of 365,000–415,000 in SDS polyacrylamide gels, have been identified. Analysis of the corresponding fibroin messenger RNAs in denaturing gels shows that mRNA size is largely correlated with polypeptide length. The mRNAs vary in size from 5.50 × 10⁶ to 6.30 × 10⁶ daltons. It has been shown elsewhere that the translation of silk fibroin mRNA in a reticulocyte cell-free system proceeds discontinuously. In this paper, I demonstrate that this discontinuous translation phenomenon can be exploited to map the location of divergent amino acid sequences in fibroin variants. SDS gel analysis of translational pause patterns shows that divergence arises internally after a relatively long amino-terminal sequence which appears to be conserved. Two-dimensional gel analysis using V8 protease digestion in the second dimension produces fingerprints of fibroin peptide fragments ordered from the amino to the carboxyl terminus of the protein. These fingerprints provide additional evidence for extensive internal divergence of the fibroins and a reduced degree of divergence near the termini. A plausible explanation for the observed genetic variability is the occurrence of relatively large unequal crossing-over exchanges in the repetitive domain of the fibroin gene.     

The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies

The keratins are a highly heterogeneous group of proteins that form intermediate filaments in a wide variety of epithelial cells. These proteins can be divided into at least seven major classes according to their molecular weight and their immunological reactivity with monoclonal antibodies. Tissue-distribution studies have revealed a correlation between the expression of specific keratin classes and different morphological features of in vivo epithelial differentiation (simple vs. stratified; keratinized vs. nonkeratinized). Specifically, a 50,000- and a 58,000-dalton keratin class were found in all stratified epithelia but not in simple epithelia, and a 56,500- and a 65-67,000-dalton keratin class were found only in keratinized epidermis. To determine whether these keratin classes can serve as markers for identifying epithelial cells in culture, we analyzed cytoskeletal proteins from various cultured human cells by the immunoblot technique using AE1 and AE3 monoclonal antikeratin antibodies. The 56,500- and 65-67,000-dalton keratins were not expressed in any cultured epithelial cells examined so far, reflecting the fact that none of them underwent morphological keratinization. The 50,000- and 58,000-dalton keratin classes were detected in all cultured cells that originated from stratified squamous epithelia, but not in cells that originated from simple epithelia. Furthermore, human epidermal cells growing as a monolayer in low calcium medium continued to express the 50,000- and 58,000-dalton keratin classes. These findings suggest that the 50,000- and 58,000-dalton keratin classes may be regarded as "permanent" markers for stratified squamous epithelial cells (keratinocytes), and that the expression of these keratin markers does not depend on the process of cellular stratification. The selective expression of the 50,000- and 58,000-dalton keratin classes, which are synthesized in large quantities on a per cell basis, may explain the high keratin content of cultured keratinocytes.     

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.