Patterns of expression of trichocytic and epithelial cytokeratins in mammalian tissues. I. Human and bovine hair follicles

The cytokeratin family of intermediate filament (IF) proteins can be grouped into the epithelial polypeptides ("soft alpha-keratins"), of which at least 19 exist in the various human epithelia, and the hair-type cytokeratins ("hard alpha-keratins"), which are typical of trichocytes, i.e., the living hair-forming cells. We have recently shown [34] that the hair follicles from diverse mammalian species contain a set of eight major cytokeratin polypeptides, four each of the acidic (type I) and the basic (type II) subfamily, which are different from all known epithelial cytokeratins. In addition, we have identified two new minor trichocytic cytokeratin polypeptides, designated Hax (type I) and Hbx (type II). Antibodies against trichocytic cytokeratins that do not crossreact with any of the epithelial cytokeratins have enabled us to study the expression of both kinds of cytokeratin in the various cell types of human and bovine hair follicles. Using immunofluorescence microscopy, we have observed intense reactions of trichocytic cytokeratins only in cells contributing to the forming hairs, i.e., hair shaft, medulla and cuticle, whereas immunostaining of the peribulbar matrix cells was weaker, if at all detectable. In contrast, epithelial cytokeratins were localized in both the inner and outer root sheath epithelia but, surprisingly, also in certain portions of the trichocyte column, notably cells of the cuticle, certain medullary cells, and trichocytes of the basalmost peripapillary cell layers. Cells coexpressing trichocytic and epithelial cytokeratins have been identified by double-label immunofluorescence microscopy. Epithelial cytokeratins of the inner and outer root sheath epithelia include, most remarkably, "simple-epithelium-type" cytokeratins 8, 18, and 19; these occur in certain peribulbar regions, in distinct patterns, but with variable frequencies. The occurrence of simple epithelial cytokeratins in hair follicles has also been confirmed by high-sensitivity immunoblotting of follicular polypeptides separated by gel electrophoresis. Vimentin-positive cells were abundantly interspersed (in some follicles, but not in all) between the trichocytes of the peripapillary cone, most of them probably being melanocytes. The cell-type complexity of the hair follicle and the different patterns of cytoskeletal protein expression in the various hair follicle cells are discussed in relation to the development and growth of this organ.     

Patterns of expression of trichocytic and epithelial cytokeratins in mammalian tissues

Cells forming hair and nail material are characterized by the synthesis of members of a particular group of alpha-keratin polypeptides (trichocytic cytokeratins. "T cytokeratins") different from epithelial cytokeratins ("E cytokeratins"). As the precursor cells to trichocytes are derived from fetal epidermal keratinocytes expressing only E cytokeratins, we have studied the patterns of expression of both T and E cytokeratins in developing human hair-and nail-forming tissues of different fetal stages, by immunocytochemistry using antibodies specific for certain T or E cytokeratins and by two-dimensional gel electrophoresis and immunoblotting. In developing hair follicles up to the early bulbous-peg stage (weeks 12-15 of gestational age), only certain E but no T cytokeratins were identified. T cytokeratins were first detected in the late bulbous-peg stage (in week-14 scalp skin) in certain cells of the central part of the hair cone. In hair-producing follicles (weeks 18-25), the lower hair matrix cells were positive for certain E cytokeratins, whereas T cytokeratins appeared in the uppermost portion of the matrix and, most prominently, in the maturing trichocytes. From the late bulbous-peg stage on. E cytokeratin antibody Ks13.1 selectively decorated the inner root sheath. In finger nail "anlagen", T cytokeratins were detected first in week 12 and 13 fetuses, specifically in cells of the lunula region. In more-advanced stages of nail formation, expression of T cytokeratins extended not only to the upper layers of the ventral nail matrix but was also found, albeit more sparsely, in cells of the whole nail-bed epithelium. Throughout these developmental stages, coexpression of T and E cytokeratins was noted in certain cells, including E cytokeratin 19. While in earlier stages E cytokeratins 10/11, characteristic of epidermal-type cornification, were noted in different regions, including the superficial stratum of the nail bed epithelium, they were later restricted to the epithelium of the proximal nail fold. The results show that terminal trichocytic differentiation starts, both in ontogeny and during the steady growth of hairs and nails, in cells expressing E cytokeratins and that coexpression of E and T polypeptides occurs in both kinds of appendages. While in the hair follicle, the change to the exclusive synthesis of T cytokeratins appears to take place relatively abruptly and simply, the development of nail structures from the ventral nail matrix seems to be more gradual and is characterized by more-complex patterns of coexpression of both kinds of cytokeratins.     

Monoclonal antibodies to human cytokeratins: application to various epithelial and mesothelial cells

Immunohistological analysis of human tissue using monoclonal antibodies against cytokeratins, which are confined to cells of epithelial origin, is a valuable technique. Using human epidermal keratins as antigen, we prepared monoclonal antibodies against cytokeratins (ZK1, ZK7, ZK61 and ZK99) and against a desmosomal protein (ZK31). Immunohistochemical staining of human skin sections using these antibodies showed a specific reaction with the epidermis: ZK1 stained the entire epidermis, ZK7 only the basal layer, ZK61 and ZK99 the suprabasal layers, and ZK31 the cellular interfaces. In order to test for antibody specificity, immunoblots with human epidermal and amnion epithelial cytokeratin polypeptides, as well as immunofluorescence microscopy of simple epithelia (glandular and simple columnar epithelia) were performed. ZK1, ZK61 and ZK99 reacted preferentially with cytokeratin polypeptides of stratified squamous epithelia and ZK7 recognized cytokeratins of stratified and simple epithelia. When the ZK antibodies were tested on mesothelial cells in pleural effusions, only ZK7 reacted with these cells. Biochemical analysis of cytokeratin accumulation in cells of primary and long-term cultures indicated that the cytokeratin pattern of mesothelial cells was quite unstable, while that of amnion epithelial cells showed only minor quantitative changes. The use of these antibodies to determine the epithelial origin of cells present in pleural effusions is proposed.     

Diversity of cytokeratins. Differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues

Epithelial cells contain a cytoskeletal system of intermediate-sized (7 to 11 nm) filaments formed by proteins related to epidermal keratins (cytokeratins). Cytoskeletal proteins from different epithelial tissues (e.g. epidermis and basaliomas, cornea, tongue, esophagus, liver, intestine, uterus) of various species (man, cow, rat, mouse) as well as from diverse cultured epithelial cells have been analyzed by one and two-dimensional gel electrophoresis. Major cytokeratin polypeptides are identified by immunological cross-reaction and phosphorylated cytokeratins by [³²P]phosphate labeling in vivo.It is shown that different epithelia exhibit different patterns of cytokeratin polypeptides varying in molecular weights (range: 40,000 to 68,000) and electrical charges (isoelectric pH range: 5 to 8.5). Basic cytokeratins, which usually represent the largest cytokeratins in those cells in which they occur, have been found in all stratified squamous epithelia examined, and in a murine keratinocyte line (HEL) but not in hepatocytes and intestinal cells, and in most other cell cultures including HeLa cells. Cell type-specificity of cytokeratin patterns is much more pronounced than species diversity. Anatomically related epithelia can express similar patterns of cytokeratin polypeptides. Carcinomas and cultured epithelial cells often continue to synthesize cytokeratins characteristic of their tissue of origin but may also produce, in addition or alternatively, other cytokeratins. It is concluded: (1) unlike other types of intermediate-sized filaments, cytokeratin filaments are highly heterogeneous in composition and can contain basic polypeptides: (2) structurally indistinguishable filaments of the same class, i.e. cytokeratin filaments, are formed, in different epithelial cells of the same species, by different proteins of the cytokeratin family; (3) vertebrate genomes contain relatively large numbers of different cytokeratin genes which are expressed in programs characteristic of specific routes of epithelial differentiation; (4) individual cytokeratins provide tissue- or cell type-specific markers that are useful in the definition and identification of the relatedness or the origin of epithelial and carcinoma cells.     

Expression of simple epithelial cytokeratins in bovine pulmonary microvascular endothelial cells.

Polypeptides of bovine aortic, pulmonary artery, and pulmonary microvascular endothelial cells, as well as vascular smooth muscle cells and retinal pericytes were evaluated by two-dimensional gel electrophoresis. The principal cytoskeletal proteins in all of these cell types were actin, vimentin, tropomyosin, and tubulin. Cultured pulmonary microvascular endothelial cells also expressed 12 unique polypeptides including a 41 kd acidic type I and two isoforms of a 52 kd basic type II simple epithelial cytokeratin microvascular endothelial cell expression of the simple epithelial cytokeratins was maintained in cultured in the presence or absence of retinal-derived growth factor, and regardless of whether cells were cultured on gelatin, fibronectin, collagen I, collagen IV, laminin, basement membrane proteins, or plastic. Cytokeratin expression was maintained through at least 50 population doublings in culture. The expression of cytokeratins was found to be regulated by cell density. Pulmonary microvascular endothelial cells seeded at 2.5 X 10(5) cell/cm2 (confluent seeding) expressed 3.5 times more cytokeratins than cells seeded at 1.25 X 10(4) cells/cm2 (sparse seeding). Vimentin expression was not altered by cell density. By indirect immunofluorescence microscopy it was determined that the cytokeratins were distributed cytoplasmically at subconfluent cell densities but that cytokeratin 19 sometimes localized at regions of cell-cell contact after cells reached confluence. Vimentin had a cytoplasmic distribution regardless of cell density. These results suggest that pulmonary microvascular endothelial cell have a distinctive cytoskeleton that may provide them with functionally unique properties when compared with endothelial cells derived from the macrovasculature. In conjunction with conventional endothelial cell markers, the presence of simple epithelial cytokeratins may be an important biochemical criterion for identifying pulmonary microvascular endothelial cells.     

Co-expression of cytokeratins and neurofilament proteins in a permanent cell line: cultured rat PC12 cells combine neuronal and epithelial features

The cytoskeleton of the rat cultured cell line PC12, which is widely used in cell biology as a model system for neuron-like differentiation, displays an unusual combination of intermediate-sized filaments (IFs). As determined by electron microscopy, immunolocalization, and biochemical analyses, these cells contain, in addition to neurofilaments, an extended meshwork of bundles of cytokeratin IFs comprising cytokeratins A and D, equivalent to human cytokeratin polypeptides Nos. 8 and 18, irrespective of whether they are grown in the presence or absence of nerve growth factor. The two IF systems differ in their fibrillar arrays, the neurofilaments being concentrated in perinuclear aggregates similar to those found in certain neuroendocrine tumors of epithelial origin. We conclude that PC12 cells permanently co-express IFs of both the epithelial and the neuronal type and thus present an IF combination different from those of adrenal medulla cells and pheochromocytomas, i.e., the putative cells of origin of the line PC12. The IF cytoskeleton of PC12 cells resembles that of various neuroendocrine tumors derived from epithelial cells. The results show that the development of a number of typical neuronal differentiation features is compatible with the existence of an epithelial type IF cytoskeleton, i.e., cytokeratins. The implications of these findings concerning the validity of the PC12 cell line as a model for neuronal differentiation and possible explanations of the origin of cells with this type of IF co-expression are discussed.     

The complement of native α-keratin polypeptides of hair-forming cells: A subset of eight polypeptides that differ from epithelial cytokeratins

Living hair-forming cells (trichocytes) were obtained from basal portions of human, bovine and ovine hair-follicles, free from contaminations of root-sheath epithelia. Their intermediate filament (IF) cytoskeleton was studied by gel electrophoresis of the native, i.e. non-S-carboxymethylated polypeptides, by peptide-map analysis of the individual components, by reconstitution experiments and by immunological methods. The IF protein complement of trichocytes from all three species is characterized by a very similar set of eight highly conserved alpha-keratin polypeptides, comprising four members of the basic (type II; Mr 56,500-60,000) and four members of the acidic (type I; Mr 41,000-44,000) cytokeratin subfamily. None of these eight trichocyte alpha-keratin polypeptides, which form heterotypic complexes and IF in vivo and in vitro, is identical to any of the epithelial cytokeratins of the same species. All the trichocyte-specific cytokeratins are native polypeptides encoded by different mRNAs, as demonstrated by in vitro translation of hair follicle mRNA. The same polypeptides are also found in mature hairs, although with different patterns of modification. Our study provides the first analysis of the native unmodified alpha-keratin polypeptides of trichocytes and hairs and therefore allows a direct comparison of these with the epithelial cytokeratins and other IF proteins from the same species. These findings indicate that, during fetal hair-follicle formation, the differentiation of trichocytes from epithelial cells involves a complete cessation of the synthesis of epithelial cytokeratins and a marked induction of the synthesis of a complex set of trichocyte-specific cytokeratins.     

Localization of cytokeratins in tissues of the rainbow trout: Fundamental differences in expression pattern between fish and higher vertebrates

Using a panel of antibodies against different cytokeratins in immunofluorescence microscopy on frozen tissue sections and two-dimensional gel electrophoresis of cytoskeletal proteins from these tissues, we have studied the tissue distribution of cytokeratins in a fish, the rainbow trout Salmo gairdneri. We have distinguished at least 14 different cytokeratin polypeptides in only a limited number of tissues, thus demonstrating the great complexity of the cytokeratin pattern in a fish species. The simplest cytokeratin pattern was that present in hepatocytes, comprising one type-II (L1) and two type-I (L2, L3) polypeptides that appear to be related to mammalian cytokeratins 8 and 18, respectively. Two or all three cytokeratins of this group were also identified in several other epithelial tissues, such as kidney. Epithelia associated with the digestive tract contained, in addition, other major tissue-specific cytokeratins, such as components D1-D3 (stomach, intestine and swim bladder) and B1 and B2 (biliary tract). With the exception of D1, all these polypeptides were also found in a cultured cell line (RTG-2). Epidermal keratinocytes contained D1 and six other major cytokeratins, termed E1-E6. The most complex cytokeratin pattern was that found in the gill epithelium. Surprisingly, antibodies specific for cytokeratins of the L1-L3 group also reacted with certain cell-sheet-forming tissues that are not considered typical epithelia and in higher vertebrates express primarily, if not exclusively, vimentin. Such tissues were (a) endothelia, including the pillar cells of the "gill filaments", (b) scale-associated cells, and (c) the ocular lens epithelium, and also several nonepithelial cell types, such as (d) fibroblasts and other mesenchymal cells, (e) chondrocytes, (f) certain vascular smooth muscle cells, and (g) astroglial cells of the optic nerve. The differences between the patterns of cytokeratin expression in this fish species and those of higher vertebrates are discussed. It is concluded that the diversity of cytokeratins has already been established in lower vertebrates such as fish, but that the tissue-expression pattern of certain cytokeratins has been restricted during vertebrate evolution. We discuss the value of antibodies specific for individual cytokeratin polypeptides as marker molecules indicating cell and tissue differentiation in fish histology, embryology, and pathology.     

Molecular characterization and expression of the stratification-related cytokeratins 4 and 15

A number of human cytokeratins are expressed during the development of stratified epithelia from one-layered polar epithelia and continue to be expressed in several adult epithelial tissues. For studies of the regulation of the synthesis of stratification-related cytokeratins in internal tissues, we have prepared cDNA and genomic clones encoding cytokeratin 4, as a representative of the basic (type II) cytokeratin subfamily and cytokeratin 15, as representative of the acidic (type I) subfamily, and determined their nucleotide sequences. The specific expression of mRNAs encoding these two polypeptides in certain stratified tissues and cultured cell lines is demonstrated by Northern blot hybridization. Hybridization in situ with antisense riboprobes and/or synthetic oligonucleotides shows the presence of cytokeratin 15 mRNA in all layers of esophagus, whereas cytokeratin 4 mRNA tends to be suprabasally enriched, although to degrees varying in different regions. We conclude that the expression of the genes encoding these stratification-related cytokeratins starts already in the basal cell layer and does not depend on vertical differentiation and detachment from the basal lamina. Our results also show that simple epithelial and stratification-related cytokeratins can be coexpressed in basal cell layers of certain stratified epithelia such as esophagus. Implications of these findings for epithelial differentiation and the formation of squamous cell carcinomas are discussed.     

Monoclonal antibodies to various acidic (type I) cytokeratins of stratified epithelia

We determined the reactivity of two monoclonal antibodies to cytokeratins that are typically expressed in certain stratified epithelia and several human squamous cell carcinomas using immunoblotting techniques and immunofluorescence microscopy. Antibody KS 8.12 reacted specifically with cytokeratin polypeptides nos. 13 and 16, and stained noncornified squamous epithelia in a rather uniform way. The examination of diverse human carcinomas showed all squamous cell carcinomas to be positively stained with this antibody, whereas all adenocarcinomas were negative. Another antibody, KK 8.60, reacted with polypeptides nos. 10 and 11, and uniformly stained the suprabasal layers of the epidermis. In several noncornified squamous epithelia (e.g., tongue, exocervix), in thymus reticulum epithelial cells, and in moderately and well differentiated squamous cell carcinomas this antibody exhibited a nonuniform labeling pattern that allowed the detection of individual cytokeratin-10/11-positive cells scattered throughout the tissue. It is concluded that antibodies KS 8.12 and KK 8.60 represent specific molecular probes for the definition of certain stages of squamous differentiation in normal development as well as in pathological processes such as squamous metaplasia and carcinogenesis. We propose the use of these antibodies in the differential diagnosis of carcinomas and their metastases.     

Intermediate filament cytoskeleton of amnion epithelium and cultured amnion epithelial cells: expression of epidermal cytokeratins in cells of a simple epithelium

Using immunofluorescence microscopy and two-dimensional gel electrophoresis, we compared the cytoskeletal proteins expressed by human amnion epithelium in situ, obtained from pregnancies of from 10-wk to birth, with the corresponding proteins from cultured amnion epithelial cells and cultures of cells from the amniotic fluid of 16 week pregnancies. Epithelia of week 16 fetuses already display tissue-specific patterns of cytokeratin polypeptides which are similar, although not identical, to those of the corresponding adult tissues. In the case of the simple amnion epithelium, a complex and characteristic complement of cytokeratin polypeptides of Mr 58,000 (No. 5), 56,000 (No. 6), 54,000 (No. 7), 52,500 (No. 8), 50,000 (No. 14), 46,000 (No. 17), 45,000 (No. 18), and 40,000 (No. 19) is present by week 10 of pregnancy and is essentially maintained until birth, with the addition of cytokeratin No. 4 (Mr 59,000) and the disappearance of No. 7 (Mr 54,000) at week 16 of pregnancy. In full-term placentae, the amnion epithelium displays two morphologically distinct regions, i.e., a simple and a stratified epithelium, both of which express the typical amnion cytokeratin polypeptides. However, in addition the stratified epithelium also synthesizes large amounts of special epidermal cytokeratins such as No. 1 (Mr 68,000), 10 (Mr 56,500), and 11 (Mr 56,000). In culture amnion epithelial cells obtained from either 16-wk pregnancies or full-term placentae will continue to synthesize the amnion-typical cytokeratin pattern, except for a loss of detection of component No. 4. This pattern is considerably different from the cytokeratins synthesized by cultures of cells from amniotic fluids (cytokeratins No. 7, 8, 18, and 19, sometimes with trace amounts of No. 17) and from several so-called "amnion epithelial cell lines." In addition, amnion epithelial cells in situ as well as amnion epithelial cell cultures appear to be heterogeneous in that they possess some cells that co-express cytokeratins and vimentin. These observations lead to several important conclusions: In contrast to the general concept of recent literature, positively charged cytokeratins of the group No. 4-6 can be synthesized in a simple, i.e., one-layered epithelium. The change from simple to stratified amnion epithelium does not require a cessation of synthesis of cytokeratins of the simple epithelium type, but in this case keratins characteristic of the terminally differentiated epidermis (No. 1, 10, and 11) are also synthesized.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytokeratins in normal lung and lung carcinomas. I. Adenocarcinomas, squamous cell carcinomas and cultured cell lines

The various epithelial cells of the lower respiratory tract and the carcinomas derived from them differ markedly in their differentiation characteristics. Using immunofluorescence microscopy and two-dimensional gel electrophoresis of cytoskeletal proteins from microdissected tissues we have considered whether cytokeratin polypeptides can serve as markers of cell differentiation in epithelia from various parts of the human and bovine lower respiratory tract. In addition , we have compared these protein patterns with those found in the two commonest types of human lung carcinoma and in several cultured lung carcinoma cell lines. By immunofluorescence microscopy, broad spectrum antibodies to cytokeratins stain all epithelial cells of the respiratory tract, including basal, ciliated, goblet, and alveolar cells as well as all tumor cells of adenocarcinomas and squamous cell carcinomas. However, in contrast, selective cytokeratin antibodies reveal cell type-related differences. Basal cells of the bronchial epithelium react with antibodies raised against a specific epidermal keratin polypeptide but not with antibodies derived from cytokeratins characteristic of simple epithelia. When examined by two-dimensional gel electrophoresis, the alveolar cells of human lung show cytokeratin polypeptides typical of simple epithelia (nos. 7, 8, 18 and 19) whereas the bronchial epithelium expresses, in addition, basic cytokeratins (no. 5, small amounts of no. 6) as well as the acidic polypeptides nos. 15 and 17. Bovine alveolar cells also differ from cells of the tracheal epithelium by the absence of a basic cytokeratin polypeptide. All adenocarcinomas of the lung reveal a "simple-epithelium-type" cytokeratin pattern (nos. 7, 8, 18 and 19). In contrast, squamous cell carcinomas of the lung contain an unusual complexity of cytokeratins. We have consistently found polypeptides nos. 5, 6, 8, 13, 17, 18 and 19 and, in some cases, variable amounts of cytokeratins nos. 4, 14 and 15. Several established cell lines derived from human lung carcinomas (SK-LU-1, Calu -1, SK-MES-1 and A-549) show a uniform pattern of cytokeratin polypeptides (nos. 7, 8, 18 and 19), similar to that found in adenocarcinomas. In addition, vimentin filaments are produced in all the cell lines examined, except for SK-LU-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tissue type-specific expression of intermediate filament proteins in a cultured epithelial cell line from bovine mammary gland

Different clonal cell lines have been isolated from cultures of mammary gland epithelium of lactating cow’s udder and have been grown in culture media containing high concentrations of hydrocortisone, insulin, and prolactin. These cell (BMGE+H), which grow in monolayers of typical epithelial appearance, are not tightly packed, but leave intercellular spaces spanned by desmosomal bridges. The cells contain extended arrays of cytokeratin fibrils, arranged in bundles attached to desmosomes. Gel electophoresis show that they synthesize cytokeratins similar, if not identical, to those found in bovine epidermis and udder, including two large (mol wt 58,500 and 59,000) and basic (pH range: 7-8) and two small (mol wt 45,500 and 50,000) and acidic (pH 5.32 and 5.36) components that also occur in phosphorylated forms. Two further cytokeratins of mol wts 44,000 (approximately pH 5.7) and 53,000 (pH 6.3) are detected as minor cytokeratins in some cell clones. BMGE+H cells do not produce vimentin filaments as determined by immunofluorescence microscopy and gel electrophoresis. By contrast, BMGE-H cells, which have emerged from the same original culture but have been grown without hormones added, are not only morphologically different, but also contain vimentin filaments and a different set of cytokeratins, the most striking difference being the absence of the two acidic cytokeratins of mol wt 50,000 and 45,500. Cells of the BMGE+H line are characterized by an unusual epithelial morphology and represent the first example of a nonmalignant permanent cell line in vitro that produces cytokeratin but not vimentin filaments. The results show that (a) tissue-specific patterns of intermediate filament expression can be maintained in permanent epithelial cell lines in culture, at least under certain growth conditions; (b) loss of expression of relatively large, basic cytokeratins is not an inevitable consequence of growth of epithelial cells in vitro. Our results further show that, during culturing, different cell clones with different cytoskeletal composition can emerge from the same cell population and suggest that the presence of certain hormones may have an influence on the expression of intermediate filament proteins.     

Low level expression of cytokeratins 8, 18 and 19 in vascular smooth muscle cells of human umbilical cord and in cultured cells derived therefrom, with an analysis of the chromosomal locus containing the cytokeratin 19 gene

Of the various intermediate filament (IF) proteins certain cytokeratins, usually a hallmark of epithelial differentiation, can also be detected in some non-epithelial cells in low amounts. We have studied a representative case of this atypical expression, the smooth muscle cells of the blood vessel walls of the human umbilical cord, at the protein and nucleic acid level, by light and electron microscopic immunolocalization, gel electrophoresis and immunoblotting of cytoskeletal proteins, and mRNA identification by Northern blotting. For the latter we have used sensitive probes for various cytokeratins, including new probes for cytokeratin 19. We also describe the chromosome 17 locus comprising the genes for cytokeratins 15 and 19, and we emphasize the occurrence of several unusual and evolutionarily stable sequence elements in the introns of the cytokeratin 19 gene. Most, perhaps all smooth muscle cells of these blood vessels, positively identified by the presence of desmin and smooth muscle type alpha-actin, are immunostained by antibodies specific for cytokeratins 8 and 18, and a subpopulation also contains cytokeratin 19. Immunoelectron microscopy indicates that these cytokeratins are arranged in IFs that are distributed differently from the majority of the IFs formed by desmin and vimentin. Gel electrophoresis of cytoskeletal proteins from microdissected vascular wall tissue shows that the amounts of cytokeratins 8 and 18 present in these tissues are very low, representing less than 1% of the total IF protein, and that cytokeratin 19 is present only in trace amounts. Correspondingly, the contents of mRNAs for cytokeratins 8, 18 and 19 in these tissues are much lower than those present in epithelial cells examined in parallel. We have also established cell cultures derived from umbilical cord vascular smooth muscles that have maintained the expression of cytokeratins 8, 18 and 19, together with vimentin and the smooth muscle type alpha-actin, but do not synthesize desmin. In these cell cultures the cytokeratins are present in much higher amounts than in the original tissue and form IFs that, surprisingly, show a similar distribution as the vimentin IFs and, upon treatment of the cells with colcemid, collapse into juxtanuclear aggregates, often even more effectively than the vimentin IFs do. We conclude that in a certain subtype of smooth muscle cells, the genes encoding cytokeratins of the "simple epithelial type", i.e., cytokeratins 8, 18 and 19, are expressed and that the low level expression of these genes is compatible with myogenic differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection of a cytokeratin determinant common to diverse epithelial cells by a broadly cross-reacting monoclonal antibody.

A monoclonal antibody derived from a mouse immunized with bovine epidermal prekeratin has been characterized by its binding to cytoskeletal polypeptides separated by one- or two-dimensional gel electrophoresis and by immunofluorescence microscopy. This antibody (KG 8.13) binds to a determinant present in a large number of human cytokeratin polypeptides, notably some polypeptides (Nos. 1, 5, 6, 7, and 8) of the 'basic cytokeratin subfamily' defined by peptide mapping, as well as a few acidic cytokeratins such as the epidermis-specific cytokeratins Nos. 10 and 11 and the more widespread cytokeratin No. 18. This antibody reacts specifically with a wide variety of epithelial tissues and cultured epithelial cells, in agreement with previous findings that at least one polypeptide of the basic cytokeratin subfamily is present in all normal and neoplastic epithelial cells so far examined. The antibody also reacts with corresponding cytokeratin polypeptides in a broad range of species including man, cow, chick, and amphibia but shows only limited reactivity with only a few rodent cytokeratins. The value of this broad-range monoclonal antibody, which apparently recognizes a stable cytokeratin determinant ubiquitous in human epithelia, for the immunohistochemical identification of epithelia and carcinomas is discussed.     

Distribution of vimentin, cytokeratins, and desmosomal-plaque proteins in human nephroblastoma as revealed by specific antibodies: co-existence of cell groups of different degrees of epithelial differentiation

The distribution of cytokeratins, desmosomal-plaque proteins (desmoplakins), and vimentin in nephroblastoma tissue was studied by immunofluorescence microscopy using specific antibodies. In undifferentiated blastema cells, desmosomes, as revealed by antibodies to desmoplakins, preceded the advent of significant amounts of cytokeratins, indicating that desmosomes are early and sensitive markers of epithelial differentiation. Cytokeratin-positive tumor cells were seen in the following distribution patterns: groups of loosely arranged and scattered cells containing only scant cytokeratin fibrils surrounded by negative stroma cells; focal accumulation of cytokeratin-positive cells with cytokeratin-specific cytoplasmic fibril meshwork staining; rosettes of cytokeratin-positive cells without formation of distinct lumina, showing concentration of cytokeratin staining in the center; tubules with distinct lumina made up of cytokeratin-positive cells, with cytokeratin staining concentrated in the subapical cell portions. In cytokeratin-positive cells, the numbers of desmoplakin-positive dots were generally increased; in well-formed tubules, enrichment of desmoplakin-positive spots, corresponding to the subapical skeletal disks, was most conspicuous. Vimentin was demonstrated in stromal areas, but also in blastema cells showing coexpression of desmosomes and vimentin filaments. Moreover, in certain blastema cells, an overlap of cytokeratin and vimentin immunostaining was observed. Epithelial cells of nephroblastoma tubules did not react with vimentin antibodies. Our results show that the appearance of desmosomal plaques, as demonstrated by antibodies to desmoplakins, may be a very early feature of epithelial differentiation, and they also emphasize the value of antibodies to desmoplakins in tumor cell typing and diagnosis.     

K562 erythroleukemia cells express cytokeratins 8, 18, and 19 and epithelial membrane antigen that disappear after induced differentiation.

The effects of differentiation-modulating drugs were studied on the expression of intermediate filaments (IFs) in the human K562 erythroleukemic cell line. The untreated cells contained typical cytoplasmic coiling bundles, positive for both vimentin and cytokeratin as judged by indirect immunofluorescence microscopy with monoclonal antibodies (Mabs). Some of the cells also showed bright immunoreactivity for epithelial membrane antigen (EMA), as revealed with a Mab and polyclonal antiserum. When exposed to hemin or to sodium butyrate, most of the cells became cytokeratin negative within 3 days and showed dispersion of vimentin fibrils. Upon exposure to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the amount of both vimentin and cytokeratin appeared to be greatly increased within 3 days and was found both in dispersed cytoplasmic fibrils, in large spherical, eccentric aggregates, as well as in cytoplasmic fibrils in cells spreading on fibronectin. TPA induced a complete loss of proliferation, as judged by immunostaining with the Mab Ki-67. The effects of TPA were found to be irreversible and could be induced by only a short exposure to the drug. Western blotting analysis and monoclonal antibodies to individual cytokeratins revealed that untreated K562 cells expressed Mr 52,000 (No. 8), 46,000 (No. 18), and 40,000 (No. 19) cytokeratin polypeptides, which disappeared when the cells were exposed to hemin or to sodium butyrate to induce erythroid differentiation but were greatly enhanced when exposed to TPA. The monoclonal anti EMA antibody reacted in K562 cells with a single Mr 320,000 polypeptide that was also revealed in MCF-7 breast carcinoma cells. Human bone marrow cells or other leukemic cell lines with erythroid differentiation capacity (HEL and KG-1) did not contain cytokeratin- or EMA-immunoreactive cells, suggesting that in K562 cells these properties may rather represent abnormal cytodifferentiation or retrodifferentiation toward early embryonic mesenchymal cells, than a more general expression of epithelial features in human leukemic cells.     

An epithelial cell line with elongated myoid morphology derived from bovine mammary gland : Expression of cytokeratins and desmosomal plaque proteins in unusual arrays

Cells of a clonal line (BMGE + HM) selected from bovine mammary gland epithelial cell cultures are described which, after reaching confluence, do not assume typical epithelioid morphology, but form elongated cells with long slender processes extending over the surfaces of other cells. However, cells of this line which display non-epithelioid morphology and are exceptionally rich in actin microfilaments are identified as epithelial cells by their synthesis of cytokeratins and desmosomal plaque proteins, as demonstrated by immunofluorescence and immunoelectron microscopy and by gel electrophoresis of cytoskeletal proteins. The cells do not produce vimentin and desmin filaments. The specific cytokeratin polypeptides of these myoid cells are identical to those present in normal epithelioid BMGE + H cells but are arranged in unusual arrays of meshworks of finely dispersed, non-fasciated filaments and granular structures. Desmosomal plaque proteins, notably desmoplakins, are abundant, but the electron microscopic appearance of the desmosomes is abnormal in that most of them are associated with a second accessory plaque formed at a distance of 0.1-0.15 micron from the normal desmosomal plaque. Both cytokeratin filaments and desmosomal structures are found throughout the whole cytoplasm, including the extended cell processes. The existence of an epithelial cell line with such an unusual morphology demonstrates the importance of non-morphological criteria in identifying epithelium-derived cells. Our findings also indicate that dramatic differences of cell shape and organization of epithelial cells need not necessarily be associated with changes in the expression of specific cytoskeletal proteins. The possible origin of this cell line from myoepithelial cells is discussed.     

Local expression of cytokeratins 8, 17 and 18 in the mesenchyme and smooth muscles in the early stages of human organogenesis

Expression of cytokeratins 7, 8, 17, 18 in human embryos and fetuses of 6.5-13 weeks was studied using light and electron immunocytochemistry and immunoelectroblotting with the monoclonal antibodies. Cytokeratins 8 and 18 were expressed in 6.5-8 week old embryos not only in epithelium but also in mesenchyme of allantois, urogenital sinus, Wolffian and Mullerian ducts, mesentery, urinary bladder and certain regions of colon, rectum and atrium cordis walls. Furthermore, starting from the 10th week smooth-muscle cells of ring layer in caudal part of rectum bound antibodies against cytokeratin 17 in addition to those against cytokeratins 8 and 18. Corresponding mesenchymal and smooth-muscle cells of adult individuals did not react with either of them. Cytokeratins were still synthesized when mesenchymal cells of embryonic intestine wall were cultivated in vitro. Intermediate filaments of these cells contain cytokeratins 8 and 18, as demonstrated by electron immunocytochemistry and immunoelectroblotting. Thus, the expression of cytokeratins is not restricted to adult and embryonic epithelial tissues but is also characteristic of mesenchyme and smooth muscle differentiation in human embryos and fetuses.     

Pair formation and promiscuity of cytokeratins: formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides

Cytokeratins are expressed in different types of epithelial cells in certain combinations of polypeptides of the acidic (type I) and basic (type II) subfamilies, showing "expression pairs." We have examined in vitro the ability of purified and denatured cytokeratin polypeptides of human, bovine, and rat origin to form the characteristic heterotypic subunit complexes, as determined by various electrophoretic techniques and chemical cross-linking, and, subsequently, intermediate-sized filaments (IFs), as shown by electron microscopy. We have found that all of the diverse type I cytokeratin polypeptides examined can form complexes and IFs when allowed to react with equimolar amounts of any of the type II polypeptides. Examples of successful subunit complex and IF formation in vitro include combinations of polypeptides that have never been found to occur in the same cell type in vivo, such as between epidermal cytokeratins and those from simple epithelia, and also heterologous combinations between cytokeratins from different species. The reconstituted complexes and IFs show stability properties, as determined by gradual "melting" and reassociation, that are similar to those of comparable native combinations or characteristic for the specific new pair combination. The results show that cytokeratin complex and IF formation in vitro requires the pairing of one representative of each the type I and type II subfamilies into the heterotypic tetramer but that there is no structural incompatibility between any of the members of the two subfamilies. These findings suggest that the co-expression of specific pair combinations observed in vivo has other reasons than general structural requirements for IF formation and probably rather reflects the selection of certain regulatory programs of expression during cell differentiation. Moreover, the fact that certain cytokeratin polypeptide pairs that readily form complexes in vitro and coexist in the same cells in vivo nevertheless show preferential, if not exclusive, partner relationships in the living cell points to the importance of differences of stabilities among cytokeratin complexes and/or the existence of extracytokeratinous factors involved in the specific formation of certain cytokeratin pairs.