Cytokeratins and Dermatology - SKINmed 2005;4(6):354-360

Claudio Jacques, MD, MS; Adriana Moura de Aquino, MD; Marcia Ramos-e-Silva, MD, PhD

From the Department of Dermatology and Post-Graduate Course in Dermatology, School of Medicine, HUCFF-UFRJ, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

Cytokeratins are fibrous intermediate-filament protein polymers present in almost all animal cells. Their function is related to epithelium structural maintenance, protection from mechanical trauma, and possibly communication between adjacent cells or cytoplasm components. Today there are 20 known cytokeratins, classified according to their molecular weight and pH as type I or acidic (cytokeratins 9–20) and type II or neutral-basic (cytokeratins 1–8). Cytokeratins are always expressed in specific pairs for each type of tissue, composed of one unit of type I and one unit of type II. Primary structural defects of cytokeratins are associated with various keratinization impairments. Two of the better characterized defects are bullous epidermolysis and epidermolytic hyperkeratosis. Anti-cytokeratin monoclonal antibodies are being used for diagnostic purposes to characterize the origin of poorly differentiated tumors and metastatic solid tumors.

The word keratin stems from the Greek word keratos, meaning horn, describing the main component of the corneal layer of skin, hair, and nails—and also of horns, hooves, wool, scales, and feathers. 1 The presence of keratins in the corneal layer leads to formation of a superadapted protective cover—chemically nonreactive, hard, waterproof in both directions, elastic, and resistant to abrasion and physical trauma.

Cytokeratin (CK) is the term used by cell biologists and pathologists to describe keratins found in the cytoplasm of epithelial cells, to distinguish them from the specialized keratins of hair and nails. In the eucaryotic cell cytoplasm, CKs constitute an important component of the cytoskeleton, responsible for the structural maintenance of the cell and tissue, protection against mechanical trauma, and probably having a role in communication with adjacent cells. 2, 3

The study of primary and secondary alterations in CKs in dermatological diseases has been of great value for better understanding the etiopathogenic aspects of those diseases and for the application of CKs in support of dermatological diagnosis.

CKs and Structure

Cytoskeleton. All eucariotic cells, both in animals and plants, carry in their cytoplasm a compound of protein filaments called cytoskeleton. 4 This structure is highly dynamic and reorganizes itself continuously, according to changing conditions in and around the cell: changes to its form, cell division, or interaction with its environment ( Figure 1 ). 5

Figure1.The cytoskeleton. A cell in culture fixed and stained with a general stain for proteins, Coomassie blue. © 1994 from Molecular Biology of the Cell by Alberts B, Bray D, Lewis J. Reproduced with permission by Garland Science/Taylor & Francis Books, Inc.

The cytoskeleton comprises three types of protein filaments, classified according to diameter: actin filaments (6 nm), intermediate filaments (IFs) (7–11 nm) and microtubules (25 nm). 6, 7 To perform their complex functions, these three components make up a dynamic set integrated in structure and function, both among themselves and also with lesser known components of the cytoskeleton. 6

The notion of “cytoskeleton” originated at the beginning of the 20th century, when the existence of a filamentary structure was observed in the cytoplasm of stained fixed cells. It was assumed at the time that its only function was to maintain the cell morphology, justifying that denomination. 8 Currently it is known that, besides maintaining form, the cytoskeleton is directly responsible for the “sliding” movement of cells, by contraction and movement of cilia and flagellae. 5 The cytoskeleton is also responsible for intracellular spatial organization, through movement and positioning of organelles in the cytoplasm. 9 Separation and movement of chromosomes during anaphase in mitosis is also a function of the cytoskeleton. 9

In addition to these functions of structural maintenance, movement, and spatial organization of the organelles, this set of filaments also seems to act as a means of communication between cytoplasm components. 5, 10

Intermediate Filaments. The IFs are fibrous protein polymers found in the cytoplasm of most animal cells that participate in cell structure maintenance without being responsible for the functions of motility and contraction, in contrast to the remaining cytoskeleton components. 9, 11 In mammalian epithelial tissue, IFs constitute about 30% of the protein found in the basal cell layer and about 85% in the horny layer. 12 They also correspond to approximately 1% of the entire body protein. 13 IFs present high resistance and durability, derived from their chemical stability and their insolubility in nonionic detergents and solutions containing high or low salt concentrations. 9, 14

IFs form a perinuclear reticular structure (nuclear lamina) whose function seems to be to position and fix the nucleus. 5 From there, IFs extend themselves in the direction of the cell periphery, passing through the desmosomes and hemidesmosomes, anchoring themselves on both sides of those structures. 9 This extension into the intercellular environment allows IFs from one cell to join themselves with IFs from adjacent cells or to the basal membrane, giving mechanical stability to the epithelial tissue. 9

IFs are classified into six types (I-VI) ( Table I ), according to the sequence of amino acids and source tissues. 15 Types I and II are found in the epithelia, hair, and nails. Type III is made up of several proteins: vimentin, synthesized by embryonic and mesenchymal cells; des-min, in smooth muscle tissue and myocardium; peripherin, expressed in neurons of the peripheral nervous system; and glial fibrillary acidic protein, found in glial cells and in astrocytes. 16 Neurofilaments and α internexin are expressed by neurons and constitute type IV. Laminins constitute type V and are expressed in all cells with nuclei. Type VI is represented by nestin, found in stem cells of the central nervous system and skeletal muscle. 4 It is known today that proteins of different IFs belong to the same multigenic family. 17

TABLE I. Intermediate Filaments: Classification, Molecular Weight, and Origin

The basic subunit of IFs is a monomer of homologous structure called the central domain with an α-helicoid configuration, formed by 310 amino acids, with exception of the laminins, which are formed by 352 amino acids. 4 This central polypeptide is subdivided into four parts ( Figure 2 ) designated 1A, 1B, 2A and 2B, joined together by three short nonhelicoid segments (linkers), identified by the abbreviations L1, L1–2, and L2. 18 In each of its extremities, the central domain is linked to terminal domains: a car-boxy terminal and an amino terminal. 18

Figure 2. Schematic representation of intermediate filaments structure. Modified from Haake AR, Holbrook K. The structure and development of skin. In: Champion RH, Wilkinson DS, Ebling FJG, et al., eds. Rook’s Textbook of Dermatology.23 Used with permission from Blackwell Publishing Ltd.

“The dermatoses studied most in respect to cytokeratins’ defects are epidermolysis bullosa and epidermolytic hyperkeratosis.”

The four subunits of the central domain present little variation concerning length and amino acid sequence, as opposed to the linkers and terminal domains. 4 The variability observed in terminal domains determines the antigen specificity of IFs. 4 Interaction with other cell components is mediated by these lateral portions of the molecule. 5

The central domain comprises a repetitive sequential pattern of seven amino acids which are identical among IFs of the same subtype at a rate of 50%–90%. Among different subtypes, identical amino acid sequences occur in approximately 30%. 10, 16

Two monomer α-helicoid structures run parallel, forming coiled-coil dimers that, at their turn, position side by side, forming protofilaments. Two protofilaments form tetramers or protofibrils. 5, 13 The final structure of IFs forms by polymerization of tetramers into a helix, resulting in a filament with a mean diameter of 10 nm ( Figure 3 ). 5

Figure3. Model of helicoidal polymerization of components of intermediate filaments structure. Modified from Haake AR, Holbrook K. The structure and development of skin. In: Champion RH, Wilkinson DS, Ebling FJG, et al., eds. Rook’s Textbook of Dermatology.23 Used with permission from Blackwell Publishing Ltd.

Classification of CKs

Keratins are the most diversified class of IF proteins, with at least 30 individual human keratins described, of which 20 are epithelial or CKs, and 10 are hair keratins, present in hair and nails. Keratin molecular weights vary from 40–70 kd. 14 Nowadays, CKs are classified according to their molecular weight, isoelectric points, and pH ( Table II ). 15

TABLE II. Classification of Cytokeratins (CKs) According to Molecular Weight (MW) and pH

In tissue, CKs are coexpressed in specific pairs, always comprising one type I unit and one type II unit. As a general rule, pairs are formed by CKs with equivalent molecular weights within each type; in other words, one CK type I of high molecular weight is always expressed together with a CK type II of high molecular weight. The same occurs for the remaining CKs, creating thus a high specificity in formation of these pairs, as shown in Table III . 19

TABLE III. General Principles of Coexpression of Cytokeratin (CK) Pairs

Expression of CKs in Epithelia

Determination of the IF type present in a certain cell or tissue can supply information concerning the origin of that cell or tissue. 7 The expression of CKs is highly heterogeneous, with polypeptide composition varying between different species, within the same species, and in the epithelia of the same individual. 20 Variations also occur in the composition of filaments influenced by environmental changes, stages of cellular differentiation, period of embryonic development, and presence or not of a pathological state. 21, 22

CKs are encoded by different genes. Most acidic CKs are regulated by genes located on chromosome 17q12–21, while genes of neutral-basic CKs are located on chromosome 12q11–13. 23

In 1982, investigators 14 published a catalog of human CKs, listing their distribution in a variety of tissues. Table III presents a summary of the coexpression of CKs in several epithelia, showing that high specificity exists between a given tissue and the coexpressed pair(s). 10

Larger CKs—CK1, CK2, CK9, CK10, and CK11— are expressed in interfollicular epidermis. 14 CK2 and CK9, however, are usually minor components, except in the palmoplantar epi-dermis. 10 CK1 and CK10 are also expressed in the upper portion of the pilosebaceous duct, continuous to the epidermis, and in the keratinized epithelium of the anal canal. 14

Stratified epithelium of the cornea expresses CK3 and CK12, and these two CKs seem to be specific for this type of differentiation. Stratified squamous nonkeratinized epithelia of the esophagus, epiglottis, jugal mucosa, and apocrine glands express CK4 and CK13 in differentiated cells. 14, 24 The majority of stratified epithelia, such as esophagus, cornea, epidermis and hair follicles, express CK5 and CK14 in cells of the basal layer and in prematurely differentiated cells. 10

The external layer of hair follicle cells and sebaceous ducts express CK5 and CK14, as well as CK6 and CK16. The latter are expressed in the palmoplantar epidermis, but are not found in normal circumstances in the interfollicular epidermis, where their presence is an indication of hyperproliferation. 25 CK17 is expressed in myoepithelial cells and in basal cells of the transitional and pseudostratified respiratory and urinary tract epithelia. 25 In the epidermis, it is found only in states of pathologic hyperproliferation, in which it seems to have the function of promoting or allowing contractibility of the keratinocytes. 25 CK15 is described as a minor component of the epidermis and of the hair follicle, but it seems to be abundant in the epithelium of the eccrine sudoriparous gland, epiglottis, and trachea. 14

Some stratified epithelia also express CK7 and CK19, and those CKs are found in the eccrine and apocrine glands, mammary gland ducts, and tracheal epithelium. CK19 is also identified as a minor CK in the epidermis and in the hair follicle, where it is specifically located in the germinative portion. The transitional epithelium of the bladder and vesicle express CK7 and CK19, besides the characteristic CKs of simple epithelia (CK8, CK18). The simple mucous epithelium of the small intestine and colon, and the simple epithelium of internal organs such as the liver and kidney, have a more simplified CK expression, consisting only of CK8 and CK18, although some expression of CK19 is also found. 26 Finally, CK20 can be found in gastrointestinal epithelium, in Merkel cells, and in the gustatory papillae of the tongue, coexpressed with CK7. 10

Diagnostic Application of Monoclonal Antibodies to IFs

Histopathologic practice is based on staining techniques that reveal components of tissues or cells or reactions to more specific staining for a particular cell, tissue, microorganism, or deposited substance. However, the arsenal of usable specific stains is limited, and therefore the correct identification of the nature of a given lesion is often dependent on the personal experience of the pathologist. 27

To improve the accuracy of histopathologic diagnosis, much effort has been made to develop new, more specific techniques. Newer reagents include antibodies that allow identification of specific cellular types in tissue, such as endothelial cells, histiocytes, monocytes, different types of lymphoid cells, epithelial, neuronal, and muscular cells. Many markers fail, nonetheless, due to the fact that poorly differentiated tumors lose expression of highly specific markers. 28

The specificity and stability of IFs in tissue are the basis for the use of antibodies to IFs with the purpose of improving characterization of tumors. 29 The first immunohistochemical studies with CKs used monoclonal or polyclonal antibodies, which reacted with several CKs at the same time. 30 Currently, monoclonal antibodies are commercially available for all 20 CKs. 10 They are particularly useful in determination of the cellular origin of undifferentiated carcinomas and in histogenetic studies of benign and malignant neoplasms. 9

In a 2002 article, 10 researchers extensively reviewed the pattern of CKs in varied neoplasms, showing that the expression profile remains almost always constant, and that anti-CK antibodies are useful in the histogenetic characterization of poorly differentiated tumors. 7, 10 Benign skin tumors present a pattern of coexpression similar to that of normal epithelium, while malignant tumors present total loss of high-molecular-weight CK expression. 31 Metastatic cells of solid tumors have CK expression patterns identical to those of the cells of the primary tumor. 7

The expression of CKs in several inflammatory dermatoses has also been studied. Hyperproliferative states are characterized by emergence of CKs 6 and 16, and a delay in the expression of CKs 1 and 10. 32, 33

IFs and the genes that codify them are useful molecular markers for the study of keratinocyte differentiation. 34 Defects in structural proteins of the skin have a causal role in several dermatoses, and most mutations are located in the central domain of the CK molecule. 35

The characterization of specific defects in CKs became possible with the advent of specific techniques for the determination of encoding genes through the study of the effects of a single mutation point—in other words, a mutation that alters a base and changes an amino acid in the sequence, altering the structure of the keratin and its function. 35

Dermatoses Primarily Related to Defects in CKs. Table IV lists dermatoses in which structural molecular changes that lead to alterations of the appearance of the CK filament and its organization are well characterized. The dermatoses studied most in that respect are epidermolysis bullosa (EB) and epidermolytic hyperkeratosis (EH).

TABLE IV. Dermatoses Primarily Related to Cytokeratin (CK) Defects

Epidermolysis Bullosa. EB comprises a group of genetically distinct dermatoses, demonstrating extreme cutaneous fragility and formation of bullae at the least mechanical trauma. 36

Minor changes in the helicoid areas of a single CK can have a dramatic effect on its structure, as demonstrated by experiments with transfection in transgenic rats. The expression of CK5 genes and defective CK14 genes leads to intense formation of bullae in the skin and oral mucous membrane of those animals, a phenotype similar to EB simplex. 37 In a study with families of bearers of EB simplex, localized mutations were demonstrated in the extremity of the helical areas both in CK5 and CK14, with mutations in the terminal areas N and C identified. 35 Those mutations are expressed in the epidermal basal layer, weakening its structure and eventuating in rupture of the basal layer, with bulla formation as a consequence of mechanical trauma. 35

EH (Congenital Bullous Ichthyosiform Erythroderma).

Another phenotype reproduced in transgenic rats, EH is a rare genodermatosis of autosomal dominant inheritance, characterized by erythroderma at birth or during childhood, accompanied by formation of bullae predominantly in traumatized areas. Later, the formation of bullae ceases and is replaced by emergence of keratotic or verrucous lesions in those areas. 36

Investigators 38 have shown that mutations in the CK10 gene lead to abnormalities in the filaments of that CK in transgenic rats, with consequent structural weakness of the supra-basal keratinocytes and formation of epidermal bullae, corresponding to a phenotype similar to that of EH. 38 Studies performed on skin samples from bearers of EH revealed abnormalities in CKs 1 and 10. 39

Dermatoses Secondarily Related to Defects in CKs. In other diseases with epidermal hyperproliferation, e.g. psoriasis, the altered expression of CK is probably a secondary event. Whenever the epidermis is traumatized, homeostasis is lost, resulting in a decrease of CK1 and CK10 and an increase of CK6 and CK16. 40

As already described, the expression of CKs is also altered in cutaneous carcinogenesis. In general, there is a tendency toward substitution of CKs of larger molecular weight (CKs 1 and 10), characteristic of adult skin, with CKs of smaller molecular weight (CKs 8, 18 and 19), found in fetal skin and in simple epithelia. 15, 35 The expression of CKs also varies with the stage of tumor differentiation. Poorly differentiated spinocellular carcinomas have decreased expression of CKs 1 and 10, while in well differentiated tumors, those CKs are expressed regularly. 41

Malignant transformation of benign cutaneous tumors is usually accompanied by a decrease in the coexpression of CKs 1 and 10. 42

Conclusions

In the last two decades, extensive new information regarding structure, expression, and regulation in CKs has become available. This information has been fundamental for understanding normal differentiation in molecular terms, as well as for elucidating structural defects of CKs in certain genetic dermatoses. These discoveries have shown the importance of CKs in dermatology, both in clarifying the etiology of certain diseases and in refining diagnostic tools for tumors and inflammatory dermatoses. Molecular and cellular biology of the skin will be a major focus for research during the coming decades.

References