Vitamin adeficiency and keratin biosynthesis in cultured hamster trachea

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

Expression of epidermal keratins and filaggrin during human fetal skin development

The major structural proteins of epithelia, the keratins, and the keratin filament-associated protein, filaggrin, were analyzed in more than 50 samples of human embryonic and fetal skin by one-dimensional SDS PAGE and immunoblotting with monoclonal and polyclonal antibodies. Companion samples were examined by immunohistochemistry and electron microscopy. Based on structural characteristics of the epidermis, four periods of human epidermal development were identified. The first is the embryonic period (before 9 wk estimated gestational age), and the others are within the fetal period: stratification (9-14 wk), follicular keratinization (14-24 wk), and interfollicular keratinization (beginning at approximately 24 wk). Keratin proteins of both the acidic (AE1-reactive, type I) and the basic (AE3-reactive, type II) subfamilies were present throughout development. Keratin intermediate filaments were recognized in the tissue by electron microscopy and immunohistochemical staining. Keratins of 50 and 58 kD were present in the epidermis at all ages studied (8 wk to birth), and those of 56.5 and 67 kD were expressed at the time of stratification and increased in abundance as development proceeded. 40- and 52-kD keratins were present early in development but disappeared with keratinization. Immunohistochemical staining suggested the presence of keratins of 50 and 58 kD in basal cells, 56.5 and 67 kD in intermediate cells, and 40 and 52 kD in the periderm as well as in the basal cells between the time of stratification and birth. Filaggrin was first detected biochemically at approximately 15 wk and was localized immunohistochemically in the keratinizing cells that surround hair follicles. It was identified 8-10 wk later in the granular and cornified cell layers of keratinized interfollicular epidermis. These results demonstrate the following. An intimate relationship exists between expression of structural proteins and morphologic changes during development of the epidermis. The order of expression of individual keratins is consistent with the known expression of keratins in simple vs. stratified vs. keratinized epithelia. Expression of keratins typical of stratified epithelia (50 and 58 kD) precedes stratification, and expression of keratins typical of keratinization (56.5 and 67 kD) precedes keratinization, which suggests that their expression marks the tissue commitment to those processes. Because only keratins that have been demonstrated in various adult tissues are expressed during fetal development, we conclude that there are no "fetal" keratins per se.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tissue distribution of keratin 7 as monitored by a monoclonal antibody

Monoclonal antibody (RCK 105) directed against keratin 7 was obtained after immunization of BALB/c mice with cytoskeletal preparations from T24 cells and characterized by one- (1D) and two-dimensional (2D) immunoblotting. In cultured epithelial cells, known from gel electrophoretic studies to contain keratin 7, this antibody gives a typical keratin intermediate filament staining pattern, comparable to that obtained with polyclonal rabbit antisera to skin keratins or with other monoclonal antibodies, recognizing for example keratins 5 and 8 or keratin 18. Using RCK 105, the distribution of keratin 7 throughout human epithelial tissues was examined and correlated with expression patterns of other keratins. Keratin 7 was found to occur in the columnar and glandular epithelium of the lung, cervix, breast, in bile ducts, collecting ducts in the kidney and in mesothelium, but to be absent from gastrointestinal epithelium, hepatocytes, proximal and distal tubules of the kidney and myoepithelium. Nor could it be detected in the stratified epithelia of the skin, tongue, esophagus, or cervix but strongly stained all cell layers of the urinary bladder transitional epithelium. When applied to carcinomas derived from these different tissue types it became obvious that an antibody to keratin 7 may allow an immunohistochemical distinction between certain types of adenocarcinomas.     

Keratin expression in rat intestinal crypt and villus cells. Analysis with a panel of monoclonal antibodies.

Seven monoclonal antibodies were prepared against cytoskeletal components of rat intestinal brush borders. In the following paper (Chandler, J. S., Calnek, D., and Quaroni, A., J. Biol. Chem. 266, 11932-11938), three of them were shown to be specific for, respectively, keratin 8 (RK4), keratin 19 (RK7), and a newly identified type I keratin (keratin 21) (RK5). With these antibodies we have investigated the changes in keratin gene expression accompanying intestinal cell differentiation. Keratin 21 was detected exclusively in differentiated villus cells and in goblet, enteroendocrine, and Paneth cells in the crypts; in the proliferative crypt cells keratin 19 was predominant. Analysis of keratins expressed by cultured rat crypt cells (IEC cells) confirmed the absence of keratin 21 in undifferentiated intestinal cells. Changes in keratin's expression similar to those observed with cell differentiation in the adult intestinal mucosa were also demonstrated during early fetal intestinal development: the stratified epithelium present at 15-16 days of gestation contained predominantly keratin 19 with only a small amount of keratin 8; keratin 21 was first detected at 18-19 days of gestation, concomitant with the appearance of a well formed brush border and an apical cytoplasmic terminal web. These results suggest that keratin tonofilaments may play a role in the morphological and structural alterations accompanying intestinal cell differentiation in vivo.     

兔角膜缘干细胞体外分离、培养和生物学鉴定的实验研究

通过体外培养兔角膜缘干细胞,观察其生物学特性,建立兔角膜缘干细胞的体外培养方法。方法0．25%胰蛋白酶消化角膜缘组织,用含15%胎牛血清的DMEM和F12(1:1)的培养液(DF)对兔角膜缘干细胞进行体外培养,形态学观察,培养的细胞早期使用AEl/AE3、晚期使用AE5角蛋白特异的单克隆抗体)作细胞免疫化学鉴定。结果:原代培养细胞48h后开始贴壁,部分细胞由圆形变为卵圆形或长梭形;10～14d形成单层,细胞呈圆形、卵圆形,类角膜上皮细胞;细胞传到第5代左右开始出现老化状态;免疫细胞化学染色:培养的细胞早期AEl/AE3呈阳性而少部分细胞AE5呈阳性,培养的细胞晚期AE5呈阳性。结论:本实验初步建立了一套兔角膜缘干细胞的体外培养方法。     

Proteolytic modification of acidic and basic keratins during terminal differentiation of mouse and human epidermis

Keratins from the living cell layers of human and neonatal mouse epidermis (prekeratins) have been compared to those from the stratum corneum (SC keratins). Human and mouse epidermis contained four prekeratins, two of each keratin subfamily: type II basic (pI 6.5-8.5; human 68 kDa, 60.5 kDa and mouse 67 kDa, 60 kDa) and type I acidic (pI 4.7-5.7; human 57 kDa, 51 kDa and mouse 58 kDa, 53 kDa,). While all four were present in equal amounts in adult human epidermis, two (67 kDa basic, 58 kDa acidic) were more prominent in neonatal mouse epidermis. Preliminary results with cell fractions (basal, spinous and granular) indicated that quantitative differences were a function of morphology, basal cells containing the smaller member of each subfamily and granular cells the larger. Mouse stratum corneum extracts contained four keratins (three in human): type II neutral-acidic (pI 5.7-6.7; human 65 kDa and mouse 64 kDa, 62 kDa) and type I acidic (pI 4.9-5.4; human 57.5 kDa, 55 kDa and mouse 58.5 kDa, 57.5 kDa). In both species, one-dimensional and two-dimensional peptide mapping (with V8 protease and trypsin respectively) indicated that while all four prekeratins were distinct gene products, similarities existed in the type II basic and the type I acidic keratin subfamilies. A strong homology also existed between type II SC keratins and the larger basic (type II) prekeratin (human 68 kDa and mouse 67 kDa) and between type I SC keratins and the larger acidic (type I) prekeratin (human 57 kDa and mouse 58 kDa). These results indicate a precursor-product relationship within each keratin subfamily, between SC keratins and the prekeratins abundant in the adjacent granular layer. This differentiation-related keratin processing was similar in mouse and human epidermis, and may represent a widespread phenomenon amongst keratinising epithelia.     

Developmentally controlled expression patterns of intermediate filament proteins in the cephalochordate Branchiostoma

Expression of cytoplasmic intermediate filament (IF) proteins starts in the gastrula with three keratins (k1, Y1, D1) and protein X1. The number of IF proteins expressed increases at the neurula and early larval stages to seven and 11, respectively, and reaches 13 in the adult. Using antibodies specific for a single IF protein the expression patterns of nine of the 13 IF proteins were analyzed at different developmental stages. Keratin k1 of the larval epidermis is replaced in the juvenile by keratin E1. Protein C1 of the larval epidermis persists only weakly and only in the most ventral part of the adult. While down-regulated in the adult epidermis k1 and C1 are major proteins in the atrial epithelium which forms in the later larva. B1 is currently the only IF protein expressed in mesodermally derived tissues such as the muscle tails and some coelomic epithelia. Two-dimensional gels confirm that keratins are the major IF proteins in the nerve cord. Immunogold electronmicroscopy shows that proteins X1 and C2 are present in epidermis and nerve cord in keratin IF.     

Altered expression of keratin and vimentin in human retinal pigment epithelial cells in vivo and in vitro.

Actively proliferating human retinal pigment epithelial (RPE) cells grown in tissue culture possess keratin-containing intermediate filaments that react with a combination of AE1 and AE3 anti-keratin monoclonal antibodies. Antibody reactivity is lost, however, from RPE cells as the cell population ceases to proliferate when it approaches confluence and attains morphological characteristics more similar to those in vivo. In contrast, clone 8.13 anti-keratin antibody stains all cells in the culture at all stages of the growth cycle and cell densities. These findings were reflected in vivo using retinal pigment epithelium taken directly from the eye. Normal non-proliferating RPE cells bound 8.13 antibody to cytoskeletal structures, as judged by indirect immunofluorescence, but did not bind AE1/AE3 antibodies. However, proliferating dedifferentiated RPE cells from the vitreous humor of patients with proliferative vitreoretinopathy possess filaments that bind both AE1/AE3 and 8.13 antibodies. Thus it appears that structures detected by AE1/AE3 antibodies only occur in actively growing RPE cells in vitro and in vivo. Keratins produced by RPE cells were identified using Western blotting. Species with molecular masses of 54 (keratin 7), 52 (keratin 8), 42 (keratin 18), and 40 (keratin 19) kiloDaltons were the most abundant in proliferating cultured cells, but cells isolated directly from the eye were found to lack keratin 7 and 19. Keratin 19 was, however, observed in proliferating RPE cells from some patients with proliferative vitreoretinopathy. The latter findings explain the differential staining observed with AE1/AE3 antibodies in cells in culture and isolated directly from the eye since these antibodies interact primarily with keratin 19 which is absent from non-proliferating RPE cells. In contrast to the presence of keratin-containing intermediate filaments in human RPE cells in vivo, there are apparently no detectable vimentin-containing cytoskeletal structures. However, all RPE cells cultured in vitro develop filaments composed of vimentin which persist in cells that have reached confluence.     

Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies

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

Transient expression of simple epithelial keratins by mesenchymal cells of regenerating newt limb

Structural proteins of the intermediate filament family are an early indicator of differentiation before organogenesis becomes apparent. Keratin intermediate filaments are characteristically expressed only by epithelial and not by mesenchymal cells. Here we show, using monoclonal antibodies, a transient expression of the keratin pair 8 and 18 in a population of mesenchymal cells in the regenerating newt limb, specifically in the undifferentiated progenitor cells (blastemal cells) which give rise to the new tissues. These keratins are also expressed in cultured limb cells that can differentiate into muscle. In contrast no reactivity with anti-keratin 8 and 18 antibodies was observed in the newt limb bud at an early stage of development, indicating a molecular difference between the developing and regenerating limb. The molecular weights of the newt proteins detected by these antibodies are very similar to those of human keratins 8 and 18, further supporting the immunocytochemical evidence that the newt homologs of these keratins are expressed in blastemal cells. This is the first demonstration of keratin expression in mesenchymal progenitor cells in an adult animal.     

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.     

Suprabasal expression of a 64-kilodalton keratin (no. 3) in developing human corneal epithelium

We have previously shown that a basic 64-kilodalton (no. 3 in the catalog of Moll et al.) and an acidic 55-kilodalton (no. 12) keratin are characteristic of suprabasal cell layers in cultured rabbit corneal epithelial colonies, and therefore may be regarded as markers for an advanced stage of corneal epithelial differentiation. Moreover, using an AE5 mouse monoclonal antibody, we showed that the 64-kilodalton keratin marker is expressed suprabasally in limbal epithelium but uniformly (basal layer included) in central corneal epithelium, suggesting that corneal basal cells are in a more differentiated state than limbal basal cells. In conjunction with previous data implicating the centripetal migration of corneal epithelial cells, our data support a model of corneal epithelial maturation in which corneal epithelial stem cells are located in the limbus, the transitional zone between the cornea and conjunctiva. In the present study, we analyzed the expression of the 64-kilodalton keratin in developing human corneal epithelium by immunohistochemical staining. At 8 weeks of gestation, the presumptive corneal epithelium is composed of a single layer of cuboidal cells with an overlying periderm; neither of these cell layers is AE5 positive. At 12-13 weeks of gestation, some superficial cells of the three- to four-layered epithelium become AE5 positive, providing the earliest sign of overt corneal epithelial differentiation. At 36 weeks, although the epithelium is morphologically mature (four to six layers), AE5 produces a suprabasal staining pattern, this being in contrast to the adult epithelium which exhibits uniform staining.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three distinct keratinocyte subtypes identified in human oral epithelium by their patterns of keratin expression in culture and in xenografts

We have characterized the cells that form the human oral epithelia by analyzing their patterns of keratin expression in culture and in transplants. Keratinocytes of all oral regions synthesized high levels of keratins K5/K14 and K6/K16,K17, as expressed by cells of all stratified squamous epithelia in culture. However, cells from different regions varied in their expression in culture of retinoid-inducible (K19 and K13) and simple epithelial (K7, K8 and K18) keratins. By these criteria, all oral cells could be classified as belonging to one of three intrinsically distinct subtypes: "keratinizing" (gingiva, hard palate), "typical nonkeratinizing" (inner cheek, floor of mouth, ventral tongue) and "special non-keratinizing" (soft palate), all of which differed from the epidermal keratinocyte subtype. Cells from fetal floor of mouth expressed a pattern of keratins in culture markedly different from that of adult floor of mouth cells but identical to that of the adult "special nonkeratinizing" subtype and similar to that of several oral squamous cell carcinoma lines. When cultures of oral keratinocytes were grafted to the dermis of nude mice, they formed stratified epithelial structures after 10 days. In some areas of the stratified structures, the basal layer recapitulated the K19 expression pattern of the oral region from which they had originated. Thus, regional differentiation of the oral epithelium is based on an intrinsic specialization of regional keratinocyte stem cells. Additionally, oral cell transformation either frequently involves reversion to the fetal keratin program or else oral cells that express this keratin program are especially susceptible to transformation.     

Expression of specific keratin markers by rabbit corneal, conjunctival, and esophageal epithelia during vitamin A deficiency

Using an in vivo rabbit model system, we have studied the morphological and biochemical changes in corneal, conjunctival, and esophageal epithelia during vitamin A deficiency. Light and electron microscopy showed that the three epithelia undergo different degrees of morphological keratinization. Corneal and conjunctival epithelia became heavily keratinized, forming multiple layers of superficial, anucleated cornified cells. In contrast, esophageal epithelium underwent only minor morphological changes. To correlate morphological alterations with the expression of specific keratin molecules, we have analyzed the keratins from these epithelia by the immunoblot technique using the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies. The results indicate that during vitamin A deficiency, all three epithelia express an AE1-reactive, acidic 56.5-kd keratin and an AE3-reactive, basic 65-67-kd keratin. Furthermore, the expression of these two keratins correlated roughly with the degree of morphological keratinization. AE2 antibody (specific for the 56.5- and 65-67-kd keratins) stained keratinized corneal epithelial sections suprabasally, as in the epidermis, suggesting that these two keratins are expressed mainly during advanced stages of keratinization. These two keratins have previously been suggested to represent markers for epidermal keratinization. Our present data indicate that they can also be expressed by other stratified epithelia during vitamin A deficiency-induced keratinization, and suggest the possibility that they may play a role in the formation of the densely packed tonofilament bundles in cornified cells of keratinized tissues.     

Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells

In this paper we present keratin expression data that lend strong support to a model of corneal epithelial maturation in which the stem cells are located in the limbus, the transitional zone between cornea and conjunctiva. Using a new monoclonal antibody, AE5, which is highly specific for a 64,000-mol-wt corneal keratin, designated RK3, we demonstrate that this keratin is localized in all cell layers of rabbit corneal epithelium, but only in the suprabasal layers of the limbal epithelium. Analysis of cultured corneal keratinocytes showed that they express sequentially three major keratin pairs. Early cultures consisting of a monolayer of "basal" cells express mainly the 50/58K keratins, exponentially growing cells synthesize additional 48/56K keratins, and postconfluent, heavily stratified cultures begin to express the 55/64K corneal keratins. Cell separation experiments showed that basal cells isolated from postconfluent cultures contain predominantly the 50/58K pair, whereas suprabasal cells contain additional 55/64K and 48/56K pairs. Basal cells of the older, postconfluent cultures, however, can become AE5 positive, indicating that suprabasal location is not a prerequisite for the expression of the 64K keratin. Taken together, these results suggest that the acidic 55K and basic 64K keratins represent markers for an advanced stage of corneal epithelial differentiation. The fact that epithelial basal cells of central cornea but not those of the limbus possess the 64K keratin therefore indicates that corneal basal cells are in a more differentiated state than limbal basal cells. These findings, coupled with the known centripetal migration of corneal epithelial cells, strongly suggest that corneal epithelial stem cells are located in the limbus, and that corneal basal cells correspond to "transient amplifying cells" in the scheme of "stem cells----transient amplifying cells----terminally differentiated cells."     

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.     

Effect of cigarette smoke condensate and vitamin A depletion on keratin expression patterns in cultured hamster tracheal epithelium. An immunohistomorphological study using monoclonal antibodies to keratins

Keratin expression in hamster tracheal epithelium was investigated during organ culture in serum-free, hormone-supplemented medium using monospecific monoclonal antibodies. Generally, tracheal basal cells expressed keratins detected by antibodies RCK102 and RCK103, while columnar epithelial cells were stained positively by RGE53, RCK103, RCK105 and HCK19. Metaplastic squamous cell foci reacted with antibodies RKSE60, RCK103 and HCK19. Early metaplastic alterations were more clearly RKSE60-positive than the mature lesions. In the vitamin A-depleted tracheas basal cells were clearly RCK102-positive. Superficial cells in the central part of areas of squamous metaplasia induced by cigarette smoke condensate expressed the basal cell keratins, and were negative for the columnar cell keratin 18 detected by the RGE53 antibody. This finding suggests that in cigarette smoke condensate-induced squamous metaplasia basal cells play an important role. The mucus-producing cells at the edges of metaplastic squamous cell foci expressed the keratins specific to columnar cells. Cigarette smoke condensate exposure accelerated epithelial keratinization compared to the vitamin A-depleted epithelium. It was concluded that not only small mucous granule cells, but also basal cells are involved in the development and maintenance of induced squamous metaplasia in tracheal epithelium. Furthermore, in vitro vitamin A-depleted epithelium did not coexpress vimentin in addition to the different keratins.     

Regulation of terminal differentiation of cultured human keratinocytes by vitamin A

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