Changes In Keratin Expression Research Articles

Alteration in keratin expression in hamster tracheal epithelial cells exposed to benzo[a]pyrene

Changes in keratin expression were documented in cultures of hamster tracheal epithelial (HTE) cells exposed to vitamin A (retinol 10(-6) M) and benzo[a]pyrene (B[a]P), two agents known to affect the differentiation of tracheal epithelial cells in vivo. Keratin protein patterns were determined after 10 days in culture in the presence and absence of B[a]P in order to determine whether the expression of these proteins was altered by this carcinogen. HTE cells maintained in the presence of vitamin A expressed four simple epithelial keratins (7,8,18 and 19) while vitamin A deficient HTE cells expressed four additional cytokeratins (5,6,14 and 17). No effect of B[a]P on keratin expression was observed in vitamin A treated cells. However, vitamin A deficient HTE cells exposed to B[a]P (0.05-10 micrograms/ml) demonstrated a decrease in the expression of the four differentiation-related keratins while the simple epithelial keratins appeared to be unaffected. These observations were verified at the RNA level employing Northern blot analysis using cDNA probes for human keratins 5,6 and 14. Results demonstrate that B[a]P alters the expression of differentiation-related genes, the cytokeratins, in cell types known to develop into tumors of the respiratory tract.

Immunohistochemical study of possible changes in keratin expression during neoplastic transformation of the uterine mucosa

The present study aimed to examine possible changes in keratin expression during neoplastic transformation of the uterine mucosa and possible differences in keratin expression between endocervical and endometrial adenocarcinomas. Routinely processed specimens with normal morphology or neoplastic changes were stained immunohistochemically using 5 commercial antibodies to keratin-filaments of molecular weight 39-58 kD: CAM 5.2, RCK 102, MCA 144, PKE and PRE. We generally found a change in keratin expression during the neoplastic transformation, consisting of pronounced heterogeneity compared with normal epithelia. In distinguishing koilocytic atypia from CIN, RCK 102 (52.5, 58 Kd) may prove helpful as it stains neoplastic cells strongly and shows no reaction in koilocytic. Staining with the antibody CAM 5.2 (reactive with 39, 43, 50 kD filaments) may aid in distinguishing between cervical and endometrial adenocarcinomas. The former is stained uniformly; the latter shows a more variable staining.

Changes in Keratin Expression of Human Vaginal Epithelium during Different Female Generation Phases

Distinct expression patterns of keratins reflect different epithelial prototypes and different epithelial differentiation pathways. In order to get evidence of the expression of keratins in a hormone-dependent tissue in man, we investigated 70 epithelial tissue specimens by means of SDS-gel electrophoresis and immunoblot. Every tissue probe belonged to different generation phases such as sexual maturity, climacteric and postmenopausal period and pregnancy. Keratins 1(avb), 4(avb), 5(avb), 10(avb), 13, 14 were detected regularly whereas keratins 6, 16, and 19 are expressed irregularly. During all generation phases, with the exception of pregnancy, the keratins 1 and 10, associated with the epidermal differentiation, and keratins 4 and 13, associated with the differentiation to a non-keratinizing stratified epithelium, are synthesized simultaneously. However in pregnancy, i.e. pregnancy at term of delivery, only K4/K13 are expressed, while K1/K10 are below the detection limit. In terms of keratin expression vaginal epithelium represents a mosaic of differentiation. Only in pregnancy the 'pure' type of a nonkeratinizing epithelium is seen, apparently as a result of the high concentration of estrogens.

Altered Keratin Expression in Benign and Malignant Skin Diseases Revealed with Monoclonal Antibodies

We studied keratin expression in benign epidermal skin diseases, and in Bowen's disease by using three monoclonal cytokeratin antibodies. In adult normal skin, these antibodies bind only to the follicular epithelium (PKK1), the basal keratinocytes (PKK2), or the suprabasal cells in interfollicular epidermis (KA5). Additionally, in fetal epidermis, the PKK1 antibody reacts with basal keratinocytes. In psoriasis and lichen planus, the PKK2 antibody distinctly revealed all epidermal cell layers by immunostaining. However, a negative basal cell-like layer was revealed in both lesions with the KA5 antibody. In pityriasis rubra pilaris, the basal cell layer was uniformly stained with the PKK2 antibody, but only some keratinocytes in upper cell layers showed fluorescence and, in chronic eczema, the 3-4 lowest epidermal cell layers were reactive. The PKK1 antibody did not stain interfollicular keratinocytes in any of the benign proliferative skin diseases studied. In Bowen's disease, a heterogeneous staining pattern with varying intensity among individual cells was seen with all of the antibodies used. Our results suggest different changes in keratin expression in chronic benign and malignant epidermal diseases that may reflect the mechanisms behind these changes.

Keratin 13 expression is linked to squamous differentiation in rabbit tracheal epithelial cells and down-regulated by retinoic acid

Rabbit tracheal epithelial (RbTE) cells in primary culture undergo at confluence a multistep program of squamous differentiation. This study examines the expression of keratins in RbTE cells in relation to this differentiation process. During the exponential growth phase RbTE cells are undifferentiated and express three major keratins, K5, K14, and K19, and two minor keratins, K6 and K16. Squamous differentiation is accompanied by increased expression of keratins K6, K16, and K19, and in particular of keratin K13, which reacts specifically with the monoclonal antibody AE8. These changes in keratin synthesis coincide with the commitment to terminal differentiation. Retinoic acid, an inhibitor of the expression of the squamous differentiated phenotype, inhibits the increase in the expression of K6, K16, and K13 and reduces the expression of K5 and K14; however, retinoic acid treatment results in increased levels of keratin K19 and K18. Retinoic acid inhibits the expression of K16 and K13 at concentrations as low as 10 −9–10 −10 M. At least some of these changes in keratins appear to be related to alterations in the cellular levels of the respective mRNAs. Our results indicate that specific changes in keratin expression, in particular keratin K13, correlate with the onset of squamous differentiation in RbTE cells. Induction of the expression of keratin K13 may function as a marker of squamous differentiation in tracheobronchial epithelial cells.

Modulation of Keratin Expression in Type II Pneumocytes by the Extracellular Matrix

The expression of specific keratin intermediate filaments during differentiation of rat type II pneumocytes in primary culture on various matrices was investigated. Changes in keratin expression were assessed using a monoclonal antikeratin antibody, 24A3, known to react strongly with alveolar epithelial cells in injured lung. Type II cell differentiation was modulated by culture on extracellular matrices known to either accelerate or retard loss of differentiated morphology and metabolic function. During culture on a plastic or fibronectin-rich surface, loss of cell differentiation correlates with increased staining with 24A3 antikeratin antibody by indirect immunofluorescence and with increased abundance of a family of acidic 46,000-dalton keratin isoforms detected in two-dimensional polyacrylamide gels of type II cell cytoskeletal extracts. Loss of type II cell differentiation is retarded or prevented by culture on substrata of purified laminin or of EHS tumor-derived basement membrane (matrigel). 24A3-linked fluorescence and expression of the 46 kDa keratins are reduced in parallel, although at 7 days in culture on matrigel or laminin, keratin expression increases. The results show that changes in type II cell differentiation effected in primary culture by the extracellular matrix correlates with changes in expression of the 24A3-reactive keratins. Loss of differentiated shape and function favors expression of these cytoskeletal antigens, which may provide quantifiable markers of the type II to type I cell transition that occurs during alveolar remodeling.

Squamous differentiation in normal and transformed rat tracheal epithelial cells

Morphological observations suggest that rate tracheal epithelial (RTE) cells undergo squamous differentiation when maintained in cell culture. The purpose of the studies presented here was to examine and define differentiation of cultured RTE cells with the help of markers previously shown to be specific for squamous differentiation. Furthermore, we wanted to determine whether neoplastic transformation of these cells causes significant disruption of their differentiation program. Our experiments showed that squamous differentiation occurs in normal primary RTE cell cultures. Epidermal transglutaminase (transglutaminase type I) activity increased approximately 20-fold in RTE cultures as a function of time. Cholesterol sulfate, another marker of squamous differentiation, increased only modestly with time. A significant number of cells formed cross-linked envelopes in cultures growth-arrested at a cell density of approximately 250 cells/mm2. However, no significant changes in keratin expression were detected. Neoplastically transformed RTE cells which exhibit a greatly increased growth capacity expressed the same three markers of squamous differentiation as normal RTE cells. However, transglutaminase type I activity was relatively low. The cross-linked envelope formation was independent of cell density in the transformed cells. Like in normal RTE cultures, cholesterol sulfate accumulation only increased moderately with increasing cell density. The keratin pattern of transformed RTE cell lines was identical to that of normal primary RTE cells. A well-differentiated squamous cell carcinoma derived from one of the neoplastic cell lines expressed in vivo keratin markers typical of keratinization (56 kd acidic keratin and 65-67 kd basic keratins). We draw the following conclusions. (i) The biochemical studies confirm that normal RTE cells undergo squamous differentiation. The pathway of terminal squamous differentiation is cell density dependent. (ii) In transformed RTE cells, growth as well as differentiation are less subject to regulation by cell density than in normal cells. (iii) Transformed RTE cells are differentiation competent; the main abnormality appears to be that in transformed cell populations proliferation and differentiation occur concomitantly.

Coordination of keratinocyte programming in human SCC‐13 squamous carcinoma and normal epidermal cells

Exploiting the sensitivity of neoplastic keratinocytes to physiological effectors, this work analyzes the degree of coordination among differentiation markers in the established human epidermal squamous carcinoma cell line SCC-13 in comparison to normal human epidermal cells. This analysis showed that overall keratin content was modulated substantially and in parallel with particulate transglutaminase activity in response to variation of calcium, retinoic acid, and hydrocortisone concentrations in the medium. The changes in keratin expression were evident primarily in the striking stimulation by hydrocortisone or calcium and the virtual suppression by retinoic acid of species in the 56-58 kd region, which have not previously been reported subject to such physiological modulation. In contrast, involucrin levels were coordinated only to a limited degree with particulate transglutaminase activity and keratin content. The very low involucrin levels observed in low calcium medium were increased 5- to 10-fold in high calcium medium. However, they were also increased 5- to 30-fold in low calcium medium by retinoic acid, a clear example of uncoupling. Activities of the tissue transglutaminase were altered considerably by the various culture conditions but were not obviously coordinated to keratinocyte markers. In normal epidermal cells, the suppressive effect of retinoic acid was much more evident with particulate transglutaminase than involucrin levels. While calcium had a large stimulatory effect on both markers, hydrocortisone had little or no influence. These results emphasize the potential importance of quantitative analysis of differentiation markers for resolving the contribution of physiological elements in coordination of cellular programming.

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

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

Keratin protein profile in squamous cell carcinoma as induced in the hamster cheek pouch with DMBA (preliminary report).

The purpose of this study is to compare the keratin profile in DMBA-induced squamous cell carcinoma with that in normal hamster cheek pouch epithelium. The pattern of keratin expression in these two groups was studied by the methods of SDS-PAGE and immunoblot analysis on the electrophoretic keratin protein. A study of the specific changes of this protein were revealed as an essential step for analyzing the consequent changes in keratin expression that occurs in the course of development of a fully malignant lesion.

Changes in Keratin Expression during the Estrogen-Mediated Differentiation of Rat Vaginal Epithelium*

While vaginal cornification occurs naturally during the estrous cycle of the adult rat, it can also be artificially induced by administering exogenous hormone to ovariectomized animals. When the maturation of rat vaginal epithelium was controlled with a timed course of estradiol (E2) injections, total cell extracts from that tissue revealed that six major keratins exhibited stage-specific increases during a 48-h period of stratification and cornification. One-dimensional polyacrylamide gel electrophoresis and electrophoretic immunoblotting showed that the expression pattern involved an earlier rise of the smaller (50,000, 51,000, and 53,000 mol wt) keratins and a later rise of the larger (57,000, 58,000, and 60,000 mol wt) keratins. Polypeptide levels were quantitated by gel densitometry, and an immunodot assay was developed to specifically measure keratin expression as a function of estrogen agonist activity. By way of comparison, four other estrogenic compounds were also evaluated after their administration under a 48-h double injection protocol. Under these circumstances, diethylstilbestrol provided a more potent stimulus than E2, while estriol, enclomiphene and zuclomiphene each produced a pattern of keratin expression and intensity levels that resembled that 24-h stage of E2 treatment. Although the same keratins were expressed by these different compounds, their synthesis was selective, since they often increased 10- to 20-fold while total protein levels only doubled. Keratin levels were correlated with the various degrees of epithelial growth and differentiation observed in parallel histological studies. These results indicate that the expression of vaginal keratins provide a useful model system for the study of estrogen action.