Keratin polypeptide expression in mouse epidermis and cultured epidermal cells

Adult mouse epidermis contains up to 11 distinct keratin polypeptides, as resolved by two-dimensional gel electrophoresis. These include both basic (Type II; 67-, 65-, 63-, 62-, and 60-kDa) and acidic (Type I; 61- to 59-, 54-, 52-, 49-, and 48-kDa) keratins that exhibit multiple isoelectric forms. Several, but not all, of these keratins, identified by immunoblotting, were found to be actively synthesized in the skin when assayed in short-term pulse-labeling experiments. When compared to the adult, newborn mouse epidermis expresses fewer keratin subunits. However, greater amounts of keratins associated with differentiated suprabasal cells and stratum corneum, which is more pronounced morphologically in the newborn, were identified. We also observed strain-specific differences in the expression of a Type I acidic keratin. This 61-kDa (pI, approx. 5.3) keratin was produced exclusively by the CF-1 mouse and, based on peptide mapping, appeared to be related to the acidic 59-kDa keratin that was identified in this strain as well as all other mouse strains. The 61-kDa keratin was not expressed in vitamin A-deficient animals, suggesting that its appearance may be related to a retinoid-dependent posttranslational modification. In comparison to keratin expression in vivo, primary mouse keratinocyte monolayer cultures maintained in low Ca2+ (less than 0.08 mM) did not express the terminal differentiation keratins of 67-kDa (basic) or 59-kDa (acidic), although enhanced synthesis of the 60-kDa (basic) and the 52-kDa and 59-kDa (acidic) keratins associated with proliferation were observed. In addition, a subpopulation of nonadherent cells was continuously produced by the primary keratinocyte cultures that expressed the 67-kDa (basic) keratin specific for terminal differentiation. When the keratinocyte cultures were induced to terminally differentiate with Ca2+, the overall pattern of keratin expression was not changed significantly. Taken together, these results provide further evidence for the variable nature of keratin expression in mouse epidermal keratinocytes under different growth conditions.     

Three cDNA sequences of mouse type I keratins. Cellular localization of the mRNAs in normal and hyperproliferative tissues

We have constructed cDNA libraries with poly(A)+ RNA from normal mouse footpad epidermis and from a squamous cell carcinoma of mouse back skin. Both libraries were screened for type I keratin clones. We present sequence data of three keratin cDNA clones which selected mRNAs coding for two 52-kDa proteins (clones pke 52 and pkSCC 52) as well as for a 50-kDa protein (clone pkSCC50). According to their carboxyl-terminal sequences, the two 52-kDa keratin proteins belong to a group of keratins with serine-rich subdomains adjacent to the alpha-helix, whereas the short carboxyl-terminus of the 50-kDa protein lacks a distinct substructure. Sequentially the two 52-kDa keratins are more closely related to each other than to any other mouse type I keratin. However, in situ hybridization with specific subclones reveals a distinctly different pattern of expression in mouse epithelia. Clone pkSCC 52 contains sequence information for a 52-kDa keratin present in basal cells of epidermis and other stratified epithelia, whereas the pke 52 cDNA encodes a keratin which is predominantly expressed in suprabasal cells of nonepidermal tissues. In terms of nucleotide sequence identities, it cannot precisely be decided which of the two mouse 52-kDa proteins is the equivalent of the human epidermal 50-kDa keratin protein (Hanukoglu, I., and Fuchs, E. (1982) Cell 31, 243-252). In the case of the bovine keratin VII, however (Jorcano, J.L., Rieger, M., Franz, J.K., Schiller, D.L., Moll, R., and Franke, W.W. (1984) J. Mol. Biol. 179, 257-281) the sequence identity values speak for an equivalence with the mouse ke 52 keratin. Obviously, in situ hybridization experiments would best be suited to unravel the precise interspecies relationship between the four highly similar keratins. The discriminatory efficacy of this technique is further emphasized by the demonstration that the mRNA for a 50-kDa keratin is present not only in hyperproliferative epithelia, but also in normal cells of hair follicles.     

Overexpression of CRABPI in suprabasal keratinocytes enhances the proliferation of epidermal basal keratinocytes in mouse skin topically treated with all-trans retinoic acid

We investigated whether ectopic expression of CRABPI, a cellular retinoic acid binding protein, influenced the actions of all-trans retinoic acid (ATRA) in transgenic (TG) mice. We targeted CRABPI to the basal vs. suprabasal layers of mouse epidermis by using the keratin 14 (K14) and keratin 10 (K10) promoters, respectively. Greater CRABPI protein levels were detected in the epidermis of adult transgenic(+) mice than in transgenic(-) mice for both transgenes. In adult mouse skin CRABPI overexpression in the basal or suprabasal keratinocytes did not cause morphological abnormalities, but did result in decreased CRABPII mRNA levels. Ectopically overexpressed CRABPI in suprabasal keratinocytes, but not in basal keratinocytes, enhanced the thickening of the epidermis induced by topical ATRA treatments (10 microM, 400 microl for 4 days) by 1.59+/-0.2-fold (p<0.05). ATRA treatment (10 microM) resulted in a 59.9+/-9.8% increase (p<0.05) in the BrdU labeling index in K10/FLAG-CRABPI TG(+) mice vs. TG(-) mice. Retinoid topical treatments reduced p27 and CYP26A1 mRNA levels in TG(+) and TG(-) mouse skin in K14 and K10/FLAG-CRABPI transgenic mice. As epidermal basal keratinocyte proliferation is stimulated by paracrine growth factors secreted by ATRA activated suprabasal keratinocytes, our results indicate that CRABPI overexpression in suprabasal keratinocytes enhances the physiological functions of ATRA.     

Transdifferentiation of corneal epithelium: evidence for a linkage between the segregation of epidermal stem cells and the induction of hair follicles during embryogenesis

Corneal epithelium transdifferentiation into a hair-bearing epidermis provides a particularly useful system for studying the possibility that transient amplifying (TA) cells are able to activate different genetic programs in response to a change in their fibroblast environment, as well as to follow the different steps of rebuilding an epidermis from induced stem cells. Corneal stem and TA cells are found in different locations - stem cells at the periphery, in the limbus, and TA cells more central. Moreover, the TA cells already express the differentiating corneal-type keratin pair K3/K12, whereas the limbal keratinocytes express the basal keratin pair K5/K14. In contrast, suprabasal epidermal keratinocytes express keratin pair K1-2/K10, and basal keratinocytes the keratin pair K5/K14. The results of tissue recombination experiments show that adult central corneal cells are able to respond to specific information originating from embryonic dermis. First, the cells located at the base of the corneal epithelium show a decrease in expression of K12 keratin, followed by an increase in K5 expression; they then proliferate and form hair follicles. The first K10 expressing cells appear at the junction of the new hair follicles and the covering corneal epithelium. Their expansion finally gives rise to epidermal strata, which displace the corneal suprabasal keratinocytes. Corneal TA cells can thus be reprogrammed to form epidermal cells, first by reverting to a basal epithelial-type, then to hair pegs and probably concomitantly to hair stem cells. This confirms the role of the hair as the main reservoir of epidermal stem cells and raises the question of the nature of the dermal messages which are both involved in hair induction and stem cell specification.     

Adult precursor cells in the tail epidermis of Xenopus tadpoles

Polyclonal antibodies were raised against Xenopus larva-specific 58 kDa keratin (PAK58) and adult-specific 63 kDa keratin (PAK63), in order to examine the origin of 63 kDa-keratin-producing cells in the tail skin. By immunofluorescent staining of the tail skin, the 58 kDa keratin was recognized in almost all of the larval epidermal cells, although a small number of PAK58-negative cells were detected at stage 64. In contrast, 63 kDa keratin was immunohistochemically recognized at stage 58, but the signal was very weak. The number of epidermal layers in the tail epidermis increased during a period from stage 58 to stage 64. At stage 64, a small number of PAK63-positive cells was clearly identified in the multilayered tail epidermis. Comparative analysis of successive sections showed that PAK63-positive cells are derived from a cell group differing from PAK58-positive cells. Immunohistochemical studies using cultured epidermal cells demonstrated that 58 kDa keratin is localized in the cytoskeletal bundles of skein cells, whereas 63 kDa keratin is produced not by skein cells but by basal cells and their descendants. These results suggest that basal cells are the adult precursor cells within the larval epidermis even in the tail area.     

The keratin polypeptide patterns in heterotypically recombined epithelia of skin and mucosa of adult mouse

Epithelial-mesenchymal interactions were investigated considering both morphologic criteria and keratin polypeptide expression in homotypic and heterotypic recombinants of adult mouse skin and oral mucosa. Two series of cross-recombinants of epithelia with different morphology and keratin patterns were chosen: (a) footpad epidermis/ear dermis and ear epidermis/footpad dermis; (b) palate epithelium/cheek connective tissue and cheek epithelium/palate connective tissue. Homotypic and heterotypic recombinants were prepared after EDTA-separation of the original tissues and then grown on syngeneic mice in subcutaneously prepared protected graft chambers. EDTA-separation is especially suited to completely separate the epidermal-dermal union, and the transplantation procedure used strictly prevents contamination with host epithelium. Five weeks after implantation keratins were analyzed by one and two-dimensional gel electrophoresis and peptide mapping. In both series, homotypic recombination of the tissues did not alter the original morphology and keratin polypeptide composition of the individual epithelial components. Ear epidermis displayed no significant changes in structure or keratin pattern in heterotypic recombinants. Recombined with ear dermis, footpad epidermis showed acquisition of some morphologic features typical for ear epidermis and slight changes in keratin composition which were, however, difficult to interpret due to the normal similarities of footpad keratin with that of ear. In contrast, the heterorecombinants of the palate/cheek series exhibited considerable alterations in their keratin patterns. Either epithelium showed suppression of distinct keratin subunits and de novo expression of subunits characteristic of the epithelium normally associated with the connective tissue component. The keratin patterns of both matches closely resembled each other and represented patterns intermediate between the normal patterns. This partial, however, significant modulation in the expression of differentiation markers was paralleled by similarly directed changes in the architecture of the heterotransplanted tissues, thus indicating that both morphogenesis and cytodifferentiation of certain adult epithelia can be influenced by extrinsic mesenchymal factors.     

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.     

Novel Rana keratin genes and their expression during larval to adult epidermal conversion in bullfrog tadpoles

The conversion of the larval to adult epidermis during metamorphosis of tadpoles of bullfrog, Rana catesbeiana, was investigated utilizing newly cloned Rana keratin cDNAs as probes. Rana larval keratin (RLK) cDNA (rlk) was cloned using highly specific antisera against Xenopus larval keratin (XLK). Tail skin proteins of bullfrog tadpoles were separated by 2-dimensional gel electrophoresis and subjected to Western blot analysis with anti-XLK antisera. The Rana antigen detected by this method was sequenced and identified as a type II keratin. We cloned rlk from tadpole skin by PCR utilizing primers designed from these peptide sequences of RLK. RLK predicted by nucleotide sequences of rlk was a 549 amino acid -long type II keratin. Subtractive cloning between the body and the tail skin of bullfrog tadpole yielded a cDNA (rak) of Rana adult keratin (RAK). RAK was a 433 amino acid-long type I keratin. We also cloned a Rana keratin 8 (RK8) cDNA (rk8) from bullfrog tadpole epidermis. RK8 was 502 amino acid-long and homologous to cytokeratin 8. Northern blot analyses and in situ hybridization experiments showed that rlk was actively expressed through prometamorphosis in larva-specific epidermal cells called skein cells and became completely inactive at the climax stage of metamorphosis and in the adult skin. RAK mRNA was expressed in basal cells of the tadpole epidermis and germinative cells in the adult epidermis. The expression of rlk and rak was down- and up-regulated by thyroid hormone (TH), respectively. In contrast, there was no change in the expression of RK8 during spontaneous and TH-induced metamorphosis. RK8 mRNA was exclusively expressed in apical cells of the larval epidermis. These patterns of keratin gene expression indicated that the expression of keratin genes is differently regulated by TH depending on the type of larval epidermal cells. The present study demonstrated the usefulness of these genes for the study of molecular mechanism of postembryonic epidermal development and differentiation.     

Transforming growth factor-beta-activated kinase 1 is essential for differentiation and the prevention of apoptosis in epidermis

Transforming growth factor-beta-activated kinase 1 (TAK1) is a member of the mitogen-activated protein (MAP) kinase family and is an upstream signaling molecule of nuclear factor-kappaB (NF-kappaB). Given that NF-kappaB regulates keratinocyte differentiation and apoptosis, TAK1 may be essential for epidermal functions. To test this, we generated keratinocyte-specific TAK1-deficient mice from Map3k7(flox/flox) mice and K5-Cre mice. The keratinocyte-specific TAK1-deficient mice were macroscopically indistinguishable from their littermates until postnatal day 2 or 3, when the skin started to roughen and wrinkle. This phenotype progressed, and the mice died by postnatal day 7. Histological analysis showed thickening of the epidermis with foci of keratinocyte apoptosis and intra-epidermal micro-abscesses. Immunohistochemical analysis showed that the suprabasal keratinocytes of the TAK1-deficient epidermis expressed keratin 5 and keratin 14, which are normally confined to the basal layer. The expression of keratin 1, keratin 10, and loricrin, which are markers for the suprabasal and late phase differentiation of the epidermis, was absent from the TAK1-deficient epidermis. Furthermore, the TAK1-deficient epidermis expressed keratin 16 and had an increased number of Ki67-positive cells. These data indicate that TAK1 deficiency in keratinocytes results in abnormal differentiation, increased proliferation, and apoptosis in the epidermis. However, the keratinocytes from the TAK1-deficient epidermis induced keratin 1 in suspension culture, indicating that the TAK1-deficient keratinocytes retain the ability to differentiate. Moreover, the removal of TAK1 from cultured keratinocytes of Map3k7(flox/flox) mice resulted in apoptosis, indicating that TAK1 is essential for preventing apoptosis. In conclusion, TAK1 is essential in the regulation of keratinocyte growth, differentiation, and apoptosis.     

The intermediate filament system of the keratinizing mouse forestomach epithelium: Coexpression of keratins of internal squamous epithelia and of epidermal keratins in differentiating cells

Summary The internal epithelium of mouse forestomach represents a fully keratinized tissue that has many morphological aspects in common with the integumental epidermis. In the present study we have, therefore, analyzed keratin expression in the total epithelium, in subfractions of basal cells and in living and dead suprabasal cells that were obtained by Percoll density gradient centrifugation of trypsin-dissociated forestomach keratinocytes. The keratin analysis revealed that basal forestomach keratinocytes synthesize the same keratin types as basal epidermal cells (60 000, 52 000 and 47 000 daltons), whereas differentiating cells contain both the epidermal suprabasal keratin pair (67 000 and 59 000 daltons) and the suprabasal keratin pair characteristic for other internal squamous epithelia (57 000 and 47 000 daltons). Indirect immunofluorescence using an antibody recognizing the members of the epidermal-type suprabasal keratin pair and in-situ-hybridization experiments using specific cDNA probes for the members of the internal-type keratin pair showed that the two keratin pairs are uniformly coexpressed in living suprabasal forestomach keratinocytes. Furthermore, it could be shown that distinct cells in the basal cell layer acquire the ability to express both the 67 000/59 000 dalton and the 57 000/47 000 dalton keratin pair and that some basal cells apparently lose the ability to synthesize mRNAs for basal keratins.     

Structural features and sites of expression of a new murine 65 kD and 48 kD hair-related keratin pair, associated with a special type of parakeratotic epithelial differentiation

In the course of studies on local keratin phenotypes in the epidermis of the adult mouse, we have identified a new 65 kD and 48 kD keratin pair. In mouse skin, this keratin pair is only expressed in suprabasal cells of adult mouse tail scale epidermis which is characterized by the complete absence of a granular layer and the formation of a remarkably compact stratum corneum. A second site in which the 65 kD and 48 kD keratin pair is suprabasally expressed and whose morphology corresponds to that of tail scale epidermis is found in the posterior unit of the complex filiform papillae of mouse tongue. The causal relationship of the expression of the 65 kD and 48 kD keratins with this particular type of a non-pathological epithelial parakeratosis is emphasized by the suppression of the mRNA synthesis of the two keratins during retinoic acid mediated orthokeratotic conversion of tail scale epidermis. Apart from tail scale epidermis and the posterior unit of the filiform papillae, the 65 kD and 48 kD keratin pair is, however, also coexpressed with "hard" alpha keratins in suprabulbar cells of hair follicles and in suprabasal cells of the central core unit of the lingual filiform papillae. The non alpha-helical domains of the two new keratins are rich in cysteine and proline residues and lack the typical subdomains into which epithelial keratins of both types can be divided. This structural resemblance of the 65 kD and 48 kD keratins to "hard" alpha keratins is supported by comparative flexibility predictions for their non alpha-helical domains. Phylogenetic investigations then show that the 65 kD and 48 kD keratin pair has evolved together with hair keratins, but has diverged from these during evolution to constitute an independent branch of a pair of hair-related keratins. In view of this exceptional position of the 65 kD and 48 kD keratins within the keratin multigene family, their expression has apparently been adopted by rare anatomical sites in which an orthokeratinized stratum corneum would be too soft and a hard keratinized structure would be too rigid to meet the functional requirement of the respective epithelia.     

New facets of keratin K77: interspecies variations of expression and different intracellular location in embryonic and adult skin of humans and mice

The differential expression of keratins is central to the formation of various epithelia and their appendages. Structurally, the type II keratin K77 is closely related to K1, the prototypical type II keratin of the suprabasal epidermis. Here, we perform a developmental study on K77 expression in human and murine skin. In both species, K77 is expressed in the suprabasal fetal epidermis. While K77 appears after K1 in the human epidermis, the opposite is true for the murine tissue. This species-specific pattern of expression is also found in conventional and organotypic cultures of human and murine keratinocytes. Ultrastructure investigation shows that, in contrast to K77 intermediate filaments of mice, those of the human ortholog are not attached to desmosomes. After birth, K77 disappears without deleterious consequences from human epidermis while it is maintained in the adult mouse epidermis, where its presence has so far gone unnoticed. After targeted Krt1 gene deletion in mice, K77 is normally expressed but fails to functionally replace K1. Besides the epidermis, both human and mouse K77 are present in luminal duct cells of eccrine sweat glands. The demonstration of a K77 ortholog in platypus but not in non-mammalian vertebrates identifies K77 as an evolutionarily ancient component of the mammalian integument that has evolved different patterns of intracellular distribution and adult tissue expression in primates.     

Demonstration of the pathogenic effect of point mutated keratin 9 in vivo

A wild type keratin 9 (K9) cDNA and a point mutated keratin 9 cDNA were injected subcutaneously into mouse skin. The hemagglutinin tag staining of the wild type K9 cDNA injected specimens mainly showed a homogeneous pattern, whereas the point mutated K9 cDNA injected specimens mainly showed a granular pattern in the suprabasal cells. Double staining of K9 and the endogenous keratin revealed the incorporation of de novo synthesized K9 into the keratin network. These results demonstrate that (1) a naked DNA transfection into mouse skin can detect the pathogenic changes of point mutated keratin in vivo and (2) the keratin 9 mutation disrupts the keratin network formation in the suprabasal cells in vivo.     

Keratin Polypeptide Analysis in Fetal and in Terminally Differentiating Newborn Mouse Epidermis

The keratin polypeptide pattern of neonatal mouse epidermis consists of eight individual polypeptides having molecular weights of between 46,000 and 67,000. Unlike the keratin patterns in adult mouse epidermis, this pattern is not subject to body site-specific alterations regarding the specific content of distinct polypeptides or the absolute number of keratin constituents.
At day 16 of fetal development the neonatal keratin pattern is only partially expressed, it being fully completed just prior to birth at day 19 of gestation. A comparative analysis of the sequential changes in epidermal morphology and keratin pattern during the last third of embryonic development confirms the view that, independent of the species, keratin polypeptides below 60,000 mol. wt. are generated by basal cells, whereas the biosynthesis of high molecular weight keratin members take place in the suprabasal cell compartments of keratinizing epithelia. The site of origin of five polypeptides (60,000, 58,000, 52,000, 49,000, 46,000) could therefore be attributed to the basal cell layer, the remaining three polypeptides (67,000, 64,000, 62,000) being synthesized in the outer metabolically active epidermal layers. Similar conclusions could be drawn after subfractionation of neonatal epidermis into living (basal, spinous, and granular) and dead cell layers (stratum corneum), and investigation of the corresponding keratin patterns.
During their progression through the epidermis, two proteins (60,000, 58,000) undergo a hitherto undescribed type of posttranslational modification characterized by a slight increase in size and a change in electrical charge. The mechanism underlying this modification is unknown and it is unclear whether the modification if functional or trivial. The largest keratin polypeptide (67,000) of the protein family - probably a product of spinous cells - disappears from the cornified layer without any evidence that it serves as a precursor for smaller keratin subunits.     

Keratin polypeptide analysis in fetal and in terminally differentiating newborn mouse epidermis

The keratin polypeptide pattern of neonatal mouse epidermis consists of eight individual polypeptides having molecular weights of between 46,000 and 67,000. Unlike the keratin patterns in adult mouse epidermis, this pattern is not subjects to body site-specific alterations regarding the specific content of distinct polypeptides or the absolute number of keratin constituents. At day 16 of fetal development the neonatal keratin pattern is only partially expressed, it being fully completed just prior to birth at day 19 of gestation. A comparative analysis of the sequential changes in epidermal morphology and keratin pattern during the last third of embryonic development confirms the view that, independent of the species, keratin polypeptides below 60,000 mol. wt. are generated by basal cells, whereas the biosynthesis of high molecular weight keratin members take place in the suprabasal cell compartments of keratinizing epithelia. The site of origin of five polypeptides (60,000, 58,000, 52,000, 49,000, 46,000) could therefore be attributed to the basal cell layer, the remaining three polypeptides (67,000, 64,000, 62,000) being synthesized in the outer metabolically active epidermal layers. Similar conclusions could be drawn after subfractionation of neonatal epidermis into living (basal, spinous, and granular) and dead cell layers (stratum corneum), and investigation of the corresponding keratin patterns. During their progression through the epidermis, two proteins (60,000, 58,000) undergo a hitherto undescribed type of posttranslational modification characterized by a slight increase in size and a change in electrical charge. The mechanism underlying this modification is unknown and it is unclear whether the modification if functional or trivial. The largest keratin polypeptide (67,000) of the protein family -- probably a product of spinous cells -- disappears from the cornified layer without any evidence that it serves as a precursor for smaller keratin subunits.     

KERATINS AND SKIN DISORDERS

The epidermal keratinocytes express two major pairs of keratin polypeptides. One pair (K5/K14) expressed specifically in basal generative compartment and the other (K1/K10) expressed specifically in the differentiating suprabasal compartment. The switch in the expression of the keratins from proliferating to differentiating compartment indicates the changes that occur in the keratin filament organization which in turn influences the functional properties of the epidermis. Proper regulation of keratin gene expression and the filament organization are absolutely necessary for normal functioning of the skin. Keratin gene mutations can influence the filament integrity thereby causing several heritable blistering disorders of the skin such as epidermolysis bullosa, bullous icthyosiform erythroderma, etc. Changes in the keratin gene expression may lead to incomplete differentiation of the epidermal keratinocyte, causing hyperproliferative diseases of the skin such as psoriasis, carcinomas, etc. This review briefly describes the changes in keratin structure or gene expression that are known to result in various disorders of the skin.     

Spatial, temporal, and hormonal regulation of epidermal keratin expression during development of the frog, Xenopus laevis.

To study the mechanism of hormone-induced keratin expression in the epidermis during Xenopus metamorphosis, a monospecific antibody was raised against a unique carboxy-terminal peptide of the 63-kDa keratin. Immunohistological analysis demonstrated that the onset of 63-kDa keratin expression showed distinct regional and temporal differences. The expression started at stage 54 in the hindlimb epidermis, at stage 57 in the head, and over 1 month later at stage 63 in the tail. The amount of 63-kDa keratin was further regulated during epidermal stratification and differentiation. The 63-kDa keratin was expressed first in basal epidermal cells before stratification began. The outer layer of the larval epidermis (periderm) did not express the 63-kDa keratin. As the cells moved out of basal layer, they stained more intensely with the anti-keratin antibody indicating that 63-kDa keratin synthesis is up-regulated during differentiation. Similar results were obtained with cultures of purified epidermal cells grown in high calcium conditions. Since we have shown that thyroid hormone (T3) induces 63-kDa keratin gene expression and hydrocortisone (HC) modulates T3 action we examined the effects of T3 and HC at the single cell level with the anti-keratin antibody. Immunostaining demonstrated that T3 alone and T3 plus HC increased the number of 63-kDa keratin-positive cells as well as the amount of 63-kDa keratin per cell. Unexpectedly these hormones had the same effects on head and tail epidermal cells even though the latter cells degenerate during metamorphosis. The major difference between tail and head cells was that the percentage 63-kDa keratin-producing cells was much greater in the head than in the tail.     

Sequential expression of mRNA-encoded keratin sets in neonatal mouse epidermis: basal cells with properties of terminally differentiating cells

The keratin pattern of newborn mouse epidermis was investigated during terminal differentiation. In highly pure fractions of basal and suprabasal cells, obtained by Percoll density gradient centrifugation, we identified two sets of mRNA-encoded proteins: a basal set of 58.5, 52, and 47 kd subunits and a suprabasal set of 67 and 60 kd subunits. The large subunits of each set were alkaline to neutral, while the small subunits were acidic. Polyclonal antibodies against the suprabasal, acidic 60 kd protein and the basal, alkaline 58.5 kd protein selectively recognized their antigens in immunoblots of NEPHGE -resolved keratins and decorated the corresponding epidermal compartments in frozen sections. The antibody to the suprabasal 60 kd protein also recognized distinct cells in the basal cell layer. Quantification of this cell population revealed a 10% cell fraction, morphologically indistinguishable from the total cell population, that, in addition to expressing basal keratin proteins, was already synthesizing suprabasal keratin subunits.