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."     

Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization

When human epidermal cells were seeded on floating rafts of collagen and fibroblasts, they stratified at the air-liquid interface. The suprabasal cells synthesized the large type II (K1) and type I (K10/K11) keratins characteristic of terminal differentiation in skin. At earlier times in culture, expression of the large type II keratins appeared to precede the expression of their type I partners. At later times, all suprabasal cells expressed both types, suggesting that the accumulation of a critical level of K1 keratin may be a necessary stimulus for K10 and K11 expression. Expression of the terminal differentiation-specific keratins was completely suppressed by adding retinoic acid to the culture medium, or by submerging the cultures in normal medium. In submerged cultures, removal of vitamin A by delipidization of the serum restored the keratinization process. In contrast, calcium and transforming growth factor-beta did not influence the expression of the large keratins in keratinocytes grown in the presence of retinoids, even though they are known to induce certain morphological features of terminal differentiation. Retinoic acid in the raft medium not only suppressed the expression of the large keratins, but, in addition, induced the synthesis of two new keratins not normally expressed in epidermis in vivo. Immunofluorescence localized one of these keratins, K19, to a few isolated cells of the stratifying culture. In contrast, the other keratin, K13, appeared uniformly in a few outer layers of the culture. Interestingly, K13 expression correlated well with the gradient of retinoid-mediated disruptions of intercellular interactions in the culture. These data suggest that K13 induction may in some way relate to the reduction in either the number or the strength of desmosomal contacts between suprabasal cells of stratified squamous epithelial tissues.     

Morphological differentiation and changes in polypeptide synthesis pattern during regeneration of human epidermal tissue developed in vitro

By incubating multilayered primary cultures of human keratinocytes in low-calcium medium the suprabasal cell layers can be stripped off leaving a basal cell monolayer. When this monolayer is re-fed normal calcium medium a reproducible series of cell kinetic, morphological, and biochemical changes takes place resulting in the reestablishment of a multilayered tissue. Analysis of cell-cycle-specific proteins indicated that, during regeneration, a large cohort of cells became synchronized undergoing DNA replication after 3 days. Examination of culture morphology at the ultrastructural level confirmed the capacity of the basal cell monolayer to gradually reestablish a multilayered, differentiated epithelium. The ultrastructural appearance at 7 days poststripping was similar to that of unstripped cultures and was indicative of a tissue in steady state. Quantitation of cornified envelope formation at different times during regeneration showed that an increasing proportion of the cells were able to undergo terminal differentiation. In general, the pattern of keratin synthesis in the original epidermal explant labelled in vitro was similar to the pattern observed in human epidermis in vivo; however, in contrast to epidermis in vivo the explant also synthesized the hyperproliferative keratins 6 and 16. The in vitro differentiated keratinocytes showed underexpression of several proteins identified as differentiation markers, whereas several basal cell markers were overexpressed compared to the original explant. In addition, the in vitro differentiated keratinocytes synthesized some new proteins, notably keratins 7, 15 and 19. The basal layer remaining after stripping mainly expressed basal cell markers; however, during recovery, some of the differentiation-specific markers (e.g. keratin 10 and 15) were again expressed together with keratin no. 19, which is also expressed during wound healing in vivo. It is suggested that the present system of regenerating epidermal tissue cultures may serve as an experimental model to investigate certain aspects of the regulation of epidermal tissue homeostasis.     

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.     

Keratin 5 knockout mice reveal plasticity of keratin expression in the corneal epithelium

We have recently demonstrated that the keratin K3 gene, which is active in the suprabasal human corneal epithelium, is missing in the genome of the mouse. We show that a normal K3 gene exists in a wide variety of mammals while in rodents the gene is converted to a pseudogene with a very strong sequence drift. The availability of K5-/- mice provides a unique opportunity to investigate type-specific keratin function during corneal differentiation in the absence of both K5 and K3. Here, we report that the deletion of K5, which in wild-type mice forms a cytoskeleton with K12, does neither cause keratin aggregation nor cytolysis in the cornea. This is due to the induction of K4 in corneal epithelial cells, normally restricted to corneal stem stem cells residing in the limbus. Using a combination of antibodies and RT-PCR, we identified additional keratins expressed in the mouse cornea including K23 which was previously thought to be specific for pancreatic carcinomas. This reflects an unexpected complexity of keratin expression in the cornea. Our data suggest that in the absence of mechanical stress, corneal differentiation does not depend on distinct keratin pairs, supporting a concept of functional redundancy, at least for certain keratins.     

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.     

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.     

Absence of differentiation-related expression of keratin 10 in earlystages of vulvar squamous carcinoma

Using specific monoclonal antibodies (DE-K10 and DE-SCK respectively), the expression of some differentiation-related epidermal keratins was studied in 38 human vulvar squamous carcinomas. In the epidermis, expression of keratin 10 (K10) strictly paralleled the extent of differentiation; it was absent in the basal layer, appeared in the first suprabasal layers and increased in concentration towards the granular layer. However, K10 was rarely detected (1 case out of 12) in early stages of vulvar squamous carcinomas (tumours less than 2 cm, clinical stage I) regardless of the tumour grade. In larger and more advanced tumours (greater than 2 cm, clinical stages II and III), K10 was detected in 21 out of 26 cases. Its expression appeared to be related to maturation of malignant keratinocytes, being preferentially detected in more-differentiated parts. Occasionally however, cells that did not show histological signs of keratinisation were also K10-positive. Modified stratum corneum keratins (recognized specifically by monoclonal antibody DE-SCK) were detected in the most keratinized areas (horn pearls and their close vicinity) of some K10-positive tumours, i.e., in a pattern close to their normal expression in terminally differentiated epidermal cells. These data suggest differences in the regulation of K10 expression during the differentiation processes in the normal keratinising squamous epithelium and in squamous carcinomas. While the normal pattern of vulvar epithelial differentiation is accompanied by an increasing expression of K10, malignant keratinocytes, also when these are histologically moderately or well differentiated, cease expressing this keratin in the early stages of tumour development.     

Commitment to differentiation and expression of early differentiation markers in murine keratinocytes in vitro are regulated independently of extracellular calcium concentrations

In the epidermis, one of the earliest characterized events in keratinocyte differentiation is the coordinate induction of a pair of keratins specifically expressed in suprabasal cells, keratin 1 (K1) and keratin 10 (K10). Both in vivo and in vitro, extracellular calcium is necessary for several biochemical and structural changes during keratinocyte differentiation. However, it has been unclear if calcium serves as a differentiation signal in keratinocytes. In these studies, expression of suprabasal keratin mRNA and protein is used to test whether the initial differentiation of primary mouse keratinocytes in vitro is dependent on changes in the concentration of extracellular calcium. K1 mRNA was expressed at low levels in cultures of keratinocytes growing on plastic in 0.05 mM calcium but in attached cells was not further induced by increases in the concentration of extracellular calcium. Suspension of the keratinocytes into semi-solid medium induced a rapid and substantial increase in both expression of K1 mRNA and in the percentage of cells expressing suprabasal keratin proteins. The induction was unaffected by the concentration of calcium in the semi-solid medium and could not be enhanced by exposing attached cells to higher calcium before suspension. The induction of K1 mRNA could be inhibited by exposure of the keratinocytes to either EGF or fibronectin. These results suggest that commitment of mouse keratinocytes to terminal differentiation is independent of extracellular calcium and may be regulated primarily by extracellular factors other than calcium.     

Adult corneal epithelium basal cells possess the capacity to activate epidermal, pilosebaceous and sweat gland genetic programs in response to embryonic dermal stimuli

Recent work has shown remarkable plasticity between neural and hematopoeitic, as well as between hematopoeitic and muscle stem cells, depending on environmental stimuli (Fuchs, E. and Segre, J. A. (2000) Cell 100, 143-155). Stem cells give rise to a proliferative transient amplifying population (TA), which is generally considered to be irreversibly committed. Corneal epithelium provides a particularly useful system for studying the ability of TA cells to activate different genetic programs in response to a change in their fibroblast environment. Indeed, corneal stem and TA cells occupy different localities - stem cells at the periphery, and TA cells more central (Lehrer, M. S., Sun, T. T. and Lavker, R. M. (1998) J. Cell Sci. 111, 2867-2875) - and thus can be discretely dissected from each other. It is well known that pluristratified epithelia of cornea and skin display distinct programs of differentiation: corneal keratinocytes express keratin pair K3/K12 and epidermal keratinocytes keratin pair K1-2/K10; moreover, the epidermis forms cutaneous appendages, which express their own set of keratins. In our experiments, central adult rabbit corneal epithelium was thus associated either with a mouse embryonic dorsal, upper-lip or plantar dermis before grafting onto nude mice. Complementary experiments were performed using adult mouse corneal epithelium from the Rosa 26 strain. The origin of the differentiated structures were identified in the first case by Hoechst staining and in the second by the detection of beta-galactosidase activity. The results show that adult central corneal cells are able to respond to specific information originating from embryonic dermis. They give rise first to a new basal stratum, which does not express anymore corneal-type keratins, then to pilosebaceous units, or sweat glands, depending of the dermis, and finally to upper layers expressing epidermal-type keratins. Our results provide the first evidence that a distinct TA cell population can be reprogrammed.     

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.     

Regulated expression of differentiation-associated keratins in cultured epidermal cells detected by monospecific antibodies to unique peptides of mouse epidermal keratins

Monospecific antibodies to mouse epidermal keratins were generated in rabbits and guinea pigs by injecting synthetic peptides of unique keratin sequences. The sequences were deduced from nucleotide sequences of cDNA clones representing basal (K14) and suprabasal (K1 and K10) cell-specific and hyperproliferative (K6) keratins of both the type-I and type-II subclasses. By applying single-and double-label immunofluorescence analysis, the expression of keratin peptides was analyzed in cultured keratinocytes maintained in the basal or suprabasal cell phenotypes. These cell types were selected by growth in medium containing 0.05 mM Ca2+ (basal cell) or 1.4 mM Ca2+ (suprabasal cell). The cultured basal cells expressed K6 and K14, but less than 1% expressed K1 and K10. Within a few hours after being placed in 1.4 mM Ca2+, K1 expression was observed, and by 24 h, 10%-17% of the cells expressed K1. K10 expression appeared to lag behind K1 expression, with only 5%-10% of cells in 1.4 mM Ca2+ exhibiting K10 immunoreactivity. Double-labeling studies indicated that virtually all K10-positive cells also expressed K1, while only about one-half of the K1-positive cells expressed K10. The treatment of basal cells with retinoic acid at pharmacological concentrations prevented the expression of K1 and K10 when cells were challenged by 1.4 mM Ca2+. Similarly, the introduction of the v-rasH oncogene into basal cells by a defective retroviral vector prevented the expression of suprabasal keratins in 1.4 mM Ca2+ medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Density-dependent modulation of synthesis of keratins 1 and 10 in the human keratinocyte line HACAT and in ras-transfected tumorigenic clones

The spontaneous human keratinocyte line HaCaT and c-Ha-ras oncogene-transfected cell clones are capable of expressing an unusually broad spectrum of keratins, not observed so far in epithelial cells. This expression is, however, strongly modulated by environmental conditions, including cell density. Both cells of the nontumorigenic HaCaT line and the tumorigenic HaCaT-ras clones, I-7 and II-3 (giving rise to benign and malignant tumors, respectively), constitutively expressed the keratins K5, K6, K14, K16 and K17, which are also common in cultures of normal keratinocytes. In addition keratins K7, K8, K18 and K19, generally associated with simple epithelia, were synthesized (to a most pronounced extent in sparse cultures), while keratins K4, K13 and K15 appeared at confluence, presumably with the onset of stratification. Moreover, in both HaCaT and HaCaT-ras clones the epidermal "suprabasal" keratins, K1 and K10, were expressed in conventional submerged cultures (at normal vitamin A levels), markedly rising with cell density, but not strictly correlated with the degree of stratification. This property was maintained in HaCaT cells up to the highest passages. According to immunofluorescence, this was due to increasing numbers of strongly stained cells, and not due to a gradual increase in all cells. Most strikingly, there was a significant delay in the appearance of K10 compared to K1, and this dissociation of expression was most evident in dispase-detached cell sheets (submerged cultures) and organotypic cultures of the ras clones (grown at the air-liquid interface). While on frozen sections bright staining for K1 was seen in some basal and virtually all suprabasal cell layers, K10 was largely restricted to the uppermost layers. Thus, obviously synthesis of K1 and K10 can be regulated independently, although generally in this given sequence. The apparent compatibility of K1 synthesis with proliferation and particularly the extended delay of K10 expression (as a postmitotic event) might be causally related to altered growth control and as such imply the significance of this disturbance. Finally, the highly preserved epidermal characteristics, in terms of expression of keratins (and other differentiation markers [5]) and their regulation, makes these cell lines excellent candidates for studying external modulators of differentiation and also underlying molecular mechanisms.     

Changes in keratin gene expression during terminal differentiation of the keratinocyte

Cells of the inner layers of the epidermis contain small keratins (46-58K), whereas the cells of the outer layers contain large keratins (63-67K) in addition to small ones. The changes in keratin composition that take place within each cell during the course of its terminal differentiation result largely from changes in synthesis. Cultured epidermal cells resemble cells of the inner layers of the epidermis in synthesizing only small keratins. The cultured cells possess translatable mRNA only for small keratins, whereas mRNA extracted from whole epidermis can be translated into both large and small keratins. As no synthesis takes place in the outermost layer of the epidermis (stratum corneum), the keratins of this layer must be synthesized earlier, but in some cases they then become smaller: this presumably occurs by post-translational processing of the molecules during the final stages of differentiation. Stratified squamous epithelia of internal organs do not form a typical stratum corneum and do not make the large keratins characteristic of epidermis. Their keratins are also different from those of cultured keratinocytes, implying that they have embarked on an alternate route of terminal keratin synthesis.     

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.     

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.     

The role of the basement membrane in differential expression of keratin proteins in epithelial cells

Extracellular matrix is considered to play an important role in determining the phenotype of cells with which it interacts. Here we have investigated the possibility that extracellular matrix is involved in specifying the pattern of keratin expression in epithelial cells. For these studies, we have developed an explant system in which epithelial cells from one type of stratified epithelial tissue, namely conjunctiva, are maintained on an extracellular matrix substrate derived from a different tissue, namely cornea. These ocular tissues are ideal for such analyses since they express distinct sets of keratins. For example, bovine conjunctival epithelium processed for immunofluorescence is not recognized by antibody preparations against keratin K3 or K12. In contrast, K3 and K12 antibodies generate intense staining in bovine corneal epithelium. At the immunochemical level, conjunctival cells in situ appear to possess no K12 and only trace amounts of K3, whereas corneal epithelial cells in situ possess both K3 and K12. When conjunctival cells are maintained on a corneal substrate with an intact basement membrane for 10 days in vitro they begin to express keratin K12 as determined by immunofluorescence. On the other hand, conjunctival cells that are maintained on a corneal substrate lacking a basement membrane fail to show staining with K12 antibodies. Conjunctival cells begin to show intense staining using K3 antibodies within about 10 days of being placed in culture regardless of their substrate. These results indicate that basement membrane can play a positive role in determining cell-specific expression of certain keratins such as K12. However, other keratins such as K3 may be "unmasked" and/or their expression may be upregulated simply by placing conjunctival epithelial cells in culture. We speculate that in conjunctiva K3 expression is influenced by certain negative exogenous factors. We discuss the possible means of regulation of keratin expression in our model system.     

Onset of re-epithelialization after skin injury correlates with a reorganization of keratin filaments in wound edge keratinocytes: defining a potential role for keratin 16

Injury to stratified epithelia causes a strong induction of keratins 6 (K6) and 16 (K16) in post-mitotic keratinocytes located at the wound edge. We show that induction of K6 and K16 occurs within 6 h after injury to human epidermis. Their subsequent accumulation in keratinocytes correlates with the profound reorganization of keratin filaments from a pan-cytoplasmic distribution to one in which filaments are aggregated in a juxtanuclear location, opposite to the direction of cell migration. This filament reorganization coincides with additional cytoarchitectural changes and the onset of re-epithelialization after 18 h post-injury. By following the assembly of K6 and K16 in vitro and in cultured cells, we find that relative to K5 and K14, a well- characterized keratin pair that is constitutively expressed in epidermis, K6 and K16 polymerize into short 10-nm filaments that accumulate near the nucleus, a property arising from K16. Forced expression of human K16 in skin keratinocytes of transgenic mice causes a retraction of keratin filaments from the cell periphery, often in a polarized fashion. These results imply that K16 may not have a primary structural function akin to epidermal keratins. Rather, they suggest that in the context of epidermal wound healing, the function of K16 could be to promote a reorganization of the cytoplasmic array of keratin filaments, an event that precedes the onset of keratinocyte migration into the wound site.     

Severe abnormalities in the oral mucosa induced by suprabasal expression of epidermal keratin K10 in transgenic mice

Previous studies have demonstrated that keratin K10 plays an important role in mediating cell signaling processes, since the ectopic expression of this keratin induces cell cycle arrest in proliferating cells in vitro and in vivo. However, apart from its well known function of providing epithelial cells with resilience to mechanical trauma, little is known about its possible roles in nondividing cells. To investigate what these might be, transgenic mice were generated in which the expression of K10 was driven by bovine K6beta gene control elements (bK6(beta)hK10). The transgenic mice displayed severe abnormalities in the tongue and palate but not in other K6-expressing cells such as those of the esophagus, nails, and hair follicles. The lesions in the tongue and palate included the cytolysis of epithelial suprabasal cells associated with an acute inflammatory response and lymphocyte infiltration. The alterations in the oral mucosa caused the death of transgenic pups soon after birth, probably because suckling was impaired. These anomalies, together with others found in the teeth, are reminiscent of the lesions observed in some patients with pachyonychia congenita, an inherited epithelial fragility associated with mutations in keratins K6 and K16. Although no epithelial fragility was observed in the bK6(beta)hK10 oral epithelia of the experimental mice, necrotic processes were seen. Collectively, these data show that the carefully regulated tissue- and differentiation-specific patterns displayed by the keratin genes have dramatic consequences on the biological behavior of epithelial cells and that changes in the specific composition of the keratin intermediate filament cytoskeleton can affect their physiology, in particular those of the oral mucosa.