Multi parameter flow cytometric assessment of regenerative epidermis with special reference to the antiproliferative effect of occlusion.

Multi parameter flow cytometrical assays permit simultaneous assessment of proliferation, differentiation, and inflammation parameters. In this study, the validation of TO-PRO-3 iodide (TP3) compared to propidium iodide (PI) and DE-K10 compared to RKSE60 were evaluated in tape stripping induced hyperproliferation. No occlusion, Duoderm (intermediate occlusion) and Blenderm (maximal occlusion) were used as a model to evaluate the effect of occlusion on epidermal regeneration. Proliferation in the keratin 10-negative compartment measured with TP3 proved to be a good approximation of proliferation measured with PI. Other epidermal subpopulations (keratin 10-dim and -bright cells) did not make a relevant contribution to hyperproliferation. DE-K10 is probable more sensitive than RKSE60 to distinguish populations that differ in degree of differentiation. Occlusion of tape stripped skin resulted in decreased proliferation and increased differentiation. This effect was most pronounced with maximal occlusion. This study showed that occlusion is a therapy, which realises normalisation of hyperproliferative skin disorders.     

Differential extraction of keratin subunits and filaments from normal human epidermis

We have investigated keratin interactions in vivo by sequentially extracting water-insoluble proteins from normal human epidermis with increasing concentrations of urea (2, 4, 6, and 9.5 M) and examining each extract by one- and two-dimensional gel electrophoresis, immunoblot analysis using monoclonal anti-keratin antibodies, and EM. The viable layers of normal human epidermis contain keratins K1, K2, K5, K10/11, K14, and K15, which are sequentially expressed during the course of epidermal differentiation. Only keratins K5, K14, and K15, which are synthesized by epidermal basal cells, were solubilized in 2 M urea. Extraction of keratins K1, K2, and K10/11, which are expressed only in differentiating suprabasal cells, required 4-6 M urea. Negative staining of the 2-M urea extract revealed predominantly keratin filament subunits, whereas abundant intermediate-sized filaments were observed in the 4-urea and 6-M urea extracts. These results indicate that in normal human epidermis, keratins K5, K14, and K15 are more soluble than the differentiation-specific keratins K1, K2, and K10/11. This finding suggests that native keratin filaments of different polypeptide composition have differing properties, despite their similar morphology. Furthermore, the observation of stable filaments in 4 and 6 M urea suggests that epidermal keratins K1, K2, and K10/11, which ultimately form the bulk of the protective, nonviable stratum corneum, may comprise filaments that are unusually resistant to denaturation.     

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.     

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.     

Abnormal expression and processing of keratins in pupoid fetus (pf/pf) and repeated epilation (Er/Er) mutant mice

The pupoid fetus (pf) and repeated epilation (Er) mutations of mice result in a failure of epidermal differentiation in homozygotes. Expression of the epidermal keratins has been followed in pf/pf and Er/Er mice by two-dimensional gel electrophoresis, and by immunohistochemistry and Western blotting using polyclonal antibodies that are monospecific for individual keratin polypeptides. Our results show that expression of the differentiation-specific keratins (K1 and K10) is delayed in both the pf/pf and Er/Er mutants and that, when these keratins do appear later in development, they are localized in the deeper layers of the thickened mutant epidermis. Conversely, K6 and K16, two keratins found in low abundance in normal epidermis, are abundant in mutant epidermis. In newborn mutant epidermis, K6 and K16 are found to be most abundant in the outermost epidermal cells, a distribution opposite to that of K1 and K10. These findings suggest that the expression of these hyperplastic keratins in mutant mice may occur to the exclusion of the differentiation-specific keratins both during development and in newborn animals. Differentiation, and an apparently normal pattern of keratin expression, occur when whole pf/pf or Er/Er skin is grafted to normal mice. These results suggest that the pf and Er genes may be expressed systemically and that transfer of the mutant skin to a "normal" environment results in the recovery of a normal phenotype.     

The use of retinoic acid to probe the relation between hyperproliferation-associated keratins and cell proliferation in normal and malignant epidermal cells

When cells from normal human epidermis and from the human squamous cell carcinoma line SCC-13 were seeded on floating rafts of collagen and fibroblasts, they stratified and underwent terminal differentiation. Although the program of differentiation in SCC-13 cells was morphologically abnormal, the cultures resembled normal epidermal raft cultures by expressing the terminal differentiation-specific keratins, K1/K10, and by restricting their proliferative capacity to the basal-like cells of the population. In addition, the differentiating cells of both normal and SCC-13 raft cultures expressed keratins K6 and K16, which are not normally expressed in epidermis, but are synthesized suprabasally during wound-healing and in various epidermal diseases associated with hyperproliferation. While the behavior of normal and SCC-13 rafts was quite similar when they were cultured over normal medium, significant biochemical differences began to emerge when the cultures were exposed to retinoic acid. Most notably, while the SCC-13 cultures still stratified extensively, they showed a marked inhibition of both abnormal (K6/K16) and normal (K1/K10) differentiation-associated keratins, concomitantly with an overall disappearance of differentiated phenotype. Surprisingly, the reduction in K6/K16 in retinoid-treated SCC-13 cultures was not accompanied by a decrease in cell proliferation. Using immunohistochemistry combined with [3H]thymidine labeling, we demonstrate that while the expression of K6 and K16 are often associated with hyperproliferation, these keratins are only produced in the nondividing, differentiating populations of proliferating cultures. Moreover, since their expression can be suppressed without a corresponding decrease in proliferation, the expression of these keratins cannot be essential to the nature of the hyperproliferative epidermal cell.     

Increased expression of keratin 16 causes anomalies in cytoarchitecture and keratinization in transgenic mouse skin

Injury to epidermis and other stratified epithelia triggers profound but transient changes in the pattern of keratin expression. In postmitotic cells located at the wound edge, a strong induction of K6, K16, and K17 synthesis occurs at the expense of the keratins produced under the normal situation. The functional significance of these alterations in keratin expression is not known. Here, we report that overexpression of a wild-type human K16 gene in a tissue-specific fashion in transgenic mice causes aberrant keratinization of the hair follicle outer root sheath and proximal epidermis, and it leads to hyperproliferation and increased thickness of the living layers (acanthosis), as well as cornified layers (hyperkeratosis). The pathogenesis of lesions in transgenic mouse skin begins with a reorganization of keratin filaments in postmitotic keratinocytes, and it progresses in a transgene level-dependent fashion to include disruption of keratinocyte cytoarchitecture and structural alterations in desmosomes at the cell surface. No evidence of cell lysis could be found at the ultrastructural level. These results demonstrate that the disruption of the normal keratin profile caused by increased K16 expression interferes with the program of terminal differentiation in outer root sheath and epidermis. They further suggest that when present at sufficiently high intracellular levels, K16, along with K6 and K17, appear capable of inducing a reorganization of keratin filaments in the cytoplasm of skin epithelial cells.     

Functional Differences between Keratins of Stratified and Simple Epithelia

Dividing populations of stratified and simple epithelial tissues express keratins 5 and 14, and keratins 8 and 18, respectively. It has been suggested that these keratins form a mechanical framework important to cellular integrity, since their absence gives rise to a blistering skin disorder in neonatal epidermis, and hemorrhaging within the embryonic liver. An unresolved fundamental issue is whether different keratins perform unique functions in epithelia. We now address this question using transgenic technology to express a K16-14 hybrid epidermal keratin transgene and a K18 simple epithelial keratin transgene in the epidermis of mice null for K14. Under conditions where the hybrid epidermal keratin restored a wild-type phenotype to newborn epidermis, K18 partially but not fully rescued. The explanation does not appear to reside in an inability of K18 to form 10-nm filaments with K5, which it does in vitro and in vivo. Rather, it appears that the keratin network formed between K5 and K18 is deficient in withstanding mechanical stress, leading to perturbations in the keratin network in regions of the skin that are subjected either to natural or to mechanically induced trauma. Taken together, these findings suggest that the loss of a type I epidermal keratin cannot be fully compensated by its counterpart of simple epithelial cells, and that in vivo, all keratins are not equivalent.     

Sequence, evolution and tissue expression patterns of an epidermal type I keratin from the shark Scyliorhinus stellaris

From the shark Scyliorhinus stellaris we cloned and sequenced a cDNA encoding a novel type I keratin, termed SstK10. By MALDI-MS peptide mass fingerprinting of cytoskeletal proteins separated on polyacrylamide gels, we assigned SstK10 to a 46-kDa protein which is the major epidermal type I ("IE") keratin in this fish and is specifically expressed in stratified epithelia. In a phylogenetic tree based on type I keratin sequences and with lamprey keratins applied as outgroup, SstK10 branches off in a rather basal position. This tree strongly supports the concept that teleost keratins and tetrapod keratins resulted from two independent gene radiation processes. The only exception is human K18 because its orthologs have been found in all jawed vertebrates (Gnathostomata) studied; in the tree, they form a common, most early branch, with the shark version, SstK18, in the most basal position. Thus, the sequences of SstK10 and SstK18 also favor the classical view of vertebrate evolution that considers the cartilaginous fishes as the most ancient living Gnathostomata. To determine the overall expression patterns of epidermal ("E") and simple epithelial ("S") keratins in this shark, we furthermore tested a panel of monoclonal anti-keratin antibodies by immunofluorescence microscopy of frozen tissue sections, and in immunoblots of cytoskeletal preparations, demonstrating that immunodetection of specific keratins is a convenient method to characterize epithelial tissues in shark.     

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.     

Cytoskeletal disorders in human keratinocytes--epidermolysis bullosa simplex]

The cytoskeletons possibly related to pathogenesis in skin disease may be limited to keratin intermediate filaments (KIF) in epidermal keratinocytes. Keratins are divided into two subclasses; 11 acidic (type I) keratins and 8 basic (type II) keratins. Combination of equimolar amounts of type I and type II can form KIF. KIFs in human epidermal basal cells consist of a pair of type I and type II keratins specifically synthesized in the basal cells, and those in spinous cells contain two pairs of keratin; a pair of basal cell keratin and another pair of keratin specific for suprabasal cells. In the first section, molecular biology and differentiation of keratins are reviewed. In the second section, epidermolysis bullosa simplex (EBS) was introduced from the view point of abnormal organization of KIFs. In the epidermis of EBS, clefts are induced in the basal cells by minor trauma or frictions consequently to produce bullae. Electron microscopy reveals small spherical aggregations of tonofilaments (KIFs) in the basal cells. In biopsies, these KIF aggregations might be caused by artifacts during procedures for biopsies, so that, in order to avoid these artifacts, we studied the KIF organization in cultured keratinocytes from a patient by immunofluorescence using anti-keratin antibodies and electron microscopy. Anti-keratin antibodies revealed a formation of small droplet-like aggregations of KIFs in many cultured cells adhering to the culture bottles, which were also suggested by electron microscopy. From these observations, it is suggested that the abnormal organization (droplets) of KIFs might be one of intrinsic factors for the pathogenesis of EBS.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Type II keratins are phosphorylated on a unique motif during stress and mitosis in tissues and cultured cells

Epithelial cell keratins make up the type I (K9-K20) and type II (K1-K8) intermediate filament proteins. In glandular epithelia, K8 becomes phosphorylated on S73 ((71)LLpSPL) in human cultured cells and tissues during stress, apoptosis, and mitosis. Of all known proteins, the context of the K8 S73 motif (LLS/TPL) is unique to type II keratins and is conserved in epidermal K5/K6, esophageal K4, and type II hair keratins, except that serine is replaced by threonine. Because knowledge regarding epidermal and esophageal keratin regulation is limited, we tested whether K4-K6 are phosphorylated on the LLTPL motif. K5 and K6 become phosphorylated in vitro on threonine by the stress-activated kinase p38. Site-specific anti-phosphokeratin antibodies to LLpTPL were generated, which demonstrated negligible basal K4-K6 phosphorylation. In contrast, treatment of primary keratinocytes and other cultured cells, and ex vivo skin and esophagus cultures, with serine/threonine phosphatase inhibitors causes a dramatic increase in K4-K6 LLpTPL phosphorylation. This phosphorylation is accompanied by keratin solubilization, filament reorganization, and collapse. K5/K6 LLTPL phosphorylation occurs in vivo during mitosis and apoptosis induced by UV light or anisomycin, and in human psoriatic skin and squamous cell carcinoma. In conclusion, type II keratins of proliferating epithelia undergo phosphorylation at a unique and conserved motif as part of physiological mitotic and stress-related signals.     

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.     

Impaired NF-kappa B activation and increased production of tumor necrosis factor alpha in transgenic mice expressing keratin K10 in the basal layer of the epidermis

Both the diversity and the precisely regulated tissue- and differentiation-specific expression patterns of keratins suggest that these proteins have specific functions in epithelia besides their well known maintenance of cell integrity. In the search for these specific functions, our previous results have demonstrated that the expression of K10, a keratin expressed in postmitotic suprabasal cells of the epidermis, prevents cell proliferation through the inhibition of Akt kinase activity. Given the roles of Akt in NF-kappa B signaling and the importance of these processes in the epidermis, a study was made into the possible alterations of the NF-kappa B pathway in transgenic mice expressing K10 in the proliferative basal layer. It was found that the inhibition of Akt, mediated by K10 expression, leads to impaired NF-kappa B activity. This appears to occur through the decreased expression of IKK beta and IKK gamma. Remarkably, increased production of tumor necrosis factor alpha and concomitant JNK activation was observed in the epidermis of these transgenic mice. These results confirm that keratin K10 functions in vivo include the control of many aspects of epithelial physiology, which affect the cells not only in a cell autonomous manner but also influence tissue homeostasis.     

Correlation of specific keratins with different types of epithelial differentiation: monoclonal antibody studies

We have prepared three monoclonal antibodies against human epidermal keratins. These antibodies were highly specific for keratins and, in combination, recognized all major epidermal keratins of several mammalian species. We have used these antibodies to study the tissue distribution of epidermis-related keratins. In various mammalian epithelia, the antibodies recognized seven classes of keratins defined by their immunological reactivity and size. The 40, 46 and 52 kilodalton (kd) keratin classes were present in almost all epithelia; the 50 kd and 58 kd keratin classes were detected in all stratified squamous epithelia, but not in any simple epithelia; and the 56 kd and 65-67 kd keratin classes were unique to keratinized epidermis. Thus the expression of specific keratin classes appeared to correlate with different types of epithelial differentiation (simple versus stratified; keratinized versus nonkeratinized).     

Keratin expression during early embryonic development of Bufo bufo gargarizans

Three anti-keratin MAbs were used to identifykeratins expressed in early embryos of Bufo bufogargarizans.MAb AF5 recognized three polypeptides ofkeratin in oocytes,fertilized eggs,up to neurula withMr of 68,65 and 60Kd respectively.At tailbud stage,three other keratins(62,58 and 54Kd)began to expressand could be detected by AF5.MAbs D10 and K12 gavedifferent results,both of them could identify four keratin-like molecules with unusual molecular weights(Mr 98,95,30 and 27 Kd).Moreover,D10 could also detect a 54 Kdkeratin in neurula and tailbud stage embryos,while K12could reveal,beside 54Kd keratin,other four more kera-tins(68,65,62 and 60 Kd).The possible interpretation ofthese results and their implications are discussed.     

Transient synthesis of K6 and K16 keratins in regenerating rabbit corneal epithelium: keratin markers for an alternative pathway of keratinocyte differentiation

Cultured rabbit corneal epithelial cells undergo three distinct stages of growth and differentiation characterized by the sequential appearance of K5/K14 keratin markers for basal keratinocytes, K6/K16 keratin markers for "hyperproliferative" keratinocytes, and K3/K12 keratin markers for corneal-type differentiation. Analyses of [35S]methionine-labeled, newly synthesized keratins revealed that K6/K16 are synthesized only briefly when the cells undergo exponential growth, and their synthesis is suppressed when the cells reach confluence and switch to synthesizing K3/K12. Transient synthesis of K6/K16 was also observed in vivo during corneal epithelial regeneration. Although K6/K16 expression in general correlates well with cellular growth, drug-induced inhibition of corneal epithelial growth and related data on human epidermal keratinocytes indicate that these two events are dissociable. These results establish clearly for the first time a reciprocal relationship, on a protein level, between the synthesis of K6/K16 and a differentiation-related keratin pair, K3/K12. Such a relationship strongly suggests a competitive mechanism controlling the synthesis of these two major classes of keratins in the suprabasal compartment. Our results also indicate that although hyperproliferation is usually accompanied by K6/K16 expression, the reverse is not always true. Taken together, the data suggest that K6/K16 are synthesized, perhaps by default, as an alternative suprabasal keratin pair under conditions that are nonpermissive for keratinocytes to express their normal, differentiation-related keratin pairs.