Inhibited morphological terminal differentiation and enhanced proliferation of cultured mouse epidermal cells at different concentrations of dimethyl sulphoxide

Dimethyl sulphoxide (DMSO), at concentrations of 1-2%, induces terminal differentiation in several different cell types in vitro and enhances the growth of newborn mouse epidermal cells in primary culture under conditions that also permit terminal differentiation. We have found that DMSO concentrations approaching 4% reversibly inhibited (with little overt toxicity) terminal differentiation of normal epidermal cells from newborn SENCAR mice. Cells cultured in medium containing 4% DMSO and calcium in excess of 1 mM did not stratify extensively or slough large amounts of keratinized debris into the medium as occurred in control cultures, nor did they form large numbers of squamous cells or keratin bundles, as revealed by light and electron microscopy. The number of detergent-insoluble cornified envelopes was similarly reduced. Long-term growth of epidermal colonies in secondary culture was optimum in 1% DMSO, this concentration also permitting normal terminal differentiation of these cells. Since DMSO had these effects on epidermal cells in vitro, it may also affect epidermal cell proliferation and terminal differentiation in vivo, an important consideration should DMSO ever be approved for topical use in the US.     

The cornified envelope of terminally differentiated human epidermal keratinocytes consists of cross-linked protein

A small proportion of the protein of stratum corneum of human epidermal callus is insoluble even when boiled in solutions containing sodium dodecylsulfate and a reducing agent. This protein is present in the cornified envelope, a structure located beneath the plasma membrane. When cornified envelopes were dissolved by exhaustive proteolytic digestion and the products analyzed by chromatography, approximately 18% of the total lysine residues were found as the cross-linking dipeptide ?-(γ-glutamyl) lysine.Labeled cornified envelope protein was synthesized by human epidermal keratinocytes allowed to differentiate terminally in culture. The extent of cross-linking, determined from the proportion of radioactive lysine in ?-(γ-glutamyl) lysine after exhaustive proteolysis, was similar to that in stratum corneum. The properties of the cornified envelopes (insolubility in detergent and reducing agents, and solubility following proteolytic digestion) are readily explained by a structure consisting of a cross-linked protein lattice.     

A new method for quantifying keratinocyte differentiation using immunofluorescent staining of involucrin and cytofluorography

Involucrin is a precursor protein of detergent-insoluble cornified envelope and a marker of terminal differentiation of epidermal keratinocytes. To quantify differentiation of cultured human keratinocytes, the population of involucrin-positive cells was estimated by immunofluorescent staining using anti-involucrin antibody and flow cytometry. Normal human keratinocytes were cultured under three conditions for induction of differentiation: low Ca2+ concentration (0.1 mM Ca2+), high Ca2+ concentration (1.8 mM Ca2+), and high Ca2+ concentration with 10% fetal bovine serum (FBS). The relationship between fluorescence intensity and involucrin synthesis was confirmed by visual examination of sorted cells. The population of involucrin-positive cells increased from 7.2 to 18.1% by elevating Ca2+ concentration and to 37.0% by adding FBS. The extent of cornified envelope formation under the same culture conditions was consistent with the estimation of involucrin-positive cells. The cytofluorographic analysis of involucrin synthesis made it possible to determine the number of differentiated cells in a large number of samples precisely and reliably. Thus, it is a useful method for quantifying keratinocyte differentiation.     

Caspase activation in the terminal differentiation of human epidermal keratinocytes

The epidermis is a multilayered squamous epithelium in which dividing basal cells withdraw from the cell cycle and progressively differentiate as they are displaced toward the skin surface. Eventually, the cells lose their nucleus and other organelles to become flattened squames, which are finally shed from the surface as bags of cross-linked keratin filaments enclosed in a cornified envelope [1]. Although keratinocytes can undergo apoptosis when stimulated by a variety of agents [2], it is not known whether their normal differentiation programme uses any components of the apoptotic biochemical machinery to produce the cornified cell. Differentiating keratinocytes have been reported to share some features with apoptotic cells, such as DNA fragmentation, but these features have not been seen consistently [3]. Apoptosis involves an intracellular proteolytic cascade, mainly mediated by members of the caspase family of cysteine proteases, which cleave one another and various key intracellular target proteins to kill the cell neatly and quickly [4]. Here, we show for the first time that caspases are activated during normal human keratinocyte differentiation and that this activation is apparently required for the normal loss of the nucleus.     

Growth of sebaceous cells in monolayer culture

Summary Rat preputial cells were grown in an epithelial cell primary monolayer culture system identical to that used for culturing epidermal cells, which were studied for comparison. Despite similar appearance when observed by phase contrast microscopy, other features identified the preputial cells as a unique epithelial cell population. Preputial cells grew as a relatively small number of large colonies, formed domes before confluence, and expressed a specific acinar keratin, K4, which had previously been found in human sebaceous glands. In addition, preputial cells formed fewer cornified envelopes than epidermal cells, too few to discern the reduction of envelope formation by retinoic acid treatment in vitro which was found in epidermal cells. Rat preputial cells in monolayer culture, therefore, are a promising model for studying the effects of hormones on sebaceous cell growth and differentiation.     

A sensitive method to quantify the terminal differentiation of cultured epidermal cells

Terminal differentiation of normal and malignant keratinocytes is routinely determined by the ability of these cells to form cornified envelopes after incubation with a calcium ionophore. We have used the human squamous cell carcinoma, SqCC/Y1, to quantify cellular differentiation by the formation of detergent-insoluble protein. The methodology developed employs the metabolic labeling of detergent-insoluble cellular protein with [35S]methionine in the presence of a calcium ionophore. The ratio of filter-retainable radioactivity to that of total cellular protein was shown to be closely correlated to the results obtained by measuring the number of envelope-competent cells when cells were induced to enter a pathway of terminal differentiation in culture by serum deprivation or by treatment with hydrocortisone, and during the inhibition of maturation by either retinoic acid (RA) or epidermal growth factor (EGF). This way of measuring the degree of terminal differentiation of epidermal cells is a relatively simple one that readily allows the simultaneous measurement of multiple samples.     

Envoplakin, a novel precursor of the cornified envelope that has homology to desmoplakin

The cornified envelope is a layer of transglutaminase cross-linked protein that is deposited under the plasma membrane of keratinocytes in the outermost layers of the epidermis. We present the sequence of one of the cornified envelope precursors, a protein with an apparent molecular mass of 210 kD. The 210-kD protein is translated from a 6.5- kb mRNA that is transcribed from a single copy gene. The mRNA was upregulated during suspension-induced terminal differentiation of cultured human keratinocytes. Like other envelope precursors, the 210- kD protein became insoluble in SDS and beta-mercaptoethanol on activation of transglutaminases in cultured keratinocytes. The protein was expressed in keratinizing and nonkeratinizing stratified squamous epithelia, but not in simple epithelia or nonepithelial cells. Immunofluorescence staining showed that in epidermal keratinocytes, both in vivo and in culture, the protein was upregulated during terminal differentiation and partially colocalized with desmosomal proteins. Immunogold EM confirmed the colocalization of the 210-kD protein and desmoplakin at desmosomes and on keratin filaments throughout the differentiated layers of the epidermis. Sequence analysis showed that the 210-kD protein is homologous to the keratin- binding proteins desmoplakin, bullous pemphigoid antigen 1, and plectin. These data suggest that the 210-kD protein may link the cornified envelope to desmosomes and keratin filaments. We propose that the 210-kD protein be named "envoplakin."     

Keratin filaments of cultured human epidermal cells. Formation of intermolecular disulfide bonds during terminal differentiation.

Human epidermal cells grown in culture synthesize abundant keratins. These keratins are similar to those of stratum corneum of human epidermal callus in their insolubility in dilute aqueous buffers, their molecular weight range of 40,000 to 60,000, their immunolgical reactivity, and their ability to assemble into 80 A tonofilaments in vitro; but there are differences in the molecular weights of some of the proteins, the number of components, and their charge heterogeneity, related at least in part to phosphorylation. About 30% of all the proteins of living cultured keratinocytes consists of keratins, compared with over 85% of stratum corneum. All the keratins of human stratum corneum were found to be cross-linked by intermolecular disulfide bonds while most keratins of the living cells were not. As the cells mature in Methocel-stabilized suspension culture, their keratins become increasingly disulfide cross-linked. When uncross-linked tonofilaments of living keratinocytes are dissolved in 8 M urea and the filaments reconstituted in vitro their keratins become disulfide cross-linked under aerobic conditions and consequently insoluble in solutions of 8 M urea or sodium dodecyl sulfate. The results indicate that the uncross-linked state of the keratins in living cells is due to the reducing intracellular environment and not to a precursor state related to the primary structure of the proteins. The disulfide cross-links stabilizing the keratin filaments must be distinguished from the epsilon-(gamma-glutamyl)lysine cross-links stabilizing the cornified cell envelope.     

Lessons from disorders of epidermal differentiation-associated keratins

A number of diseases have been associated with mutations in genes encoding keratin intermediate filaments. Several of these disorders have skin manifestations, in which histological changes highlight the role of various different keratins in epidermal differentiation. For example, mutations in either K1 or K10 (the major keratin pair expressed in differentiated keratinocytes) usually lead to clumped keratin filaments and cytolysis. Furthermore, the precise nature of the mutation has direct implications for disease phenotype. Specifically, mutations in the H1 and alpha-helical rod domains of K1/K10 result in bullous congenital ichthyosiform erythroderma, underscoring the critical role for this keratin filament domain in maintaining cellular integrity. However, a lysine to isoleucine substitution in the V1 domain of K1 underlies a form of palmoplantar keratoderma, which has different cell biological implications. Keratins are cross-linked into the cornified cell envelopes through this particular lysine residue and the consequences of the mutation lead to changes in keratin-desmosome association and cornified cell morphology, suggesting a role for this keratin subdomain in cornified cell envelope formation. Recently, to extend genotype-phenotype correlation, a frameshift mutation in the V2 region of the K1 tail domain was identified in ichthyosis hystrix (Curth-Macklin type), in which keratin filaments show a characteristic shell-like structure and fail to form proper bundles. In this case, the association of desmosomes with loricrin was also altered, implicating this keratin domain in organizing the intracellular distribution of loricrin during cornification. Collectively, these mutations in K1/K10 provide a fascinating insight into both normal and abnormal processes of epidermal differentiation.     

Rat esophageal and epidermal keratinocytes: intrinsic differences in culture and derivation of continuous lines

Serially cultivated with 3T3 feeder layer support as colonies of stratified squamous epithelium, rat epidermal and esophageal epithelial cells were readily distinguishable by three criteria. First, the epidermal colonies, exhibiting extensive piling up of squames in the centers, were more stratified than esophageal colonies. Second, in sparse culture 70 to 90% of the esophageal cells but as few as 1 to 5% of the epidermal cells were competent in cross-linked envelope formation upon treatment with the ionophore X537A. After reaching confluence, up to 90% of the cells of both types formed envelopes upon ionophore treatment. Third, epidermal cells in suspension culture reached maximal levels of spontaneously cross-linked envelopes in 1 day or less, while esophageal cells required about 4 days in suspension to reach maximal levels. A reproducible finding with both cell types was that initial colony-forming efficiencies of less than 1% increased to about 40% upon serial passage with consequent derivation of continuous lines. Sparse cultures of esophageal cells with high colony-forming ability retained a high degree of envelope competence (70 to 90%), indicating these two properties are not mutually exclusive. The derived lines exhibited reduced dependence upon feeder layer support at clonal density, but in suspension culture the cells did not grow and lost colony-forming ability with a half-time of several hours. We conclude that cells from these keratinized rat epithelia exhibit intrinsic differences in culture and become continuous lines expressing characteristic regulation of envelope competence and loss of germinative capability in suspension.     

Extracellular products of Staphylococcus aureus reversibly inhibit the terminal differentiation of cultured mouse epidermal cells

The effect of extracellular products from Staphylococcus aureus on the differentiation of mouse epidermal cells was studied using an in vitro cell culture system. The extracellular products from a clinical strain of S. aureus isolated from human skin lesions reversibly inhibited the Ca++-induced terminal differentiation of epidermal cells, as determined by their morphology and the extent of cornified envelope formation. This suggests that a similar modification of cell differentiation is involved in the pathogenesis of S. aureus-induced skin disease.     

Incomplete epidermal differentiation of A431 epidermoid carcinoma cells

Summary A431 malignant keratinocytes, although derived from a muco-cutaneous carcinoma of the vulva, fail to achieve terminal epidermal differentiation in culture as shown by their inability to form cornified envelopes. Even after culture in a serum-free medium (MCDB 153) containing no retinoic acid and a high (10⁻³ M) calcium concentration (conditions known to facilitate epidermal differentiation), the cells do not become competent as shown by the fact that subsequent treatment with a calcium ionophore is unable to provoke the formation of cornified envelopes. Nevertheless, A431 cells are able to synthesize the envelope precursor involucrin. The block in formation of cornified envelopes is thus not due to a lack in involucrin. The results described here suggest that the absence of cross-linking of this molecule is due to a lowered epidermal membrane-bound transglutaminase activity in A431 cells, enhances involucrin accumulation in these cells, although in normal human keratinocytes it stimulates growth and reduces involucrin synthesis. These results suggest that involucrin synthesis is triggered by the arrest of growth. EDITOR'S STATEMENT The A431 cell line has been used extensively in the study of EGF receptors and effects, and recently has been employed in studies of surface membrane receptors for other factors, as well as studies of extracellular matrix synthesis and deposition and tumor promoter activities. The expanding use of A431 cells calls for a more thorough understanding of the cell type it represents and the degree to which it represents a general in vitro model of normal or neoplastic epidermal cells. This article addresses some of these questions.     

A model for the hierarchical structure of the human epidermal cornified cell envelope

The epidermal cornified cell envelope (CE) is a 15 nm thick layer of highly insoluble protein that is assembled on the intracellular surface of the cell membrane during terminal differentiation, and comprises about 10% of the mass of the cornified dead layers of the tissue. The CE consists of a complex amalgam of several known proteins that are crosslinked by isodipeptide bonds formed by the action of transglutaminases, but little is known about their order of accretion during CE assembly, or how they are crosslinked. In this paper, CEs purified from human foreskin epidermis were examined by immunogold electron microscopy before and after digestion with proteases.The mass fractions of the proteins remaining in CE remnants during digestion were estimated from the amino acid compositions by mathematical modelling. Together, the data support a new model for the complex hierachical structure of the CE. The cytoplasmic surface of intact purified CEs consists of filaggrin, loricrin, SPRs and keratin intermediate filaments. The bulk of the CE consists of a mixture of loricrin (75%) and SPRs (5%). Following removal of most of these, the novel protein elafin is exposed, which contributes about 6% of CE mass. The protein material on the inner CE 'core' adjacent or attached to the lipid envelope consists of cystatin alpha (5%), involucrin (2%), keratin filaments (3%) and possibly other as yet unidentified protein(s)(2-5%). This model supports but considerably extends an earlier extant hypotheis for CE structure, and thus provides the basis for further detailed biochemical and ultra-structural studies.     

The reorganization of microtubules and microfilaments in differentiating keratinocytes

Using immunofluorescence techniques, we have examined the microtubules and microfilaments in colonies of terminally differentiating human keratinocytes in tissue culture. The undifferentiated keratinocytes contained numerous microtubules, which radiated from a centrosomal organization center (MTOC). Differentiating keratinocytes, which leave the basal layer and begin to synthesize involucrin, displayed an altered cytoskeleton. Thick mats and coils of microtubules formed throughout the cytoplasm of the differentiated squames, and microfilaments were no longer visible after staining with phalloidin. Instead, only scattered stipples of phalloidin-stained material were observed. The results suggest that the terminal differentiation of epidermal cells involves a reorganization not only of the keratin filaments but of the entire cytoskeleton.     

Sodium butyrate selectively antagonizes the inhibitory effect of retinoids on cornified envelope formation in cultured human keratinocytes

Sodium butyrate affects cell differentiation in confluent epidermal keratinocyte cultures by considerably increasing the spontaneous formation of cross-linked envelopes in normal human keratinocytes (NHK). It also favors the development of envelope competence in the Simian virus-40 (SV-40)-transformed human foreskin keratinocyte line SV-K14. It completely abolishes the inhibitory effect of serum and retinoic acid on the expression of plasma membrane-associated transglutaminase. However, other markers of epidermal differentiation that are also under the control of retinoids such as keratins or the enzyme cholesterol sulfotransferase are not affected by butyrate. The level of the cellular retinoic acid binding protein (CRABP) is considerably increased in its presence. Butyrate does not interfere with the binding of retinoids to their cellular binding proteins. Our observations suggest that sodium butyrate stimulates cornified envelope formation via the induction of the plasma membrane-associated transglutaminase required for cornified envelope synthesis and, additionally, by abolishing the inhibitory effect of retinoids on the expression of this enzyme.     

Calcium regulation of cell-cell contact and differentiation of epidermal cells in culture. An ultrastructural study

Calcium modulation of keratinocyte growth in culture was studied by both transmission (TEM) and scanning electron microscopy (SEM). Under standard culture conditions (1.2-1.8 mM calcium), cells were connected by desmosomes and stratified to 4-6 cell layers. Many aspects of in vitro epidermal maturation were analogous to the in vivo process, with formation of keratohyalin granules, loss of nuclei, formation of cornified envelopes and shedding of cornified cells containing keratin filaments. When the medium calcium concentration was lowered to 0.02-0.1 mM, the pattern of keratinocyte growth was strikingly changed. Cells grew as a monolayer with no desmosomal connections and proliferated rapidly, shedding largely non-cornified cells into the medium. Large bundles of keratin filaments were concentrated in the perinuclear cytoplasm. The elevation of extracellular calcium to 1.2 mM induced low calcium keratinocytes to stratify, keratinize and cornify in a manner analogous to that seen when plated in standard calcium medium. The earliest calcium-induced ultrastructural change was the asymmetric formation of desmosomes between adjacent cells. Desmosomal plaques with associated tonofilaments were observed 5 min after calcium addition; symmetric desmosomes were formed within 1-2 h. This system is presented as a useful model for the study of the regulation of desmosome assembly and disassembly.     

Differentiation of human scalp hair follicle keratinocytes in culture

The morphology of human scalp hair follicle keratinocytes, cultured on the bovine eye lens capsule, is studied by light and electron microscopy. The hair follicle keratinocytes in the stratified cultures are characterized by the presence of numerous tonofilaments, desmosomes and lysosomes and by the presence of glycogen accumulations. The cells in the upper layers develop a cornified envelope. Moreover, an incomplete basal lamina is found between the capsule and the basal cells. However, some features of epidermal keratinocytes in vivo, such as keratohyalin granules and stratum corneum formation, are absent. Analysis of the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis also reveals differences between the cultured hair follicle cells and epidermis, whilst the patterns of cultured cells and hair follicle sheaths are similar. The morphological and protein biosynthetic aspects of terminal differentiation of the keratinocytes in vitro are correlated. These results are discussed in the light of the findings with cultured epidermal keratinocytes, reported in the literature.     

Differentiation-dependent changes in the solubility of a 195-kD protein in human epidermal keratinocytes

We have prepared a monoclonal antibody, AE11, that recognizes specifically a 195-kD protein (pI 5.4) of human keratinocytes. This antigen constitutes approximately 0.01-0.1% of total protein in keratinocytes of skin, esophagus, and cornea, and is readily detectable in these cells by immunofluorescent staining and immunoblotting. However, it is barely detectable in MCF mammary carcinoma cells and HeLa cells, and is undetectable in nonepithelial cell types. Results from serial extraction experiments have shown that this protein exists in two distinct pools: a Tris-soluble, and a Tris-insoluble but urea- or SDS-soluble one. The distribution of the 195-kD protein between these two pools appears to be differentiation-related, since relatively undifferentiated cells selected by a low-calcium medium contain primarily the soluble form, while highly differentiated cells contain mainly the insoluble form. Data from immunofluorescent staining and trypsin-sensitivity experiments suggest that the soluble form is cytoplasmic, whereas the insoluble form is submembranously located at the cell periphery of upper, differentiated cells. The insoluble, cell peripheral form of the 195-kD antigen increases progressively during epidermal differentiation; its insolubility appears to be related to the formation of disulfide-bond(s). These results indicate that the 195-kD protein, which has recently been suggested to be involved in cornified envelope formation (Simon, M., and H. Green, 1985, Cell, 36:827-834), undergoes significant changes in its solubility characteristics and intracellular location during keratinocyte maturation.     

Effects of retinoids on differentiation, lipid metabolism, epidermal growth factor, and low-density lipoprotein binding in squamous carcinoma cells

The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13-cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis, resulting in changes in the plasma membrane composition. Hydrocortisone stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. Retinoic acid, arotinoid ethylsulfone, and hydrocortisone had no effects on LDL binding and only minor effects on LDL degradation. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity.