Keratinocyte-specific transglutaminase of cultured human epidermal cells: relation to cross-linked envelope formation and terminal differentiation

The predominant form of the cross-linking enzyme, transglutaminase, in cultured normal human epidermal keratinocytes, is found in cell particulate material and can be solubilized by nonionic detergent. It elutes as a single peak upon either anion-exchange or gel-filtration chromatography. Monoclonal antibodies raised to the particulate enzyme cross-react with one of two transglutaminases in the cell cytosol. The second cytosolic transglutaminase, which has distinct kinetic and physical properties from the first, does not cross-react and is not essential for formation of the keratinocyte cross-linked envelope in vitro. The anti-transglutaminase antibodies stain the more differentiated layers of epidermis in a pattern similar to that given by anti-involucrin antiserum. These observations support the hypothesis that the transglutaminase so identified is involved in cross-linked envelope formation in vivo.     

Retinoid suppression of transglutaminase activity and envelope competence in cultured human epidermal carcinoma cells

Growth of SCC-13 squamous carcinoma cultures in the presence of retinoids considerably reduced the expression of two differentiation markers, the cellular capability to form cross-linked envelopes, and the enzyme transglutaminase required for cross-linking. A limited survey of retinoids showed that all-trans retinoic acid, 13-cis retinoic acid, and arotinoid Ro 13-6298 were highly effective in the absence of hydrocortisone and were only slightly antagonized by its presence in the medium. In contrast, retinyl acetate, retinol, and retinol bound to its plasma binding protein were quite active in the absence of hydrocortisone but were essentially inactive in its presence. Dexamethasone was also highly effective in antagonizing the suppressive action of retinyl acetate on envelope formation, while the corticosteroid antagonists cortexolone and progesterone were inactive. These results suggest that there are separate pathways, which are differentially regulated by hydrocortisone, for either the metabolism or action of retinol and retinoic acid in SCC-13 cells.     

Ceramide-dependent regulation of human epidermal keratinocyte CD1d expression during terminal differentiation

Human keratinocytes (KC), when cultured under conditions to remain undifferentiated or to terminally differentiate, changed their cellular distribution of CD1d. As studied by confocal microscopy, undifferentiated KC had a pool of cytoplasmic CD1d, whereas after terminal differentiation, this molecule localized in the cell membrane, which recapitulates CD1d expression in vivo. A comparison of undifferentiated and differentiated cultured KC did not reveal any differences in the association with beta(2)-microglobulin, invariant chain of class II MHC, or patterns of glycosylation, suggesting that these biochemical properties are not regulating the cellular distribution of CD1d. Time-course studies of CD1d gene expression indicated that KC slowly increased gene expression with CaCl(2)-induced terminal differentiation. Increased CD1d gene expression was dependent on ceramide synthesis, because fumonisin B1, a ceramide synthetase inhibitor, blocked the increase in CD1d gene expression during terminal differentiation. Similarly, exogenous ceramide or the ceramidase inhibitor, B13, induced CD1d gene expression by undifferentiated, but not terminally differentiated, KC. A protein kinase C-zeta (PKC-zeta) inhibitor (a pseudosubstrate oligopeptide), but not a PKC-alphabeta inhibitor, significantly decreased CD1d gene expression by undifferentiated or ceramide-stimulated cultured, undifferentiated KC. As expected, downstream signaling events of PKC-zeta (JNK phosphorylation and NF-kappaBeta accumulation in the nucleus) were also attenuated. The calcineurin phosphatase inhibitor cyclosporine A, which blocks KC terminal differentiation, also blocked CD1d gene expression by cultured KC. In conclusion, this novel function of cellular ceramides extends the importance of this class of biologically active lipids beyond that of terminal differentiation and barrier function in normal human skin.     

Chromosomal rearrangements during human epidermal keratinocyte differentiation

Undifferentiated human epidermal keratinocytes are self‐renewing stem cells that can be induced to undergo a program of differentiation by varying the calcium chloride concentration in the culture media. We utilize this model of cell differentiation and a 3D chromosome painting technique to document significant changes in the radial arrangement, morphology, and interchromosomal associations between the gene poor chromosome 18 and the gene rich chromosome 19 territories at discrete stages during keratinocyte differentiation. We suggest that changes observed in chromosomal territorial organization provides an architectural basis for genomic function during cell differentiation and provide further support for a chromosome territory code that contributes to gene expression at the global level. J. Cell. Physiol. J. Cell. Physiol. 221: 139–146, 2009. © 2009 Wiley‐Liss, Inc.     

A novel tumor suppressor protein promotes keratinocyte terminal differentiation via activation of type I transglutaminase

Tazarotene-induced protein 3 (TIG3) is a recently discovered regulatory protein that is expressed in the suprabasal epidermis. In the present study, we show that TIG3 regulates keratinocyte viability and proliferation. TIG3-dependent reduction in keratinocyte viability is accompanied by a substantial increase in the number of sub-G1 cells, nuclear shrinkage, and increased formation of cornified envelope-like structures. TIG3 localizes to the membrane fraction, and TIG3-dependent differentiation is associated with increased type I transglutaminase activity. Microscopic localization and isopeptide cross-linking studies suggest that TIG3 and type I transglutaminase co-localize in membranes. Markers of apoptosis, including caspases and poly(ADP-ribose) polymerase, are not activated by TIG3, and caspase inhibitors do not stop the TIG3-dependent reduction in cell viability. Truncation of the carboxyl-terminal membrane-anchoring domain results in a complete loss of TIG3 activity. The morphology of the TIG3-positive cells and the effects on cornified envelope formation suggest that TIG3 is an activator of terminal keratinocyte differentiation. Our studies suggest that TIG3 facilitates the terminal stages in keratinocyte differentiation via activation of type I transglutaminase.     

Hoxb13 up-regulates transglutaminase activity and drives terminal differentiation in an epidermal organotypic model

Hox genes act to differentiate and pattern embryonic structures by promoting the proliferation of specific cell types. An exception is Hoxb13, which functions as a proapoptotic and antiproliferative protein during development of the caudal spinal cord and tail vertebrae and has also been implicated in adult cutaneous wound repair. The adult epidermis, which expresses several Hox genes including Hoxb13, is continually renewed in a program of growth arrest, differentiation, and a specialized form of apoptosis (cornification). Yet little is known about the function(s) of these genes in skin. Based on its role during embryogenesis, Hoxb13 is an attractive candidate to be involved in the regulation of epidermal differentiation. Here, we demonstrate that Hoxb13 overexpression in an adult organotypic epidermal model recapitulates actions of Hoxb13 reported in embryonic development. Epidermal cell proliferation is decreased, apoptosis increased, and excessive terminal differentiation observed, as characterized by enhanced transglutaminase activity and excessive cornified envelope formation. Overexpression of Hoxb13 also produces abnormal phenotypes in the epidermal tissue that resemble certain pathological features of dysplastic skin diseases. Our results suggest that Hoxb13 functions to promote epidermal differentiation, a critical process for skin regeneration and for the maintenance of normal barrier function.     

Participation of membrane-associated proteins in the formation of the cross-linked envelope of the keratinocyte

Cultured keratinocytes, like those in natural squamous epithelia, form submembranous protein envelopes cross-linked by cellular transglutaminase. During the cross-linking, the cytosolic protein involucrin becomes incorporated into the envelope and can no longer be extracted by detergents. We show here that when transglutaminase is activated in cultured keratinocytes, at least six other proteins also become nonextractable. In contrast to involucrin, these proteins are associated with membranes. Two of the proteins (210 and 195 kd) are differentiated products specific to the keratinocyte; like involucrin, they are absent from small keratinocytes and fibroblasts, but appear in larger keratinocytes during the course of their terminal differentiation. The other proteins that become nonextractable cannot be destined exclusively for envelope formation since they are also present in fibroblasts. Transglutaminase is used by the mature (large) keratinocyte to make a detergent-resistant envelope from what appears to be a mixture of differentiation-specific and nonspecific proteins, both membrane-bound and cytosolic.     

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 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.     

Specific epidermal protein markers are modulated during calcium-induced terminal differentiation

Extracellular calcium concentration has been shown to be an important determinant of proliferation rate in a number of cell culture models. Recently, the role of calcium as a regulator of cellular differentiation has also become apparent. This effect of calcium was exemplified by the discovery that keratinocytes of mouse or human origin grew as a proliferating monolayer in medium with a calcium concentration of 0.02-0.09 mM but that proliferation ceased and cells stratified and cornified when calcium was increased greater than 0.1 mM. While the morphological and biological effects of changes in calcium concentration are dramatic in keratinocyte cultures, it has been difficult to identify specific protein changes associated with the modulation of maturation. In vivo, however, several proteins that are markers for stratified squamous epithelia have been identified by specific autoimmune sera. Pemphigoid antigen is a 220-kdalton protein found in the basement membrane and closely associated with the plasma membrane of the basal cell. Pemphigus antigen is a 130-kdalton glycoprotein found on the cell surface of stratifying epithelial cells. Immunofluorescence staining of cells cultured in low Ca2+ or cells switched to high Ca2+ for 48 h before staining demonstrated that pemphigoid antigen was detected in low Ca2+ cultures but was diminished or absent in high Ca2+ cultures and that pemphigus antigen was seen only in high Ca2+ cultures. The synthesis of each antigen was studied in immunoprecipitates of cell lysates radiolabeled with 14C-amino acids or D-[1-14C]glucosamine. Pemphigoid antigen was synthesized mainly by proliferating cells in low Ca2+ medium and its synthesis was decreased by greater than 90% in cells switched to high Ca2+ medium. In contrast, synthesis of pemphigus antigen was detected only in stratifying cells cultured in high Ca2+ medium. These studies indicate that extracellular calcium concentrations which modulate the transition between proliferating and stratifying epidermal cells also modulate, in parallel, the synthesis of specific marker proteins for these cell types.     

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.     

Plasma membrane transglutaminase and cornified envelope competence in cultured human keratinocytes

When confluent cultures of the transformed human keratinocyte line SV-K14 are shifted to serum-free medium the cells achieve, within 4 days, the ability to synthesize a cornified envelope after challenge with the Ca2+ ionophore A23187. During these 4 days the enzyme transglutaminase (EC 2.3.2.13), which catalyses the cross-linking of different envelope precursor proteins, is partially transferred from the cytosolic pool into the plasma membrane. The association of the enzyme with the plasma membrane proves to be an essential step in the envelope formation since a direct correlation between plasma membrane-bound transglutaminase and envelope competence is observed. Retinoids block the insertion of the enzyme and therefore prevent envelope formation.     

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.     

The roles of ABCA12 in epidermal lipid barrier formation and keratinocyte differentiation

ATP-binding cassette (ABC) transporters form a large superfamily of transporters that bind and hydrolyze ATP to transport various molecules across limiting membranes or into vesicles. The ABCA subfamily members are thought to transport lipid materials. ABCA12 is a keratinocyte transmembrane lipid transporter protein associated with the transport of lipids via lamellar granules. ABCA12 is considered to transport lipids including ceramides to form extracellular lipid layers in the stratum corneum of the epidermis, which is essential for skin barrier function. ABCA12 mutations are known to underlie the three major types of autosomal recessive congenital ichthyoses: harlequin ichthyosis, lamellar ichthyosis and congenital ichthyosiform erythroderma. ABCA12 mutations result in defective lipid transport via lamellar granules in the keratinocytes, leading to ichthyosis phenotypes from malformation of the stratum corneum lipid barrier. Studies on ABCA12-deficient bioengineered models have revealed that lipid transport by ABCA12 is required for keratinocyte differentiation and epidermal morphogenesis. Defective lipid transport due to loss of ABCA12 function leads to the accumulation of intracellular lipids, including glucosylceramides and gangliosides, in the epidermal keratinocytes. The accumulation of gangliosides seems to result in the apoptosis of Abca12^−/− keratinocytes. It was reported that AKT activation occurs in Abca12^−/− granular-layer keratinocytes, which suggests that AKT activation serves to prevent the cell death of Abca12^−/− keratinocytes. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

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.     

Retinoic acid induces transglutaminase activity but inhibits cornification of cultured epidermal cells

In cultured mouse epidermal basal cells, retinoic acid is a potent inducer of transglutaminase, the enzyme responsible for isodipeptide bond formation in protein cross-linking in the production of the cornified membrane during terminal differentiation. Paradoxically retinoic acid also inhibits the formation of the cross-linked envelope and greatly reduces the level of dipeptide bond formation in epidermal cells induced to differentiate by calcium. These results suggest a novel mechanism by which retinoids can modify transglutaminase activity and epidermal differentiation.     

Terminal differentiation of cultured human epidermal cells.

Three aspects of terminal differentiation of the epidermal keratinocyte have been studied in cell culture—the development of detergent-insoluble cytoplasmic filaments, the formation of a cornified cell envelope and the destruction of the cell nucleus.In the presence of lethally irradiated 3T3 cells, single human epidermal keratinocytes grow into stratified colonies. After the colonies become confluent, the culture enters a steady state in which the upper cells are shed from the surface of the cell layer like stratum corneum cells in vivo and are replaced by the proliferation of dividing cells in the basal layer. The cells shed into the medium are flattened and elongated squames, and are insoluble in solutions of sodium dodecylsulfate. Since the squames usually detach before their nuclei are digested, the cultures behave like some wet-surfaced, stratified squamous epithelia in that they possess little or no anucleate stratum corneum. The rates of proliferation and squame detachment in confluent cultures are increased by the presence of epidermal growth factor.Most of the squames harvested from the medium are permeable to trypan blue. The permeable squames may or may not have a visible nucleus, but squames not permeable to trypan blue nearly always possess a nucleus. When freshly detached squames containing nuclei are incubated in medium containing serum, their nuclei are digested and disappear within a few days. On the other hand, if the squames are washed and incubated in serum-free medium, their nuclei are not digested. This suggests that the permeable cell membrane permits a serum component essential for nuclear digestion to enter the cytoplasm.When growing colonies of epidermal keratinocytes are disaggregated and the cells suspended in medium containing methyl cellulose, they cannot multiply, but within a few days the cells become permeable to trypan blue and insoluble in sodium dodecylsulfate. This insolubility is due to disulfide linking of the proteins of the abundant cytoplasmic filaments, for the filaments are dissolved when β-mercaptoethanol is added as well, leaving the emptied cornified cell envelopes. Nuclear digestion follows some days later. In the absence of serum, cells become permeable and develop detergent-insoluble filaments and a cornified envelope, but, as in the case of spontaneously detached squames of surface cultures, their nuclei are not destroyed. Purified plasminogen supports nuclear destruction, whereas serum depleted of plasminogen does not.Earlier studies on intact skin have suggested that chemical gradients between epidermis and dermis might be responsible for the differentiation of the epidermal cells. In surface culture, basal cells multiply and nonbasal cells undergo terminal differentiation, even though all the cells are bathed in the same medium and the terminally differentiating cells have, if anything, better access to the medium than do the basal cells. Differentiation also begins in virtually all singly suspended cells uniformly exposed to the medium. The program of differentiation is therefore independent of the orientation of any chemical gradients in the cellular environment. Cell-cell contacts are not required for the development of detergent insolubility, the formation of the cornified envelope or the process of nuclear digestion, although they are essential for the formation of flattened squames. Unlike proliferation, which is strongly dependent upon fibroblast products, terminal differentiation proceeds in the absence of fibroblast support.