2,3,7,8-Tetrachlorodibenzo-p-dioxin: examination of biochemical effects involved in the proliferation and differentiation of XB cells

XB, a cell line derived from a mouse teratoma, differentiates into stratified squamous epithelium when incubated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To examine the possible biochemical mediators of this response, we compared the effects produced by TCDD to those elicited by other compounds which stimulate epidermal proliferation and/or differentiation in mice. XB/3T3 cultures keratinize when incubated with cholera toxin, epidermal growth factor (EGF), or TCDD, but not 12-0-tetradecanoylphorbol-13-acetate (TPA). Incubation of XB cells with TCDD (10(-9)M) for 48 hours produces a 20% increase in thymidine incorporation, a response which is neither as large nor as rapid as that produced by cholera toxin, TPA, or EGF. Although both cholera toxin and TCDD stimulate differentiation and thymidine incorporation in XB/3T3 cultures, cholera toxin increases cAMP 30-fold in these cells, while TCDD does not affect cAMP accumulation at any of the times studies (15 min to 120 hours). Inhibitors of arachidonic acid metabolism, which block epidermal proliferative responses to TPA in vivo, do not prevent the differentiation of XB cells in response to TCDD. In XB/3T3 cultures, TPA stimulates arachidonic acid release at all times tested (1,6, and 24 hours) and increases the incorporation of 32Pi into total phospholipids and phosphatidylcholine after 3 hours. In contrast, TCDD affects neither arachidonic acid release nor the turnover of phosphatidylinositol or phosphatidylcholine at any of the times tested. Although we examined biochemical effects which have been suggested as part of the mechanism of TCDD and which are produced by other epidermal proliferative compounds in XB cells, no mediator of the TCDD-produced differentiation of XB/3T3 cultures was observed.     

Cholera toxin stimulates division of 3T3 cells.

Cholera toxin was used in an attempt to inhibit epidermal growth factor stimulated 3T3 cell division. Instead, cholera toxin alone at low concentrations (10(-10) M), was able to stimulate cell division and could augment EGF stimulated cell division. The mitogenic effect of cholera toxin can occur despite a dramatic increase in the intracellular levels of cAMP in 3T3 cells. Cholera toxin stimulated mitogenesis could not be mimicked by choleragenoid, the binding but inactive subunit of cholera toxin, or by other agents which elevate cAMP levels in 3T3 cells.     

Involucrin synthesis and tissue assembly by keratinocytes in natural and cultured human epithelia

Different stratified squamous epithelia, whether they bear a stratum corneum or not, are shown by immunofluorescence to possess the precursor protein of the cross-linked envelope that is characteristic of epidermal s. corneum. This protein, involucrin, is not present in the deepest epithelial cells but appears in the course of their outward migration. The boundary at which involucrin first appears can sometimes by correlated with a visible boundary between zones of large and small cells. Cultured keratinocytes, derived from all stratified squamous epithelia (epidermal, corneal, conjuctival, esophageal, lingual, and vaginal), form colonies that grow together to form a stratified epithelium. The cells of the basal layer are nearly always free of detectable involucrin, but, in contrast to the natural epithelium, this protein usually makes its appearance in the cells immediately above the basal layer. When a cultured epithelium derived from epidermal keratinocytes is detached and applied as a graft to animals, the cells flatten and the distinctness of the basal layer is at first reduced; but with time the organization of the epithelium becomes more characteristic of epidermis. Cell size and shape become more orderly along the cell migration pathway, and involucrin first appears at some distance from the basal layer, instead of in immediately suprabasal cells, as in the cultured epithelium. The progeny of dissociated and cultured keratinocytes are therefore able, when grafted, to reassemble an epidermis in which the timing of specific gene expression is restored to that of the original tissue.     

Modulation of hepatocyte growth factor induction in human skin fibroblasts by retinoic acid

Topical treatment of skin with all-trans-retinoic acid (ATRA), the major biologically active form of vitamin A, results in hyperproliferation of basal keratinocytes, leading to an accelerated turnover of epidermis cells and thickening of the epidermis, probably via induction of production of paracrine growth factors for keratinocytes in epidermal suprabasal keratinocytes and/or dermal fibroblasts. Since hepatocyte growth factor (HGF) is a factor mitogenic to epidermal keratinocytes secreted from dermal fibroblasts, the effect of ATRA on basal and induced HGF production in human dermal fibroblasts in culture was examined. ATRA alone did not induce HGF production, but it significantly enhanced HGF production induced by the cAMP-elevating agent cholera toxin or the membrane-permeable cAMP analog 8-bromo-cAMP. Cholera toxin-induced activation of cAMP responsive element (CRE)-binding protein (CREB) was enhanced by pretreating cells with ATRA for 24 h. In contrast, HGF production induced by epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) was potently inhibited by ATRA. These modulatory effects of ATRA were different from the effects of transforming growth factor-beta1 (TGF-beta) and dexamethasone, both of which inhibited HGF production induced by all of the four inducers. Up-regulation of HGF gene expression by cholera toxin and EGF was also enhanced and inhibited, respectively, by ATRA. Both 9-cis-retinoic acid (9-cis-RA) and 13-cis-retinoic acid (13-cis-RA), which are stereo-isomers of ATRA, showed a modulatory effect on HGF induction similar to that of ATRA. These results suggest that ATRA augments the induction of HGF production caused by increased intracellular cAMP.     

The effect of growth-promoting agents on replication and cell cycle withdrawal in cultures of epidermal keratinocytes

Summary Epidermal keratinocytes grow in culture to form a stratified squamous epithelium. These cultures contain a replicating as well as a terminally differentiating population and undergo surface desquamation. Epidermal growth factor (EGF) and cholera toxin are usually employed as growth-promoting agents because they reduce the population doubling time; that is, the period required to increase the total cell number twofold. There are three ways in which this reduction in population doubling time could be achieved: (a) the time for one cell cycle or the cell cycle length may be shortened; (b) the number of cells that withdraw from the cell cycle and terminally differentiate may be reduced; or (c) the number of cells that desquamate into the medium over a set period of time may be reduced. We have explored these possibilities in growing cultures of epidermal keratinocytes using a newly developed double-label assay. This assay gives a measure of both cell length and cell cycle withdrawal. Results show that the growth enhancement induced by EGF and cholera toxin can be attributed primarily to a reduction in cell cycle withdrawal and, to a lesser degree, to a reduction in cell cycle length. EGF and cholera toxin have no significant effect on the rate of desquamation. A linear correlation was noted between cell cycle lengths and withdrawal, suggesting an interconnection between the rate of cell renewal and the likelihood of undergoing terminal differentiation. This research was supported by grant DE04511 from the National Institute of Dental Research, Bethesda, MD, and gifts from the University Hospital Auxilliary, Health Sciences Center, SUNY Stony Brook, and the Suffolk County Volunteer Firefighter Fund.     

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.     

Differentiating cells of murine stratified squamous epithelia constitutively express plasminogen activator inhibitor type 2 (PAI-2)

 In stratified squamous epithelia a critical balance among cell proliferation, differentiation, and death must be maintained in order for these tissues to fulfill their barrier function. Previous studies have demonstrated that plasminogen activator inhibitor 2 (PAI-2) is a product of differentiating epidermal keratinocytes, suggesting a role for this inhibitor during squamous differentiation. Furthermore, in certain tumor cell lines, overexpression of PAI-2 confers resistance to the induction of programmed cell death, suggesting cytoprotective function(s). In the present study we demonstrate that PAI-2 mRNA and protein are constitutively and uniquely expressed in differentiating cells of murine stratified squamous epithelia, including epidermis, esophagus, vagina, oral mucosa, and tongue. PAI-2 immunohistochemical localization patterns suggest a predominantly cytosolic distribution, consistent with biochemical identification of the major PAI-2 species as a 43-kDa, presumably non-glycosylated protein. Functional analysis shows that the majority of epithelial PAI-2 is active. In contrast to the high levels of PAI-2 expression in stratified squamous epithelia, little or no PAI-2 is detectable in simple epithelia. These findings suggest that epithelial PAI-2 may mediate inhibition of intracellular proteinases associated with events during terminal differentiation and death that are unique to stratified squamous epithelia. Accepted: 29 June 1998     

Interleukin-6 promotes human epidermal keratinocyte proliferation and keratin cytoskeleton reorganization in culture

We have studied the effects of interleukin-6 (IL-6) on human epidermal keratinocytes by using serum-free culture conditions that allow the serial transfer, differentiation, and formation of well-organized multilayered epithelia. IL-6 at 2.5 ng/ml or higher concentrations promoted keratinocyte proliferation, with an ED₅₀ of about 15 ng/ml and a maximum effect at 50 ng/ml. IL-6 was 10-fold less potent than epidermal growth factor (EGF) or transforming growth factor-α (TGF-α) and supported keratinocyte growth for up to eight cumulative cell generations. IL-6-treated keratinocytes formed highly stratified colonies with a narrower proliferative/migratory rim than those keratinocytes stimulated with EGF or TGF-α; confluent epithelial sheets treated with IL-6 also underwent an increase in the number of cell layers. We also examined the effect of IL-6 on the keratin cytoskeleton. Immunostaining with anti-K16 monoclonal antibodies showed that the keratin network was aggregated and reorganized around cell nucleus and that this was not attributable to changes in keratin levels. This is the first report concerning the induction of the reorganization of keratin intermediate filaments by IL-6 in human epidermal keratinocytes.This work was supported in part by CONACyT grant nos. 1314P-N9507 and G28272-N.     

The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies

The keratins are a highly heterogeneous group of proteins that form intermediate filaments in a wide variety of epithelial cells. These proteins can be divided into at least seven major classes according to their molecular weight and their immunological reactivity with monoclonal antibodies. Tissue-distribution studies have revealed a correlation between the expression of specific keratin classes and different morphological features of in vivo epithelial differentiation (simple vs. stratified; keratinized vs. nonkeratinized). Specifically, a 50,000- and a 58,000-dalton keratin class were found in all stratified epithelia but not in simple epithelia, and a 56,500- and a 65-67,000-dalton keratin class were found only in keratinized epidermis. To determine whether these keratin classes can serve as markers for identifying epithelial cells in culture, we analyzed cytoskeletal proteins from various cultured human cells by the immunoblot technique using AE1 and AE3 monoclonal antikeratin antibodies. The 56,500- and 65-67,000-dalton keratins were not expressed in any cultured epithelial cells examined so far, reflecting the fact that none of them underwent morphological keratinization. The 50,000- and 58,000-dalton keratin classes were detected in all cultured cells that originated from stratified squamous epithelia, but not in cells that originated from simple epithelia. Furthermore, human epidermal cells growing as a monolayer in low calcium medium continued to express the 50,000- and 58,000-dalton keratin classes. These findings suggest that the 50,000- and 58,000-dalton keratin classes may be regarded as "permanent" markers for stratified squamous epithelial cells (keratinocytes), and that the expression of these keratin markers does not depend on the process of cellular stratification. The selective expression of the 50,000- and 58,000-dalton keratin classes, which are synthesized in large quantities on a per cell basis, may explain the high keratin content of cultured keratinocytes.     

The serine protease marapsin is expressed in stratified squamous epithelia and is up-regulated in the hyperproliferative epidermis of psoriasis and regenerating wounds

The trypsin-like serine protease marapsin is a member of the large protease gene cluster at human chromosome 16p13.3, which also contains the structurally related proteases testisin, tryptase epsilon, tryptase gamma, and EOS. To gain insight into the biological functions of marapsin, we undertook a detailed gene expression analysis. It showed that marapsin expression was restricted to tissues containing stratified squamous epithelia and was absent or only weakly expressed in all other tissues, including the pancreas. Marapsin was constitutively expressed in nonkeratinizing stratified squamous epithelia of human esophagus, tonsil, cervix, larynx, and cornea. In the keratinizing stratified squamous epidermis of skin, however, its expression was induced only during epidermal hyperproliferation, such as in psoriasis and in murine wound healing. In fact, marapsin was the second most strongly up-regulated protease in psoriatic lesions, where expression was localized to the upper region of the hyperplastic epidermis. Similarly, in the hyperproliferative epithelium of regenerating murine skin wounds, marapsin localized to the suprabasal layers, where keratinocytes undergo squamous differentiation. The transient up-regulation of marapsin, which closely correlated with re-epithelialization, was virtually absent in a genetic mouse model of delayed wound closure. These results suggested a function during the process of re-epithelialization. Furthermore, in reconstituted human epidermis, a model system of epidermal differentiation, members of the IL-20 subfamily of cytokines, such as IL-22, induced marapsin expression. Consistent with a physiologic role in marapsin regulation, IL-22 was also strongly expressed in re-epithelializing skin wounds. Marapsin's restricted expression, localization, and cytokine-inducible expression suggest a role in the terminal differentiation of keratinocytes in hyperproliferating squamous epithelia.     

Evidence for intracellular cleavage of plasminogen activator inhibitor type 2 (PAI-2) in normal epidermal keratinocytes

Plasminogen activator inhibitor type 2 (PAI-2) is a serine proteinase inhibitor (serpin), present in high quantities in stratified squamous epithelia. Detergent extracts of human epidermis or cultured keratinocytes contain primarily active, nonglycosylated PAI-2. In keratinocytes, the vast majority of PAI-2 is retained within the cell, supporting the hypothesis that PAI-2 may serve specific intracellular function(s) through interaction with an unknown cytoplasmic proteinase. During interaction with the target proteinase, cleavage of PAI-2 within its reactive site loop leads to the formation of a more stable, "relaxed" conformation (PAI-2r). Using a monoclonal antibody specific for PAI-2r, we demonstrate here that PAI-2r is present in keratinocytes of the granular and basal layers of normal human epidermis. In addition, PAI-2r is detectable in cultured human epidermal keratinocytes, where it is concentrated in a detergent-insoluble fraction within differentiating cells. These data provide evidence for the presence of an endogenous, keratinocyte-derived proteinase that constitutively cleaves intracellular PAI-2 in normal human epidermal keratinocytes. Cleavage of PAI-2 by this proteinase may reflect specific intracellular action of PAI-2 in normal cells. Finally, we demonstrate that a commercially available anti-PAI-2 monoclonal antibody (#3750, American Diagnostica, Greenwich, CT), under native experimental conditions, preferentially recognizes the uncleaved, active form of PAI-2 and does not efficiently detect PAI-2r.     

Differentiation-dependent expression of lectin binding sites on normal and neoplastic keratinocytes in vivo and in vitro.

We used lectins as probes to demonstrate the composition of membrane carbohydrates of canine keratinocytes in various functional stages and various degrees of differentiation. Keratinocytes during normal epidermal turnover were compared by lectin immunohistochemistry to cells of hyperplastic epidermis and neoplastic keratinocytes. Three types of epidermal tumors and oral squamous cell carcinomas were examined. In addition, two in vitro tissue culture systems for keratinocytes were studied and compared with in vivo epithelium. In normal skin, PNA reacted only weakly with basal cells, whereas in hyperplastic skin basal cells bound this lectin strongly, demonstrating increasing expression of PNA binding sites with increasing thickness of the stratified squamous epithelium. ConA bound to basal cell tumors only. In oral squamous cell carcinomas, the expression of distinct lectin binding sites correlated with certain histological growth patterns, e.g., UEA-I reacted with highly invasive tumors but not with tumors showing a solid growth pattern. Using cell surface iodination and polyacrylamide gel electrophoresis, distinct differences in cell membrane protein expression were demonstrated between normal and neoplastic keratinocytes. SDS-polyacrylamide gel electrophoresis of cultured normal and neoplastic keratinocytes revealed several cell surface proteins that are specific for either cell type. Neoplastic cells specifically express a 140 KD lectin binding cell surface glycoprotein. The results of this study show that lectin binding patterns of keratinocytes are dependent on the functional state and the degree of differentiation of the cells and demonstrate correlation of some histological growth patterns with distinct lectin binding phenotypes, suggesting association of expression of cell membrane carbohydrate moieties with growth patterns. In addition, close similarities between "lifted cultures" grown at the air-liquid interface and native tissue demonstrate the value of this culture system as a model for differentiated stratified squamous epithelium.     

Temporal and spatial sequence expression of cytokeratin K19 in cultured human keratinocyte

The unique cytokeratin K19 specifically expresses in simple epithelial cells, basal cells of non-keratinized stratified squamous epithelium, epidermal cells during the embryonic stage and squamous carcinoma cells, but it is not expressed in adult epidermis. Interestingly, when epidermal cells are cultured in vitro, K19 is re-expressed in the supra-basal layer. K19 expression was used as a marker for epidermal cell growth and differentiation. In order to clarify the temporal and spatial sequential expression in cultured keratinocyte, two-stage human keratinocyte culture systems were used to examine K19 expression in keratinocytes in a proliferation and differentiation stages through immunoblotting and immunohistochemistry assay. According to our results, K19 was not expressed in cultured human keratinocytes in the proliferation stage but was re-expressed in keratinocytes three days after the cultured medium was changed to a differentiation medium. Immunohistochemical observation revealed that K19 was persistently expressed in the supra-basal layer of cultured keratinocytes during first three weeks of culturing, but none was detectable in the basal cell layer. When keratinocytes were cultured with an "inserted cultured dish," K19 was persistently expressed in all layers of keratinocytes nourished by medium both from an inner chamber and an outer chamber. The different expression of K19 in these two different culture systems seemed to indicate that down regulation of K19 expression in keratinocyte was related to the direction of medium supply.     

Association of EGF and LDL receptors with the cytoskeleton of cultured keratinocytes

In this paper we demonstrate that isolated cytoskeletons of normal keratinocytes cultured under differentiation inducing conditions exhibit a high level of epidermal growth factor (EGF) binding. This binding is approximately 300% higher than the binding of intact cells. In contrast, various squamous carcinoma cell lines or normal keratinocytes cultured under differentiation retarding conditions exhibit EGF binding to isolated cytoskeletons which is around 10-20% of the binding to intact cells. Incubation of normal keratinocytes in the presence of arotinoid ethyl sulfone resulted in a marked decrease of the ability of the cells to differentiate, and a decrease of EGF binding to isolated cytoskeletons. These results suggest a close relationship between the differentiation capacity of the cells and the presence of cytoskeleton-associated EGF receptors. Similar results were obtained for low density lipoprotein (LDL) binding.     

Delta Np63 alpha expression is regulated by the phosphoinositide 3-kinase pathway

p63 is a homologue of p53 that functions to maintain progenitor cell populations in stratified epithelia. Delta Np63 alpha is overexpressed in epithelial cancers and has been shown to have oncogenic properties. We have previously reported that inhibition of epidermal growth factor receptor signaling results in a decrease in Delta Np63 alpha expression. Here, we demonstrate Delta Np63 alpha is a target of the phosphoinositide-3-kinase (PI3K) pathway downstream of the epidermal growth factor receptor. Treatment of keratinocytes with epidermal growth factor results in an increase in Delta Np63 alpha expression at the mRNA level, which is abrogated by inhibition of PI3K but not mitogen-activated protein kinase signaling. Small interfering RNA-mediated knockdown of the p110 beta catalytic subunit of PI3K results in a decrease in Delta Np63 alpha protein levels in keratinocytes. The results presented herein suggest that regulation of Delta Np63 alpha expression by the PI3K pathway plays a critical role in the survival and proliferative capacity of squamous epithelia.