Mouse epidermal cell renewal after topical treatment with different concentrations of the epidermal peptide pyroGlu-Glu-Asp-Ser-GlyOH

Earlier work has shown that epidermal cells contain a peptide, pyroGlu-Glu-Asp-Ser-GlyOH, that induces a moderate but long-lasting inhibition of epidermal cell proliferation when given at low (picomol) doses ip in vivo and in vitro. In the present study, the epidermal pentapeptide was applied topically to the back skin of hairless mice at different concentrations and in a water-miscible cream. A single topical application of either high (0.25% wt/wt) or low (0.004% or 0.02% wt/wt) doses of the pentapeptide was followed by oscillations in epidermal DNA synthesis and G2-M cell flux (mitotic rate). In general, epidermal cell proliferation was inhibited during the first 10-day period after treatment with the two lower doses, while the highest concentration of pentapeptide (0.25%) stimulated epidermal cell proliferation. In spite of the effects on epidermal cell proliferation the size of the epidermal cell population in the treated area (number of nucleated cells and epidermal thickness) showed no corresponding alterations. The results could imply that the epidermal pentapeptide modifies epidermal cell proliferation and terminal differentiation in such a way that the two are balance with each other.     

Mouse epidermal cell renewal after topical treatment with different concentrations of the epidermal peptide pyroGlu-Glu-Asp-Ser-GlyOH

Earlier work has shown that epidermal cells contain a peptide, pyroGlu-Glu-Asp-Ser-GlyOH, that induces a moderate but long-lasting inhibition of epidermal cell proliferation when given at low (picomol) doses ip in vivo and in vitro. In the present study, the epidermal pentapeptide was applied topically to the back skin of hairless mice at different concentrations and in a water-miscible cream. A single topical application of either high (0.25% wt/wt) or low (0.004% or 0.02% wt/ wt) doses of the pentapeptide was followed by oscillations in epidermal DNA synthesis and G₂-M cell flux (mitotic rate). In general, epidermal cell proliferation was inhibited during the first 10-day period after treatment with the two lower doses, while the highest concentration of pentapeptide (0.25%) stimulated epidermal cell proliferation. In spite of the effects on epidermal cell proliferation the size of the epidermal cell population in the treated area (number of nucleated cells and epidermal thickness) showed no corresponding alterations. The results could imply that the epidermal pentapeptide modifies epidermal cell proliferation and terminal differentiation in such a way that the two are balance with each other.     

Adaptive response is differently induced depending on the sensitivity to radiation-induced cell death in mouse epidermal cells

We investigated the relationship between induction of radio-adaptive response and cell death in mouse normal and neoplastic epidermal cells. Mouse normal primary keratinocytes (PK), cancer-prone cells [v-ras ^Ha-transfected mouse keratinocytes (ras-PK), and line 308 cells (mouse skin papilloma cells which have activatedras ^Ha gene with A-to-T transversion at codon 61) were primed with a low dose of γ-rays (0.01 Gy), and were challenged with a high dose (4 Gy) after a 4 or 7 h interval. The induction of cell death in PK was 2–10 times higher and was also more rapid in PK than in ras-PK or 308 cells. Low-dose pretreatment with a 4 h interval decreased cell death, and this adaptive response was prominent in PK, whereas it was less obvious in the cases of ras-PK and 308 cells. The response of each protein kinase C (PKC) isozymes to high-dose radiation, especially PKCα, PKCδ, PKCε, and PKCη, were different between the normal andras oncogene-activated neoplastic keratinocytes; translocation of these isozymes to membrane occurred more rapidly in normal than in neoplastic cells. Furthermore, low-dose pretreatment did not induce the translocation of PKCδ in PK significantly more than in ras-PK and 308. Thus, the difference in the induction of radio-adaptive responses between mouse normal and neoplastic epidermal cells reflects difference in the rapidity of cell death, and responsiveness of PKC may affect this adaptive response. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.     

Growth kinetics in newborn mouse epidermis: response to epidermal chalone.

Epidermal DNA synthesis, the epidermal mitotic rate, and the responsiveness to the epidermal G1 and G2 inhibitors were examined in newborn mice at different times after birth. The rate of epidermal cell renewal was in general low during the first two weeks of life. Later the two growth parameters increased and reached very high values at 32-33 days after birth. The rate of epidermal cell proliferation then decreased to a level comparable with that found in adult hairless mouse epidermis at 40-45 days. A single i.p. injection of skin extract containing the two epidermal growth inhibitors induced varying types of responses. The epidermal G2 inhibitor stimulated the mitotic rate on day 2 and day 10, but inhibited it on all other days. The epidermal G1 inhibitor brought about an increase in epidermal DNA synthesis on day 6 and possibly on the following days. No response at all seen at 2, 4, 17, and 32 days after birth. At the other examined times the inhibition was similar to that found in adult mice. These findings differed from those made in vitro on separated newborn mouse epidermal cells (our own unpublished data), and we suggest that the variability of newborn mouse epidermis could be an expression of the immaturity of the skin as a whole, and that dermis in some way modifies the response of epidermis to exogenous epidermal chalone. Our study did not support the theory that the nonresponsiveness of newborn mouse epidermal at certain times could be due to the presence of nonresponsive stem cells in epidermis.     

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.     

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.     

Ultraviolet B irradiation induces epidermal regeneration with rapidly cycling cells

The left flank of hairless mouse skin was irradiated with a minimal erythema dose of ultraviolet B (UVB) light at 297 nm (25 mJcm-2), while the right flank served as untreated control. The alterations in epidermal growth kinetics induced by this UVB dose were studied with the percentage of labelled mitoses (PLM) technique during the period of increased proliferation. Thirty hours after irradiation, when a large cohort of cells appears in S phase, each animal was injected intra-peritoneally with 50 microCi tritiated thymidine [( 3H]-TdR). The number of labelled basal and suprabasal cells, as well as their localization in epidermis were registered in histological sections at short intervals up to 48 h after the [3H]-TdR pulse. Labelled mitoses were also counted in the same specimens. The results showed a four-fold increase of the high initial number of labelled cells in UVB-exposed epidermis within 18 h of the pulse injection, and a six-fold increase after 36 h. In control epidermis, where the starting value of the labelling index was much lower, there was only a three to four-fold increase in the number of labelled cells during the period studied. The PLM and the labelling index data were consistent with an average cell cycle time of approximately 10-12 h for UVB-exposed cells, in contrast to about 30 h for the fastest cycling population in control epidermis. The PLM curve also indicated a prolonged S phase duration in UVB-exposed epidermis compared with controls. In addition, labelled cells were seen in the suprabasal layer as early as 6 h after the [3H]-TdR injection and within 36 h labelled cells had reached the outermost layer of nucleated cells, indicating a reduced transit time through epidermis. The present study shows that a minimal erythema dose of UVB light at 297 nm induced a period of increased transit time through the S phase, combined with rapid cell proliferation, leading to an overall shortening of the epidermal cell cycle time. The cohort of cells labelled with [3H]-TdR 30 h after irradiation seemed to proceed as a wave of partially synchronized cells through the cell cycle for more than two rounds, which is comparable with the cell kinetic perturbations observed in regenerating mouse epidermis.     

Ultraviolet B irradiation induces epidermal regeneration with rapidly cycling cells

Abstract. The left flank of hairless mouse skin was irradiated with a minimal erythema dose of ultraviolet B (UVB) light at 297 nm (25 mJcm^-2), while the right flank served as untreated control. The alterations in epidermal growth kinetics induced by this UVB dose were studied with the percentage of labelled mitoses (PLM) technique during the period of increased proliferation. Thirty hours after irradiation, when a large cohort of cells appears in S phase, each animal was injected intra-peritoneally with 50 /iCi tritiated thymidine ([³H]-TdR). The number of labelled basal and suprabasal cells, as well as their localization in epidermis were registered in histological sections at short intervals up to 48 h after the [³H]-TdR pulse. Labelled mitoses were also counted in the same specimens. The results showed a four-fold increase of the high initial number of labelled cells in UVB-exposed epidermis within 18 h of the pulse injection, and a sixfold increase after 36 h. In control epidermis, where the starting value of the labelling index was much lower, there was only a three to four-fold increase in the number of labelled cells during the period studied. The PLM and the labelling index data were consistent with an average cell cycle time of approximately 10–12 h for UVB-exposed cells, in contrast to about 30 h for the fastest cycling population in control epidermis. The PLM curve also indicated a prolonged S phase duration in UVB-exposed epidermis compared with controls. In addition, labelled cells were seen in the suprabasal layer as early as 6 h after the [³H]-TdR injection and within 36 h labelled cells had reached the outermost layer of nucleated cells, indicating a reduced transit time through epidermis. The present study shows that a minimal erythema dose of UVB light at 297 nm induced a period of increased transit time through the S phase, combined with rapid cell proliferation, leading to an overall shortening of the epidermal cell cycle time. The cohort of cells labelled with [³H]-TdR 30 h after irradiation seemed to proceed as a wave of partially synchronized cells through the cell cycle for more than two rounds, which is comparable with the cell kinetic perturbations observed in regenerating mouse epidermis.     

Retinoic acid-induced transglutaminase in mouse epidermal cells is distinct from epidermal transglutaminase

Elevated transglutaminase activity and formation of cornified envelopes are markers of terminal differentiation in mouse epidermal cells. Epidermal transglutaminase catalyzes cornified envelope formation and in cultured cells is inducible by calcium ion or phorbol ester tumor promoters. Retinoic acid also induces transglutaminase activity but inhibits cross-linked envelope formation. This apparent paradox might be resolved by the observation that the retinoic acid-induced transglutaminase appears to be either a different enzyme or a markedly altered form of the epidermal enzyme. The retinoic acid-induced transglutaminase is soluble in aqueous buffers, is thermolabile at pH 9.0, 37 degrees C, and elutes from an anion exchange column at 0.4 M NaCl. In contrast, the epidermal enzyme is particulate and requires detergent for solubilization, is relatively thermostable, and elutes from the anion exchanger at 0.25 M NaCl. The retinoic acid-induced enzyme is probably identical with the "tissue" transglutaminase present in liver and in other cells. It is proposed that the transglutaminase induced by retinoic acid may play a role in the inhibition by retinoids of calcium and tumor promoter-induced differentiation.     

Free and protein-conjugated polyamines in mouse epidermal cells. Effect of high calcium and retinoic acid

We have investigated polyamine metabolism in primary cultures of mouse epidermal cells. These cells, which grow at low Ca2+ levels as a monolayer with characteristics of basal cells, terminally differentiate when the extracellular Ca2+ level is raised above 1 mM. The cellular levels of free polyamines were measured, and, after incubation of cell cultures with [3H]putrescine, the distribution of label in both acid-soluble and acid-insoluble cellular components was examined. Free polyamine levels were reduced in cells induced to differentiate. Treatment with retinoic acid, which prevents differentiation and causes increased proliferation, resulted in an increase in free putrescine. Upon adjustment of the calcium concentration to a level that induces differentiation, the enzyme transglutaminase was activated, and a concomitant increase in the level of both protein-bound mono- and bis-gamma-glutamyl derivatives of putrescine and spermidine was observed. Isolation of a material of apparent molecular weight about 6000 which contains only mono-gamma-glutamylpolyamines and the finding of both mono- and bis-gamma-glutamylpolyamines in the protein fraction containing cornified cell envelopes provided the basis for speculation on polyamines in envelope formation. Our data suggest that polyamines play a role during epidermal cell differentiation through transglutaminase-mediated post-translational modification.     

Decreased epidermal growth factor binding in cells growth arrested in G1 by nutrient deficiency

Previous studies have shown that the nontransformed AKR-2B mouse embryo derived cell line may growth arrest by two separate mechanisms in the G₁ phase of the cell cycle-growth factor deficiency arrest (G₀) and low molecular weight nutrient deficiency arrest. An examination of epidermal growth factor (EGF) receptors under the different resting or growth conditions has shown that rapidly growing cells or cells arrested due to growth factor deficiency have the expected amount of ¹²⁵I-EGF binding with approximately 10⁵ receptors per cell being present in G₀ arrested cells. In contrast, cells arrested due to nutrient deficiency show a reduction in ¹²⁵I-EGF binding to 10--20% of that observed under the other conditions. This effect appears to be due to decreased receptor number and not to a change in the affinity of the receptor. Stimulation of DNA synthesis by nutrient replenishment causes a tenfold increase in EGF binding 20 hours later, with some increase in binding being detectable as early as six hours. The increase in binding is inhibited by cycloheximide and actinomycin D. This suggests that new mRNA synthesis as well as increased protein synthesis is required for the increase in EGF binding.     

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.     

Nuclear transport in 3T3 fibroblasts: effects of growth factors, transformation, and cell shape

Nucleocytoplasmic transport of fluorescent-labeled macromolecules was investigated in transformed and nontransformed 3T3 fibroblasts. Insulin and epidermal growth factor enhanced transport three-fold after 1-2-h incubation with nontransformed adhering fibroblasts; no enhancement of transport was observed for spherical unattached fibroblasts. The concentration of growth factor for maximal enhancement was 3-10 nM. Nuclear transport for Kirsten murine sarcoma virus-transformed BALB/c 3T3 fibroblasts, however, was maximally enhanced before addition of growth factors; addition of insulin or epidermal growth factor causes no additional transport enhancement. Transformation also minimizes cell shape effects on macromolecular nuclear transport. These results provide evidence that protein growth factors and oncogenic transformation may use a similar mechanism for activation of nuclear transport.     

Effect of epidermal chalones on the growth of transplantable tumors]

The ethanolic extract of rat skin contains epidermal G1- and G2-chalones which having been added to cell suspension of transplantable squamous-cell cornified carcinoma of mice cervix, inhibits its growth in recipients by 72,6 per cent. The same extract failed to inhibit the growth of transplantable mouse tumours of other histogenesis (hepatoma 22a, leukemia L-1210 and sarcoma 180). When added to cell suspension of transplantable carcinoma of mouse skin which had been anaplized during a long period of transplantation (over 10 years) this extract inhibits its growth by 39,2 per cent only.     

Analysis of the cellular heterogeneity in the basal layer of mouse ear epidermis: an approach from partial decomposition in vitro and retroviral cell marking in vivo

In the thin epidermis, the existence of epidermal proliferation units was hypothesized. Each unit is supposed to be partitioned into each column of polygonal-shaped cornified plates, estimated to contain a central stem cell in its basal layer. We attempted to verify this hypothesis in vitro by analyzing the partially decomposed fragment of mouse ear epidermis and in vivo using retroviral cell marking. Partially decomposed fragments of the mouse ear epidermis, mostly composed of cytokeratin 14-expressing basal keratinocytes, formed multicellular colonies in vitro. They were composed of heterogeneously shaped cells, morphologically resembling the cells in each single cell-derived colony, including potential stem cells with great proliferative potency in vitro. The estimated frequency of the candidates of stem cells in the fragments was much lower than the prediction from the representative hypothesis. Retroviral cell marking with nuclear localizing LacZ protein in vivo suggested the existence of a large clonal cellular unit for epidermal renewal. From these in vitro and in vivo observations, we propose a new model for the epidermal proliferation unit.     

Differentiation of human epidermal cells transformed by SV40

Human epidermal cells were transformed with DNA from wild-type SV40 virus or with DNA from a temperature-sensitive A mutant (tsA209). The SV40-transformed cells differed from nontransformed cells in their morphologic appearance, growth properties, and expression of certain characteristics associated with differentiation. The transformed cells were more variable in size and shape than their nontransformed counterparts and were less stratified and less keratinized. While the growth properties of the cells were similar under optimal growth conditions, the transformed cells could be propagated under stringent growth conditions that did not support the growth of nontransformed human epidermal cells. The transformants still required a 3T3 feeder layer for growth, remained anchorage dependent as assayed in soft agar, and were not tumorigenic in athymic nude mice. The expression of certain differentiated functions of the human epidermal cell, the presence of keratins and cross-linked envelopes, was decreased in the transformed cells, and these functions could be restored at the nonpermissive temperature in the tsA209 transformed cells.     

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.     

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.