Role of intracellular-free calcium in the cornified envelope formation of keratinocytes: differences in the mode of action of extracellular calcium and 1,25 dihydroxyvitamin D3

Extracellular calcium (Cao) and the steroid hormone 1,25(OH)2D, induce the differentiation of human epidermal cells in culture. Recent studies suggest that increases in intracellular free calcium (Cai) levels may be an initial signal that triggers keratinocyte differentiation. In the present study, we evaluated cornified envelope formation, the terminal event during keratinocyte differentiation, and correlated it with changes in the Cai levels during differentiation of keratinocytes in culture induced by Cao or 1,25(OH)2D. Keratinocytes were grown in different Cao concentrations (0.1 or 1.2 mM) or in the presence of 1,25(OH)2D (10(-11) to 10(-7) M), and the Cai levels were measured using the fluorescent probe Indo-1. Our results suggest that the induction of cornified envelope formation is associated with an increase in Cai level during calcium-induced differentiation. Cao and the calcium ionophore ionomycin acutely increased Cai and cornified envelope formation. In contrast, the effect of 1,25(OH)2D on increasing Cai levels and stimulating cornified envelope formation was long-term, requiring days of treatment with 1,25(OH)2D. Our data are consistent with other recent studies and support the hypothesis that Cao regulates keratinocyte differentiation primarily by acutely increasing their Cai levels. The role of calcium in the mechanism of action of 1,25(OH)2D on keratinocyte differentiation is less clear. The increase in Cai of keratinocytes during 1,25(OH)2D induced differentiation may be essential for or subsequent to its prodifferentiation effects.     

Lanthanum influx into cultured human keratinocytes: effect on calcium flux and terminal differentiation.

Trivalent cation lanthanum (La) binds to calcium binding sites of cells and either mimics the properties of calcium or inhibits the effects of calcium by displacing calcium from its binding sites. Extracellular calcium induces differentiation of human epidermal keratinocytes in culture, in part by increasing the intracellular calcium levels (Cai). Therefore, in this study we determined the effect of La on differentiation and intracellular calcium levels of keratinocytes. We observed that La inhibited the production of cornified envelopes, a marker for terminal differentiation of keratinocytes. La inhibited the calcium requiring envelope cross-linking enzyme, transglutaminase, in a direct manner, presumably, by displacing calcium from its binding site on the enzyme. La inhibited the influx and the efflux of 45Ca from keratinocytes. Paradoxically, extracellular La appeared to increase the Cai levels of keratinocytes as measured by the fluorescent probe indo-1. However, subsequent experiments revealed that indo-1 bound La with a higher affinity than Ca and emitted fluorescence in the same wavelength as the Ca bound form. Using this probe, we observed that La enters keratinocytes in a dose-dependent fashion and achieves concentrations exceeding 80 nM when the external La concentration is raised to 300 microM. This fully accounted for the apparent increase in Cai when La was added to the cells. Treatment of cells with ionomycin increased indo-1 fluorescence maximally in the presence of La indicating influx of La via this Ca specific ionophore. Our results indicate that La enters cells and inhibits calcium mediated keratinocyte differentiation both by blocking Ca influx and by blocking calcium regulated intracellular processes such as transglutaminase directed cornified envelope formation.     

Calcium regulation of growth and differentiation of normal human keratinocytes: modulation of differentiation competence by stages of growth and extracellular calcium

In this study we examined the different aspects of the pathway leading to the differentiation of keratinocytes as a function of time in culture and calcium concentration of the culture medium. Human neonatal foreskin keratinocytes were grown in a serum-free, defined medium containing 0.07, 1.2, or 2.4 mM calcium and assayed for the rate of growth and protein synthesis, involucrin content, transglutaminase activity, and cornified envelope formation at preconfluent, confluent, and postconfluent stages of growth. We observed that keratinocytes grown to postconfluence in all calcium concentrations showed an increased protein/DNA ratio and an increased rate of membrane-associated protein synthesis. Extracellular calcium concentrations did not have a clear influence on these parameters. However, preconfluent and confluent keratinocytes grown in 0.07 mM calcium showed markedly retarded differentiation at all steps, i.e., involucrin synthesis, transglutaminase activity, and cornified envelope formation. Within 1 week after achieving confluence, these keratinocytes began synthesizing involucrin and transglutaminase and developed the ability to form cornified envelopes. Cells grown in 1.2 and 2.4 mM calcium synthesized involucrin and transglutaminase prior to confluence and were fully competent to form cornified envelopes by confluence. Thus external calcium-regulated keratinocyte differentiation is not an all or none phenomenon, but rather it is the rate at which keratinocytes differentiate that is controlled by calcium. We conclude that either or both higher extracellular calcium concentration and the achievement of cell-cell contacts lead to a coordinate increase of at least two precursors--involucrin content and transglutaminase activity--required for cornified envelope formation. We speculate that a critical level of cytosolic calcium, achieved by increased extracellular calcium or by achievement of intercellular communication established by cell-cell contact, may trigger mechanisms required for initiation of keratinocyte differentiation.     

Buffering of intracellular calcium in response to increased extracellular levels in mortal, immortal, and transformed human breast epithelial cells.

Extracellular levels of calcium at 1.05 mM or higher induce terminal differentiation and senescence in the mortal (MCF-10M) line of human breast epithelial cells, but does not retard the growth or induce differentiation in the immortal (MCF-10A) and oncogene transformed (MCF-10AneoT) lines. Intracellular levels of calcium and inositol triphosphate were determined in MCF-10M, MCF-10A, and MCF-10AneoT, under conditions of low and high extracellular calcium. We hereby report that increases in extracellular calcium is translated into significant increases in intracellular levels of calcium and inositol triphosphate in MCF-10M, but not in MCF-10A and MCF-10AneoT. This difference in the apparent calcium buffering capacity between the mortal and the immortalized human breast epithelial cells could account for the latter's unperturbed growth potential in high extracellular calcium environment.     

Role of protein kinase C alpha in calcium induced keratinocyte differentiation: defective regulation in squamous cell carcinoma

Calcium induces both involucrin and transglutaminase-K in normal keratinocytes (NHK) but not in squamous carcinoma cell lines (SCC). The protein kinase C (PKC) agonist phorbol myristoyl acetate potentiates and the PKC antagonist Ro31-8220 blocks the ability of calcium to stimulate the involucrin promoter in normal human keratinocytes but not in SCC4. We thus examined the ability of calcium to regulate the levels of five PKC isozymes in NHK and two SCC. In the normal keratinocytes, the levels of PKC [alpha], PKC [delta], PKC [eta], and PKC [zeta] increased over the first one to two weeks in a calcium-and time-dependent manner. PKC [epsilon] decreased in a time-and calcium-dependent fashion over the three-week period. All five isozymes showed little change during culture in SCC4 at any calcium concentration. Calcium and time of culture had partial effects on SCC12B2, a carcinoma that shows partial differentiation characteristics. Since PKC [alpha] is the only calcium responsive PKC isozyme in keratinocytes and most likely to be directly involved in calcium induced differentiation, we evaluated the effect of inhibiting its production with antisense oligonucleotides on calcium-regulated markers of differentiation. We found that the PKC [alpha] specific antisense oligonucleotide blocked calcium stimulated involucrin promoter activity as well as PKC [alpha], involucrin, and transglutaminase protein production, whereas the sense oligonucleotide control did not. We conclude that although a number of PKC isozymes are regulated during calcium-induced differentiation, PKC [alpha] plays a necessary role in mediating calcium-induced differentiation. Failure to regulate PKC [alpha] in SCC4 may underlie at least part of the failure of calcium to promote differentiation in these cells.     

Differentiation of cultured human keratinocytes: Effect of culture conditions on lipid composition of normal vs. malignant cells

Summary Differentiation in keratinocytes can be experimentally modulated by changing the culture conditions. When cultured under conventional, submerged conditions, the extent of cellular differentiation is reduced in the presence of low calcium medium and is enhanced in medium containing physiologic calcium concentrations. Moreover cultures grown at the air-medium interface or on a dermal substrate, or both, differentiate even further. Herein we report the effect of culture conditions on lipid composition in normal human keratinocytes and three squamous carcinoma cell (SCC) lines that vary in their capacity to differentiate as assessed by cornified envelope formation. Under submerged conditions, the total phospholipid content was lower, triglyceride content higher, and phospholipid: neutral lipid ratio lower in direct correlation to the degree of differentiation in these cultures. When grown at the air-medium interface on the-epidermized dermis, evidence of further morphologic differentiation was found only for well-differentiated SCC cells and normal keratinocytes. Similarly, the phospholipid content remained high in poorly differentiated SCC cells and it, decreased modestly in well-differentiated SCC cells and markedly in normal keratinocytes. In all cell lines the triglyceride content was increased and cholesterol content decreased when compared to parallel submerged cultures, but these differences were most pronounced in well-differentiated cell lines. Acylceramides and acylglucosylceramides were found only in normal keratinocytes and only under the most differentiation-enhancing conditions. These studies demonstrate differentiation-related changes in the lipid content of both normal and neoplastic keratinocytes. This work was supported in part by NATO Scientific Award (RG 8510056).     

Epidermal growth factor receptor expression related to differentiation capacity in normal and transformed keratinocytes

Epidermal growth factor (EGF) and Ca2+ have been indicated to play a major role in skin development. We have used normal keratinocytes, SV40-transformed keratinocytes (SVK14) and various squamous carcinoma cell (SCC) lines as in vitro model system to study the effect of the extracellular Ca2+ concentration of EGF-receptor expression in relation to the capability of cells to differentiate. The cell lines used exhibit a decreasing capacity to differentiate in the order of keratinocytes approximately SVK14 greater than SCC-12F2 greater than SCC-15 greater than SCC-12B2 greater than SCC-4, as judged from Ca2+-ionophore-induced cornified envelope formation. Under normal Ca2+ conditions, all cell lines (except for SCC-15) exhibited two classes of EGF-binding sites. The number of low-affinity binding sites increased considerably as cells were less able to differentiate, while the apparent dissociation constant (kd) was similar in all cell lines. In contrast, the properties of high-affinity EGF binding varied in the various cell lines without a clear relationship to the degree of differentiation capacity. Lowering the extracellular Ca2+ concentration to 0.06 mM resulted in a decrease of Ca2+ ionophore-induced cornified envelope formation, demonstrating the decreased ability to differentiate under these conditions. The decreased ability to differentiate was accompanied by a marked increase in the number of EGF-binding sites, but without a change of the kd. Furthermore, no high-affinity EGF-binding sites were detectable under these conditions. Finally, addition of Ca2+ to low Ca2+-cultured cells caused a rapid decrease of EGF binding in all cell lines, most prominently in normal keratinocytes and SCC-12F2 cells. The data presented demonstrate: The combination of normal keratinocytes, SVK14 and the various SCC lines provides an attractive model system to study differentiation in vitro; EGF-receptor expression is related to the state of differentiation, both phenomena being sensitive to the external Ca2+ concentration; and EGF-receptor expression is related to the capability of cells to differentiate.     

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.     

Vitamin D regulated keratinocyte differentiation: role of coactivators

1,25 Dihydroxyvitamin D (1,25(OH)(2)D) regulates the differentiation of keratinocytes. 1,25(OH)(2)D raises intracellular free calcium (Cai) as a necessary early step toward stimulating differentiation. 1,25(OH)(2)D induces the calcium sensing receptor (CaR) in keratinocytes and enhances the calcium response of these cells. Activation of the CaR by calcium increases intracellular free calcium by a mechanism involving phospholipase C (PLC) cleavage of phosphatidylinositolbisphosphate into inositoltrisphosphate (IP(3)) and diacylglycerol (DG). 1,25(OH)(2)D induces the family of PLCs. PLC-gamma1 has a DR6 VDRE in its promoter which binds and is activated by VDR/RAR rather than VDR/RXR. The involucrin gene, which encodes a critical component of the cornified envelope, contains a DR3 VDRE in its promoter that acts in conjunction with a nearby AP-1 site. The sequential regulation of these genes is critical for the differentiation process. In undifferentiated keratinocytes, the VDR binds preferentially to the DRIP complex of coactivators. However, with differentiation DRIP 205 is no longer produced, and the VDR switches partners to the SRC family (SRC2 and 3). These studies suggest that at least part of the sequential activation of genes required during keratinocyte differentiation is regulated by the change (availability) of these different coactivator complexes.     

Localization and quantitation of calcium pools and calcium binding sites in cultured human keratinocytes

Calcium plays a crucial role in regulating the growth and differentiation of cultured keratinocytes. However, the mechanism(s) of this regulation is not clear. Prior studies have shown that intracellular free calcium (Cai) increases with keratinocyte differentiation. In this study, in order to evaluate the role of cytosolic free calcium and organelle-bound calcium in keratinocyte differentiation, we quantitated and localized calcium pools in keratinocytes, utilizing the fluorescence probe indo-1 and ion-capture cytochemistry, respectively. Cai of undifferentiated keratinocytes was 80–120 nM, whereas Cai of differentiated keratinocytes was 200–300 nM depending on the extent of differentiation. The Cai of individual cells in an undifferentiated colony was heterogeneous (60–160 nM) with larger cells displaying higher Cai. Heterogeneity also was observed in the intracellular calcium-containing precipitates in the different layers of stratifying keratinocyte cultures using the cytochemical technique. Calcium precipitates were abundant in the lower cell layers, progressively decreasing apically, with the uppermost layer devoid of precipitates. Calcium-containing precipitates appeared as fine-tocoarse electron-dense granules on the plasma membrane, within the cytosol, mitochondria, nucleus, and vacuolar organelles. Whereas ionomycin in the presence of extracellular calcium increased the amount of intracellular calcium precipitates, EGTA removed calcium precipitates from organelles. Unlike intact epidermis, keratinocytes displayed no extracellular calcium reservoirs. Putative calcium binding sites, visualized by trivalent lanthanum (La) binding, were abundant on cell membranes and desmosomes of basaloid cells, but decreased in the upper cell layers. These studies revealed differences in the distribution of free ionic calcium (as determined by the fluorescence technique) and organelle-bound calcium (as determined by the cytochemical technique). Striking differences were also observed in calcium localization between intact epidermis and cultured epidermal cells. The localization pattern of calcium in cultured keratinocytes may reflect the hyperproliferative state of these cells, as in psoriatic epidermis, and/or the absence of a normal permeability barrier in these submerged cultures. © 1993 Wiley-Liss, Inc.     

Increased intracellular calcium is associated with progression of HPV-18 immortalized human keratinocytes to tumorigenicity.

Studies were conducted using normal and human papillomavirus Type 18 (HPV-18) immortalized human keratinocytes to assess possible alterations in the differentiation process as a consequence of increased intracellular calcium concentration. Normal keratinocytes exposed to increased extracellular calcium or the phorbol ester TPA, exhibited terminal differentiation characteristics. However, late passage HPV-18 immortalized keratinocytes (designated FEP-1811) were resistant to such terminal differentiation signals. Flow cytometric analyses of 1811 cells at various stages of passage in culture revealed progressively higher levels of intracellular calcium in the immortalized cells with passage in culture when compared to normal, primary keratinocytes. Furthermore, 1811 cells isolated from tumors which developed in irradiated nude mice contained the highest level of intracellular calcium of all the cells examined. These results suggest that an increase in the concentration of intracellular calcium is associated with progression of HPV-18 immortalized keratinocytes to tumorigenicity.     

Diminished calcium influx in lectin-stimulated T cells from old mice

T lymphocytes from aged donors function poorly, but the biochemical basis for the defect remains uncertain. We tested the hypothesis that T cells from old mice had a diminished ability to transmit extracellular signals into the cytoplasm, by measuring intracellular free calcium concentrations (Cai) in T cells stimulated by the polyclonal activator concanavalin A (Con A). Using the second-generation fluorochrome indo-1 as a reporter of Cai, we found that the Con A-induced elevation of Cai levels is reduced both in rate and extent in old T cells, as compared to T cells from young mice. Flow cytometric analysis showed that this age-sensitive change represents a decline, with age, in the number of T cells that can respond to Con A by increasing their Cai above resting baseline levels (100-120 nM). These results thus show that defects in activation are manifested by T cells from old donors within the first 5 minutes of the activation process, and suggest that aging may lead to alterations either in the surface molecules that receive extracellular signals, or in the sequence of coupled events by which these extracellular signals bring about alterations in the intracellular ionic milieu.     

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.     

Regulation of goblet cell differentiation by calcium in embryonic chick intestine

The potential role of calcium as a regulator of goblet cell differentiation was tested by culturing duodenal explants from 14-day chicken embryos in media containing a range of calcium concentrations. Extracellular calcium in vitro approximated the range of plasma calcium concentration that was found to rise by 16% during the third week of embryonic development. As ionic calcium was increased from 0.9 to 2.0 mM in 48-h cultures, the number of goblet cells per 100 ridges increased progressively from 29 +/- 2.2 to 158 +/- 6.9 while attaining a distribution on the previllous ridges similar to that in ovo. The rate of goblet cell differentiation in vitro exceeded that in ovo when extracellular calcium was increased to 1.1 mM and was maximal at 1.6-2.0 mM calcium. Neither the growth of previllous ridges nor gross morphology of the tissue was altered as extracellular calcium was increased. Goblet cell differentiation in 1.3 mM calcium was stimulated by 0.1 microM calcium ionophore and inhibited by the calmodulin antagonist trifluoperazine, indicating an intracellular site for calcium action. These results indicate that calcium plays a primary role in regulating goblet cell differentiation in embryonic intestine and suggest that rising levels of plasma calcium influence the rate of differentiation in ovo.     

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.     

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.