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

JOURNAL OF CELLULAR PHYSIOLOGY 146:94-100 (1991)

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 D, SREEKUMAR PILLAI* AND DANIEL D. BIKLE

Department of Medicine, University of California, San Francisco 94 143, and Division of Endocrinology and Dermatology, Veterans Administration Medical Center,

San Francisco 94 12 I , California

Extracellular calcium (Cao) and the steroid hormone 1,25(OH),D, 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),D. Keratinocytes were grown in different Cao concentrations (0.1 or 1.2 mM) or in the presence of 1,25(OH),D ( lo- ' ' to lo-' 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),D on increasing Cai levels and stimulating cornified envelope formation was long-term, requiring days of treatment with 1,25(OH),D. Our data are consistent with other recent studies and support tl-le 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),D on keratinocyte differentiation is less clear. The increase in Cai of keratinocytes during 1,25(OH),D induced differentiation may be essential for or subsequent to its prodifferentiation effects.

Extracellular calcium concentration (Cao) has a pro- found influence on the growth and differentiation of keratinocytes in culture. Low levels of Cao (0.03- 0.1 mM) permit cell proliferation, whereas higher levels of Cao (0.3-1.8 mM) permit stratification and differentiation (Hennings et al., 1980; Boyce and Ham, 1983; Milstone, 1987). Physiologic levels of Cao are required for these cells to express a number of proteins required for differentiation, such as membrane bound transglutaminase, involucrin, and desmosomal components (Hennings et al., 1981, 1983a; Watt et al., 1984). The requirement for calcium in activating several enzyme systems potentially involved in cell proliferation and differentiation is well established, although the precise molecular mechanisms involved in the calcium regulated cellular changes in keratinocytes remain largely unknown. 1,25 dihydroxyvitamin D [1,25(OH),DI, a major regulator of calcium homeosta- sis, also influences the differentiation of a variety of tissues (Abe et al., 1981; Colston et al., 1981; Majeska and Rodan, 1982; Murao et al., 19831, including the epidermis (Hosomi et al., 1983; Smith et al., 1986; Pillai et al., 1988). 1,25(OH)2D causes a dose-dependent growth inhibition and morphologic differentiation of 0 1991 WILEY-LISS. INC.

human and murine keratinocytes (Hosomi et al., 1983; Smith et al., 1986). Transglutaminase activity, involucrin content, and cornified envelope content of keratinocytes grown in physiological levels of Cao were enhanced by treatment with 1,2f1(0H)~D (Pillai et al., 1988). Changes in intracellular free calcium (Cai) levels may be one of the mechanisms by which this hormone exerts its effects on cell differentiation (Bar, 1986; Baran, 1986; Smith and Holick, 1987; Hruska et al., 1988).

Recent studies on the mobilization of Cai during agonist-induced signal transduction have spurred in- terest in the role of Cai in biological processes (Watson et al., 1986; Wollheim and Biden, 1986; Hamilton and Sims, 1987). Such changes in Cai coupled to increased phosphoinositide turnover occur during cell differentiation (Klip and Ramlal, 1987) and in response to agents inducing growth and differentiation such as growth

Received June 22, 1990; accepted October 5, 1990. *To whom reprint requestsicorrespondence should be addressed at Veterans Administration Medical Center, Department of Derma- tology (1901, 4150 Clement Street, San Francisco, CA 94121.

CALCIUM AND VITAMIN D, IN KERATINOCYTE DIFFERENTIATION 95

factors (Hepler et al., 1987) or antibodies (Pezzuto, 1987). Increases in phosphoinositol turnover accompa- nied by a rise in inositol triphosphate level is observed during keratinocyte differentiation induced by calcium (Ziboh et al., 1984; Tang et al., 1988), 1,25(OH)2D (Tang et al., 19871, or ionomycin (Jaken and Yuspa, 1988). Optimal expression of keratin, filaggrin, and cornified envelope precursor genes occurs only at Cao levels between 0.1 and 0.16 mM in murine keratinocytes (Yuspa et al., 1989). Assuming these specific changes in Cao reflect analogous changes in Cai, Cai levels may dictate the expression of the differentiation phenotype of normal keratinocytes (Yuspa et al., 1988).

Increases in Cai activate the calcium-dependent transglutaminase to form the cornified envelope (Yuspa et al., 1980). Likewise, increments in Cai along with diacyl glycerol activate protein kinase C (Yuspa et al., 1988) or in conjunction with calmodulin activate calmodulin stimulated protein kinases. The observa- tion in vivo that the more differentiated granular cell layers have a higher intracellular calcium content than the less differentiated basal cells also supports the importance of Cai in epidermal differentiation (Menon et al., 1985). All these studies suggest that increases in Cai may be the initial event triggering keratinocyte differentiation. In the present study we directly evaluated this possibility by measuring the Cai levels using the fluorescent probe Indo-1 in keratinocytes grown under conditions which inhibit differentiation (low calcium) or conditions that enhance differentiation (high calcium and 1,25(OH)2D).

MATERIALS AND METHODS Culture conditions

First assage keratinocytes isolated from neonatal

Medium (KGM) obtained from Clonetics Corporation (San Diego) as described previously (Pillai et al., 1988). Briefly, keratinocytes were isolated from newborn human foreskins using 0.25% trypsin; the cells were grown to confluence in DMEM/5%FCS and then pas- saged into KGM media containing bovine pituitary extract (BPE) and a variety of growth factors (Pillai et al., 1988) and either 0.1 mM calcium or 1.2 mM calcium. Cells were grown for various lengths of time in the respective media prior to measurement of Cai and selected markers of growth and differentiation. In some experiments, keratinocytes were incubated with various concentrations of the active vitamin D metabolite, 1,25(OH),D, 24 hr after plating. Fresh 1,25(OH)2D was added with each change of medium.

Measurement of Cai Keratinocytes were dispersed from the plate using

0.1% trypsin/O.Ol% EDTA, and the trypsin was subse- uently neutralized with soybean trypsin inhibitor.

8ells resuspended in KGM containing either 0.1 or 1.2 mM calcium were incubated with 3 pM Indo 1 AM (Molecular probes, Eugene, OR) at 34" C for 1 hr. The cells were washed once and then resuspended in basal KGM (without BPE and growth factors) containing 0.03 mM calcium or different amounts of calcium as described in the figure legends. Fluorescence was re- corded with a Perkin Elmer 650-40 fluorimeter using

human P oreskins were grown in Keratinocyte Growth

360 nm and 405 nm for excitation and emission wave- lengths, respectively, with a thermostated (37°C) cuvette, equipped with a magnetic stirrer. Fluorescence signals were calibrated for each sample by the addition of (final concentrations) 10 pM ionomycin (F max) followed by 0.1 9% triton X-100 and 10 mM EGTA/Tris (pH 8.3) (F mid. Cai was calculated from the following formula: Cai = Kd (F-Fmin)/(Fmax-F), where Kd is 250 nM (Grynkiewicz et al., 19851, as calculated from a calcium standard curve using Indo -1 free acid form. Results were corrected for changes in autofluorescence in each experiment.

Growth and differentiation markers Cell numbers were determined by counting the cells

in a hemocytometer. DNA was assayed as described by Labarca and Paigen (1980), using the fluorescent re- agent, bis benzimidazole.

Cornified envelope content, a marker for differentiated keratinocytes, was determined by the method described by Sun and Green (1976). Cells from each well were dissolved in 2% SDSI2O mM DTT, sonicated lightly to reduce viscosity, and the optical density of the turbid solution determined at 340 nm.

Rate of cornified envelope formation was determined by a modification of the method of King et al. (1986). In this method, the cornified envelope precursor proteins of keratinocytes were prelabelled with 35S-methionine by incubating cultures with 35S L-methionine (2 pCi/ml) for 48 hr. The rate of formation of the envelope precursors in the 48-hr period was then determined by accelerating their cross-linking by the calcium-dependent enzyme transglutaminase. This was achieved by raising the Cai levels of the cells acutely by incubating the cells with a calcium ionophore, ionomycin, in the presence of adequate amounts of extracellular calcium (1.2 mM) at 34°C for up to 6 hr. Aliquots of 1 ml were withdrawn at various time points into 1 ml 4%SDS/40 mM DTT, boiled for 10 min, and the cpm incorporated into the insoluble material was quantitated in a filtration assay.

RESULTS Cai levels of keratinocytes

Loading of confluent keratinocytes with 1-3 pM Indo-1 AM provided optimal baseline fluorescent signals (Fig. 1). Higher concentrations tended to reduce the calculated level of Cai. Addition of 10 pM ionomycin acutely increased this signal to its maximum in a sustained way in the presence of extracellular calcium. This maximum Cai level was higher than the Fmax obtained with Triton X 100 alone, since triton X-100 quenched the Indo-1 fluorescence in our system. Che- lating the calcium with excess EGTA (10 mM final concentration) in tris pH 8.3, after the cells have been lysed with 0.1 % triton X-100, reduced the fluorescence to basal levels. Addition of calcium, ionomycin, or triton X-100 to the medium alone had no effect on the fluorescence signal. However, control keratinacytes not exposed to Indo-1 showed a low level of autofluorescence, which was reduced slightly by the addition of triton X-100 and EGTA (Fig. 1). Therefore, control unloaded cells were included in each experiment and

96 PILLAl AND BlKLE

4 + c W

i I 0

I ’- Fmin

L Ti m e

Fig. 1. Confluent keratinocytes grown in 1.2 mM calcium containing medium were loaded with Indo-1 AM as described in Materials and Methods, 1 x lo6 cells/ml were added into a quartz cuvette for the Cai measurement in a Perkin-Elmer 650-40 fluorescent spectrofluorime- ter as described (A). Two p1 of 5 mM ionomycin was added at 6 min (arrow) to measure F max; 10 p1 of 1% triton X-100 and 100 p1 of 100 mM EDTA in 100 mM Tris pH 8.3 was then added (arrow) to measure F min. Control keratinocytes (1 x lo6 cellsiml) not loaded with Indo-1 were treated in the same fashion, and their fluorescence profile is shown in the lower part of the figure (B).

the changes in autofluorescence following the addition of triton X-100 and EGTA were used to correct the Cai by subtracting the control autofluorescence from the test samples.

Cell density did not affect fluorescent signals over a range of 0.6 to 3 x lo6 cells/ml. In contrast, lower numbers of cells gave variable results. Therefore, in all subsequent studies, we used 1-1.5 X lo6 cells/ml. Measurement of Cai with Fura-2 gave comparable results to Indo-1 (287 ? 8 nM vs 312 2 6 nM, respectively), when parallel preparations of the same keratinocyte lines were loaded with the same concentration (3 pM) of these probes and studied under otherwise identical conditions. Indo-1 leakage from the cells was minimal in the first 1 hr (less than 3%). However, with long incubation times (3 hr or more) substantial amounts of Indo-1 leaked out of the cells (10-15%) as observed by quenching of the fluorescence by EGTA in “conditioned medium.” But this leakage was indepen- dent of growth conditions (8% for cells grown in 0.07 mM Cao vs. 6.8% for cells grown in 1.2 mM Cao at 2 hr) or differentiation states of cells (6.8% for confluent cells vs. 7.5% for 2-weeks postconfluent cells grown in 1.2 mM Cao by 2 hr). Moreover, acute changes in Cao did not influence the Indo-1 leakage from the cells. To minimize any artifact from the extracellular Indo-1, all experiments were carried out within 1-2 hr of Indo-1 loading of the cells.

Cai changes during spontaneous cornified envelope formation

The development of cornified envelopes marks the terminal differentiation of keratinocytes. When the cells were grown in 1.2 mM calcium, cornified envelope

TABLE 1. Cai changes and cornified envelope formation during spontaneous differentiation of keratinocytesl

0.1 mM calcium Days in culture CE Cai(nM) CE Cai(nM) I 0.068 f .02 172 f 34 0.110 f .03 164 f 29 10 0.048 +c .01 154 + 39 0.128 f .06 238 + 12 13 0.078 f .02 156 f 16 0.220 f .05 235 f 10 17 0.310 f .08 212 f 8 0.750 f .12 291 f 28 25 0.500 f .14 425 f 82 0.840 f .09 523 f 91

1.2 mM calcium

‘First passage keratinocytes were grown in KGM containing either 0.1 mM or 1.2 mM Cao for up to 25 days. Cells achieved confluenceby day 10. On days 7,10,13,17, and 25, duplicate wells were harvested and loaded with Indo-1. The Cai levels of these cells were measuredas described in Materialsand Methods. The cells after the Cai measurement weredissolvedin 2%SDS/20mM DTl’,and thecomifiedenvelope content (CE) was measured by the optical density method. The results of cornified envelope content are expressed as the OD at 340 nm/106 cells * range of duplicate dishes; the result of Cai measurements are expressed as mean SD of 3 determinations.

6ool 500

/ .-

zoo ”:.’ l o o ! . I , I . I , I . I

0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 .0

Cornifled Envelope Content

Fig. 2. Correlation of Cai levels with cornified envelope (CE) content. Combined data from Tables 1 and 2 were used to plot the correlation between CE formation and Cai levels. The data were fit in a linear fashion using a computer program. The high level of correlation (R = 0.8) suggests an increase of CE with Cai in a coordinated fashion.

formation increased 10-fold following confluence. This increase in cornified enveloDe formation was associated with a rise in Cai from 164 2 29 to 523 2 91 nM (Ta- ble 1). Keratinocytes grown in 0.1 mM Cao also formed cornified cells during postconfluence, but at a reduced level (less than 30% by 1 week postconfluence, day 13) than cells grown in 1.2 mM calcium. Keratinocytes maintained in 0.1 mM Cao had a lower Cai than keratinocytes grown in 1.2 mM Cao up to 1 week postconfluence, but their Cai levels increased to levels comparable to cells grown in 1.2 rnM Cao by 2 weeks postconfluence. The increase in Cai during postconfluence in both low and normal calcium grown cells correlated (R = 0.8) with the increase in cornified envelope formation regardless of the Cao (Fig. 2).

Cai changes during calcium-induced cornified envelope formation

When keratinocytes grown in low calcium medium (0.1 mM) were switched to 1.2 mM Cao, cornified envelopes began to develop within the first 24 hr. By 3


TABLE 2. Cai and cornified envelope formation of keratinocytes after calcium switch’

Days after calcium switch [Cao] mM 3 7 15 init final CE Cai CE Cai CE Cai

0.1 0.1 0.078 f .02 156 f 16 0.310 f .08 212 f 8 0.500 f .14 425 f 82 0.1 1.2 0.176 f .03 186 f 38 0.680 f .15 312 f 62 0.850 f .28 563 * 28 1.2 1.2 0.220 f .05 235 f 10 0.750 f .12 291 f 28 0.840 f .09 523 + 91

‘Keratinocytes weregrownin KGMmediacontainingeither0.1 mM or1.2mMCaoasdescribedinTablel.Onday 10, when the cells were confluent, one set of cells in 0.1 mM Cao was switched to 1.2 mM Cao. The 3 groups of cells were grown for 15 more days in their respective medium. On days 3.7, and 15 after the switch, the Cai levels (nM) and the cornified envelope content (CE, OD at 340 nm/106 cells) of the cells were measured a s described for Table 1. Values are the mean of triplicate determinations f SD.

CailnM) 630- 580-

f ’ 440-

215- --r f ’

U

1 min

- Fmin

Fig. 3. Keratinocytes were grown in 0.07 mM Cao to confluence and then loaded with Indo-1 as described. The cells were then suspended in KGM medium containing 0.03 mM Cao, and the basal fluorescence was noted. The cells were then challenged with increasing amounts of Cao by adding 2 ~1 of a 250 mM stock CaCl, solution to 2 ml of cells in the cuvette. The changes in fluorescence profile were noted, and the Cai levels were calculated from Fmax and Fmin values obtained after the addition of ionomycin and TritoniEGTA, respectively (see Fig 1.) The figure is representative of experiments repeated at least 3 times.

days, such “switched cells’’ displayed both cornified envelope and Cai levels that were intermediate between those of cells grown in 0.1 mM Cao and 1.2 mM Cao (Table 2). By 7 days, the calcium-switched cells contained similar amounts of cornified envelopes and Cai as did cells grown in 1.2 mM Cao from the beginning. Moreover, the calcium switched cells were indistinguishable morphologically from 1.2 mM Cao grown keratinocytes and displayed similar Cai levels (291 f 28 nM for 1.2 mM calcium grown cells vs. 312 2 62 nM for calcium-switched cells). However, by 2 weeks postconfluence, 0.1 mM Cao grown cells also formed cornified envelopes (0.54 ? .12) with higher Cai (425 * 82) levels.

Using cells grown to confluence in 0.07 mM calcium, we observed that increasing the extracellular calcium to 2.4 mM in a stepwise fashion increased Cai acutely from 215 nM to 580 nM (Fig. 3). The response to Cao was curvilinear reaching saturation by 1.75 mM Cao. Postconfluence keratinocytes cultured in 1.2 mM Cao did not response to increases in Cao (data not shown).

TABLE 3. Effect of 1,25 dihydroxyvitamin D on growth, differentiation, and Cai levels of keratinocytes’

1,25(OH)2D DNA CE formation Cai (M) (uddish) (com/uc! DNA) (nM) 0 16.7 f 0.5 3760 f 854 212 f 18

10-11 10-9 10-7

12.1 f 0.9 7158 f 1612 310 f 12 11.2 f 1.2 6881 + 1953 282 f 13 8.9 f 0.6 8925 f 2498 332 rt 20

‘Keratinocytes were plated in KGM media containing 1.2 mM Cao; 24 h after plating, the indicated amounts of 1,25(OH)zD were added to the medium from an ethanolic stock solution. The ethanol concentrations were kept below 0.05%. and 0.05% of ethanol was added in the controls. Fresh 1.25(OH)a or ethanol were added to thecells witheverychangeofmedium.Onesetofwellswerelabel1ed with 1 uCi=S metbionine for 24 h on day7andthecells were harvestedonday 8. when thecontrol wells were confluent. Cai levels, cornified envelope formation, and DNA content of the cells were measured as described in Materials and Methods. Results are expressed as mean + S.D of triplicate dishes.

Cai changes during 1,25(OH),D-induced cornified envelope formation

1,25(OH)2D, the hormonally active form of vitamin D, is a potent inducer of keratinocyte differentiation (Hosomi et al., 1983; Smith et al., 1986). Consistent with previous studies, lo-’ to M 1,25(oHl2D inhibited keratinocyte growth in a dose-dependent fashion (up to 50% growth inhibition for cells grown in

M 1,25(OH),D) in media containing 1.2 mM Cao (Table 3). Cai levels and cornified envelope formation were higher in keratinocytes grown in the presence of 1,25(OH)2D at all concentrations tested. In contrast to the acute effects of calcium (Fig. 3) or the calcium ionophore, ionomycin, 1,25(OH)2D did not exert an acute effect on Cai (Fig. 4) or cornified envelope formation (Fig. 5). Ionomycin acutely increased Cai and cornified envelopes in a dose-dependent fashion with maximal effect a t 10 p,M (Figs. 4, 5). The increase in cornified envelope formation was seen as early as 15 min, and by 2 hr there was a 60-70-fold increase in cornified envelope formation in ionomycin-treated cells.

DISCUSSION Since the study of Hennings et al. (1980) demonstrat-

ing the relationship between extracellular calcium and keratinocyte growth and differentiation, a number of studies have confirmed and extended these original observations (Hennings et al., 1981, 1983a; Watt and Green, 1982; Boyce and Ham, 1983; Dale et al., 1983; Watt et al., 1984; Stanley and Yuspa, 1984; Milstone, 1987). In serum-free medium, keratinocytes grown in

98 PILLAI AND BIKLE

0.01 1.0 100 1,25(OH)zD (nM) * * *

I

Time

Fig. 4. Acute effects of ionomycin (A) and 1,25(OH),D (B) on Cai levels of keratinocytes. Confluent cultures of keratinocytes grown in 1.2 mM Cao were loaded with Indo-1 and challenged with ionomycin (0.1,1.0,10.0 and 10.0 pM) or 1,25(OH),D (W7 to lo-''). The concentrations shown for the ionomycin and 1,25(OH),D additions represent the incremental increase in final concentrations following each addition. The fluorescence emission at 405 nm was noted. Each trace is representative of experiments repeated at least 3 times. The Cai level of keratinocytes at the start of this experiment was 186 nM.

0.1 mM calcium maintain basaloid morphology and display a higher rate of proliferation than do cells grown in 1.2 mM calcium (Pillai et al., 1988). In contrast, in the presence of 1.2 mM calcium keratinocytes develop higher levels of involucrin, transglutaminase and cornified envelope (Pillai et al., 1988). These results correlate with morphological observations that show that cells grown in 1.2 mM calcium were more stratified, generating a greatest number of cornified cells, keratohyalin granules, tonofilaments, and lamellar bodies than do cells grown in 0.1 mM calcium (Pillai et al., 1988). Previous studies indicate that a gradient of extracellular calcium exists in vivo, with greater amounts of extracellular calcium being present in the upper, differentiated cell layers (Menon et al., 1985). Moreover, these in vivo studies also demonstrate displacement of calcium from extracellular and organelle reservoirs into the cytosol coincident with terminal differentiation. Thus calcium gradients appear to regulate differentiation in vivo as well as in vitro.

Calcium-sensitive fluorescent dyes have been used effectively to measure Cai levels of keratinocytes (Wheeler and Sach, 1989). Cai measurements obtained using Indo-1 are comparable to those obtained with Fura-2 indicating that either are suitable for measuring Cai levels in keratinocytes. Although some leakage of Indo-1 from the cells was determined, this had little effect on our results. Changes in Cao (from 0.03 to 1.2 mM) would not have changed the fluorescence of extracellular dye because the 0.03 mM Cao of the medium should already saturate extracellular Indo 1 (Kd of Indo-1 for calcium is 250 nM). Furthermore, in the time period the measurements were carried out (2 hr) the amount of Indo-1 leaked into the medium was minimal and would not have contributed substantially to the measured Cai levels of the cells.

0 0 .-

e c 5 - w 0

. .....p#ppp ~ <--== . r ~ ~ ~ ~ ~ - - . ~-

1 2 i 6 TIME ( h r )

Fig. 5. Acute effects of ionomycin and 1,25(OH),D on cornified envelope formation of keratinocytes. 80% confluent cultures of keratinocytes were prelabelled with 35S methionine and harvested by trypsinization. The cells were then exposed in suspension to 10 pM ionomycin (0) or M 1,25(OH)2D for (W different periods of time. The amount of cornified envelopes present at each time point was measured in duplicate dishes by dissolving the cells in SDSlDTT and counting the undissolved fraction. The data are expressed as the mean of the fold stimulation above controls. Control cells (0 time) incorporated 670 cpm 35S methionine into cornified envelopes of lo6 cells. After 6 hours of treatment with M 1,25(OH),D or 10 pM ionomycin, the 35S methionine incorporation into envelopes were 850 cpm/106 cells and 516000 com/106 cells, respectively.

Keratinocytes adapt to changes in Cao in the culture media. The difference in the Cai level of cells grown to confluence in low calcium and high calcium medium from plating is about 50% (154 2 39 nM in low calcium cells vs. 238 It_ 12 nM in high calcium cells). However, when confluent cells grown in low calcium were challenged acutely with high Cao (1.25 mM) (Fig. 31, Cai increased from 215 to 550 nM (156% increase). This suggests a desensitization mechanism by which keratinocytes grown in high calcium have lost their Cai response to changes in Cao. The high levels of Cai in postconfluent keratinocytes (400-600 nM) are not a result of leaky cells. These postconfluent keratinocytes exclude trypan blue and continue to synthesize protein and DNA. Furthermore, unlike preconfluent keratinocytes grown in 0.1 mM Cao, postconfluent keratinocytes placed in a calcium-free medium do not increase their Cai when acutely challenged by the addition of 2.4 mM Cao, which one would expect of a leaky cell. However, exposure of these cells to ionomycin increases their Cai levels to the same degree as that of preconfluent keratinocytes.

Increased cornified envelope formation during growth in culture of keratinocytes was associated with an increase in Cai. This suggests a role for Cai during terminal differentiation of keratinocytes, although the possibility that the increase in Cai is subsequent to other events occurring during terminal differentiation cannot be entirely ruled out. It is also unclear at present whether this increase in Cai with differentiation is due to influx of calcium from outside or due to intracellular redistribution of calcium. Acute increases in Cai by calcium ionophore in the presence of Cao (within seconds) and the resulting increase in cornified envelope formation within minutes further supports the contention that Cai regulates cornified envelope formation. However, transient increases of Cai induced


by ionomycin or ATP in the absence of Cao do not result in an increase in cornified envelope formation. Iono- phore-induced cornified envelope formation, although not a physiological maturation and differentiation phe- nomenon, still represents one of the important terminal differentiation processes.

Two recent studies evaluate changes in Cai during keratinocyte differentiation are in agreement with the present study and strengthen our hypothesis that changes in Cai regulate keratinocyte differentiation. Sharpe et al. (19891, using human keratinocytes at- tached to dishes have shown a 2-2.5-fold increase in Cai associated with changes in Cao from 0.07 to 1.0 mM. Moreover, the Cai levels of differentiated cells in the middle of a stratified colony of cells were higher than the Cai of undifferentiated cells at the edge of the colony. Hennings et al. (1989), using normal and neoplastic mouse keratinocytes, reported a 2-3-fold increase in Cai in response to increases in Cao. These recent studies and our present study using keratinocytes from different sources cultured under different conditions and measuring Cai using different tech- niques have arrived a t the same conclusion that Cao increases Cai and induces keratinocyte differentiation. The significance of the high Cai levels of postconfluent keratinocytes are not clear at present. It is possible that post confluent keratinocytes need a higher Cai to generate and stabilize the cornified envelo es.

entiation of a variety of cells including keratinocytes (Abe, 1981; Colston, 1981; Majeska and Rodan, 1982; Hosomi et al., 1983; Murao et al., 1983; Smith et al., 1986; Piilai et al., 1988). 1,25(OH),D has also been shown to alter Cai of a variety of cells including keratinocytes (Bar, 1986; Baran, 1986; Lieberhen, 1987; Smith and Holick, 1987; Hruska et al., 1988). In some, but not all studies, 1,25(OH),D acutely raised Cai (Baran, 1986; Lieberhen, 1987; Smith, 1987). 1,25(OHI2D acutely increased Cai and stimulated phosphoinositide turnover in preconfluent keratinocytes grown in serum-free medium containing 1.8 mM Cao (MacLaughlin et al., 1990). However, we did not ob- serve such an acute effect of 1,25(OH),D on either Cai or cornified envelope formation in the present study. Rather, longer incubation times of keratinocytes with 1,25(OH),D (days) were necessary to inhibit growth, induce cornified envelope formation, and increase Cai. The reason for this difference is not clear a t present. However, other cells, such as HL-60 cells require hours or days of exposure to 1,25(OH),D before changes in Cai and differentiation are observed (Hruska et al., 1988). Conceivably, the mechanism by which 1 ,25(OH),D acts on keratinocyte differentiation is via increasing Cai level, but the increased Cai observed in keratinocytes treated with 1,25(OH),D may be a result of differentiation rather than its cause. In other classical systems of 1,25(OH)2D action such as intestinal calcium absorp- tion, the effect of 1,25(OHI2D takes hours (Bikle, 1990). As in the intestinal epithelial cell, 1,25(OH)2D may regulate calcium transport across the keratinocyte cell membrane by both genomic and non-genomic mechanisms (Bikle, 1990).

In summary, our data suggest a difference in the molecular mechanisms by which Cao and 1,25(OH),D

1,25(OH),D inhibits proliferation and in c f uces differ-

increase Cai and stimulate cornified envelope formation in keratinocytes. Cao may mediate its effects primarily by raising Cai acutely and thus activating the cross-linking of envelope precursor proteins. 1,25(0H),D increases Cai more slowly, possibly by modulating the calcium transport mechanism itself. Cai may act via coordinate expression of a variety of calcium requiring systems involved in cellular growth and differentiation.

ACKNOWLEDGMENTS This work was supported by grants from the NIH

(AR-383860 and AR-39448) and a Merit Review from the Veterans Administration.

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Documents

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