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

JOURNAL OF CELLULAR PHYSIOLOGY 143294-302 (1990)

Calcium Regulation of Growth and Differentiation of Normal Human

Keratinocytes: Modulation of Differentiation Competence by Stages of Growth and

Extracellular Calcium SREEKUMAR PILLAI,* DANIEL D. BIKLE, MARIA-LAURA MANCIANTI, POLLY CLINE,

AND MARA HINCENBERGS Departments of Medicine (S.P., D.D.B.1 and Dermatology (M.-L.M., P.C., M.H.), University

of Cdlrfornra; and Veterans Administration Medicdl Center, San Francisco. CA 94 12 7

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 m M 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. Ex- tracellular calcium concentrations did not have a clear influence on these parameters. However, preconfluent and confluent keratinocytes grown in 0.07 m M calcium showed markedly retarded differentiation at all steps, i.c., involucrin synthesis, transglutaminase activity, and cornified envelope formation. Within 1 week after achieving confluence, these kerati nocytes began synthesizing i nvol u- crin and transglutaminase and developed the ability to form cornified envelopes. Cells grown in 1.2 and 2.4 m M 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 i s 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 spec- ulate 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.

The discovery that extracellular calcium modulates the differentiation of epidermal cells in culture (Hen- nings et al., 1980) provides a model to study the differentiation events that occur in normal epidermis. In culture, keratinocytes in low calcium conditions (0.03 -0.1 mM) do not differentiate and are phenotypically similar to basal epidermal cells (Yuspa, 1985). Differentia- tion-specific marker expression is enhanced at restricted calcium concentrations in the range of 0.1 to 0.16 mM in murine keratinocytes (Yuspa et al., 1989). Increasing extracellular calcium levels above 0.1 mM induces differentiation of human keratinocytes, and they achieve morphological characteristics of the su- prabasal epidermal cell. In addition to the calcium regulation of keratinocyte differentiation, a relationship

also exists between the stages of differentiation and the metabolism of keratinocytes (Milstone, 1987). For ex- ample, cell cycle time (Alberts et al., 1986) and production and response of keratinocytes to growth factors (Pentland and Needleman, 1986; Okada et al., 1982; Pillai e t al., 1988a,b; Milstone et al., 1982; Pittelkow et al., 1988; Hawley-Nelson et al., 1980) depend on the differentiation stage of the cells. The sequence of events following the addition of calcium to low- calcium-grown cells (“calcium switch”) prior to conflu-

Received September 8, 1989; accepted January 3, 1990.

*‘To whom reprint requestskorrespondence should be addressed.

c) 1990 WILEY-LISS. INC

CALClUM REGULATION OF KERATINOCYTE UIPFERENTIATION 295

ence or a t confluence has been characterized in some detail in cultures of murine and human keratinocytes (Watt and Green, 1982; Hennings et al., 1981, 1989; Hennings and Holbrook, 1982, 1983; Watt and Phil, 1983). Not much attention has been given to the temporal relationship of keratinocyte differentiation with extracellular calcium concentration. As a part of our studies on the intracellular mechanism of keratinocyte differentiation, we compared the molecular events that occur during keratinocyte differentiation induced by calcium to those occur during postconfluent growth in low calcium medium.

Of the various markers of keratinocyte differentiation, the best characterized is the cornified envelope formation. A 40-50-fold increase in cornified cells is observed by 24 hours after calcium switch (Hennings and Holbrook, 1982). Proteins involved in cornified envelope formation, involucrin content and transglutaminase activity, are also increased by calcium in cultured keratinocytes. Epidermal transglutaminase activity increases within 6 hours of calcium switch and is responsible for cross-linking involucrin and other envelope precursors into the highly insoluble cornified envelope (Hennings et al., 1981). Involucrin appears in the spinous layers and is found in greatest concentration in the cells of granular layer and inner stratum corneum (Warhol et al., 1985). In culture, involucrin and its mRNA are not found in the small basal keratinocytes but appear in the larger, more differentiated cells (Watt and Green, 1981, 1982).

In these studies, we examined both the temporal response of the keratinocytes to calcium and the sensitivity of the keratinocyte response to calcium with re- spect to parameters of both growth and differentiation. For these studies we used cells grown in serum-free, defined medium in the absence of feeder cells. We found that either increased extracellular calcium or the achievement of intercellular communication during postconfluent growth lead to the coordinate increase of involucrin content, transglutaminase activity, and cornified envelope formation.

MATERIALS AND METHODS

Cell culture Human keratinocytes were isolated from newborn

human foreskins by overnight treatment at 4°C with 0.25% trypsin. Primary cultures were established in DMEM containing 5% FCS in the presence of mitomy- cin C-treated 3T3 cells (Rheinwald and Green, 1975). First passage keratinocytes were then grown in serum- free keratinocyte growth medium (KGM) obtained from Clonetics corporation (San Diego, CA) a t different calcium concentrations by seeding the cells at 1-2 x lo5 cells per well in six-well multiwell plates (Pillai et al., 1988b). The cells were fed every 2 or 3 days with the respective media and grown for up to 20 days. Cells were harvested on day 5, 7, or 20 either by trypsinization (Pillai e t al., 1988b) or by scraping into PBS for the various assays.

Assay of markers of growth and differentiation “‘S-methionine labeling of the cells was performed

by incubating the cultures of keratinocytes in their respective fresh media with 10 pCi of cell culture grade

“‘S-L-methionine (Specific activity, 1,300 Ciimmol, Amersham) for 24 hours. Preliminary studies indicated linearity of labeling up to 24 hours. After labeling, the wells were rinsed three times with PBS, and the cells were harvested by scraping into PBS. Cell homoge- nates were prepared by sonication (three times for 15 seconds each). Cell supernatant fractions were prepared by centrifuging the cell homogenate a t 12,OOOg for 1 hour. The pellet was solubilized in 2%; SDS/20 mM DTT by boiling a t 100°C for 10 minutes to prepare the SDS soluble fraction. SDS insoluble pellets were processed to determine the rate of formation of cornified envelopes.

The rate of protein synthesis in the 12,OOOg supernatant fraction was quantitated by determining the amount of radioactivity incorporated into the TCA pre- cipitate of an aliquot. The rate of synthesis of keratins was quantitated by determining the radioactivity incorporated into the SDS soluble fraction of the 12,OOOg pellet of the cell homogenate (Fuchs and Green, 1981).

The rate of formation of cornified envelopes was determined by the method of King et al. (1986). The SDS insoluble pellet of the “%-labeled cells was washed re- peatedly in 10 pm pore sized filters; the amount of radioactivity incorporated into the cross-linked, deter- gent insoluble, cornified envelope was then quantitated. A fraction of the cells in each condition was incubated for up to 2 hours with 10 p.M ionomycin, a calcium ionophore, prior to solubilization. The amount of radioactivity incorporated into cornified envelopes in the absence of ionomycin was taken as a measure of spontaneous differentiation; the amount of radioactivity incorporated into cornified envelopes in the presence of 10 pM ionomycin was taken as the measure of differentiation competence.

Cornified envelope content of keratinocytes was determined by the method of Rice and Green (1979). Briefly, the cells harvested by trypsinization were di- vided into two fractions. The total number of cells in the fractions was determined by counting in a hemocytometer. One aliquot was dissolved immediately in 2% SDSi 20 mM DTT solution by boiling for 5 minutes at 100°C. A second aliquot was resuspended in KGM medium containing 1.2 mM calcium and treated with 10 pM ionomycin for 2 hours a t 37°C prior to boiling in SDSi DTT solution as before. The envelopes were counted by phase-contrast microscopy with a hemocytometer.

Involucrin, a precursor of the cornified envelope, was determined with a solid phase enzyme-linked immu- noassay as described by Parenteau et al. (1987) using a polyclonal rabbit antihuman involucrin antibody and purified human involucrin as standard (gifts from Dr. Robert Rice). The 12,OOOg supernatant of the appropri- ate cell homogenate was incubated overnight a t 4°C with the involucrin antiserum in a 96 well microtiter plate. Aliquots from each well were then transferred to another plate containing solid-phase adsorbed standard involucrin and incubated a t room temperature for 30 minutes. After washing, the amount of involucrin antibody bound to the plate was quantitated using an alkaline phosphataseeconjugated goat antirabbit IgG antibody. This assay has a sensitivity of 0.025 ng of human involucrin.

Transglutaminase, the enzyme catalyzing the y-glu- tamyl (e-lysyl) cross-link, was determined using the

296 PILLAI ET AL

TABLE 1. Effect of extracellular calcium levels on the cornified envelooe content and cell number at confluence'

Corniced envelone (% 1

Ca (mM) SDontaneous Induced Cell Nos. (10") 0.03 >1 4, 10 48,80 0.07 >1 8, 12 128, 132 n 1 5 I. 1 40.52 188. 132 _._I 1.2 5, 9 71; 75 200; 240 2.4 6, 6 80, 76 185,230

'First pasgage keratinocytes were grown in complete MCDR 163 medium containing the indicated amounts of extracellular calcium. At confluence the cells from duplicate wells were processed for cornified envelope content or cell numbers as detailed in Materials and Methods.

TABLE 2. Protein and DNA content of keratinocytes at different stages of growth'

Ca Days in DNA Protein ProteiniDNA (mM) culture (pgidish) (pgidish) ratio

5 14.6 2 2.5 562 i 65 38.5 i 2.1 .07 7 50.8 -t 2.4 1,444 f 51 28.4 ? 2.4

20 111.6 f 1.6 4,814 f 215 43.1 f 3.2 5 35.3 2 3.1 1,210 i 42 34.3 i 2.0

1.2 7 72.5 i 3.2 2,600 t 169 35.9 -C 3.9 20 135.0 t 8.6 6,400 -t 679 47.8 + 13 5 33.5 i 4.2 1,152 i 56 34.7 i 6.0

2.4 I 62.5 2 7.4 2,708 k 390 43.9 i 11.5 20 99.8 t 5.0 6,600 i 424 66.0 t 5.3

'Keratinocytes were grown for 6 , 7 , or 20 days in medium containing (1.07, 1.2, or 2.4 mM calcium. Cells grown in 0.07 mM calcium were 40-hO'k cmfluent by day 6 and 90% confluent by day 7. Cells grown in 1.2 and 2.4 mM calcium were 60-70% confluent by day 5 and 100% confluent hy day 7. By day 20, cells in all calcium concentrations were in their postconfluent stages of growth. Data are expressed as mean -r SD of triplicate dishes.

method of Schmidt et al. (1985). Membrane-bound enzyme activity, associated with the cell pellet (12,00Og, 30 minute pellet), was resuspended in Tris-HC1, pH 8.0, containing 5 mM EDTA and 0.1% Triton X 100. Then 100 11.1 of this suspension was incubated with 600 p1 of 50 mM Tris-HC1 buffer containing 10 mM calcium chloride, 5 mM DTT, 540 pg dimethyl casein, 1 mM putrescine, and 2.5 pCi 'H-putrescine for 30 minutes a t 37°C. The reaction was stopped by 10% TCA, and the 'H-putrescine associated with the precipitated casein was quantitated by scintillation spectroscopy. The specific activity was calculated as cpm putrescine incorporated into caseinipg protein.

DNA was determined using the fluorescent reagent bisbenzimidazole as described by Labarca and Paigen (1980). Protein was measured by the BCA protein assay reagent, available from Pierce Chemical Company (1985). Assays were performed using cells grown in duplicate wells for each growth stage and each calcium concentration. Each assay was performed in duplicate or triplicate from each well.

RESULTS To determine the effect of different calcium concen-

trations on growth and differentiation of keratinocytes, cells were plated in media containing 0.03, 0.07, 0.15, 1.2, or 2.4 mM calcium and grown for 7 days. At this time, the total number of cells and both spontaneous and ionomycin-induced cornified envelope formation were measured. As shown in Table 1, the number of keratinocytes was maximal when grown in 1.2 mM cal-

cium. A further increase in calcium concentration to 2.4 mM had no significant effect on cell number. Kera- tinocytes grown in low calcium (.03 to 0.15 mMI failed to make significant amounts of cornified envelopes spontaneously. When induced with ionomycin, cells in 0.03 and 0.07 mM calcium showed only minimal increases in cornified envelope formation (7 and lo%, respectively), but cells in 0.15 mM calcium produced abundant amounts of cornified envelope (46%~) similar to that of cells grown in 1.2 and 2.4 mM calcium (73 and 7896, respectively). These data suggest that keratinocytes grown in 0.15 mM calcium acquire the ability to differentiate as they approach confluence, although spontaneous differentiation is retarded. Since our aim was to determine the molecular events required to achieve differentiation competence, for subsequent studies described here, we grew keratinocytes in 0.07 mM (low calcium) and compared their growth and differentiation with those grown in 1.2 (physiological levels) and 2.4 mM (high) calcium.

Markers of keratinocyte growth The cells grown in 1.2mM calcium were 60-70%

confluent by day 5 and 100% confluent on day 7. By 12 days postconfluence, these cells stratified into multi- layered islands (three to five layers), surmounted by two to four layers of enucleated cells with thickened cornified envelopes (Pillai et al., 1988a,bI. Kerati- nocytes in 0.07 mM calcium grew as a monolayer and reached confluence by day 9. Beyond confluence, they also stratified, although to a much lesser extent than cells in 1.2 or 2.4 mM calcium.

DNA and protein synthesis Table 2 shows the changes in total protein content,

DNA content, and the ratio of proteiniDNA of keratinocytes grown to different stages in 0.07, 1.2, and 2.4mM calcium. Total protein and DNA content increased with time in culture a t all calcium concentrations. Whereas the increases in DNA content were less striking between confluence and postconf luence (1.6- 1.9-fold), the increases in protein content after confluence were more pronounced (2.5-3.0-fold). This is re- flected in the proteidDNA ratio, which increased at all calcium concentrations in the postconfluent stage. The protein/DNA ratio was highest for postconfluent keratinocytes grown in 2.4 mM calcium, suggesting that these cells are the most differentiated. Table 3 shows the differences in the rate of "S-methionine incorporation into 12,OOOg supernatant proteins, SDS soluble proteins (keratins and other membrane proteins), and SDS insoluble fraction (cornified envelopes). The fraction of SDS insoluble counts represents the highly cross-linked cornified envelopes formed spontaneously by these cells at that stage of growth. The distribution of radioactivity incorporated into 12,OOOg supernatant fraction and keratins indicated a shift from synthesis of cytosolic proteins t o keratins as the cells reached confluence. This relationship was observed for keratinocytes grown in all three calcium concentrations. Ap- proximately 80% of the newly synthesized proteins were in the cell supernatant of preconfluent cells, whereas only 20-40% of the newly synthesized pro-

CALCIUM REGULATION OF KERATINOCYTE DIFFERENTIATION

TABLE 3. Rate of synthesis of proteins in different cellular fractions of keratinocytes grown to different stages of growth'

297

A Ca Days in (mM) culture

5 0.07 7

20 5

1.2 7 20 5

2.4 7 20

12,ooog Supernatant

79.1 -t 8.0 50.3 i 3.5 22.4 5 3.8 72.1 i 9.8 31.0 5 4.9 26.9 i- 6.6 81.1 -t 2.8 53.6 2 6.4 46.8 i 5.6

SDS soluble fraction

18.6 f 2.8 49.3 2 1.4 76.4 2 6.1 28.2 2 2.0 66.9 2 4.7 71.1 f 7.6

44.9 f 3.2 51.5 -t 5.2

18.6 2 1.0

SDS insoluble fraction

0,008 + ,003 0.355 + ,011 0.931 + .500 0.280 f ,030 1.560 2 .320 1.800 2 ,016 0.221 2 .om 0.870 2 ,024 1.380 f ,250

'Keratinocytes were grown in the same conditions as in Table 2. .'?-rnethionine labeling of the cells was carried out a s described in Materials and Methods. The percentage of radioactivity incorporated into the different cellular fractions wils quantitated. Data are expressed as the :'sSS-methionine incorporation into cach fraction as the percentage of total incnrporation. Each value is the mean of triplicate dishes t SI).

teins were in this fraction of postconfluent keratinocytes. Calcium did not have a clear influence on this shift from supernatant to keratin fraction. The shift in the protein synthesis from preconfluence to confluence was significant, but the changes from confluence to 2 weeks postconfluence were generally not significant. The rate of synthesis of cornified envelope fraction was lowest in preconfluent cultures in 0.07 mM calcium and highest in confluent and postconfluent keratinocytes in 1.2 mM calcium.

Markers of differentiation Figure 1 shows the spontaneously formed cornified

envelope content and the cornified envelope content after induction with ionomycin of keratinocytes grown to different stages in .07, 1.2, and 2.4 mM calcium. Ke- ratinocytes in their preconfluent and confluent stages of growth contained few cornified cells. After confluence, cells grown in 1.2 and 2.4 mM calcium stratified, and the upper layers of individual colonies formed enucleated squamous cells that contained cornified cells (data not shown). The number of spontaneously ap- pearing cornified cells in postconfluent cultures increased with the calcium concentration of the media. Cultures in 0.07 mM calcium contained 10% cornified cells, whereas cultures in 1.2 and 2.4 mM calcium contained 40% and 55% cornified cells, respectively (Fig. 1A). When these cultures were induced to form cornified envelopes by a calcium ionophore, ionomycin, cells at all three stages of growth in 1.2 and 2.4 mM calcium formed substantial quantities of cornified envelopes (Fig. 1B; up to 70% of cells in 1.2 mM calcium and 50-95% of the cells in 2.4 mM calcium). However, preconfluent cells formed fewer cornified envelopes than confluent or postconfluent cells. Keratinocytes grown in 0.07 mM calcium contained few cornified cells in the preconf luent and confluent stages of growth even when induced by ionomycin, but were able to form significant numbers (60%) of cornified envelopes in their postconfluent stage of growth with ionomycin induction. These data indicate that keratinocytes grown in 0.07 mM calcium are not capable of cornified envelope formation prior to confluence, unlike keratinocytes grown in 1.2 or 2.4 mM calcium.

To investigate further the role of calcium in cornified envelope formation we examined the incorporation of

s 20

0

100 1

0 . 0 7 1 . 2 2 . 4

Calcium (mM)

80 Q) Q 2 60 Q) > c w 40

s 20

0 0 . 0 7 1 . 2 2 . 4

Calcium (mM)

Fig. 1. Cornified envelope content of keratinocytes grown in 0.07, 1.2, or 2.4 mM calcium for 5,7 , or 20 days. Keratinocytes were grown in KGM containing the different calcium concentrations for the indicated time periods. The numbers of cornified cells spontaneously formed (A) and of cornified envelopes formed when these cells were induced by a calcium ionophore (B) were quantitated by phase microscopy as described in Materials and Methods. The results are expressed as the percentage of the total number of cells in the dish that formed cornified envelopes. The results are the means of two dishes, each done in duplicate. Closed bars, preconfluent (cells grown for 5 days); open bars, confluent (cells grown for 7 days); dotted bars, 2 weeks postconfluent (cells grown for 20 days).

35 S-methionine-labeled precursor into spontaneously occurring and ionomycin-induced cornified envelopes of keratinocytes grown to three stages of confluence in 0.07, 1.2, and 2.4mM calcium (Fig. 2A,B). Although the general pattern of cornified envelope formation with this approach is similar to that achieved by counting the cornified envelopes (Fig. lA,B), some subtle differences emerge. Preconfluent keratinocytes a t all calcium concentrations had low rates of cornified envelope formation, in agreement with total cornified envelope content. However, a t confluence, keratinocytes grown in 1.2 and 2.4 mM calcium showed near-maximal rates of cornified envelope formation (increased by four- to six-fold over preconf luent cells), although

298 PILLAI ET AL

A

3.0 1 W 0 C .- 2.0

P 0

E

8 1.0

0.0 0 . 0 7 1 . 2 2 . 4

Calcium (mM)

B

3.0 T

w 0

E .- = 2.0

.& 1.0

P 0

0.0 0 . 0 7 1 . 2 2 . 4

Calcium (mM)

Fig. 2. Cornified envelope formation of keratinocytes grown in 0.07, 1.2, or 2.4 mM calcium for 5, 7, or 20 days. Keratinocytes in their respective media were labeled with ""S-rnethionine for 24 hours prior to harvest. The amount of radioactivity incorporated into the newly formed cornified envelope was quantitated by dissolving the cells in 2% SDS/2O mM DTT and measuring the radioactivity in the undis- solved envelopes. The rates of formation of spontaneously formed cornified envelopes (A) and that induced by ionomycin (B) were quantitated. The data (mean of triplicate dishes ? SD) are plotted as the percentage of total incorporated radioactivity that was incorporated in the cornified envelope fraction. Closed bars, preconfluent: open bars, confluent; dotted bars, 2 weeks postconfluent.

envelope content, as shown in Figure lA, a t confluence was quite low (5%) of total cells). This observation suggests that the synthesis of envelope precursors (which would incorporate the ""S-methionine) is increased prior to the spontaneous production of the envelope and that this synthesis is dependent on adequate amounts of extracellular calcium.

This conclusion is supported by the results obtained with ionomycin induction of cornified envelope formation. Ionomycin induction of cornified envelope formation was maximal for confluent cells grown in 1.2 and 2.4 mM calcium and postconfluent cells in all calcium concentrations (Fig. 2B). This suggests that these cells already contained adequate amounts of the "S-methio-

nine-labeled precursor of cornified envelope, namely, involucrin. Preconfluent cells a t all calcium concentrations and confluent cells in 0.07 mM calcium do not synthesize involucrin.

To determine directly the effect of calcium on the synthesis of the precursors of the cornified envelope, we determined the involucrin content of keratinocytes grown to different stages of confluence in 0.07,1.2, and 2.4 mM calcium (Fig. 3). Involucrin is produced in abundant quantities (0.5-0.7 nglpg protein) by keratinocytes grown in 1.2 and 2.4 mM calcium even prior to confluence. These levels increased (up to 2 ngikg protein) as the cells grew past confluence, reaching their highest levels in the cells grown in 2.4 mM calcium. However, keratinocytes grown in 0.07 mM calcium produce significant amounts of involucrin only during postconfluent stages of growth and at all times have less involucrin than keratinocytes grown in 1.2 or 2.4 mM calcium. These results parallel the ability of the keratinocytes to form cornified envelopes (compare Fig. 3 with Figs. lB , 2B). Thus one explanation for the ability of calcium to regulate cornified envelope formation is its ability to regulate involucrin production.

A second mechanism by which calcium regulates cornified envelope formation is through the synthesis or activation of transglutaminase, the enzyme responsible for the cross-linking of involucrin and other minor precursor proteins into the cornified envelopes. We measured transglutaminase activity in the keratinocytes grown to different stages of confluence in the different calcium concentrations and found that it par- alleled that of involucrin and envelope competence (Fig. 4). Transglutaminase activity was higher in keratinocytes grown in 1.2 and 2.4 mM calcium at all stages of growth. Highest activity was observed for cells in the confluent stage of growth and thereafter decreased slightly in the postconfluent stage for cells grown in 1.2 and 2.4mM calcium. In keratinocytes grown in 0.07 mM calcium, significant amounts of transglutaminase activity were observed only in the postconfluent cells when the cells reached envelope competence.

In Figure 5 involucrin content and transglutaminase activity are correlated with cornified envelope content under the various conditions of calcium concentrations and degree of confluence. The high degree of correlation (0.78 for transglutaminase activity and 0.85 for involucrin content) suggests that as the cells differentiate (by increased extracellular calcium or increased time in culture) involucrin protein and transglutaminase activity, and/or protein are increased in a coordinate fashion with the onset of cornified envelope formation. A critical level of transglutaminase activity (40 units) and involucrin protein (0.5 ngikg protein) appear to be sufficient for adequate synthesis of cornified envelopes. Further increases in these markers did not result in substantially greater increase in cornified envelope content.

DISCUSSION The sequence of events following the addition of cal-

cium to low calcium grown keratinocytes, a t or near confluence, has been characterized in some detail for mouse and human keratinocytes with several morphological and immunocytochemical criteria (Hennings et

CALCIUM REGULATION OF KERATINOCYTE DIFFERENTIATION

3-0 1

160 - 140 - 120 - 100 -

299

1 T

2.0

1.5

1 .o

0.5

0.0 0 . 0 7 1 . 2 2 . 4

Calcium (mM)

Fig. 3. Involucrin content of keratinocytes grown in 0.07, 1.2, and 2.4 mM calcium for 5, 7, or 20 days. The involucrin content of cells grown under the different conditions was determined by a solid phase enzyme-linked immune assay as described. The involucrin content of

1

60

40

20

0

I

0 . 0 7 1 . 2 2 . 4

Calcium (mM)

Fig. 4. Transglutaminase activity of keratinocytes grown in 0.07, 1.2, and 2.4 mM calcium for 5, 7, or 20 days. Transglutaminase activity of the membrane fraction was determined as described. Enzyme activity was expressed as cpm of ‘H-putrescine incorporated into dimethyl caseinihouripg of the membrane protein. Each data point is the mean of triplicate determinations of two separate dishes ? SD. Closed bars, preconfluent; open bars, confluent; dotted bars, 2 weeks postconfluent.

al., 1980, 1981; Yuspa, 1985; Watt and Green, 1982; Hennings and Holbrook, 1982, 1983; Watt and Phil, 1983). Previous studies have shown that desmosome formation begin within 5 minutes and is complete by 2 hours. Cell proliferation decreases after 5 hours, reach-

the cell cytosolic fraction was expressed as ng involucrinikg of the cytosolic protein. Each data point is the mean of triplicate determinations on two separate dishes 4 SD. Closed bars, preconfluent; open bars, confluent; dotted bars, 2 weeks postconfluent.

ing minimum levels by 24 to 36 hours. Protein and RNA synthesis decrease gradually over 3 days. Epider- mal transglutaminase activity increases within 6 hours of calcium switch and remain elevated for 1 to 2 days. Cross-linked cornified envelope formation occurs by 24 hours, with a resulting 40-50-fold increase of cornified cells seen 2 days after the calcium switch. No major changes in the synthesis of proteins has been shown to occur in the first 42 hours. Keratinocytes grown in 0.1 mM calcium to confluence do not appear to require new protein synthesis for cornified envelope formation, since cycloheximide does not block terminal differentiation of these cells (Rice and Green, 1978). Most of these studies have been carried out in cells that were grown in serum containing medium using 0.1 mM calcium as “low calcium medium.” Such a low calcium condition may be sufficient to inhibit stratification of keratinocytes (Hennings and Holbrook, 19821, but not sufficient enough to inhibit the synthesis of cornified envelope precursor molecules (Watt and Green, 1982).

The onset of synthesis of involucrin is an early marker of terminal differentiation of keratinocytes. In the normal epidermis, involucrin is not synthesized in the basal layer (Rice and Green, 1979); cells migrate several layers upward before starting to make involu- win. However, in culture, involucrin synthesis begins immediately above the basal layer (Banks-Schlegel and Green, 1981). As cells migrate beyond the basal layer, they enlarge in size (Watt and Green, 1981). In low calcium medium (0.1 mM), epidermal cells do not stratify, but still contain small basaloid and large squamoid cells; involucrin is present only in the latter (Watt and Green, 1981).

Transglutaminase, the other major epidermal differentiation marker, is present as two major immunolog- ically distinct forms in the epidermis (Thacher and Rice, 1985): a cytosolic form, which is identical to the

300 PILLAI ET AL.

creased extracellular calcium was found to be growth stimulatory to keratinocytes in medium containing low levels of growth factors and inhibitory in medium containing high levels of growth factors (Tong et al., 1988). The presence of other divalent cations in the medium, such as strontium, reduced the sensitivity of these cells to calcium (Praeger et al., 1987). Keratinocytes grown in serum containing medium differentiated to a lesser degree than cells grown in a serum free medium of similar calcium concentrations (Pillai et al., 1988b). In addition, the effect of calcium on cultured human keratinocytes is quantitatively and qualitatively different from its effect on mouse keratinocytes (Milstone,

0 1 2 1987). Physiological calcium concentrations permit proliferation of human keratinocytes but not mouse keratinocytes (Hawley-Nelson et al., 1980; Hennings et al., 1980). Stratification and clonal growth occurs in mouse keratinocytes a t calcium concentrations as low as 0.05-0.07 mM (Hennings et al., 1980), whereas human keratinocytes require higher calcium levels (0.3 mM) (Hawley-Nelson et al., 1980).

v) Most studies of the calcium induction of the molecu- g 8 0 - lar markers of keratinocyte differentiation have fo-

cused on the calcium switch (from 0.1 mM to 1-2 mM) 0

of cells grown to confluence in a serum containing me- Q

dium. The effects of lower levels of calcium on kerati- * 60 - E Q

'z1 40- nocytes grown in serum free media have received little Q 0 attention. Our initial experiments indicated that hu- 3 man keratinocytes grown to confluence in 0.15 mM 'CI c 20 - calcium already contain relatively high levels of mo-

lecular precursors required for terminal differentia-

0 1 0 0 2 0 0 to the cultures a t confluence fails to block cornified envelope formation (Rice and Green, 1978). These results are in agreement with the recent studies by Yuspa et al. (1989) in which murine keratinocytes in a

El 100 -4

v) Q 80 - P

A lnvolucrin content

13 100 - CI

-

- 0 I 1 tion. Thus it is not surprising that cycloheximide added

Transglutaminase activity 6

Fig. 5. Correlation of envelope competence of keratinocytes (ability to form cornified envelopes) with involucrin content (A) and transglutaminase activity (€3) of keratinocytes grown in different calcium concentrations or to different stages of growth. Data from Figure 1B (induced envelope content) were plotted against involucrin content (Fig. 3) and transglutaminase activity (Fig. 4). The correlation between envelope competence and involucrin content ( R = 0.85) and transglutaminase activity (R = 0.78) was equivalent irrespective of how the cells differentiated (by increased calcium or by increased time in culture).

low concentration Of o'1-o'16 mM expressed maximum levels of Protein and mRNA of keratins, cornified envelope precursors, and fillagrin. At higher or lower concentrations of calcium these markers were diminished or undetected.

To study the molecular events that must Occur in order for the keratinocytes to reach envelope competence, we reexamined both the effects of calcium concentration and degree of confluence (time) on the de- velopment of envelope competence using defined

tissue transglutaminase of liver, and a specific plasma conditions k e . , no serum or feeder cells). We found that membrane-bound epidermal transglutaminase, which keratinocytes grown in 0.07 mM calcium synthesized is involved in the cross-linked envelope formation dur- very little involucrin and had low levels of transglu- ing terminal differentiation. Calcium and retinoic acid taminase activity (a precursor molecule and enzyme, stimulation of transglutaminase activity was demon- required for the formation of cornified envelopes, re- strated by Yuspa et al. (1981, 1982) in cultures of spectively) until the cells were well past confluence. mouse epidermal cells. Retinoids stimulate the cyto- This explains the inability of these cells to form corni- solic transglutaminase and inhibit cornified envelope fied envelopes in the presence of ionomycin up to con- formation; calcium and the phorbol ester TPA stimu- fluence. Postconfluent cells in 0.07 mM calcium and late both plasma membrane epidermal transgluta- even preconfluent cells in 1.2 and 2.4 mM calcium con- minase and cornified envelope formation (Lichti and tained adequate amounts of these precursors and were Yuspa, 1988). induced to differentiate terminally by ionomycin. In-

Several recent observations suggest that the influ- creases in calcium beyond 1.2 mM had only modest in- ence of calcium on growth and differentiation of kera- cremental effects on the ability of keratinocytes to dif- tinocytes varies with the stage of cell growth and with ferentiate. Thus keratinocytes grown in 0.07 mM the composition of the medium in which the cells are calcium remained undifferentiated even when conflu- grown. Optimum calcium for clonal growth of kerati- ent, but, beyond confluence, even they gradually nocytes is inadequate for growth a t higher seeding den- achieved envelope competence by synthesizing involu- sities or confluent cultures (Price et al., 1983). In- crin and transglutaminase.

CALCIUM REGULATION OF KERATINOCYTE DIFFERENTIATION 301

One important change when a monolayer of keratinocytes reaches confluence and establishes cell-cell contact is the formation of desmosomes and gap junctions (Hennings et al., 1980; McNutt and Weinstein, 1973). Extracellular calcium as well as stages of con- f luence determine desmosome formation (Hennings et al., 1980; Hennings and Holhrook, 1983). Monolayers of undifferentiated keratinocytes in low calcium medium contain abundant amounts of gap junctions (Kam et al., 1987). With the formation of such junctions comes the establishment of intercellular communications in the form of movement of small ions and molecules between the cells (Finbow and Pitts, 1981). One such molecule relevant to keratinocyte differentiation is the cytosolic free calcium ion. Since the formation of gap junctions precedes differentiation, i t is conceivable that the intercellular movement of calcium ions between the cells through the gap junctions facilitate the differentiation of keratinocytes.

Extracellular calcium may regulate the temporal sequence of differentiation events via an effect on intracellular free calcium levels. We have previously re- ported that the cytosolic free calcium levels increase with differentiation (Pillai and Bikle, 1989). This increase would be more in cells grown in 1.2 mM or 2.4 mM calcium than in cells grown in 0.07 mM calcium. It is conceivable that a critical level of cytosolic calcium is required for initiation of the differentiation events, and cells in 0.07 mM extracellular calcium do not achieve this level until after confluence. Establish- ment of gap junctions and intercellular communications may facilitate this increase in intracellular calcium levels. Thus both increases in extracellular calcium and intercellular communications by gap junctions may regulate the differentiation process by influ- encing intracellular calcium levels. Further investiga- tion to determine the exact mode of signal transduction by these two regulatory pathway should prove fruitful.

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ACKNOWLEDGMENTS We appreciate the support of Dr. Peter Elias in help-

ful discussions. This work was supported by grants AR- 383860 and PO1 AR 39448 from the NIH.

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