Suspension-Induced Murine Keratinocyte Differentiation Is Mediated by Calcium

Luowei Li, Tamar T elmenbaum, and Stuart H . Yuspa Laboratory of Cellular Carcinogenesis and T umor Promotion, Division of Cancer E tiology, National Cancer Institute , Bethesda, M aryland , U .S.A.

Modulating extracell~lar Ca2 + (Cao ) and suspension culture are two 'frequently used methods to induce maturation of cultured human and mouse keratino­cytes. To determine if the two methods shar,e a common mechanism, changes in Ca2 + metabolism were studied in suspension cultures of mouse kerati­nocytes. Spontaneopsly detached and suspension­cultured keratinocytes in 0.05 mM Ca2 + medium express markers of suprabasal differentiation, while 0.05 mM Ca2 + is not 'permissive for marker expression by attached keratinocytes. Intracellular free Ca2+ (Ca;) increased rapidly after placing keratinocytes in suspension in 0.05 mM Ca2 +, reaching levels up to 3-to 4-fold higher than Caj in attached cells after 4-5 h. In suspended cells, the increase in Caj was associated

The proliferation and differentiation of cuJtured mouse keratinocy, tes can be selectively regulated by chang­ing extraceUular Ca2 + concentration (Cao ) (Hen­nings et ai , 1980). Elevation of Cao from 0.05 to 0.1 ruM or higher inhibits proliferation and induces th e

formation of desmosomes, cell stratification, and cornification. At the molecular level this is associated with sequential changes in gene expression, including first the upregulation of the epidermal spinous keratins 1 (Ki) and 10 (Ki0) and induction of granular layer proteins filaggrin, keratinocyte transglutamillase, and loricrin (YUSp;J el aI, 1989; Roop et ai, 1987).

Studies from several laboratories have inoicated that increases in intracellul ar free Caz+ , (Ca,) and phosphatidylinositol turnover were cor'related to the differen tiation response inauced by extra­cellular Ca2 + or 1,25(OH)2 vitamin D 3 , another inducer of matu­ration, in cultured hunlan and mouse keratinocytes (Kruszewski et ai, 1991; J ensen el ai, 1990; MacLaughlin el ai, 1990; Sharpe et ai, 1989; PiUai and Bikle, 1991; J aken and Y uspa, 1988; Tang et ai, 1988;, Pun non en t;t ai, 1993), Furthermore, pharmacological agents that specifically ~odifY, intracellular Ca2 + interfere with the induc­tion of epi dermal differentiation by > 0.1 mM CaZ-I- hledium (Li cl

ai, 1995a; Jones, and Sharpe, 1994; Li et ai, 1995b), suggesting that intracellular Ca2+ tllediates the response to changes in extracellular Ca2

+ , T he discov,ery of a Ca2 + -sensitive enhancer element on the human Kl gene reinforced this concept (Huff e/. ai, 1993 ; Rothnagel

Manuscript r!"ceived July 16, 1995; revised October 3, 1995; accepted for publication October 9, 1995.

Reprint requests to: Dr. Stuart l-l, Yuspa, National Cancer Institute , Building 37, R oom 3B25, 37 Convent Drive MSC 4255, Bethesda, MD 20892-4255,

with a 2- to 6-fold increase hi Ca2 + transport across plasma membrane as well as depletion of intracellu­lar Ca2 + -stores. Differentiation marker expression and terminal differentiation were inhibited in suspen­sion-cultured keratinocytes by preventing the rise of Gaj using either 1,2-bis(o-aminophenoxy)-ethane­N ;N,N' ,N' -tetraacetic acid to chelate intracellular Ca2+ or ethyleneglycol-bis(f3-aminoethyl ether)­N,N,Nt ,N' -tetraacetic acid to reduce Cao ' Together, these results indicate that a rise in Caj is a common mechanism controlling differentiation in cultured mouse keratinocytes, and suspension of keratino­cytes enhances Ca2 + transport and alters intracellular Ca2 + sequestration producing a rise in Ca j • ] [II"est Dermatol 106:254-260, 1996

e( (/1, 1993). Analyses of both rodent and human epidennis ill llivo have identifi ed a g radient of both intracellular and extracellular Ca2 + whereby concentrations in the basa l compartment are below serum levels and concentrations in the ,granular cell compartment are high (M enon ct ai, 1985; Forslind el ai, 1984; M almquist el ai, 1984). Together these studies suggest that C;i2+ is a physiological regulator of keratinocyte m aturation,

Several other experimen tal m odels have been described where keratinocyte differentiation is regulated ill IJitro (Rheinwald and Beckett, 1980; Watt et ai, 1988; Bell e( ai, 1982). Among these, the suspensio n of cultured human keratinocytes in semisolid medium has been actively studied . In tlli s model, suspension induces the expression of involucrin , downregula tes {31 integrin, and causes cornification and temlinal differen tiatio n (Adams and Watt, 1990; Rice ;U1d Green, 1979). Expression of K1 and K10 was also no ted in keratinocytes placed in suspension , and this was reported to be independent of extracellular Caz+ concentrations (Drozdoff and Pledger, 1993; Poumay and Pittelkow, 1995) . T hus, de tachment of ceils from a substrate, in addition to Ca2 + , m ay be an important differentia tion signal for cul tured keratinocytes. To determine if there is a relationship between Ca2 + and suspension-induced differentiatio n signals, we examined the role ofCa2 + in suspension­induced maturation of mo use keratinocytes. T he results indicate th<1t suspension-induced differentiatiOli is m ediated by an in crease in in tracellular CaZ-I- .

M ATERIALS AND METH ODS

Chemicals and Antibodies Fura-2IAM, 1,2-bis(O- aminophenoxJ)­ethane-N,N ,N ' ,N ' -terraacetic acid (BArTA)1 AM and pluro nic F-127 were purchased from M olecular Probes Inc. (Eugene, OR), Jonom yci n was purchased fi'om Calbiochem Jnc. (San Diego, CAl, CaCI" e thyleneglycol­b is(J3-aminocthyl. ether)-N.N,N' ,N' -tetraacetic acid (EGTA) , and other

0022-202X/96/S10,50 • Copyright © 1996 by T he Society fo r Investigative Dermatology, lnc.

254

VOL. 106, NO.2 FEBR.UARY 1996

chemicals or reagents were from Sigma Chemical Co. (St. Louis, MO) . Monospecific antisera to epidermal protein markers K1, KI0, keratin 14 (K14), and loricrin were prepared in rabbits by injecting unique synthetic peptides corresponding to carboxyl-te rminal amino acid sequences deduced from the nucleotide sequen ces of cDNA clones for K :I . Kl0 . K14 , and loricrin (Roop cf ai, 1984; Mehrcl ct ai, 1990). The antisera to filaggrin was produced against a synthetic peptide corresponding to residues 24-39 in the partial sequence of filaggrin (Rothnagel ef ai , 1987). The antibodies w ere affin.ity purifi ed using the syntheti c peptide, coupled to activated Sepharose.

Cell Culture Primary kera tinocytes from newborn BALB/c m o use epi­de nnis were pre pared by a trypsin flota tion ptocedure (Heni1ings el al.

1980). The keratinocytes were plated in Eagle's minimum essential medium (Ca2+ and M g2+ free, Whittaker Bioproducts. \Valkersvllle, MD) supple­mented with 8% Chelex (Bio-Rad Laboratories, Richmond, CAl-treated fe tal bovine se rum (Upstate Biotechnology Inc., Lake Placid , NY) and 0.05 roM Ca2 + (Hennings Cl ai, 1980). The cell s were maintained at 36°C in a humidified incubator with 7% CO2 for 6 to 9 days. Fresh medium was supplied daily for the flIst 3 days and every 2 days thereafter. Spontaneously detaching ce lls were co llected from washed monolayers over 24-48 h. Cormfied envelopes were quantitated as previously described (Nagae ef ai, 1987).

To study cultured suspended ceils, keratinocytes were pla ted in 100-m111 tissue cul ture dish es , grown to confluence in 0.05 mM Ca2+ m edium and trypsinized by treatment with 0.1 % trypsin. Equal cell numbers introduced iu to 17 X 20 111m sterile po lystyrene tubes in appropriate medium. T he ce lls were mixed every 3 h and incubated at 36°C in a humidified incubator with 7% CO2 for the time indicated in figure legends. For Ca2 + imaging studies , keratinocytes were placed or cultured on glas~ coverslips in a specially con structed culture chamber fro m a plastic culture dish (35 mm, Corning Glass Works , Corning. NY) as described (Kruszewski el ai, 1991 ) . Cell viability assay was de termined by the MTT assay (Promega. Madison , WI).

InImunoblotting Primary mouse keratinocytes were grown in 12-well tissue culture plates in 0.05 mM C a2 + medium. washed carefully with phosphate-buffered saline (PBS) , and detacl1ing cells in the m edium w ere co llected over a 24- to 48-h period . Detached cell s, attached ce lls, or cell s cultured in suspension were lysed into 200 J.LI of Lacmmti buffer, and the Iysates were heated for 15 min at 90°C. Aliquots of Iysates w ere subjected to electrophoresis on 8.5'X, sodium dodecyl sulfate-po lyacrylamide gel and transferred onto nitrocellulose filters (Schleicher & Schuell Inc., Keene, NH). Equal loading was achieved by adjusting volumes to equalize Po nceau staining of filters and confirmed by K14 immunostaining. Monospccifi c ra bbit antibodies against mouse K1 , Kl 0, K14, fi laggrin. and loric,·in were u sed to d etec t specifi c bands. and the second antibody was horseradish­peroxidase- conjugated goat anti-rabbit antibody (Bio-Rad Laboratories . Richmond, C Al at 1 to 5000 dilution . T he immunoblots were v'isualized by e nhanced chemiluminescence (Dul)o nt N E N. Boston. MA) . T be Same mtrocellulose fIlt er was reblotted after stripping in a buffe r contai~ing 50 mM Tris, pH 6.S, 2%, sodium dodecyl sulf.1 te, and 0.7'% J3-m ercaptoethano l at 65°C fo r 30 min.

Determining the Concentrations of Intracellular Free CalciUln Analysis of Ca; in single ce ll s w as accompli shed by using fluo rescence video microscopy with PC-based digital imaging ana lysis as previo usly described (Kruszewski ef al. 1991) . Primary keratinocytes w ere l o~ided w ith fura-21 AM (25 J.LM) in a serum-free Krebs/N- 2-hyd roxyetbylpipera zine-N ' - 2-ethanesulfonic acid (HEPES) bufrer: 10 mM H EPES, pH 7.4, 120 mM NnC I. 4 mM KC l, 1 111M KH2PO." 1 mM MgC I2' 5 111M glucose, and amino acids (SO X . BRL) w ith 0.05 mM Ca2 + fo r 30 min at room temperature and then washed with the same buffer. A dispersing agent, Pluronic F-1 27, was included in th e loading m edium to f:lC ili tate the efticient loading of fura-2/ AM. The loaded ce ll s w ere held in this m edium fo r 30 min before ex perimental analys is to allow intracellular dc-es terifi ca tio n of fura-21 AM. For suspended cell s, the loading and de-este rification of fura-21 AM were perform ed in test tubes, 'Illd cells were allowed to settle o n glass coverslips for 15 min before the imaging anal ysis. Fura-2-loaded ce lls were imaged in 0. 05 mM C a2 + Krebs/HEPES buffe r or appropriate m edia. T he total ionomycin-sensitive Ca2+ - stores were released by the addition of 10 J.LM ion omycin in the presence of 1 mM EGTA to chelate extracellular C a2

+ .

The imaged fie lds wcre rando m ly se1ectedin an inverted fluo rescence microscope, and each fi e ld contained 15 to 25 cell s. The final Ca; represents the mean va lue :t SO from aLl ceLls in the field.

4' Ca2+ Uptake Attached keratinocytes were incubated w ith 0.05 mM Ca2 + m ediulll containing 0.5 J.LC i/ ml .,sCa2+ for 5 min at room tempera­ture , and the reaction was terminated by rcmoval of the medium and washing w ith icc-cold washing buffer (Li ef al. 199 5a) . Cell number per c ulture vva s d cternlincd in parallel cultures. To exanlin c the .ISe a2+ u ptake

DIFFER.ENTIATIO N OF MURINE KERATINOCYTES 2S5

Ca2+(mM)

K1

K10

Filaggrin

Loricrln

K14

It) N 0 .... .... N It)

0 0 0 0 0

I ---~

1

--~--

Detached cells

C! ....

II 1

I 1

.7

1---.. ---:-1 Attached

cells

Figure 1. Cao

requirement for expression ofkeratinocyte differen­tiation n,arkers differs in attached and detached cells. Monolayer cultures of mouse keratin-ocytes were incnbated in m edia with various concentra ti on of C a2+ for 40 h. Both spo ntaneously detaching cells and the attached cell s were lysed and subjected to Western analysis using antibodies specific for K14 and epidernial suprabasal markers K1, K10, fila ggrin. and

loricrin.

in suspension-cultured cell s. attached keratinocytes were tryp sini zed and 10" cell s we re aliquoted to rubes and incubated in 0.05 m[VI Ca2

+. riledinl11 for 4 h . "sC a2 + was -added to the test tube to a fi,ial specifi c acti vity of D.5 J.LC i/ ml , and after 5 min the cells were tran sferred to \V hitman 'glassfiber filte rs (Whitman, International Ltd, Maidstone, UK), fi ltered and washed w ith the icc-cold washing buffer. To determine 4'Ca2 + uptake , cell s were lysed with 0.5 N NaOH. and the rad ioactivity was determined by scintil­

lation counter.

Immunofluorescence Keratinocytes were suspended in m eqiul11 \viti, 0 .05 111M C a2 + for 3 or 4 h in the presence or absen ce of BAPTA, and 100,000 cells per sample were affixed to glass slides in a c)'tosp in apparatu.s. Cvtospin sa'~lpl es were also prepared from trypsinized keratinocytes that h~d been cul tured as attached cell s in 0.05 mM Ca2 + o r 0.12 mM C a2 + m edium for 24 h . Sbdes were fixed in methanollacetone (1:1 vollvol) for 5 min , air dri ed , rehydrated in PBS fo r 5 min , and incupated wit~ 12% bovine serum alb LUll in for 20 ni in to reduce nonspecifi c binding. Slides were tI,en incubated witll a mixture of primary antibodies: guinea pig antibo dy to K14 (1 :500) together with rabbit antiboc;ly to K1 (1:500) . or 10 licrin (1 :50) , overnight at 4°C in 12'% bovine semm albumin in PBS. For detection of "'''0 antigens simultaneou sly, slides were incubated Witll biotinylated anti-guinea pig antibody (1:200 . Vector La bo rato ries. Burlington . CAl for 20 min. Following aID-min wash in PBS. slides were incubated fo r 20 min with a mixture of fluorescein iso thiocyanate- conjugatcd swine anti-rabbit anti­body (1 :50) and strcptavidin Texas- red (1 :SOO). After washing w ith PBS and water, slides were air dri ed and coverslips were mounted with pol)'mount (Polysciences , Warrington , PAl . Fluorescence was detected u sing a Zeiss Axiophot fluorescence microscope.

RESULTS

The Expression of Differentiation Markers in Spontane­ously Detached Keratinocytes Is Independent of Cao T h e

e xpression ofKl, Kl0 , loricrin"and fIlaggrin, suprabasal marke r s of epidet:mis, w a s evaluate d b y W es te rn blots in substrate-attached cells and floating cell s collected from the c ulture m e dium over 40

h (spontane ously detac hing cell s) (Fig 1). These m arkers were not d e tected in attach e d cells c ulture d in 0 .05 111M Ca

2+ m edium , but

w e r e readily d e tected in attached cells cultured in 0.1-0.2 111M

Ca2 + medium. In this population marke r expression was poor jn

256 LI ET AL T HE J OU RNAL O F INV ESTI GATIVE DERMATOLOGY

Table I. Comparison of Intracellular Free Ca2 + Levels in Attached, Spontaneously Deta ched, and Suspension-Cultured Keratinocytes"

Cao

Experinlcn t (mM) Cells

A 0.05 Spontaneously Detached 0.05 Spontaneously Detached 0.05 Attached 0.05 Attached 0.05 Attached

B 0.05 Suspended 0.05 Spontancously Detached

C 0.05 Suspended 0.05 BAPTAIAM 0.05 EGTA

D 1.0 Suspended 1.0 BAPTA/AM 1.0 EGTA

T une in Suspension (h)

24 24

1 24

4 4 4

5 5 5

Dasal

403.5 :!: 124.2 (1 8) 520.9 :!: 51.2 (21) 47.5 :!: 10.4 (2 1) 53.3 :!: 14.2 (17) 48.7 :!: 9.6 (16)

86.3 :!: 23.4 (81) 11 8.6 :!: 30.7 (45)

125.8 :!: 19.4 (99) 62 .1 :!: 14.7 (88) 38 .5 :!: 8.7 (89)

158.7 :!: 33.8 (90) 95 .2 :!: 22.7 (88)

110.5 :!: 22.8 (74)

Ca; (nM)

El l

ND 233.4 :!: 38.1 (21)

ND ND

152.3 :!: 24.0 (16)

166. 7 :!: 31.1 (45) 105.1 :!: 28.7 (21)

104.7 ::!: 19.1 (45) 41.7 :!: 11.1 (44) 44.0 :!: 7.5 (46)

147. 2 :!: 35.0 (45) 92.3 :!: 20.0 (50) 92.1 :!: 21.9 (52)

II T he b asal le ve l o f C al in fu ra- 2-1oad c d sin gle ce lls was d etermined b y d igital image ~lI1 a l ys is o n b.oth attach ed :lI1d suspen de d cell s as descr ibe d in AI/aleri ll l .,' (J ll ri M e/fwds. Suspen ded cells were loaded with fura-2/ AM for 30 min , w;1shcd and held for 15 min . T he cells were then allowed to settle on glass covc rslips for 15 min prior to m C;l su rc m CJl[ ofCaj. The amount of Ca2+ in intracellular Ca2+ - stores was detected after addi tion of ionom ycin .lIld EGTA. Dat.a arc fro m fo ur indepe ndent cxperirncnts. T ile cul ture cond itions and the in cubntion time are also indi cated . The number of, cells analyzed is indicated in the parenthesis. D ata presented arc mean ± SO. Basa1. basal le ve l of C Ol i: El l. the peak value of the intracellular free CoH levels aftcr addi tion of ionomyci n (10 J.LM ) and EGTA (2 mM). NO. not determined .

0 .5 or 1.0 rnM C a2 + at 40 h as previo usly rep orted (Yuspa et ai, 1989). In contrast , express ion of K1 4, a basal ce ll inarker that is constitutively expressed in cul tured ke ratinocytes, was not affected by the le vel of Ca2 + in culture Imidii.nn . In de tached cell s, the fo ur suprab asal m arkers w ere expressed as well in 0.05 mM C a2 + as in 0.1-0.2 mM Ca2 + m edium, but these protein's w ere undetectable in 0. 5 and 1.0 mM Ca2 + . The reduce d amoun t of K14 in ex tracts from 0 .5 and 1.0 mM Ca2 + cul tures was p arallel to th e redu ced am o un t of total pro tei.n. Very few spon taneously detach ed cells were collected fro m 0 .5 and 1.0 mM Ca2+ m edium since the cells form a stratified sheet: T hese results indica te that supra basa l m arker expression in spontaneously d e taching keratinocytes occurs under conditions o f extracellular Ca2

+ too low to induce exp ression in attach ed ceLIs. T hus, m arker expression in detached ceLI s appears to be independent of extracellular Ca2

-!- tmtil higher le vels w h en few cells de tach , and m arker expressio n is also re duced in attached cells.

Since inductio n of kera tinocyte diffe ren tiation by Ca2-!- o r

1,25(O H) 2 vitamin D3 is associated with an increase of in tracelluiar Ca2

+ , w e asked if de tachment of keratinocytes w ill increase in tracellular Ca2 + independent o f extracellular Ca2 + . After 24 h in suspension , the free intracellular Ca2+ (Ca;) in spontan eously detached ceLIs in 0.05 mM Ca2 + medium w as almost 10-fold higher than ill attached cells (Table I, Experiment A) , and the ionomycin releasible Ca2 + is deple ted. Thcse results sugges t that de tached cell s increase Ca2+ uptake and lose intracellular Ca2 + - stores. T his increase ill Ca; observed in detached keratinocytes could co ntribute to the inductio n of d ifferentiation or di fferentiation of detached cell s could enhance Ca2+ uptak e independently, perhaps thro ugh plasma membrane chan ges.

BAPTA Inhibits Marker Expression in Spontaneously De­tached Keratinocytes To determine if an increase o f Ca; was causally re lated to the induction of diffe ren tiatio n o r a consequen ce of differentiation, keratinocytes w ere loaded wi th the intrace llular C a2

-!- chelator BAPTA / AM (Li et ai, 1.995a) in 0 .05 mM C a2+ medium and attach ed and spontaneously detached cells w ere collec ted afte r 24 h (Fig 2). T he detached control ce lls expressed bo th spino us and granular prote ins as expected , but BAPT A treatm ent inhibi ted the expression of these markers. Attached cells ill 0 .05 mM Ca2

+ m edium with and w ith out BAPTA trea tment did no t express these m arkers. These results in dicate that intrace llular

K1

K10

Filaggrin

'0 .b c: o (J

c:( I-

~ m

e .... c: o (J

wE] DD B3

-Loricrin DI ~ . DlJ

K14 B8l EB Attached Detached

cells cells Figure 2. BAPTA blocks the expression o f epidermal differentia­tion markers ill spontaneously detached cells. Cells were loaded with 50 JLM B APTAI AM and then incubated in 0.05 111 M Ca2 + medium. Both attached cells and the spontaneously detached cel.l s were collected after 48 h and subjected to il11l11unoblotting with Kl , Kl 0. fi laggrin , loricrin, and K1 4 antibodies.

VOL. 106. N O . 2 FEBR UAR Y 1996

a Suspended cells

Time 6 hrs 24 hrs 48 hrs II

en N en N en N ~ ~ "": 0 en "": 0 en "": 0 ci 0 ci ci ci ci ci ci

- -'I --.. --- --- .

K10

Fi laggrin

Loricrin ~========~~~~~~~~~.~I K141 ' 4U1 ___ aws_ru:-__ ~I

b Suspended cells ..!!! ..!!! Q3 Q3

2· () Ca (mM) 0.05 0.5 ()

'0 '0 Q) Q)

~ ~ ~ ~ Q) « Q) ~ () ()

e a. l- e a. ItS Q) -0 « (!) 0 « (!) - Q) -Z III UJ Z III UJ « Cl

K1 I-------~ K10

1 - I Filaggrin

Loricrin f

K14 ~E ~~::::~::;::;~~ Figure 3. Suspension culture of keratinocytes induces the expres­sion of differentiation markers, and EGTA and BAPTA block the induction. a) Kerntinocytcs cultured in 0 .05 mM Ca2 + medium were trypsinized and placed into suspensio n culture at 10" cells per tube in 0.05. 0.12, 0.5. and 1.4 mM Ca2+ m edium for 6. 24. or 48 h . At the end of incubac;o n period , the expression of Kl. KIO, f. laggrin, and loricrin was evaluated by inllllunobiotting. b) Differentiation marker expression was also examined in cells loaded with BAPTA I AM (50 J.LM) o r cultured in the presence ofEGTA (I 111M) in suspension.

Ca2 + is an important mediator of detachment-associated differen­tia tion and marker expression.

Suspension Culture Induces Differentiation Marker Expres­sion Independent of Cao but Dependent on Cal Since Kl and KI0 expression is detected in some cells in the attached population (Roop et til, 1987), it is possible that cells commit to di.fferentiate independently of extracellular Ca2 + while attached , an d th.is resu lts i.n detachment and subsequent in creases in Ca;. To determine if Ca2 + is required to initiate differentiation in de tached cells, basal cells cultured in 0 .05 mM Ca2 + medium were trypsin ized and suspended in m edium containing 0.05. 0.12, 0.5 , or 1.4 mM Ca2 + for up to 48 h . As shown in Fig 311 , keratinocytes express suprabasa l markers with increasing time in suspension.

D IFFERENTIATION OF MURINE KER.ATINOCYTES 257

Filaggrin expression seems particul arly susceptible to suspension, being upregulated as early as 6 h . T he timing and magnitude of expression is regulated to some extent by extracellular Ca2 + .

Higher Ca2 + accelerates expression of KJ 0 a.nd loricrin and enhances K1 and fi laggrin . T he expression of markers in suspended cells in high Ca2 4 medium supports the conclusion 5'om Fig 1 that lower e xpression of m arkers in detached cells indicates a lower nlllnber of detached cells in high Ca2 + m edium. To confirm the contributio n of Ca2 + to marker expression , cells were loaded with BAPTA or suspended in m edium containing EGTA for 24 h, to

buffer intracellular or extracellular Ca2 + , respectively (Fig 3"). Marker expression was inhibited by both BAPTA and EGTA in suspended cells, indicating that influx of extracellular Ca2 + across the plasma m embrane and the rise in intracellular Ca2 + contribute to the e.\.ll ress ion of differentiation m arke rs. As seen previously. Kl appears to be less sensitive to the effects of Ca2 + than the other markers. Neither BAPTA nor EGTA d epress ovel'a ll leucine in cor­poration in suspended cells (not shown), indicating that inhibiti on of m arker expressio n is not due to an effect of these agents 011

overall protein synthesis.

Cal and 45Ca2+ Influx Increase in Both Spontaneously Detached and Suspension-Cultured Keratinocytes To de­termine if forced detachment resul ted in an in crease of intracellular Ca2 + similar to spontaneous detachment, keratinocytes cultured in 0.05 mM Ca2 + m edium were trypsinized and intracellul ar Ca2 + was assayed immediately or after 4 -5 h in suspension (Table I). One hour is required for fura- 21 AM to be loaded into ce lls in suspension before the cells were placed on a g lass coverslip for Ca2 + image analysis. In 0.05 mM Ca2 + m ed ium, Ca; in creases within 1 h in newly trypsinized cell s w hen compared w ith the still attached population, and the io nomycin releasable Ca2 + - stores remain intact (Table I, Expcri lll clIl B) . Spon taneo usly detached cell s from these same cultures show higher Ca; and depleted Ca2 + -stores (Table I, Expl'rilllCIIf B) but lower levels than detected in Experi ­ment A. After 4 h of suspension in 0.05 mM Ca2

+ , Ca; is 50% higher than at time 0, aJld Ca2

+ - stores are depl eted (Table I. Experi lll ellt C). Both BAPTA and EGTA prevented the increase in Ca; in suspended cells. Afte r 5 h of suspension in 1.0 mM Ca- + (Table I , Expcrilllellt D) , Ca; was higher than cells suspended for 4 h in 0.05 mM Ca2 + . T his compensatory increase in Ca; by 1.0 mM Ca2 + m edium may explain the accelerated and en.hanced expres­sion of markers in suspended cells in higher Ca2 + media. Both BAPTA and EGTA prevented the increase in Ca; produced by 1.0 mM Cao '

Studies of 4SCa2 + uptake indicated that Ca2 + flux across the plasma m embrane increases rapidly in suspended keratinocytes (Fig 4) . Intracellular accumulation of ·15 Ca2 + increases by 2- to 3- fold in cells in suspension shortly after trypsiniza tion w hen compared with attached cells. After 4 h in suspension, there is a 6- to 7-fo ld increase. W hile BAPT A partially prevents this increase, EGT A completely inhibits accumulation of intracellul ar "5Ca 2 + . T hus. BAPTA reduces intrace llul ar Ca2 + mainly by chelating Ca2 + intracellular1y, w hile EGTA preven ts influx of Ca2 + across the plasma m embrane . Since both of these agents prevent the rise of intracellular Ca2 + and inhibit expression of differentiation markers, these twO even ts are likely to be related.

Expression of Differentiation Markers Is Detected within 3 h When Keratinocytes Are Placed in Suspension To determine if differentiation markers were expressed early after suspension w hen a rise in Ca; is detected, cells were trypsin ized . suspended in 0.05 mM Ca2 + medium, collected by cytospin after 3 h , and the expression of Kl and loricrin (Fig 5). and KI0 and fi laggrin (not shown) was d etermined by immunostaining. In attached cells in 0. 12 mM Ca2 + for 24 h . many intensely stained K I positive cells are detected . In tensely sta in ed Kl positive cells and less intense positive cells are also detected in suspended cultures after 3 h . In the presence of BAPTA, the n um ber of intensely stained cells is reduced . Since few loricrin positive cells are detected after 24 h in th e attached population (Yuspa et ti l. 1989). only

258 1I ET AL

1000

C 750 cu 'E ~

1'1:1 Lt') -..... c.~ ::l Qi

u 500 + .. N 0

1'1:1 -U ..... 11\ '0 .. E

250 ~

II • ~ c None BAPTA EGTA Qi 0 . ;;; u c ~ Gl

Cell in suspension CD 0-.. ~

" for 4 hours u .. !! .= <

~ "0 U

Figure 4. "Ca'+ uptake increases in suspension cultured keratino­cytes. 45Ca2+ uptake was examined in monolayer cultured cells and cells in suspension culture at 0 and 4 h in 0.05 mM Ca2 + medium with either EGTA in the medium or prcloaded with BAPTA. 45Ca2+ (0.5 p.Cilml) was added to cells for 5 min just prior to termination of cul ture. The cell associated radioactivity was determined by scintillation counting as described in Materials al/d Methods . En'or bars, SEM (n = 3).

suspended cells were studied. Loricrin positive cells were also detected after only 3 h of suspension, and the number of strongly positive cells is diminished by BAPTA. A similar pattern was seen for KIO and filaggrin.

Induction of Cornification in Suspension-Cultured Kerati­nocytes is Ca2 + Dependent To determine if the differentiation program proceeded beyond expression of markers, cornified cells were counted after 48 h of placing keratinocytes in suspension (Fig 6). Abundant cornified envelopes were detected in attached cells cultured in 1.4 mM Ca2 + medium. Spontaneously detached cells had a modest increase in cornification in 0.05 mM Ca2 + . Suspend­ing cells also increased cornification after 48 h, but the extent was dependent on Cao ' The most substantial increase was detected in suspended cells in 1.4 mM CaH medium. The extent of cornifi­cation was partially reduced by BAPTA in medium with low extracellular Ca2 + . EGTA was more effective in inhibiting cornifi­cation, indicating this process is Ca2 + dependent; however, neither BAPTA nor EGTA prevented the loss of viability in suspension detected over 48 h by the MTT assay (not shown).

DISCUSSION

It is now well established that a rise 111 intrace llular Ca2 + is associated with keratinocyte d ifferentiation i ll IIi/ro and ill "illo (Pillai and BikJe, 1991; Kruszewski et aI, 1991; Sharpe e/ aI, 1989; Menon c/ ai, 1985) . Recent studies indicate that chelating in tracellul ar Ca2

+ in differentiating keratinocytes (Li et aI, 1995a) or a ltering Ca2+ - stores Oones and Sharpe, 1994; Li cl aI, 1995b) can inhibit differentiation, suggesting a causal association for increased intra­cellular Ca2

+ and progression through the differentiation program. Si.nce a rise in extracellular Ca 2 + can induce both terminal differ­entiation and an increase of Ca j in cultured mouse and human keratinocytes, it fo llows that at least part of the l'i se in Ca j is due to flux across the plasma membrane. 45Ca2 -'- studies have con firmed that influx increases in differentiating keratinocytes (Li ct aI, 1995a) . At least three ion channels have been identi.fied on keratinocytes, and a voltage dependent C I-channel is activated by raising extra­cellular Ca2

+ (Mauro el al, 1993). Furthermore, a plasma mem-

THE J O URNAL O F INVESTIGAT IV E DEr~MATOLOGY

CaCI2 (mM) K1 K14

0.05

AU. . 0.12

• • .. r 'It . ~o! :; , • ~ ..:. .. . I . " , .. ·,i· : ~ , '. .. .. .., .'O . ~ ' ., ",

0.05

. ' ~ . # • . , - '1" " , : 'If ,

03 ' . :J. • . . ~ . ' • , • • ,." . < ~ ~ _ 0 ; ,. . . . .,

0" ".. ~ ....

" . . .. + BAPTA .' ,

.. e ,. , ~

Susp. ' .'" \I . . Lor. K14

o - .'

0.05 . " • • J "' •

,f"- •. ~ O· .

+ BAPTA

Figure 5. Differentiation marker expression in suspension cultured keratinocytes is detected by imnlUl1ofluoresccllce. Trypsinized at­tached keratinocytes in 0.05 or 0.12 111 M Ca2 + medium (Au.) and cells in suspension culture (10 5 cells) for 3 or 4 h (SI/Sp.) were affixed to glass slides in a cytospin apparatus. Cells were incubated with a mixture of guinea pig al1ti -K.14 antibody and either rabbit anti-K1 or anti-Ioricrin antibody overnight at 4°C. Simul taneous detection utilized biotinylated anti-guinea pig antibody and streptavidin Texas-red or fluorescein isothiocyanate­conjugated swine anti-rabbit antibody and fluorescence microscopy. Scale bal'. 100 p.m.

brane Ca2'" -receptor, simi lar to that characte rized in parathyroid

ceJl s (Brown et aI, 1993) , ha s been identified on human keratino­cytes. I

W hile a rise in in tracellul ar Ca2 + is linked to i.nduction of differentiation by extracell u lar Ca2 + and 1,25(OHh vitamin 0 3 ,

the genera li ty of this chan ge has not been explored for other stiI1luli that induce keratinocyte differe ntiation. Among these are suspen­sion cu lture and high cell density in the absence of growth f.1ctors (Orozdoff and Pledger, 1993; Poul11ay and Pittelkow, 1995) where it has been proposed that extracellular Ca2 + is not a major factor for regulating differentiation . We now present data confirming that a rise in extracellul ar Ca2 + is not required for upregu lating expres­sion of differentiation markers in kerati nocytes maintained in suspension or spontaneously detached fi'ol11 the culture substratum. However, thi s loss of dependence for increased Ca" is associated with a rapid increase ofCa 2

-'- transport across th e plasma m embrane

, ll.atnal11 A. 13ikle D: Regulation of the calciul11 receptor and mRNA levels during kcratinocytc differentiation. J II/ pest Dcrmat"f 104:592, 1995 (abst,)

VOL. 106. NO. 2 FE13R.UAR Y 1996

A ttached Cells Suspended Cells

30 ~--------~----------------------~

ltC -Z 20 0 l-e( ()

u. Z 10 a: 0 ()

a d s abc d abc d abc d

BAPTA EGTA

Figure 6. F ormation of cornified envelopes in suspension cultured keratinocytes is Ca'+ dependent. Keratinocytes were cul tu red in sus­pension in 0.05 . 0.12, 0.5. and 1.4 mM Ca2 + medium for 48 h in the presence of EGTA- or I3APTA-loaded ce ll s. Corni fication in attached cell s cultured in 0.05 mM Ca2 + or .1.4 mM Ca ' + medium for 48 hand spontaneously detached cell s in 0.05 mM Ca2 + m edium was also eva luated . The number of ce ll s in each sample was determined by coulter counter and the number of corn ified envelopes was determined in sodium dodccyl sulfate/J3-mercaptoethnol lysed ce ll s in a hem ocytometer. T he amount of cornified envelopes was expressed as the I)(!rccn t;' ge o f tota l ccllnulIlbcr. a. 0.05 mM Ca,, ; b, O. '12 mM Cn,,; c. 0.5 111M Cae'; d, '1 .4 mM Ca,, ; s, spontaneously detached cell s in 0.05 111M C a". Error bars, SEM (n = 3).

in suspended cell s in low Ca2 + m edium . T he notion that thi s rise in intracellular Ca2 + is crucial for expression of differen tiation markers is supported by the suppressio n of m arker expressio n by either EGTA O J' BAPTA. EGTA prevents C a2

-1 influx and BAPTA chelates in trace llul ar Ca2

-!- , both havi.ng the same inhibitory con­seq uences o n differentiati on of suspended cells as they do on atta ched cells.

Our data indica te that suspensio n culture of keratinocytes is also associated with a I'eduction in ER Ca' + - stores when viewed as a proportion of total cellular Ca2

-!- (Table I) . Release of Ca2 + from endopl asmic reticutum Ca2 + -s tores could also contribute to the elevation of Ca; in suspended ce lls. In creases in Ca; have been documen ted in many cell types undergoing volume changes, and this is attributed to both an increase in flux ac ross the pla sm a membrane and release of stored ea2 + (McCarty and O'Neil , 1992). Release of stored C a2 + resul tin g in an increase in C a; is also documented in cultured endothelial ceIJ s indu ced to undergo a rounding phenotype by treatment w ith antibod ies to E-selecti n (Kaplanski ci aI, 1994) . T hus, chan ges in cell shape arc associated with changes in Ca; through both plasma membrane transport and intracellular re lease mechanisms .

T he results reported here indicate that the link between Ca2 +

an d keratinocyte diffe rentia tion is detined by changes in intracellu­lar Ca' + . Increases in extra cellular Ca2

t- that induce difFe re ntia tio n i" v itro arc one m echani sm to raise in tracellular Ca2

+ , and this may also be a regula tory m echanism i" lIir", (Menon cf aI, 1985; Malmquist el (/1, 1984; Me non c( aI, 1994). T his conclusio n may clarify ambiguities in the li te rature on the ro le of extracellular Ca'+ in keratinocyte differentia tion. For exampl e, previous studies indi­cated th at rounded keratinocytes, unable to attach firmly to a matrix, express markers of differentiation (Watt ct aI, 1988). Other studies concluded that human ke ratin ocytes cul tured at subconflu­ent densities in the absence of growth f.1cto rs express diffe rentia tion ma rkers in respo nse to a ri se in extracellul ar Ca' -!- , but postconflu­ent tightly packed , smalJ keratinocytes arc mu ch less Ca 2 + respon-

D IFFERENTIAT ION OF MURIN E KERATINOCVTES 259

sive (Killackey el aI, 1989) . Similarly, keratinocytes fi'om postcon­flu ent cul tures already expressing differentiation markers do not increase m arker expression in suspension (Poumay and Pittelkow, 1995) . It is likely that shape changes in postconfluent keratinocyte cul tures and poorly attached keratinocytes are suffic ient to raise imracellul ar Ca 2 + and cause phenotypic changes witho ut a requ ire­ment for increased extracellul ar Ca2 + . In contrast, less confluent or tightl y attached cells m ay require an external stim ulu s to raise intrace llular Ca2 + .

When placed in suspension , keratin ocytes experience both a rap id ri se in in traceLlul ar Ca2 + and an accelerated expression of both spinous and granular celJ ma.rkers. At the cellular level, loricrin protein can be detected within 3 h b y immullofluorescence, and at the population level Kl, K10, loric6n , and fi laggrin are increased by 24 h on Western blots. T his rapid onset of expression of late differen tiatio n m arkers indica tes that pathways dow nstream from in tracellul ar Ca2

+ must also be engaged in suspended cell s. Among these, prote in kinase C activation is critica l to late m arker expression (Chakraval'th y et aI, 1995 ; D lugosz and Yuspa, 1993; Denning ef aI, 1995) . T hus, the rapid rise in intra cellul ar Ca 2 + .is likely to initiate a cascade of intracellul ar activities leading to gene expression and terminal diffe renti ation.

I'lie lI,is lt to r/lllllk Dr. U/rikc Lirltri!or /, er hdJ~fi" dis(IIssiolls alld Mr. CcO/gc Z lt ao

./e)l· his Itelp ,"irlt i",,"""obloui"g.

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