Ca2+-Dependent and Ca2+-Independent Regulation of the Thyroid Epithelial Junction Complex by Protein...

EXPERIMENTAL CELL RESEARCH 225, 1–11 (1996)ARTICLE NO. 0151

Ca2/-Dependent and Ca2/-Independent Regulation of the ThyroidEpithelial Junction Complex by Protein Kinases

MIKAEL NILSSON,1 HENRIK FAGMAN, AND LARS E. ERICSON

Institute of Anatomy and Cell Biology, Goteborg University, Goteborg, Sweden

pending on the duration of the preceding low Ca2/ pe-riod. The trypsin-induced displacement of ZO-1 in H-The integrity of epithelial cell junctions is controlled7-treated cells in low Ca2/ suggests that the localiza-by E-cadherin-mediated (Ca2/-dependent) cell–cell ad-tion of ZO-1 to the tight junction is not necessary forhesion. In thyroid follicular cells the dissociation ofthe maintenance of junctional tightness. q 1996 Academicjunctions induced by transfer to low Ca2/ mediumPress, Inc.(Ca2/ switch) is prevented by thyrotropin acting via

cyclic AMP/protein kinase A (cAMP/PKA) (Nilsson etal., Eur. J. Cell Biol. 56, 308–318, 1991). In MDCK kid-ney epithelial cells protein kinase inhibitors elicit a INTRODUCTIONsimilar response which, however, is cadherin-indepen-dent (Citi, J. Cell Biol. 117, 169–178, 1992; Citi et al., J.

Epithelial cells are characterized by firm cell-to-cellCell Sci. 107, 683–692, 1994). As such inhibitors alsoadhesion required for the epithelial sheet to resist me-may interfere with PKA, we examined in a single cellchanical tension and, indirectly, to maintain a diffusiontype, filter-cultured pig thyrocytes, the effects and pos-barrier for transepithelial solute gradients. The mainsible interactions of the cAMP/PKA agonist forskolinepithelial adhesion mechanism so far characterized in(or thyrotropin) and the kinase inhibitor H-7 in Ca2/

detail is that mediated by E-cadherin, a single-span-switch experiments. We found that the epithelial bar-ning transmembrane protein which binds homo-rier dysfunction, comprising loss of transepithelial re-philically to complementary molecules on adjacent cellssistance, increased transepithelial flux of [3H]inulin[1]. Although present along the entire lateral plasmaand redistribution of junction proteins (cadherin andmembrane E-cadherin is concentrated in the adherensZO-1), which follows Ca2/ removal were inhibited by

TSH, forskolin, and H-7. All agents were also able to junction (AJ) but excluded from the tight junction (TJ)induce recovery of resistance in low Ca2/. The maximal [2]. However, not only AJ but also TJ and desmosomesrecovery effects of forskolin and H-7 were additive seem to require E-cadherin to be structurally and func-when given simultaneous with Ca2/ chelator. In con- tionally organized [3–5]. E-cadherin has a critical roletrast, forskolin-induced recovery initiated 10 min after in cellular differentiation of the epithelial phenotype,Ca2/ removal was antagonized by H-7. The protection including the development of cell polarity and epithe-of junctions by forskolin in low Ca2/ was rapidly abol- lial tightness [4–7]. Its importance is further empha-ished by light trypsinization (0.001%), whereas the sized by the fact that E-cadherin frequently is lost orsame concentration of trypsin had little or no effect down-regulated in epithelial tumor cells and experi-on the corresponding action of H-7 or staurosporine, mentally transformed cells [8–11].another potent kinase inhibitor. In H-7-treated cells The adhesive function of E-cadherin, like that of allkept in low Ca2/, trypsin caused redistribution of ZO- members of the cadherin superfamily, depends strictly1 from the plasma membrane to the cytoplasm while

on the presence of Ca2/ outside the cells [1]; specificthe transepithelial resistance remained high. TakenCa2/-binding sites have been identified in the extracel-together, the data indicate that TSH via cAMP/PKAlular domain of the molecule [12]. Ca2/ switch experi-and the protein kinase inhibitor H-7 reinforce the thy-ments have provided new insights into the dynamicroid epithelial barrier under low Ca2/ conditions byturnover and regulation of E-cadherin as well as otherdistinct although interacting mechanisms. The highjunction components. For instance, when extracellularsensitivity to proteolysis in the absence of Ca2/ sug-Ca2/ is reduced to micromolar levels the binding func-gests that the cAMP-regulated mechanism is cadherin-tion of E-cadherin is immediately lost and the wholedependent. H-7 promotes or inhibits the cAMP/PKA-junctional complex is gradually disintegrated [13–15].mediated recovery of transepithelial resistance de-The subsequent transfer of cells to normal Ca2/ concen-tration enables the synchronized monitoring of E-cad-1 To whom reprint requests should be addressed at Institute ofherin reassociation and the de novo assembly and for-Anatomy and Cell Biology, Goteborg University, Medicinaregatan 3,

S-413 90 Goteborg, Sweden. Fax: /4631-773 33 22. mation of epithelial junctions [4, 16–18].

1 0014-4827/96 $18.00Copyright q 1996 by Academic Press, Inc.

All rights of reproduction in any form reserved.

AID ECR 3167 / 6i0e$$$121 05-02-96 13:42:08 ecal AP: Exp Cell

2 NILSSON, FAGMAN, AND ERICSON

The cytoplasmic domain of E-cadherin is also re- including PKA, but with different inhibition con-stants [32]. An obvious matter is whether epithelialquired for adhesion [19]. It is linked to the cortical

cytoskeleton via a group of proteins, a-, b-, and g- junctions are independently influenced by PKA andanother H-7-sensitive kinase(s), which then wouldcatenins, which are believed to exert regulatory func-

tions [20, 21]. The cadherin–catenin complex is in act on distinct effector mechanisms, or if differentkinase pathways converge on a common mechanism,turn associated with other molecules which may in-

fluence cadherin-mediated adhesion. For instance, presumably involving the microfilament system. Thepresent study was undertaken to compare TSH/for-the receptor for epidermal growth factor binds to and

phosphorylates b-catenin [22], a transmembrane- skolin and protein kinase inhibitors, mainly H-7, intheir ability to reinforce epithelial junctions in onetype tyrosine phosphatase is connected to E-cadherin

[23], and members of src kinase family are concen- cell type, pig thyrocytes in Transwell culture. Trans-epithelial resistance and cellular distribution oftrated in the submembranous area of junctions [24].

Thus, the epithelial junction complex contain not cadherin and ZO-1 were analyzed in Ca2/ switch ex-periments. We found that all agents induce protec-only the adhesion components but also molecules in-

volved in signal transduction. This close relationship tion and recovery of the epithelial barrier in Ca2/-depleted cells, but the individual responses have dif-may be functionally important in cell behavior like

migration and invasion in which disassembly of AJ ferent kinetics, are additive at maximal stimulation,and show different sensitivity to cell surface trypsin-and TJ likely occurs. However, mechanisms by which

epithelial junctions are positively and negatively con- ization. Specifically, the TSH/forskolin-induced pro-tection of junctions in low Ca2/ is rapidly abolishedtrolled by external and cytoplasmic signals are still

largely unknown. by trypsin, whereas that induced by H-7 or stauro-sporine is not. Together, the data indicate that TSH/The role of protein kinases in the regulation of epi-

thelial junctions was recently examined in a series of forskolin and the kinase inhibitors work via distinctmechanisms. Taking into consideration the biochem-papers by Citi and co-workers [25–28]. Well-known ki-

nase inhibitors like H-7 and staurosporine were found ical properties of cadherins, which resist proteolysisin the presence of Ca2/ but require only a very lowto counteract the junction disassembly produced by

switching to low Ca2/ medium [25]. This suggests that concentration of trypsin to be cleaved in Ca2/-freeconditions [1, 33, 34], the tight junction barrier inthe dissociation of Ca2/-depleted E-cadherin at AJ is

converted to an intracellular signal which involves ki- thyrocytes may be stabilized by an adhesion-promot-ing effect of TSH via cAMP/PKA on E-cadherin.nase activity and is functionally related to the break-

down of the whole junctional complex. However, sev-eral observations indicated that the mechanism by MATERIALS AND METHODSwhich the kinase inhibitors promote junctional integ-rity is cadherin-independent: the junctions were also

Chemicals. Collagenase (type II) was obtained from Worthingtonmaintained in low Ca2/ when cadherin was degraded Biochemical Corp. (Freehold, NJ); Eagle’s MEM, fetal calf serumby external trypsin and the junction-disrupting effect (FCS), penicillin, streptomycin, and fungizone were purchased from

Gibco Ltd. (Paisly, Scotland); forskolin was from Calbiochem (Laof cytochalasins, which is indirect via cytoskeletal de-Jolla, CA); H-7, staurosporine, ethylene glycol-bis(b-aminoethylrangement, was neutralized [26]. As H-7 also directlyether)-N,N*-tetraacetic acid (EGTA), thyrotropin (TSH; bovine), andaffects the actin-based cytoskeleton [28, 29], it was pro-trypsin were from Sigma Chemical Co. (St. Louis, MO); Collagen

posed that this drug inhibits junction disassembly by S (type I; calf skin) was from Boehringer (Mannheim, Germany);preventing the actin filaments associated with AJ from [3H]inulin was obtained from Amersham International (Amersham,

England). H-7 purchased from Sigma is known to be 1[5-isoquinolyl-recoiling into the cytoplasm, which typically occurs insulfonyl]-3-methylpiperazine [35] and not, as stated in the manufac-Ca2/-depleted cells [13–15], rather than stabilizing theturer’s data sheet, the 2-isomer.cadherin-mediated adhesion.

Follicle preparation and culture conditions. The methods of folli-The kinase(s) regulating this effect on junctionscle isolation and subsequent culture have previously been described

remains to be identified. We have previously found [15]. Briefly, pig thyroid glands were minced and subjected to re-in filter-cultured thyroid epithelial cells [30] that the peated collagenase digestions and mechanical disintegrations. The

digest was rinsed from single cells by repeated washings (centrifuga-disruptions of junctions which follow Ca2/ removaltion; 400 rpm) and remnants of connective tissue were removed byor cytochalasin treatment are both prevented by thepassing the follicle suspension through nylon filters of diminishingacute stimulation with thyrotropin (TSH), thus mim- pore size. Isolated follicles, of which almost all were in a ruptured

icking the action of protein kinase inhibitors [25, 26]. state, were suspended in Eagle’s MEM supplemented with 10% FCS,TSH also rapidly induces dramatic changes in the penicillin (200 U/ml), streptomycin (200 mg/ml), and fungizone (2.5

mg/ml) and plated at a final density of about 50 follicles/mm2 on theorganization of actin filaments in thyrocytes [31].filter (pore size: 0.4 mm) of bicameral chambers (Transwell; CostarThese effects of TSH are reproduced by forskolin andCorp., Cambridge, MA). The filters were precoated with collagen typeare therefore, like most TSH-regulated functions, I (0.3 mg/ml) in order to promote attachment of cells. The follicle

mediated by a cyclic AMP(cAMP)-dependent protein segments were reorganized into a tight monolayer after culture for4 to 5 days.kinase (PKA). H-7 is known to inhibit many kinases,


3REGULATION OF THYROID EPITHELIAL JUNCTIONS

Ca2/ switch experiments. Experiments were carried out 6 to 10 to previous protocols [30]. Replacement of the basal me-days after plating in Tyrode’s salt solution containing 10 mM Tris– dium with Ca2/-free solution containing EGTA caused aHCl, pH 7.3, at 377C; the Ca2/ concentration in complete solution

rapid decrease of RTE toõ100 Vrcm2 (Fig. 2, dotted line)was 1.8 mM. As the integrity of the thyroid junctional complex pre-and a simultaneous increase of the transepithelial fluxviously was shown to depend primarily on Ca2/ present basolaterally

[15], only the basal medium (500 ml) was exchanged for Ca2/-free of [3H]inulin (Fig. 3), indicating paracellular leakage.solution containing 1 mM EGTA (‘‘Ca2/ switch’’); the chelator was Morphologically, this effect was accompanied by a grad-needed to rapidly disrupt junctions as lowering the Ca2/ concentra- ual disintegration of the linear ZO-1 immunofluorescencetion to 1.8 mM caused only a slow and modest impairment of the

(Fig. 1C), which eventually became diffusely distributedepithelial barrier function within the longest time (60 min) studied.in the cytoplasm without any distinct localization to theAgents (forskolin; TSH; H-7; staurosporine; trypsin) were added be-

fore, simultaneous with or after Ca2/ switch as detailed under Re- plasma membrane (not shown). In addition, the cadherinsults. Each type of experiment was performed on triplicate cultures immunoreactivity at AJ changed but at a slower rateand repeated at least three times in independent platings with con- than that observed for ZO-1. After 20 min in low Ca2/,forming results.

when RTE was already maximally reduced, cadherin wasMeasurement of transepithelial electrical resistance (RTE). RTEstill mainly present at the cell borders but the linearacross the thyrocyte monolayer was monitored by the use of a com-fluorescence was focally disconnected typically in the in-bined millivolt/ohmmeter equipped with Ag/AgCl electrodes (Millicell

ERS; Millipore Corp., Bedford, MA). In experiments RTE was inter- tersection area of three or more cells (Fig. 1D). Prolongedmittently measured at room temperature by passing a current pulse incubation (ú30 min) in Ca2/-reduced conditions causedacross the cell layer/filter. Presented data of RTE are subtracted by a general dissociation of AJ and a gradual transfer ofthe bare transfilter resistance, which on average was 100 Vrcm2.

cadherin immunoreactivity from the surface to the inte-Transepithelial flux (FTE) of [3H]inulin. Alterations of paracellu-rior of the cell (not shown).lar permeability in Ca2/ switch experiments were evaluated by re-

cording the flux of [3H]inulin in apical-to-basal direction. The radio- Forskolin added 10 min before Ca2/ chelation dose-tracer was added at a final activity of 1 mCi/ml in Tyrode–Tris buffer, dependently prevented the EGTA-induced loss of RTEpH 7.3, to the apical medium (200 ml). After incubation for 10 min (Fig. 2) while FTE[3H]inulin remained low (Fig. 3); similarat 377C the basal medium (500 ml) was collected and the radioactivity

results were obtained with TSH (not shown here, butdetermined in a Wallac liquid scintillator.see [30]). This indicates that activation of the adenylateImmunofluorescence. Cultures were fixed in ice-cold absolute eth-cyclase/cAMP/PKA pathway antagonized the junction-anol for 30 min, washed twice in PBS, and preincubated for 5 min

in dilution buffer consisting of PBS, pH 7.4, supplemented with 5% disrupting effect of Ca2/ removal. Forskolin by itself re-fat-free milk, 0.1% gelatin, 7.5% sucrose, and 0.02% sodium azide. duced RTE to a new steady-state level of ú2000 Vrcm2

Before the addition of antibodies the cultures were also incubated (Fig. 2, before Ca2/ chelation, and Fig. 4). This change iswith a biotin/avidin blocking kit (Vector Laboratories, Burlingame,not due to leaky junctions but reflects an increased so-CA) to reduce nonspecific staining. E-cadherin was immunolocalizeddium permeability in the apical plasma membrane [37,by a pan-cadherin antibody (dilution 1:200) raised in rabbit against

a fusion protein containing a conserved portion of the cytoplasmic 38]. In fact, the protective effect of forskolin on the barrierdomain which is common to most cadherin subtypes (Sigma); a mono- function was stronger at the doses which gave the mostclonal against the extracellular domain of human E-cadherin (DE- pronounced reduction of RTE prior to Ca2/ chelation (Fig.CMA-1; Sigma) which stains human thyroid cells [36] did not react

2). The linear distribution of ZO-1 (Fig. 1E) and cadherinwith the pig thyrocyte cultures. ZO-1 was immunostained by a rabbitpolyclonal antibody (1:400) directed to a fusion protein containing a (Fig. 1F) at the level of the junctional complex was notZO-1 sequence shared by the two ZO-1 isoforms (Zymed Laboratories, changed by EGTA in forskolin-stimulated cells.Inc., San Francisco, CA). Primary antibodies, which were incubated In accordance with original observations in MDCKat room temperature for 60 min, were detected by biotinylated goat

epithelial cells [25], the protein kinase inhibitor H-7 (50anti-rabbit IgG (Amersham; 1:400; 30 min) followed by FITC-strep-mM) antagonized the negative effect of Ca2/ removal ontavidin (Amersham; 1:300; 30 min); each incubation step was ended

by washing three times in dilution buffer. Filters with immunola- the barrier function of the thyrocyte monolayers (Fig.beled cell monolayers were cut out from the Transwell inserts, 4). RTE decreased to about 4000 Vrcm2, which is consid-mounted on glass with diazobicyclooctane (DABCO; Sigma), and erably higher than values corresponding to even aviewed and photographed in a Nikon FXA light microscope with

small paracellular leakage. In addition, H-7 fully inhib-epifluorescence equipment.ited the accelerated transepithelial flux of [3H]inulin(Fig. 3) and counteracted the redistribution of ZO-1RESULTS(Fig. 1G) and cadherin (Fig. 1H) taking place in lowCa2/. Thus, H-7 had the same ability as TSH/forskolinParacellular Leakage Induced by Removal ofto promote the tight junction barrier when added priorExtracellular Ca2/ Is Prevented by Thyrotropin,to Ca2/ removal.Forskolin, and H-7

Pig thyrocytes at confluence in Transwell culture Forskolin and H-7 Induce Recovery of the Epithelialchambers are known to establish a high RTE, in general Barrier under Low Ca2/ Conditions: Bothú6000 Vrcm2 [15]. As previously described [37], the cell Synergistic and Antagonistic Interactionsborders were outlined by ZO-1 (Fig. 1A) and cadherin(Fig. 1B), indicating the position of the junctional com- The effects of TSH, forskolin, and H-7 on thyroid

epithelial tightness were further compared in experi-plex. Ca2/ switch experiments were performed according



FIG. 1. Immunofluorescence of ZO-1 and cadherin in Transwell-cultured pig thyrocyte monolayers. (A, B) Untreated cultures and beloware cultures depleted of Ca2/ in the basal medium for 20 min without (C, D) or with prestimulation with forskolin (Fo; 50 mM) (E, F) or H-7 (50 mM) (G, H) for 10 min. Ca2/ in the basal medium was depleted by changing to Ca2/-free solution containing 1 mM EGTA; thistreatment is referred to ‘‘EGTA’’ in this as well as the following graphs. Note dissociation of ZO-1 and cadherin (arrows) in EGTA-treatedcells (C, D) and that this is prevented by forskolin (E, F) and H-7 (G, H). Bar, 10 mm.

ments in which their ability induce recovery of RTE in when EGTA was present alone, but already a few min-utes later RTE began to rise although at a slower ratelow Ca2/ medium was examined. First, the stimuli

were added simultaneously with EGTA (Fig. 5). In than that characterizing the initial drop (Fig. 5, leftand middle panels). Maximal recovery of RTE was in-TSH/forskolin-exposed cultures, RTE was almost in-

stantly reduced to a very low level, similar to that seen duced by forskolinú10 mM. H-7 also reversed the effect



FIG. 4. Changes in transepithelial resistance (RTE) by switch toFIG. 2. Forskolin-induced inhibition of epithelial barrier dys-low Ca2/ (EGTA) medium in cultures prestimulated for 10 min withfunction caused by Ca2/ removal from pig thyrocyte monolayers inforskolin (Fo; 50 mM) or H-7 (50 mM). H-7 does not affect RTE on itTranswell culture. Forskolin (Fo), added at different concentrationsown, whereas forskolin immediately reduces RTE to an intermediate(0.1–50 mM) to the basal medium 10 min prior to Ca2/ chelation,level (see text for comments). Both forskolin and H-7 inhibit theprevented dose-dependently the loss of transepithelial resistanceEGTA-induced reduction of RTE. Mean { SD (n Å 3).(RTE); dotted line marks the instant drop of RTE in response to EGTA

alone. Note the intrinsic reducing effect of forskolin on RTE between0 and 5 min; see text for comments. Mean { SD (n Å 3).

of low Ca2/ on RTE (Fig. 5, right panel). The H-7-in-duced recovery often had an earlier onset (Fig. 5) andconsistently reached higher peak levels (Fig. 6A) thanthose caused by TSH or forskolin. Importantly, whencultures were simultaneously stimulated with for-skolin and H-7 at concentrations (50 mM) which gavemaximal recovery for each agent, an additive effect wasobserved (Fig. 6A).

When Ca2/ depletion was allowed for 10 min beforeaddition of forskolin and H-7, the recovery responsesgenerally had a lower magnitude than those earlierinduced (Fig. 6B, compare with 6A). However, anotherdifference was that H-7 was less effective than for-skolin in increasing RTE, except very early during re-covery (Fig. 6B). In fact, the H-7-induced recovery wasinterupted after a short time and diverged from theslow but continuous increase of RTE appearing afterforskolin. Additionally, there was no additive effect butinstead the forskolin-stimulated recovery was com-pletely inhibited by H-7 (Fig. 6B). Prolonged incubation

FIG. 3. Effects of forskolin (Fo) or H-7 on transepithelial flux (ú90 min) under Ca2/-reduced conditions gradually at-(FTE) of [3H]inulin in Ca2/-depleted cultures with or without subse- tenuated all recovery responses (not shown).quent trypsinization. Agents (Fo or H-7, EGTA, and trypsin) were

Taken together, the recovery data obtained underadded in sequence as indicated at 10-min intervals. Following theterminal treatment of each group [3H]inulin flux for 10 min was low Ca2/ conditions suggest that TSH/forskolin andanalyzed. The increase in FTE[3H]inulin caused by Ca2/ removal H-7 act via different although interacting mecha-(EGTA) is prevented by both forskolin and H-7. Trypsin (0.001%) nisms to reinforce the thyroid junctional complex.fully antagonized RTE protection induced by forskolin (Fo; 50 mM)

This possibility was further examined in the next setbut not that by H-7 (50 mM). Trypsin itself had no effect on FTE[3H]-inulin. Mean { SD (n Å 3). of experiments.



FIG. 5. Recovery of RTE induced by different doses of thyrotropin (TSH; left), forskolin (Fo; middle) or H-7 (right) under low Ca2/

conditions. Stimuli were added from the start of Ca2/ depletion. RTE is nearly abolished before restitution takes place in TSH/forskolin-treated cells. EGTA-induced reduction of RTE is retarded by simultaneously added H-7. Mean { SD (n Å 3).

Trypsin Reduced the Junction-Protecting Effect ofTSH/Forskolin but Not That of H-7 orStaurosporineTrypsin-induced proteolysis is classically used to dis-

tinguish between Ca2/-dependent (cadherin-mediated)and Ca2/-independent cell–cell adhesion mechanisms[33, 34]. Cadherins are largely insensitive to trypsin inthe presence of Ca2/, but once Ca2/ is removed only avery small amount (0.001%) of trypsin are needed toimmediately cleave the extracellular domain of themolecule. Higher trypsin concentrations are requiredto degrade Ca2/-independent adhesion molecules [33,34]. It was therefore interesting to examine the effectof trypsin on thyrocyte monolayers in low Ca2/ mediumin which the epithelial barrier was maintained by for-skolin or H-7.

As shown in Fig. 7, 0.001% trypsin present in thebasal medium with normal Ca2/ concentration for 30min did not reduce RTE; higher trypsin concentrations(0.01-0.1%) were also ineffective (not shown). In addi-tion, the junction-protecting effects of forskolin and H-7 was not influenced by limited trypsinization for 10min preceding Ca2/ removal (Fig. 7). However, clearlydifferent responses were obtained when trypsin wasadded after Ca2/ depletion to cultures in which thebarrier function was protected (Fig. 8). In TSH/for-skolin cultures RTE was abolished (Fig. 8, top) andFTE[3H]inulin was substantially increased (Fig. 3). Thelowest effective trypsin concentration to obliterate RTE

varied among cultures from different batches of start-FIG. 6. Interactions of forskolin (Fo; 50 mM) and H-7 (50 mM) in ing tissue at plating but was for some as low as

the restitution of epithelial barrier function. (A) Exposure to agents 0.0001%. In marked contrast, RTE in cultures pre-from the start of Ca2/ depletion. The recovery responses to forskolin treated with H-7 and then transferred to Ca2/-free me-and H-7 on RTE are additive. (B) Ca2/ depletion preceds the addition

dium resisted trypsinization unless high concentra-of forskolin and H-7 by 10 min; incubation then continues in Ca2/-deprived medium. Recovery of RTE is attenuated and retarded com- tions (0.01%) were used (Fig. 8, middle). Staurosporine,pared to that seen when stimulation occurred simultaneously with another kinase inhibitor, was even more potent thanthe addition of EGTA; dotted line indicates the forskolin-induced H-7 to protect the epithelial barrier during trypsiniza-recovery in (A). Recovery responses to forskolin and H-7 are not tion in the absence of Ca2/ (Fig. 8, bottom). The lowsynergistic. In contrast, the H-7-induced recovery levels off and,

sensitivity of H-7-treated cultures to trypsin was con-when combined with forskolin, exerts a dominant negative effect.Mean { SD (n Å 3). firmed in FTE[3H]inulin recordings (Fig. 3).



tures previously challenged with H-7 did not affect thelinear cadherin immunoreactivity at cell–cell contacts(Fig. 9F). However, despite a lasting epithelial barrierin H-7-treated cultures (1028 { 73 Vrcm2; n Å 3), ZO-

FIG. 7. Effect of trypsin on transepithelial resistance (RTE). Tryp-sin (0.001%) present in the basal medium with normal Ca2/ concen-tration for 30 min does not influence RTE. Neither are the protectiveeffects of forskolin (Fo; 50 mM) and H-7 (50 mM) influenced by trypsin-ization when performed before the addition of EGTA; the partialreduction of RTE in the forskolin- and H-7-treated cultures are compa-rable to those observed without trypsin, as shown in Fig. 2. Mean {SD (n Å 3).

Trypsin Induces a Loss of ZO-1 at the Cell Border inLow Ca2/ Despite a Maintained Epithelial Barrierin H-7-Treated Cells

Cultures sequentially exposed to low Ca2/ and tryp-sin were prepared for immunolocalization of ZO-1 andcadherin (Fig. 9). Trypsin (0.001%) potentiated theEGTA-induced redistribution of ZO-1, which dissa-peared completely from the cell periphery and accumu-lated as a diffuse staining in the central portions of thecytoplasm (Fig. 9A; compare with ZO-1 pattern afterCa2/ chelation without trypsin in Fig. 1C). In contrast,cadherin remained to a large extent at the cell borders(Fig. 9B), indicating that the protein although probablylacking the extracellular domain after trypsinizationwas still bound to the plasma membrane. However, thefocal dissociation of cadherin immunofluorescence atcell corners was more frequent than after Ca2/ deple-

FIG. 8. Effect of trypsin on junctional integrity protected by TSHtion alone (see Fig. 1B for comparison). and protein kinase inhibitors in low Ca2/. Cultures (n Å 3) were incu-

In Ca2/-depleted cultures preexposed to forskolin bated in the following sequence (all reagents added to the basal me-dium): TSH (10 mU/ml), H-7 (10 mM), or staurosporine (Stauro; 10 mM)and then trypsinized, RTE had almost dissappeared (50for 10 min; Ca2/ depletion for 10 min (along with continuous exposure{ 14 Vrcm2; nÅ 3) at the time of fixation for immunola-to TSH or kinase inhibitors); trypsin (0.001 or 0.01%) for 30 min underbeling. Along with this, ZO-1 was completely redistrib- ongoing low Ca2/ conditions. The ability of TSH to inhibit RTE is in-

uted from the plasma membrane to the perinuclear stantly abolished by 0.01% trypsin and gradually deteriorated at thecytoplasm (Fig. 9C), similar to that seen in trypsin- lower trypsin concentration (top). RTE in H-7-treated cultures resists

0.001% trypsin and is only slowly and not completely reduced by 0.01%treated controls. Cadherin immunoreactivity remainedtrypsin (middle). Staurosporine-induced protection of RTE is essentiallylargely at the cell periphery (Fig. 9D), but, strikingly,maintained also after exposure to the highest dose of trypsin for 30the dissociation of neighboring cells was general and min (bottom). Dotted lines indicate RTE in control cultures for each

more pronounced than that found in controls (compare panel, treated in the same way as the others except for trypsinization.Mean { SD.with Fig. 1B). In contrast, the same treatment of cul-



FIG. 9. Distribution of ZO-1 (A, C, E) and cadherin (B, D, F) after trypsinization under low Ca2/ conditions. Control cultures (A, B)and cultures preexposed to forskolin (Fo; 50 mM) (C, D) or H-7 (50 mM) (E, F) were incubated in low Ca2/ for 30 min. Trypsin (0.001%) waspresent during the last 20 min, after which the cells were fixed and immunostained. At the time of fixation only cultures pretreated withH-7 (50 mM) retain a high RTE (see text). Cadherin at cell–cell contacts is dissociated (arrows, asterisk) in control (B) and after prestimulationwith forskolin (D), but is unchanged by trypsin in H-7-treated cells (F). ZO-1 is retracted into the perinuclear cytoplasm by trypsin irrespectiveof pretreatment (A, C, E). Bar, 10 mm.

1 was disconnected from the plasma membrane by tryp- tion-protective effect in MDCK epithelial cells switchedto low Ca2/ conditions [25], but the mechanism is Ca2/-sinization in low Ca2/ medium (Fig. 9E).independent [26–28]. In order to further characterizethe regulation of the thyroid junctional complex we ex-DISCUSSIONamined the effect of the protein kinase inhibitor, H-7,

TSH, the main regulator of thyroid hormone produc- and its possible interaction with that of TSH or for-tion, is known to promote the organization of thyro- skolin, a cAMP-generating agent, in primary culturedcytes into follicles [40–42], but the mechanism at the pig thyrocytes.molecular level for this morphoregulatory effect is not We found several differences in the ability of TSH/yet identified. We have previously found [30] that TSH forskolin and H-7 to influence the thyroid epithelialvia cAMP inhibits the dissociation of TJ and AJ in barrier. All agents were able to induce recovery of RTE

filter-cultured thyrocytes deprived of extracellular under low Ca2/ conditions. However, early after Ca2/

Ca2/, suggesting that Ca2/-dependent cell–cell adhe- removal the H-7-induced recovery was more rapid andsion may be positively regulated by TSH. Protein ki- stronger than that elicited by forskolin. Moreover,

when H-7 and forskolin were added together at concen-nase inhibitors (H-7 and others) have a similar junc-



trations which gave the highest individual responses criterion of Ca2/-dependent cell–cell adhesion. Re-cently, long-term TSH stimulation was found to in-an additive effect was obvious. This is in marked con-

trast to the recovery induced 10 min after Ca2/ deple- crease the expression of E-cadherin mRNA and proteinin cultured thyroid cells [43]. The present findingstion, when H-7 was less potent that forskolin. In fact,

in H-7-treated cultures the delayed recovery was short strongly suggest that TSH via the cAMP/PKA signaltransduction pathway in addition rapidly stabilizes theand transient, whereas in the same time interval for-

skolin caused a progressive increase of RTE. Another function of cadherin. Further studies are required toconfirm this and to identify the mechanism at the mo-important feature of the delayed recovery was that H-

7 had a dominant negative effect when the drugs were lecular level. There are several possibilities. One is aconformational change of E-cadherin transduced to thecombined. Thus, depending on the time in low Ca2/

preceding the initiation of recovery H-7 and forskolin extracellular domain which alters the binding proper-ties and makes it resist Ca2/ reduction. This is sup-are either synergistic or antagonistic in action. Alto-

gether, these data strongly suggest that the mecha- ported by the observation in MDCK cells [44], that for-skolin inhibits the ability of anti-uvomorulin antibod-nisms by which TSH/forskolin and H-7 reinforce the

thyroid epithelial barrier are not the same. Moreover, ies to dissociate the junctional complex including TJ,which may be due to a disappearance of epitope(s). ItH-7, known to be a nonselective kinase inhibitor [32],

does not interfere with the TSH/forskolin effect, which is also known that the three-dimensional structure ofE-cadherin varies depending on whether Ca2/ is pres-likely is PKA-dependent, unless Ca2/ depletion pre-

vails for a certain time before the addition of the drugs. ent [12]. Therefore, another mechanism might be thatthe native conformation of E-cadherin is stabilized soThe effect of trypsin on the barrier function in low

Ca2/ conditions further indicated that different mecha- that Ca2/ is no longer required for binding. It is im-portant to note, however, that the precise role of Ca2/nisms were influenced. The experiments were carried

out in view of the fact that trypsin treatment enables in the molecular mechanism of adhesion is still an openquestion [21]. Although the importance of extracellulara distinction between Ca2/-dependent and Ca2/-inde-

pendent cell–cell adhesion. As originally identified in Ca2/ is well documented, recent observations indicatethat also the intracellular Ca2/ level is rapidly changedan aggregation assay of dispersed cells [33, 34], Ca2/-

dependent (cadherin-mediated) adhesion largely re- in Ca2/ switch experiments [16, 45, 46]. Mobilizationof intracellular Ca2/ stores (or apical Ca2/ influx; seesists trypsin in normal culture medium but is rapidly

destroyed (cleavage of the extracellular domain of cad- ref. 46) may thus be another pathway by which epithe-lial junctions can be regulated. TSH has been shownherin) by very low concentrations of trypsin in low Ca2/

medium [33, 34]. We found that H-7 and staurosporine, to increase cytosolic Ca2/ in pig thyrocytes [47]. How-ever, the TSH-induced Ca2/ mobilization is not mim-another potent kinase inhibitor, were able to keep the

barrier function even though the Ca2/-depleted thyro- icked by forskolin [47], suggesting that intracellularCa2/ is less likely to be involved in the action of TSHcytes were exposed to 0.01% trypsin. This confirms pre-

vious findings in MDCK cells [25] and, provided that on thyroid junctions.The present study also shows that the dissociationthe biochemical properties of Ca2/-dependent and -in-

dependent adhesion accounts also for epithelial cell of ZO-1 from TJ in Ca2/-depleted thyrocytes is highlyaugmented by trypsinization. Interestingly, this occursmonolayers, suggests that the kinase inhibitors do not

affect cadherin binding. Indeed, the location and gen- also in H-7-treated cells in which the barrier functionis still maintained. ZO-1, the first identified specificeral morphology of the junctional complex in H-7-

treated lens epithelial cells are unchanged under low component of TJ [48], binds to the cytoplasmic domainof a transmembrane protein, occludin [49], which isCa2/ conditions despite the fact that N-cadherin is

readily cleaved by subsequent trypsinization [26]. As believed to exert the gate and fence functions of TJ[50]. ZO-1 seems to be required for a correct localizationH-7 has pronounced effects on the organization of the

cytoskeleton [28, 29] and also inhibits the paracellular of occudin during the formation of the junctional com-plex [49], but the precise role of ZO-1 in intact TJ isleakage induced by cytochalasins [26], it is hypothe-

sized that kinase inhibitors make epithelial junctions unknown. Provided that occludin is the single or mainTJ barrier molecule, the observation in H-7-treatedCa2/-independent by preventing the AJ-associated ac-

tin ring from being retracted into the central cytoplasm cells implicates that ZO-1 and occludin may be physi-cally disconnected without an immediate increase ofin response to Ca2/ depletion [26, 28]. It is possible

that H-7 and staurosporine act on a similar mechanism the paracellular permeability. Consequently, ZO-1 isnot necessary to maintain the thyroid epithelial bar-in filter-cultured pig thyrocytes.

In marked contrast, the junction-protective effect of rier. An obvious question is then why ZO-1 is discon-nected from the plasma membrane by cell surface tryp-TSH/forskolin was rapidly abolished by light trypsin-

ization (0.001%). This response, demonstrating a very sinization in low Ca2/. With reference to immunoblotdata from studies on lens cells [26] and thyrocytes (ownhigh sensitivity to trypsin-mediated proteolysis only

in the absence of extracellular Ca2/, fulfills a major preliminary results; unpublished) treated with H-7, it



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Received November 14, 1995Revised version received March 1, 1996


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Ca2+-Dependent and Ca2+-Independent Regulation of the Thyroid Epithelial Junction Complex by Protein...

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