Opposing effects of PKCalpha and PKCepsilon on basolateral membrane dynamics in intestinal epithelia

Jaekyung Cecilia Song1, Patangi K. Rangachari2, and Jeffrey B. Matthews1

1 Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267; and 2 Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8S 4L8


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PKC is a critical effector of plasma membrane dynamics, yet the mechanism and isoform-specific role of PKC are poorly understood. We recently showed that the phorbol ester PMA (100 nM) induces prompt activation of the novel isoform PKCepsilon followed by late activation of the conventional isoform PKCalpha in T84 intestinal epithelia. PMA also elicited biphasic effects on endocytosis, characterized by an initial stimulatory phase followed by an inhibitory phase. Activation of PKCepsilon was shown to be responsible for stimulation of basolateral endocytosis, but the role of PKCalpha was not defined. Here, we used detailed time-course analysis as well as selective activators and inhibitors of PKC isoforms to infer the action of PKCalpha on basolateral endocytosis. Inhibition of PKCalpha by the selective conventional PKC inhibitor Gö-6976 (5 µM) completely blocked the late inhibitory phase and markedly prolonged the stimulatory phase of endocytosis measured by FITC-dextran uptake. The PKCepsilon -selective agonist carbachol (100 µM) induced prolonged stimulation of endocytosis devoid of an inhibitory phase. Actin disassembly caused by PMA was completely blocked by Gö-6850 but not by Gö-6976, implicating PKCepsilon as the key isoform responsible for actin disruption. The Ca2+ agonist thapsigargin (5 µM) induced early activation of PKCalpha when added simultaneously with PMA. This early activation of PKCalpha blocked the ability of PMA to remodel basolateral F-actin and abolished the stimulatory phase of basolateral endocytosis. Activation of PKCalpha stabilizes F-actin and thereby opposes the effect of PKCepsilon on membrane remodeling in T84 cells.

protein kinase C isoforms; cytoskeleton; intestinal mucosa; calcium


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PROTEIN KINASE C (PKC) is a family of at least 10 different serine/threonine isoforms (20) that have been implicated in a variety of cellular responses such as membrane trafficking, migration, ion transport, and cell differentiation. Each isoform has distinct enzymological properties, tissue distribution, and subcellular localization (22), implying that each affects a unique complement of biological functions. The basis for this functional diversity and for the selective regulation of individual isoforms is not well understood.

PKC isoforms are usually subdivided into three groups, conventional (cPKC), novel (nPKC), and atypical (aPKC), on the basis of their activation requirements, which, in turn, reflect the structure of their regulatory domains (20, 21). The regulatory domain of cPKC isoforms (alpha , beta I, beta II, and gamma ) contains two common regions, C1 and C2. The C1 region mediates diacylglycerol (DAG) and phorbol ester binding. The presence of a C2 region makes the cPKC isoforms distinct from other subfamilies in that they require Ca2+ for activation. The nPKC isoforms (delta , epsilon , eta , and theta ) contain a C1 region, and, therefore, DAG and phorbol ester binding activate these isoforms. However, nPKC are Ca2+ independent because they lack the C2 region. The aPKC isoforms (zeta  and iota /lambda ) are independent of DAG or Ca2+ and, as a general rule, cannot be directly activated by phorbol esters such as PMA. This structural heterogeneity implies that intracellular Ca2+ is a key determinant of the specific pattern of PKC isoform activation.

We previously showed that in model polarized T84 human intestinal epithelia, PMA induces a dramatic increase in the rate of basolateral fluid-phase endocytosis without affecting endocytosis at the apical membrane (28). Of the four PKC isoforms identified in T84 cells (alpha , epsilon , delta , and zeta ), PMA induced early activation of two novel isoforms, PKCepsilon and PKCdelta , followed by late activation of the conventional isoform PKCalpha (27). PMA was also shown to induce biphasic effects on basolateral endocytosis characterized by an early stimulation period (stimulatory phase) followed by a later return to baseline rates (inhibitory phase) (28). Selective inhibition of PKCepsilon completely abolished the early stimulatory phase, suggesting that PKCepsilon is the key isoform responsible for PMA-induced stimulation of basolateral endocytosis by a mechanism that appeared to involve localized actin disassembly. The basis for the inhibitory phase was not determined, but preliminary data reported in abstract form suggested a possible role for PKCalpha (29). PKCdelta did not appear to be involved in either the stimulatory or inhibitory phase, on the basis of insensitivity to the PKCdelta -specific inhibitor rottlerin.

There have been several reports describing antagonistic effects of different PKC isoforms on the regulation of the same biological function (2, 5, 6, 12, 33). For example, in rat fibroblasts, PKCalpha and PKCdelta were shown to have opposite effects on epidermal growth factor receptor-mediated transformation and phospholipase D activity. PKCbeta I and PKCbeta II had opposite roles in vascular smooth muscle cell proliferation. PKCbeta and PKCzeta mediated opposing effects on proximal tubule Na+/K+-ATPase activity. These considerations suggested to us the possibility that, in T84 cells, PKCalpha could play a counterregulatory role to PKCepsilon in control of basolateral membrane dynamics.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. T84 human intestinal epithelial cells obtained from Dr. Kim Barrett (University of California, San Diego, CA) were grown to confluence as described previously (27).

Fluid-phase endocytosis. Uptake of fluorescein isothiocyanate-dextran (FITC-dextran, MW 12,000, 0.73 mol fluorescein/mol dextran) from the basolateral aspect of confluent T84 monolayers grown on collagen-coated permeable supports (4.7 cm2, 3.0-µm pore size) was measured as described previously (27).

Subcellular fractionation. T84 cells grown to confluence on collagen-coated permeable supports (4.7 cm2) were fractionated into the cytosolic and membrane fractions as described previously (28). Briefly, monolayers were scraped with the cold homogenization buffer (HB) containing 20 mM Tris · HCl, pH 7.5, 250 mM sucrose, 4 mM EDTA, 2 mM EGTA, and Complete protease inhibitor cocktail tablets. The cells were homogenized on ice, and the homogenate was ultracentrifuged at 86,000 g for 50 min at 4°C (TLA 45 rotor, TL-100 Ultracentrifuge; Beckman). The supernatant was designated the cytosolic fraction. The pellet was resuspended in HB containing 0.5% (vol/vol) Triton X-100 and incubated in ice for 30 min. At the end of the incubation period, the samples were centrifuged at 14,000 g for 20 min at 4°C. The resulting supernatant was designated the membrane fraction.

In vitro kinase assay. Confluent T84 monolayers grown on 4.7-cm2 permeable supports were treated with various PKC agonists, and proteins were extracted with the lysis buffer containing 50 mM Tris · HCl, pH 7.5, 150 mM NaCl, 1% (vol/vol) Triton X-100, 2 mM EDTA, 1 mM EGTA, 30 mM sodium pyrophosphate, 50 mM NaF, 100 µM Na3VO4, and Complete protease inhibitor cocktail tablets (27). Polyclonal antibodies against cPKCalpha (2 µg), nPKCepsilon (4 µg), or nPKCdelta (2 µg) were added to each lysate for overnight rotation at 4°C. After incubation, immune complexes were precipitated with the use of protein A-agarose beads, resuspended in kinase buffer (35 mM Tris · HCl, pH 7.5, 10 mM MgCl2, 0.5 mM EGTA, 10 µCi [gamma -32P]ATP, 60 µM cold ATP, and 1 mM Na3VO4), and incubated with myelin basic protein (MBP) as a substrate at 30°C for 30 min. After incubation, the reaction was terminated with Laemmli sample and subjected to SDS-PAGE (15% gels). The gel was then dried and subjected to autoradiography.

Gel electrophoresis and Western blotting. Equal amounts of protein (~50 µg/sample) were subjected to SDS-PAGE and Western blot as described previously (27). Briefly, proteins were separated on 8% gels, transblotted to nitrocellulose membranes, and incubated with the polyclonal antibodies to different PKC isoforms for 1 h. After brief washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody for 1 h, washed, and visualized with enhanced chemiluminescence (ECL) detection reagent.

Immunofluorescence and microscopy. Monolayers grown on 0.33-cm2 permeable supports were treated with various agonists and prepared for confocal microscopy as described previously (27). Briefly, cells were fixed and permeabilized with 0.1% (vol/vol) Triton X-100. Cells were then incubated with the blocking buffer (1% normal goat serum, 3% BSA in PBS) followed by the primary antibody against PKCalpha . After overnight incubation in a moisture chamber at 4°C, monolayers were incubated in rhodamine-conjugated goat anti-rabbit polyclonal IgG along with FITC-phalloidin for F-actin staining. Confocal images were acquired with a Zeiss inverted microscope equipped with MRC-1024 and Lasersharp software (Bio-Rad).

Materials. Tissue culture reagents and protein A-agarose beads were purchased from Invitrogen. Gel electrophoresis and Western blotting reagents were from Bio-Rad, with the exception of ECL detection reagent, which was purchased from Amersham. Complete protease inhibitor cocktail tablets were from Roche. Anti-PKCalpha was obtained from Sigma and Santa Cruz Biotechnology for Western blotting and immunostaining, respectively. Anti-PKCepsilon was purchased from Santa Cruz Biotechnology. Secondary antibodies were obtained from Bio-Rad and Jackson Laboratories for Western blotting and immunostaining, respectively. Vectashield mounting medium was from Vector Laboratories. The PKC inhibitors Gö-6976, Gö-6850, and rottlerin were obtained from Calbiochem. [gamma -32P]ATP with specific activity of 3,000 Ci/mmol was purchased from NEN. All other chemicals were from Sigma.

Statistical analysis. Data are reported as means ± SE. Data were analyzed by one-way ANOVA with Bonferroni/Dunn's post hoc test for comparison with control.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of PKCepsilon and PKCalpha are temporally associated with stimulation and inhibition of basolateral endocytosis, respectively. Our previous results (28) suggested that PMA elicits biphasic effects on basolateral uptake of FITC-dextran in T84 monolayers. This was confirmed in repeated experiments, shown in Fig. 1A, that extended the time course of observation over 150 min. During the initial stimulatory phase, 100 nM PMA progressively increased basolateral endocytosis, reaching a peak at ~60 min (Fig. 1A). Subsequently, during the inhibitory phase, basolateral endocytosis slowly returned to its basal level. To elucidate the specific PKC isoform that may be involved in each phase of the endocytosis, we compared this response to the time course of activation of the conventional isoform PKCalpha and the novel isoform PKCepsilon . The results of these isoform translocation and in vitro kinase assays, shown in Fig. 1, B and C, are similar to findings in our earlier report (27). For example, in a new set of experiments, 100 nM PMA initially induced translocation of PKCepsilon from the cytosolic to the membrane fraction during the 30- to 60-min window that corresponds to the initial stimulatory phase of endocytosis (Fig. 1B), whereas translocation of PKCalpha lagged ~1 h behind the activation of PKCepsilon and was temporally associated with the inhibitory phase. In vitro kinase assays for PKCepsilon and PKCalpha were consistent with these translocation data (Fig. 1C) and with our earlier reported results (29).


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Fig. 1.   Activation of PKCepsilon and PKCalpha is temporally associated with stimulation and inhibition of basolateral endocytosis, respectively. A: basolateral uptake of FITC-dextran (BLFD) was measured to assess the rate of basolateral endocytosis in T84 monolayers grown on 4.7-cm2 permeable supports. Treatment with 100 nM PMA elicited biphasic effects on basolateral endocytosis. During the initial stimulatory phase, PMA progressively increased basolateral uptake, reaching a peak at ~60 min. During the subsequent 2 h of inhibitory phase, basolateral uptake returned to the basal level (each n = 3). *P < 0.05 compared with control. B: cytosolic (C) and membrane (M) fractions of T84 homogenates were subjected to SDS-PAGE and blotted with PKC isoform-specific antibodies to examine subcellular redistribution of PKC isoforms in response to PMA. PKCepsilon promptly translocated from the cytosolic to membrane fraction as early as 15 min after PMA (100 nM) addition. Translocation of PKCalpha , however, was not evident until 60 min after treatment. Activation of both PKCepsilon and PKCalpha continued for at least 4 h without significant degradation. Similar experiments were confirmed in triplicate experiments. C: monolayers were treated with 100 nM PMA and subjected to in vitro kinase assay for direct assessment of PKCepsilon and PKCalpha activity. Both isoforms were immunoprecipitated with the isoform-specific antibodies and subjected to kinase reaction with myelin basic protein (MBP). PMA time-dependently increased the activities of PKCepsilon as shown by the steady increase in intensity of the 19-kDa MBP band. Activation of the conventional isoform PKCalpha was not evident until 2 h after PMA addition. Representative blots are shown from the 3 separate experiments.

To begin to determine whether activation of PKCepsilon and PKCalpha functionally correlated with these two phases of endocytosis elicited by PMA, we used two PKC-specific inhibitors, Gö-6976 and Gö-6850, for which we have previously validated the isoform selectivity profile (27). Gö-6976 is largely selective for (Ca2+ dependent) cPKC isoforms in vitro, whereas Gö-6850 blocks both cPKC and nPKC (14, 34). In T84 cells, we found that Gö-6976 at 5 µM exerted a slight inhibitory effect on PKCdelta in addition to PKCalpha , but there was no evidence of inhibition of PKCepsilon .

We found that inhibition of PKCalpha with pretreatment with Gö-6976 had a potentiating effect on basolateral endocytosis triggered by PMA. When Gö-6976 was added before PMA treatment, FITC-dextran uptake from the basolateral buffer was greater than when PMA was added alone (Fig. 2A). By 60 min of treatment with PMA, PKCalpha began to appear at the membrane fraction (Fig. 1B) and inhibition of PKCalpha activation by Gö-6976 caused further enhancement of endocytosis induced by PMA. Furthermore, inhibition of PKCalpha by Gö-6976 completely blocked the late inhibitory phase of endocytosis and markedly prolonged the stimulatory phase of endocytosis. This effect was not seen with the PKCdelta -specific inhibitor rottlerin (Fig. 2B) at a concentration demonstrated by kinase assay to block PKCdelta activity, excluding a role for this isozyme in the inhibitory phase. Thus the delayed activation of PKCalpha by PMA temporally and functionally correlates with the decline in basolateral endocytosis to basal levels after 2 h of PMA treatment. We previously showed that inhibition of both PKCepsilon and PKCalpha by Gö-6850 blocked both the stimulatory and inhibitory phase of PMA-elicited response (28).


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Fig. 2.   PKCepsilon is stimulatory whereas PKCalpha is inhibitory for basolateral endocytosis. Monolayers were pretreated with 1 of the 3 PKC inhibitors---Gö-6976 for inhibition of the conventional isoform, Gö-6850 for inhibition of both the conventional and novel isoforms, and rottlerin for inhibition of PKCdelta ---before PMA addition, and basolateral uptake of FITC-dextran was subsequently measured from the triplicate Transwell inserts. A: Gö-6976 (5 µM) completely blocked the late inhibitory phase and markedly prolonged the stimulatory phase of endocytosis, suggesting that late activation of PKCalpha is responsible for returning the basolateral uptake to the basal level. Gö-6850 (5 µM), on the other hand, blocked both the stimulatory and inhibitory phases of the PMA-elicited response, indicating that PKCepsilon is the stimulator for endocytosis. B: comparison of Gö-6976, Gö-6850, and rottlerin for their effects on the late inhibitory phase of the PMA-elicited endocytosis (2 h PMA). Rottlerin (10 µM) had no effect on the late inhibitory phase compared with control. *P < 0.05 compared with control. dagger P < 0.05 compared with PMA alone.

These findings strongly suggest that PKCepsilon stimulates, whereas PKCalpha inhibits, basolateral endocytosis in T84 cells. Experiments with a second PKC agonist, carbachol (CCh), indirectly supported this concept. We previously reported that CCh, unlike PMA, activates only PKCepsilon and not PKCalpha in this model (Fig. 3A) (27, 28) and that the stimulation of endocytosis by CCh was blocked by Gö-6850 but not Gö-6976 (28). However, CCh was noted to induce a more strikingly sustained stimulatory phase than PMA, as shown in Fig. 3B. CCh-elicited stimulation of endocytosis did not diminish until 6 h after treatment (data not shown). In the case of CCh, the termination of the endocytosis response was not associated with activation of PKCalpha but, rather, deactivation of PKCepsilon determined by translocation assay (Fig. 3A).


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Fig. 3.   Carbachol (CCh)-stimulated endocytosis is sustained because of the absence of PKCalpha activation. A: monolayers were treated with 100 µM CCh for the times indicated and subjected to subcellular fractionation and Western blot with antibodies against PKCepsilon and PKCalpha . PKCepsilon rapidly translocated to the membrane in response to CCh and remained active for at least 2 h. In contrast, PKCalpha remained associated with the cytosolic fraction. Representative blots of 3 separate experiments are shown. B: monolayers were pretreated with either 5 µM Gö-6976 or 5 µM Gö-6850 before CCh addition, and basolateral uptake of FITC-dextran was measured from the triplicate Transwell inserts after incubation with CCh for the various times indicated. Unlike PMA, CCh-induced stimulation of endocytosis was sustained for at least 2 h and was unaffected by Gö-6976. Gö-6850 completely abolished stimulation of endocytosis, implicating PKCepsilon as the key isoform for stimulation of uptake. *P < 0.05 compared with control.

PMA induces actin rearrangement via activation of PKCepsilon , not PKCalpha . We previously showed that PMA increases basolateral endocytosis via disruption of actin cytoskeleton (28). As shown in Fig. 4, PMA induced significant remodeling of basolateral F-actin and condensation of staining around the cell periphery. We examined sensitivity of PMA-elicited cytoskeletal remodeling to isoform-selective PKC inhibitors. Pretreatment with Gö-6850 but not Gö-6976 attenuated this actin remodeling, suggesting that PKCepsilon is the isoform responsible for actin disassembly caused by PMA. Inhibition of PKCalpha by Gö-6976 did not attenuate, and instead appeared to qualitatively exacerbate, the degree of disruption of the actin cytoskeleton.


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Fig. 4.   PMA disrupts actin cytoskeleton via activation of PKCepsilon , not PKCalpha . Monolayers treated with 100 nM PMA in the presence or absence of PKC inhibitors were stained with FITC-phalloidin and subjected to confocal microscopy. Three fields from each slide were chosen at random and en face images of basal stress fibers were obtained from the optical section of ~6 µm above the basal membrane. Representative images from the multiple experiments are shown. In control monolayers (A), basal stress fibers appeared as randomly dispersed homogeneous filaments. After 1 h of exposure to 100 nM PMA (B), F-actin was displaced toward the periphery of cells and basal stress fibers were significantly attenuated. Pretreatment with Gö-6976 (C) failed to prevent PMA-induced actin rearrangement, and, in fact, basal stress fibers appear more severely disrupted. Pretreatment with Gö-6850 (D), in contrast, completely blocked actin disassembly caused by PMA.

Thapsigargin prevents stimulation of basolateral endocytosis by PMA. We wondered whether the combination of PMA plus the Ca2+-ATPase inhibitor thapsigargin would accelerate activation of PKCalpha and allow us to more directly address the potential opposing actions of PKCalpha and PKCepsilon . Indeed, this proved to be the case. Thapsigargin alone had no effect on PKCalpha or PKCepsilon . However, the combined addition of PMA and thapsigargin accelerated activation of PKCalpha . As shown in Fig. 5A, PKCalpha had already translocated to the membrane by 30 min. Activation of PKCepsilon by PMA, in contrast, was not affected by thapsigargin. Moreover, the combined addition of thapsigargin and PMA failed to stimulate basolateral endocytosis (Fig. 5B); stated differently, thapsigargin blocked the ability of PMA to stimulate endocytosis. The simultaneous activation of PKCalpha and PKCepsilon by costimulation with thapsigargin and PMA led to inhibition of the early stimulatory effects of PMA on basolateral endocytosis. In contrast to thapsigargin, the Ca2+ agonist CCh, which does not induce early activation of PKCalpha either alone or in combination with PMA, did not antagonize the effects of PMA on basolateral endocytosis. In fact, combined addition of CCh and PMA exaggerated the initial stimulatory phase (data not shown). Although CCh may have other effects that may interfere with PMA's ability to induce basolateral endocytosis, these data further strengthen the possibility that PKCalpha may be involved in inhibition of endocytosis.


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Fig. 5.   Simultaneous activation of PKCalpha with PKCepsilon prevents increase in basolateral endocytosis by PMA. A: PMA and thapsigargin (Tg) were added either separately or simultaneously to T84 monolayers, and translocation of PKC isoforms was examined. As shown previously, PKCalpha remained inactive after 30 min of PMA. Elevation of intracellular Ca2+ by Tg had no effect on PKCalpha when added alone. However, when PMA and Tg were added simultaneously, PKCalpha translocated to the membrane as early as 30 min after treatment. Translocation of PKCepsilon was unaffected by Tg. Representative blots of 3 separate experiments are shown. B: Tg alone had no effect on basolateral uptake of FITC-dextran. However, when Tg was added together with PMA, the ability of PMA to increase basolateral endocytosis was completely abolished, suggesting that activation of PKCalpha inhibits the effect of PKCepsilon to stimulate basolateral endocytosis. *P < 0.05 compared with control. dagger P < 0.05 compared with PMA alone.

To further address the mechanism whereby thapsigargin blocks PMA-elicited endocytosis, we examined the role of Ca2+ entry pathways. To do so, we removed extracellular Ca2+ from either the apical or basolateral buffer during the combined treatment with PMA and thapsigargin. As shown in Fig. 6A, removal of apical Ca2+ did not alter the translocation of PKCalpha by PMA and thapsigargin. However, removal of basolateral Ca2+ completely blocked translocation of PKCalpha , suggesting that basolateral Ca2+ entry is required for PKCalpha activation. Removal of basolateral Ca2+ also prevented the ability of thapsigargin to inhibit PMA-stimulated endocytosis (Fig. 6B). Because thapsigargin is known to activate store-operated Ca2+ channels (SOCs) (24) that are thought to be restricted to the basolateral membrane of T84 cells (11), we examined whether the SOC inhibitor La3+ (1) would affect membrane translocation of PKCalpha . As shown in Fig. 6C, membrane translocation of PKCalpha by thapsigargin and PMA was completely abolished by the presence of the SOC-specific inhibitor La3+, and, as evidenced by data not shown, thapsigargin did not block PMA-stimulated endocytosis in the presence of basolateral La3+.


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Fig. 6.   Basolateral Ca2+ entry via store-operated Ca2+ channels (SOCs) is required for activation of PKCalpha by PMA and Tg. A: PKC translocation by PMA and Tg was examined after removal of extracellular Ca2+ from either the apical or basolateral buffer. The absence of apical Ca2+ had no effect on membrane translocation of PKCalpha or PKCepsilon induced by PMA and Tg. However, removal of basolateral Ca2+ completely blocked translocation of PKCalpha , suggesting that basolateral Ca2+ is critical for its activation. Translocation of PKCepsilon was unaffected by removal of basolateral Ca2+. B: removal of basolateral Ca2+ prevented the ability of Tg to inhibit PMA-stimulated increase in basolateral endocytosis. In fact, endocytosis by PMA and Tg was further stimulated in the absence of basolateral Ca2+. Removal of apical Ca2+ had no effect. *P < 0.05 compared with control. dagger P < 0.05 compared with PMA alone. C: the SOC-specific inhibitor La3+ (5 µM) was added 15 min before addition of Tg and PMA, and PKCalpha translocation was examined. Inhibition of basolateral Ca2+ entry via SOCs completely inhibited translocation of PKCalpha by PMA and Tg, suggesting that elevation of local Ca2+ at the basolateral domain is important for translocating PKCalpha . All experiments were performed in triplicate.

PKCalpha rapidly translocates to the basal membrane upon addition of thapsigargin with PMA. We previously showed that inactive PKCalpha is localized in the basal cytoplasm of T84 cells (27). Vertical images captured from confocal microscopy revealed that PKCalpha is found in basal zone of the cell in a diffuse cytoplasmic pattern in control monolayers (Fig. 7A). When PMA was added alone, PKCalpha moved apically and was found in the apical domain (Fig. 7B). In striking contrast, with PMA plus thapsigargin, PKCalpha rapidly cleared from the basal cytoplasm and relocated to the basal membrane (Fig. 7D), suggesting that Ca2+ influx in the basal region could redirect the subcellular localization of PKCalpha from the apical domain (without thapsigargin) to the basal membrane (with thapsigargin). Thapsigargin alone without PMA-induced elevation of DAG had no effect on PKCalpha distribution (Fig. 7C).


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Fig. 7.   PKCalpha translocates to basal membrane upon addition of Tg with PMA. T84 monolayers grown on 0.33-cm2 permeable supports were treated with PMA and Tg, and translocation of PKCalpha was visualized by immunolabeling and confocal microscopy. PKCalpha was stained red by addition of anti-PKCalpha with the rhodamine-conjugated secondary antibody. Red bars on the right side of each image denote regions of PKCalpha localization. F-actin was stained green by FITC-phalloidin to outline the cell boundary. Both the apical and basal boundaries of the monolayers are indicated by blue arrows (base line). Three representative images are shown for each treatment condition. A: in control monolayer, PKCalpha was dispersedly localized at the basal cytoplasm in a diffuse cytoplasmic pattern. PKCalpha was mostly localized above the basal boundary formed by F-actin staining. B: after 1 h of treatment with PMA, PKCalpha became clearly localized to the apical membrane and subapical cytoplasmic domain. C: Ca2+ influx by Tg alone did not affect distribution of PKCalpha . D: simultaneous addition of PMA with Tg cleared PKCalpha from the basal cytoplasm. Staining was restricted to the basal line of F-actin, implicating association of PKCalpha with the basal membrane.

PMA-mediated actin rearrangement is prevented by thapsigargin. Because thapsigargin prevented the PMA-elicited increase in basolateral endocytosis, we anticipated that thapsigargin would also inhibit PMA-induced actin disassembly. Indeed, this was the case, as shown in Fig. 8D. Pretreatment with the PKCalpha inhibitor Gö-6976 abolished this effect of thapsigargin (Fig. 8E). This finding strongly suggests that thapsigargin-induced translocation and/or activation of PKCalpha to the basal membrane (in the context of PMA treatment) inhibits PKCepsilon -mediated actin disassembly as well as stimulation of basolateral endocytosis.


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Fig. 8.   PKCepsilon -mediated actin disassembly is prevented by early activation of PKCalpha . Monolayers were treated with PMA and Tg in the presence or absence of Gö-6976. Cells were then fixed, permeabilized, and incubated with FITC-phalloidin for F-actin staining. Representative images from the multiple experiments are shown. A: control monolayer with intact basal stress fibers. B: 30 min of treatment with Tg alone did not affect F-actin arrangement. C and D: PMA-induced disruption of F-actin (C) was prevented by simultaneous addition of PMA with Tg (D). E: pretreatment with Gö-6976 abolished the effect of Tg on stabilization of F-actin and augmented PMA-induced disruption of actin.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In our earlier report, we found that a variety of physiological and pharmacological activators of the novel Ca2+-independent PKCepsilon isoform rapidly induced actin remodeling and enhanced endocytosis at the basolateral aspect of polarized T84 monolayers (27). In the present study, we developed evidence to suggest that activation of the conventional Ca2+-dependent PKCalpha isoform opposes this action of PKCepsilon . Treatment of T84 monolayers with PMA was observed to sequentially activate PKCepsilon and PKCalpha in temporal association with an early stimulatory and a later inhibitory phase of basolateral endocytosis. The inhibitory phase was eliminated by concurrent treatment with Gö-6976, a selective Ca2+-dependent PKC inhibitor that has been validated in our system, at the concentration used, to be largely selective for PKCalpha . Cytoskeletal remodeling induced by PMA was prevented by the PKCepsilon /PKCalpha inhibitor Gö-6850 but was exacerbated by Gö-6976. Although the Ca2+-ATPase inhibitor thapsigargin did not by itself affect PKC isoform activity, addition of thapsigargin plus PMA induced simultaneous rather than sequential activation of PKCepsilon and PKCalpha . Moreover, thapsigargin prevented PMA-elicited stimulation of basolateral endocytosis and actin remodeling. Interestingly, this action of thapsigargin depended on the presence of Ca2+ in the basolateral bath and was abrogated by the SOC inhibitor La3+, suggesting that basolaterally restricted capacitive Ca2+ entry may play a key strategic role in activation of PKCalpha and in regulation of dynamic basolateral membrane/cytoskeletal remodeling. This concept was further supported by the observation that CCh, in contrast to PMA, activates only PKCepsilon and not PKCalpha . The different pattern of isoform activation between PMA and CCh appears to account for the different time courses of their effects on basolateral endocytosis. CCh induced an extended stimulation of fluid-phase tracer uptake without the subsequent Gö-6976-sensitive inhibitory phase observed with PMA attributable to PKCalpha .

The subcellular localization of inactive PKC isoforms and their dynamic translocation to other subcellular compartments upon activation likely accounts for specificity in regulation of various biological processes (3, 8, 15). Spatial constraints may therefore determine the relative availability or accessibility of key activating cofactors to the PKC isozymes. Thus the location of inactive cPKC or nPKC isoforms in reference to sites of DAG generation in response to phospholipase-coupled membrane receptors could determine their degree of activation. In our initial report (28), we found that the DAG mimetic PMA sequentially activated PKCepsilon and then PKCalpha . CCh, an acetylcholine analog that induces DAG generation via basolaterally located M3 muscarinic receptors, was also shown to activate PKCepsilon ; however, there was no evidence of activation of PKCalpha by CCh (27). We were puzzled by this observation, given the known ability of CCh to generate DAG and to increase cytosolic Ca2+ from phosphoinositol-sensitive stores. One possible explanation is that DAG production in response to CCh occurs only in the limited vicinity of the basolateral M3 receptor and is available only to PKCepsilon within this microdomain, whereas PMA from the bulk solution can diffuse throughout the cytoplasm in essentially unlimited capacity and can reach both PKCepsilon and PKCalpha . Alternatively, the increase in cytosolic Ca2+ elicited by CCh could be spatially restricted to a subcellular localization in which either DAG is not available or inactive PKCalpha is scarce.

Intracellular Ca2+ concentration ([Ca2+]i) plays a central signaling role for a variety of cellular functions. In polarized epithelial cells such as pancreatic acinar cells, Ca2+ signaling has been shown to occur in a highly compartmentalized fashion (35). Inositol 1,4,5-trisphosphate (IP3)-elicited [Ca2+]i release begins in an apical "trigger zone" (9, 10, 32), where the vast majority of IP3 receptors are localized (13, 19, 36). Spreading of Ca2+ through the cytoplasm is modulated by activation of ryanodine receptors localized at the basal pole of acinar cells (30). Ca2+ spreading is also dependent on agonist concentration (31). When cells are stimulated to low levels, Ca2+ spikes remain restricted to the apical pole. Greater levels of stimulation cause Ca2+ waves to propagate toward the basal pole. These findings indicate that agonist-specific regulation of Ca2+ signaling and compartmentalization are likely to exist in polarized cells and that activation of Ca2+-dependent PKC isoforms could be precisely regulated on the basis of agonist type.

Thapsigargin prevents refilling of Ca2+ stores by inhibiting Ca2+-sequestering ATPase pumps in the endoplasmic reticulum membrane. This slowly depletes Ca2+ stores and thereby activates SOCs. We have found that, in T84 cells, the SOCs activated by thapsigargin are functionally restricted to the basolateral membrane domain (26). Although CCh is known to activate SOCs in other cell systems, we have found no evidence of sustained activation of basolateral membrane SOCs by CCh in T84 cells (Ref. 26 and unpublished data). Thus, whereas both thapsigargin and CCh induce an increase in [Ca2+]i, only thapsigargin is associated with sustained activation of basolateral SOCs. In the present study, we were able to use the Ca2+-ATPase inhibitor thapsigargin to selectively manipulate the timing of activation of PKCalpha after PMA stimulation. We found that thapsigargin prevented PMA-stimulated endocytosis, an effect that was inhibited by the PKCalpha -selective inhibitor Gö-6976 and that appeared to require basolateral Ca2+ entry via a La3+-sensitive pathway.

Translocation of specific PKC isoforms to distinct intracellular structures following activation likely represents one mechanism for determining their unique physiological roles (17). Targeting of activated PKC isoforms involves association with anchoring proteins such as receptors for activated C-kinase (RACKs). RACKs are thought to increase PKC phosphorylating efficiency by stabilizing the active kinase near its target substrate (25). RACKs specific for different PKC isoforms have been identified (4, 18). Both PMA and CCh appear to induce PKCepsilon translocation specifically to the basolateral domain. Although the specific RACK(s) governing this response remains uncertain, F-actin has been shown to represent a PKCepsilon -specific RACK (23); in addition, beta '-COP, which has been associated with caveolae, may also be a RACK for PKCepsilon (4).

We previously showed that stimulation of basolateral endocytosis by PMA and CCh involved remodeling of the F-actin cytoskeleton via PKCepsilon and the actin cross-linker MARCKS (myristoylated alanine-rich C kinase substrate) (28). In the present study, we found that thapsigargin prevents PMA-induced actin remodeling and stimulation of endocytosis in association with activation of PKCalpha , an effect blocked by Gö-6976. The detailed basis for PKCalpha antagonism of PKCepsilon -induced actin disassembly is unclear. The small GTPase protein RhoA, a well-known regulator of stress fiber formation in many cell types, is a candidate target for PKCalpha (7, 16). One possible scenario, therefore, is that thapsigargin, by inducing basolateral Ca2+ influx via SOCs, allows PMA to activate PKCalpha and, in turn, RhoA, which then prevents remodeling of F-actin cytoskeleton by PKCepsilon .

In summary, we have found that activation of PKCalpha , whether by PMA alone or in conjunction with thapsigargin, antagonizes the ability of PKCepsilon to disassemble basolateral F-actin and stimulate basolateral membrane endocytosis in a model intestinal epithelium. Our data suggest that cytoskeletal and membrane structure may be dynamically modulated by a balance between nPKCepsilon and cPKCalpha that appears to depend on subtleties in agonist-regulated subcellular redistribution of the isoenzymes and the microorganization of Ca2+ signaling.


    FOOTNOTES

Address for reprint requests and other correspondence: J. B. Matthews, Dept. of Surgery, Univ. of Cincinnati College of Medicine, 231 Albert B. Sabin Way, PO Box 670558, Cincinnati, OH 45267-0558 (E-mail: Jeffrey.Matthews{at}uc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

July 24, 2002;10.1152/ajpcell.00105.2002

Received 7 March 2002; accepted in final form 17 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aussel, C, Marhaba R, Pelassy C, and Breittmayer JP. Submicromolar La3+ concentrations block the calcium release-activated channel, and impair CD69 and CD25 expression in CD3- or thapsigargin-activated Jurkat cells. Biochem J 313: 909-913, 1996[ISI][Medline].

2.   Chen, C, and Mochly-Rosen D. Opposing effects of delta and xi PKC in ethanol-induced cardioprotection. J Mol Cell Cardiol 33: 581-585, 2001[ISI][Medline].

3.   Chen, ZZ, McGuire JC, Leach KL, and Cambier JC. Transmembrane signaling through B cell MHC class II molecules: anti-Ia antibodies induce protein kinase C translocation to the nuclear fraction. J Immunol 138: 2345-2352, 1987[Abstract/Free Full Text].

4.   Csukai, M, Chen CH, De Matteis MA, and Mochly-Rosen D. The coatomer protein beta'-COP, a selective binding protein (RACK) for protein kinase Cepsilon. J Biol Chem 272: 29200-29206, 1997[Abstract/Free Full Text].

5.   Efendiev, R, Bertorello AM, and Pedemonte CH. PKC-beta and PKC-zeta mediate opposing effects on proximal tubule Na+,K+-ATPase activity. FEBS Lett 456: 45-48, 1999[ISI][Medline].

6.   Gusovsky, F, and Gutkind JS. Selective effects of activation of protein kinase C isozymes on cyclic AMP accumulation. Mol Pharmacol 39: 124-129, 1991[Abstract].

7.   Hall, A. Rho GTPases and the actin cytoskeleton. Science 279: 509-514, 1998[Abstract/Free Full Text].

8.   Halsey, DL, Girard PR, Kuo JF, and Blackshear PJ. Protein kinase C in fibroblasts. Characteristics of its intracellular location during growth and after exposure to phorbol esters and other mitogens. J Biol Chem 262: 2234-2243, 1987[Abstract/Free Full Text].

9.   Ito, K, Miyashita Y, and Kasai H. Kinetic control of multiple forms of Ca2+ spikes by inositol trisphosphate in pancreatic acinar cells. J Cell Biol 146: 405-413, 1999[Abstract/Free Full Text].

10.   Kasai, H, and Augustine GJ. Cytosolic Ca2+ gradients triggering unidirectional fluid secretion from exocrine pancreas. Nature 348: 735-738, 1990[ISI][Medline].

11.   Kerstan, D, Thomas J, Nitschke R, and Leipziger J. Basolateral store-operated Ca2+-entry in polarized human bronchial and colonic epithelial cells. Cell Calcium 26: 253-260, 1999[ISI][Medline].

12.   Konishi, H, Matsuzaki H, Takaishi H, Yamamoto T, Fukunaga M, Ono Y, and Kikkawa U. Opposing effects of protein kinase C delta and protein kinase B alpha on H2O2-induced apoptosis in CHO cells. Biochem Biophys Res Commun 264: 840-846, 1999[ISI][Medline].

13.   Lee, MG, Xu X, Zeng W, Diaz J, Wojcikiewicz RJ, Kuo TH, Wuytack F, Racymaekers L, and Muallem S. Polarized expression of Ca2+ channels in pancreatic and salivary gland cells. Correlation with initiation and propagation of [Ca2+]i waves. J Biol Chem 272: 15765-15770, 1997[Abstract/Free Full Text].

14.   Martiny-Baron, G, Kazanietz M, Mischak H, Blumberg P, Kochs G, Hug H, Marme D, and Schachtele C. Selective inhibition of protein kinase C isozymes by the indolocarazole Gö6976. J Biol Chem 268: 9194-9197, 1993[Abstract/Free Full Text].

15.   Masmoudi, A, Labourdette G, Mersel M, Huang FL, Huang KP, Vincendon G, and Malviya AN. Protein kinase C located in rat liver nuclei. Partial purification and biochemical and immunochemical characterization. J Biol Chem 264: 1172-1179, 1989[Abstract/Free Full Text].

16.   Meacci, E, Donati C, Cencetti F, Romiti E, and Bruni P. Permissive role of protein kinase C alpha but not protein kinase C delta in sphingosine 1-phosphate-induced RhoA activation in C2C12 myoblasts. FEBS Lett 482: 97-101, 2000[ISI][Medline].

17.   Mochly-Rosen, D, Henrich CJ, Cheever L, Khaner H, and Simpson PC. A protein kinase C isozyme is translocated to cytoskeletal elements on activation. Cell Regul 1: 693-706, 1990[ISI][Medline].

18.   Mochly-Rosen, D, Smith BL, Chen CH, Disatnik MH, and Ron D. Interaction of protein kinase C with RACK1, a receptor for activated C- kinase: a role in beta protein kinase C mediated signal transduction. Biochem Soc Trans 23: 596-600, 1995[ISI][Medline].

19.   Nathanson, MH, Fallon MB, Padfield PJ, and Maranto AR. Localization of the type 3 inositol 1,4,5-trisphosphate receptor in the Ca2+ wave trigger zone of pancreatic acinar cells. J Biol Chem 269: 4693-4696, 1994[Abstract/Free Full Text].

20.   Newton, AC. Protein Kinase C: structure, function and regulation. J Biol Chem 268: 28495-28498, 1995.

21.   Newton, AC. Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem Rev 101: 2353-2364, 2001[ISI][Medline].

22.   Nishizuka, Y, and Nakamura S. Lipid mediators and protein kinase C for intracellular signalling. Clin Exp Pharmacol Physiol 22, Suppl1: S202-S203, 1995[Medline].

23.   Prekeris, R, Hernandez RM, Mayhew MW, White MK, and Terrian DM. Molecular analysis of the interactions between protein kinase C-epsilon and filamentous actin. J Biol Chem 273: 26790-26798, 1998[Abstract/Free Full Text].

24.   Putney, JW, Jr, and Bird GS. The inositol phosphate-calcium signaling system in nonexcitable cells. Endocr Rev 14: 610-631, 1993[ISI][Medline].

25.   Ron, D, Chen CH, Caldwell J, Jamieson L, Orr E, and Mochly-Rosen D. Cloning of an intracellular receptor for protein kinase C: a homolog of the beta subunit of G proteins. Proc Natl Acad Sci USA 91: 839-843, 1994[Abstract].

26.   Song, J, Rangachari PK, and Matthews JB. Ca2+-dependent Cl- secretion in T84 epithelia: role of SOCs and Na+-K+ ATPase (Abstract). Gastroenterology 120, Suppl 1: A527, 2001.

27.   Song, JC, Hanson CM, Tsai V, Farokhzad OC, Lotz M, and Matthews JB. Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms. Am J Physiol Cell Physiol 281: C649-C661, 2001[Abstract/Free Full Text].

28.   Song, JC, Hrnjez BJ, Farokhzad OC, and Matthews JB. PKC-epsilon regulates basolateral endocytosis in human T84 intestinal epithelia: role of F-actin and MARCKS. Am J Physiol Cell Physiol 277: C1239-C1249, 1999[Abstract/Free Full Text].

29.   Song, JC, and Matthews JB. Opposing effects of PKCalpha and PKCepsilon on basolateral membrane dynamics in intestinal epithelia (Abstract). Gastroenterology 118, Suppl 2: A871, 2000.

30.   Straub, SV, Giovannucci DR, and Yule DI. Calcium wave propagation in pancreatic acinar cells: functional interaction of inositol 1,4,5-trisphosphate receptors, ryanodine receptors, and mitochondria. J Gen Physiol 116: 547-560, 2000[Abstract/Free Full Text].

31.   Tinel, H, Cancela JM, Mogami H, Gerasimenko JV, Gerasimenko OV, Tepikin AV, and Petersen OH. Active mitochondria surrounding the pancreatic acinar granule region prevent spreading of inositol trisphosphate-evoked local cytosolic Ca2+ signals. EMBO J 18: 4999-5008, 1999[Abstract/Free Full Text].

32.   Toescu, EC, Lawrie AM, Petersen OH, and Gallacher DV. Spatial and temporal distribution of agonist-evoked cytoplasmic Ca2+ signals in exocrine acinar cells analysed by digital image microscopy. EMBO J 11: 1623-1629, 1992[Abstract].

33.   Weller, SG, Klein IK, Penington RC, and Karnes WE, Jr. Distinct protein kinase C isozymes signal mitogenesis and apoptosis in human colon cancer cells. Gastroenterology 117: 848-857, 1999[ISI][Medline].

34.   Wenzel-Seifert, K, Schächtele C, and Seifert R. N-protein kinase C isozymes may be involved in the regulation of various neutrophil functions. Biochem Biophys Res Commun 200: 1536-1543, 1994[ISI][Medline].

35.   Xu, X, Zeng W, Diaz J, and Muallem S. Spatial compartmentalization of Ca2+ signaling complexes in pancreatic acini. J Biol Chem 271: 24684-24690, 1996[Abstract/Free Full Text].

36.   Yule, DI, Ernst SA, Ohnishi H, and Wojcikiewicz RJ. Evidence that zymogen granules are not a physiologically relevant calcium pool. Defining the distribution of inositol 1,4,5-trisphosphate receptors in pancreatic acinar cells. J Biol Chem 272: 9093-9098, 1997[Abstract/Free Full Text].


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