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Am J Physiol Gastrointest Liver Physiol 291: G331-G344, 2006. First published March 30, 2006; doi:10.1152/ajpgi.00356.2005
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MUCOSAL BIOLOGY

Chronic PKC-beta2 activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane CFTR targeting

James R. Broughman,1 Limin Sun,1 Shahid Umar,1 Joseph H. Sellin,2 and Andrew P. Morris1,2

1Department of Integrative Biology and Department of Pharmacology and Physiology and 2Division of Gastroenterology, Hepatology, and Nutrition, Department of Internal Medicine, University of Texas Health Science Center Medical School, Houston, Texas

Submitted 28 July 2005 ; accepted in final form 16 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We investigated the effects of chronically applied PKC-stimulating phorbol esters on subcellular CFTR expression and localization in polarized HT-29 Cl.19A monolayers. Modulation of PKC activity with the PKC-beta-specific agonist 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) or nonisoform-selective PMA altered monolayer CFTR immunofluorescence. A decrease in the CFTR signal within the luminal cellular pole was noted with both phorbol esters. Volumetric analysis of the intracellular CFTR signal revealed that both compounds promoted CFTR accumulation into punctate vesicle-like structures found adjacent to the cellular tight junction [labeled with zona occludens (ZO)-1 antibody], extending basally (DOPPA) into the cell. Puncta were more frequent with DOPPA and larger in size with PMA. DOPPA also promoted ZO-1 accumulation at tricellular corners associated with enhanced CFTR puncta number. The observed loss of CFTR immunofluorescence signal induced by low-dose PMA was related to CFTR sequestration into fewer cytoplasmic puncta and correlated with larger increases in PKC substrate phosphorylation. Both phorbol esters downregulated steady-state cellular CFTR mRNA levels by 70%. However, the effects of DOPPA and PMA were largely independent of CFTR biosynthesis: expression levels were 80–85% of control, and the glycosylation status of immunoprecipitated protein remained largely unchanged. Thus changes in cellular CFTR localization correlated with our companion study showing that PMA-induced inhibition of transcellular cAMP-dependent short-circuit current (ISC) was accompanied by cytoplasmic PKC-beta2 accumulation and modest activation of PKC-beta1 and PKC-{epsilon}. The inhibitory effect of DOPPA on ISC was related solely to increased cytoplasmic PKC-beta2 levels. Thus PKC-beta2 is hypothesized to participate in the regulation of CFTR apical plasma membrane targeting within the constitutive cellular biosynthetic pathway.

protein kinase C; immunofluorescence; short-circuit current; cystic fibrosis transmembrane conductance regulator; phorbol ester

THE CELL BIOLOGY of CFTR expression in vitro is dominated by reports demonstrating a negative role of phorbol ester-sensitive PKC signaling on channel mRNA levels (4, 9, 25), with prolonged (24 h) exposure causing the degradation of CFTR protein by as much as 50% (9, 25). In addition, a recent antisense oligonucleotide study (16) has identified Ca2+-independent, diacylglycerol-stimulated PKC-{epsilon} as a stimulator of cellular halide influx in Calu-3 airway cell monolayers. Finally, cell biological evidence from polarized epithelial cell lines has shown that PKC activation enhances constitutive vesicle movement within the cellular biosynthetic pathway (10, 17). We have proposed that this mechanism consequently enhances apical plasma membrane targeting of CFTR (21).

Our laboratory investigated the effects of PKC-stimulated proliferation on CFTR expression in vivo (27) and found that PKC-beta activation promotes increased cellular CFTR expression at the mRNA and protein levels (28). However, accompanying changes in transepithelial cAMP-dependent Cl– secretion were not as large as predicted, leading us to speculate that cellular homeostatic mechanisms preventing excessive secretory capacity were active. Abrogation of PKC activity in vivo abolished all changes. In our companion study (9a), we demonstrated that in basolateral permeabilized monolayers, isoform-specific PKC activation was required for the inhibition of apical membrane-generated cAMP-dependent short-circuit current (ISC-cAMP). Chronic downregulation of PKC-beta1 and PKC-{epsilon} mass led to a recovery of this current. However, the most dramatic changes recorded were increases in PKC-beta2 cytoplasmic partitioning apparently unrelated to apical plasma membrane ISC inhibition. To determine the role of PKC-beta2 in CFTR-mediated transcellular Cl– secretion across intact monolayers, we used a novel experimental approach that allowed us to monitor the geography of CFTR protein localization and mRNA expression in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. FBS, DMEM, penicillin-streptomycin, and trypsin-EDTA solutions were procured from GIBCO-BRL (Grand Island, NY). Isoform-specific anti-PKC-{alpha}, -beta1, -beta2, and -{gamma} polyclonal antibodies were purchased from Santa Cruz Biotechnology and Upstate Biotechnology (CA). 12-Deoxyphorbol 13-phenylacetate 20-acetate (DOPPA), Gö6976, and PMA were obtained from Alexis (San Diego, CA). All other chemicals were purchased from Sigma (St. Louis, MO) unless otherwise specified.

Cell culture. The human colon carcinoma cell line HT-29 Cl.19A was the generous gift of C. L. Laboisse (1, 3) (Institut National de la Santé et de la Recherche Médicale, Paris, France) and was cultured in DMEM supplemented with 4.5 g/l glucose, 10% FBS, 100 U/ml penicillin, and 100 µg/l streptomycin. Cells were grown at 37°C in a humidified atmosphere of 5% CO2-95% air.

Treatment of cells with PKC modulators. Cells were grown to near confluence on sterile 12-mm-diameter filters (0.4-µm pore, Millicell-HA, Millipore; Bedford, MA) in DMEM with 10% FBS. The culture medium was replaced with serum-free DMEM 2 h before experimental treatment and then replaced with 1 ml of identical serum-free medium containing PMA (0.01–1 µM) in DMSO, DOPPA (1–500 nM), or Gö6976 (1–5 µM) for 10 min to 48 h. To ensure that significant cellular metabolism of DOPPA to the nonspecific activator 12-deoxyphorbol-13-phenylacetate (DOPP) did not occur (15), we exchanged DOPPA incubation media every 4–6 h.

Northern blot analysis and RT-PCR. Poly(A)+ mRNA was isolated from treated HT-29 Cl.19A cells using TRIzol reagent (GIBCO-BRL) or the micro Fast Track kit (Invitrogen; San Diego, CA) according to the manufacturer's instructions. For Northern blot analysis, 2.5 µg poly(A)+mRNA was denatured and fractionated on a 1% agarose gel containing formaldehyde. RNA was then transferred to GeneScreen Plus nylon membranes (DuPont-New England Nuclear). The blot was hybridized at 60°C in 10% dextran sulfate, 1 M NaCl, 1% SDS, and 100 µg/ml denatured salmon testes DNA with the use of a [{alpha}-32P]dCTP-labeled probe encompassing the regulatory domain of CFTR (bases 1773–2654, 2 x 106 counts·min–1·ml–1) and subsequently with a probe against GAPDH (bases 163–608, 1 x 106 counts·min–1·ml–1). The latter was used to normalize mRNA levels. The probe for CFTR detection was generated by a PCR of full-length CFTR cDNA, and the GAPDH probe was generated by RT-PCR from mouse colonic RNA (28). Both sequences were confirmed by oligonucleotide sequencing before random primer labeling.

Immunoprecipitation of CFTR. Cells were lysed in 500 µl of ice-cold buffer consisting of (in mM) 10 Na-HEPES, 150 NaCl, and 1 EDTA with 1% Nonidet P-40 (pH 7.0). The lysate was cleared by centrifugation for 5 min at 4°C, and the supernatant was diluted with a 1:4 volume of 250 mM Tris, 120 mM sodium deoxycholate, 750 mM NaCl, 5% Triton X-100, and 0.5% SDS [pH 7.5; 5x radioimmunoprecipitation (RIPA) buffer]. Total protein was measured before the addition of 5x RIPA buffer by the bicinchoninic acid (BCA) assay (Pierce Chemical). CFTR was immunoprecipitated with 1.5 µg of monoclonal antibody directed against the regulatory domain of CFTR (Genzyme; Cambridge, MA) and protein G-Sepharose, according to standard techniques. As the control, the CFTR antibody was substituted with 1.5 µg of nonimmune mouse IgG (Pierce). Immunoprecipitates were then washed, resuspended in 50 µl of 50 mM Tris (pH 7.5), 10 mM MgCl2, and 0.01% BSA, and phosphorylated at 30°C by the addition of 10 µCi of [{gamma}-32P]ATP (3,000 Ci/mmol, DuPont-New England Nuclear) and 1 µl of 50 ng/µl PKA.

PKC activity assay. PKC activity was measured in cytosolic and membrane fractions prepared using the GIBCO-BRL PKC assay system. Subcellular fractions were prepared by homogenization of HT-29 Cl.19A cells in 1.5 ml of ice-cold homogenization buffer [50 mM Tris·HCl (pH 7.5) containing 5 mM EDTA, 10 mM EGTA, 0.3% mercaptoethanol, 10 mM benzamidine, 50 µg/ml PMSF, and 50 µg/ml leupeptin] using a Tekmar Tissuemizer (Tekmar; Cincinnati, OH) rotating at 20,000 rpm for five 40-s periods. Cytosolic and membrane fractions were separated by sedimentation for 30 min at 100,000 g. The pellet was then detergent extracted in Tris-buffered saline (TBS) containing (in mM) 10 Tris·HCl, 140 NaCl, 25 KCl, 5 MgCl2, 2 EDTA, and 2 EGTA with 1% Triton X-100 (pH 7.5), protease inhibitor cocktail, and the solubilized membrane fraction collected by centrifugation. The protein content was determined using the Bio-Rad DC protein assay kit. PKC activity was quantified by the transfer of the terminal phosphate of [{gamma}-32P]ATP (Amersham; Arlington Heights, IL) to myelin basic protein synthetic peptide. PKC specificity was determined using a PKC pseudosubstrate inhibitor peptide (PKC19–36) provided by the manufacturer. Incorporated radioactivity was determined by scintillation counting, and activities are expressed as nanomoles of phosphate transferred per minute per milligram of protein.

Densitometric analysis of PKC isoform expression by Western blot analysis. Crude homogenates were prepared from the HT-29 Cl.19A cell monolayers by homogenization in detergent-containing buffer [composed of (in mM) 50 Tris·HCl, 250 sucrose, 2 EDTA, 1 EGTA (pH 7.5), and 10 2-mercaptoethanol with 0.5% Triton X-100 plus protease inhibitors] followed by a low-speed spin (15,000 g for 15 min). The clear supernatant was saved as the total cell extract. The protein concentration was measured by BCA assay before electrophoresis. Mouse brain homogenates and purified PKC isoforms acted as positive controls for the immunoblotting assay. The total cell extract (30 µg protein/lane) was subjected to 10% SDS-PAGE and electrotransferred to nitrocellulose membranes. The efficiency of electrotransfer was checked by back staining gels with Coomassie blue and/or by reversible staining of the electrotransferred protein directly on the nitrocellulose membrane with Ponceau S solution. No variability in transfer was noted. Destained membranes were blocked with 5% nonfat dried milk in TBS (20 mM Tris·HCl and 137 mM NaCl; pH 7.5) for 1 h at room temperature and then overnight at 4°C. Immunoantigenicity was detected by incubating the membranes for 2 h with relevant, isoform-specific PKC antibodies (0.5–1.0 µg/ml in TBS containing 0.1% Tween 20). After being washed, membranes were incubated with horseradish peroxide-conjugated goat anti-rabbit IgG (Sigma) secondary antibodies and developed using the ECL detection system (Amersham; Arlington Heights, IL) according to the manufacturer's instructions. Blots were then stripped and reprobed for beta-actin. Autoradiographs were digitized at 16 bits using a Fuji FinePix S-2 Pro Digital Camera (Fuji; Edison, NJ), and densitometric signal intensity was determined using Metamorph one-dimensional gel scanning software (Molecular Devices; Sunnyvale, CA).

Immunofluorescence: quantification of total cellular CFTR intensity in monolayers. Monolayers grown to confluence on optically transparent 280-mm filters were rinsed in Ringer solution and fixed in methanol (25 min at –20°C). They were then blocked with 1% BSA before the addition of primary and secondary antibodies and washed with four changes of PBS. All antibodies were diluted in blocking solution. Monolayers were sequentially incubated with murine anti-CFTR regulatory domain antibody (1:25, Genzyme; Cambridge, MA) and goat anti-mouse IgG-FITC (1:10) for 40 min at 37°C. After a final wash, monolayers were mounted on glass coverslips in 0.1% phenylene diamine in 1:9 PBS-glycerol. Fluorescence was viewed using a Noran confocal laser-scanning microscope (Noran Instruments; Middleton, WI) equipped with an argon laser and appropriate optics and filter modules for fluorophore detection (27). Digital images were obtained at x400, x800, and x1,200 (Nikon 40 x 1.4 numerical aperture lens). A Z-axis motor attached to the inverted microscope stage was calibrated to move the plane of focus at 0.1- to 1.0-µm steps through the sample. Eight- or sixteen-bit images collected at 512 x 480 resolution were then stored on a mass storage device (optical hard disk) and volumetrically reconstructed using Image-1/Metamorph three-dimensional software module (Universal Imaging; Brandywine Parkway, PA). When Z-axis stacks were captured of <20 x-y image planes, laser power was maintained at threefold higher levels than those used for larger stack captures, and a 10-µm slit width was chosen for optimal optical resolution. In all other instances, the expanded slit width was 25 µm. This action maintained image detection intensity and ensured that photobleaching was minimal, but some resolution was lost within the vertical optical plane. As an additional precaution, Z-axis stacks were collected starting both above and below the monolayer.

Subcellular CFTR immunofluorescence visualized during multiple fluorophore labeling. Similar monolayer preparations to those used for monolayer CFTR immunofluorescence (IMF) intensity calculation (see Immunofluorescence: quantification of total cellular CFTR intensity in monolayers) but containing additional fluorescent markers for the cellular tight junction [zonula occludens (ZO)-1] and nucleus (TO-PRO-3) were employed for these studies. Cells were fixed with 4% formaldehyde in 0.01 M PBS for 30 min at 4°C and permeabilized with 0.5% Triton X-100 for 30 min. After being washed three times with PBS, slides were incubated with a rabbit polyclonal primary antibody to ZO-1 (Zymed; San Francisco, CA), blocked with mouse and rabbit serum, and then incubated with mouse anti-CFTR TAM-18 IgM antibody (Labvision Neomarkers; Union City, CA). Monolayers were then washed three times with PBS and coincubated overnight at 4°C with secondary Cy2-labeled goat anti-rabbit (Jackson Labs; West Grove, PA) and donkey anti-mouse Cy3-conjugated (Jackson Labs) antibodies. Before being mounted onto coverslips, monolayers were labeled with the dimeric cyanine nuclear acid stain TO-PRO-3 (Molecular Probes; Eugene, OR). After membranes had been washed and the autofluorescence quenched by treatment with 1 mg/ml NaBH4 in PBS (Sigma), monolayers were mounted in Vectashield mounting medium (Alexis PLATFORM; San Diego, CA), and the overlying coverslip was sealed with clear nail polish. Samples were then imaged with a Zeiss LSM 510 META confocal microscope using a x63 oil-immersion objective (Carl Zeiss Optical; Thornwood, NY) in multitrack scanning mode with excitation wavelengths set at 488 nm (Argon laser) and 543 and 633 nm (HeNe lasers). Emission wavelengths were 505–530, 560–615, and >650 nm for Cy2, Cy3, and TO-PRO-3 signal detection, respectively. Single optical slices were set to 0.8 µm, and Z-stack slices were set to 0.38 µM. Collected images were processed using a LSM Image three-dimensional bit plane and VisArt software (version 3.2, Carl Zeiss) and exported in a 16-bit TIFF RGB format.

Data analysis. All summary results are presented as arithmetic means ± SE. The differences between control and treatment data were analyzed using Student's t-test (Excel, Microsoft; Redmond, WA). The probability of making a type I error <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PKC activation status during chronic phorbol ester exposure. When PKC activity was measured in both cytoplasmic- and membrane-associated fractions by radiolabeled phosphate incorporation into a synthetic substrate peptide (see MATERIALS AND METHODS), 24-h DOPPA preincubation increased membrane-bound PKC activity by 2.2-fold (P < 0.001, n = 6) and cytosolic activity by 1.1-fold (Fig. 1A, bars i and ii, respectively). When the levels of each immunodetectable PKC isoform were determined from the same DOPPA-treated cellular extracts by Western blot analysis (Fig. 1B), we found that the levels of PKC-{alpha}, -{gamma}, and -{epsilon} were unchanged relative to control (carrier-treated monolayers), whereas a modest decrease in PKC-beta1 (73% of control, P < 0.05) and a significant increase in total PKC-beta2 (122% of control, P < 0.01) levels were recorded. In our companion study (9a), we showed that DOPPA pretreatment of the same monolayers failed to induce PKC-{alpha}/beta1/{gamma} or PKC-{epsilon} membrane translocation. DOPPA did, however, induce a significant decrease in the PKC-beta2 membrane translocation ratio by disproportionately increasing cytosolic PKC-beta2 levels while modestly increasing the size of the membrane-associated pool. Thus increased PKC catalytic activity in monolayers during DOPPA exposure (Fig. 1A) was caused by selective PKC beta2-isoform activation. The accompanying increase in PKC-beta2 cytoplasmic translocation and chronic upregulation (Fig. 1B) further indicated that cytoplasmic PKC-beta2 protein in postactivation status was less susceptible to degradation than PKC-beta1 [whose cytoplasmic levels decreased rather than increased (9a)]. The PKC beta-isoforms in active and inactive conformation therefore appeared to associate with different signaling partners (see DISCUSSION in Ref. 12).


Figure 1
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  		Fig. 1. Modulation of cellular phorbol ester-sensitive PKC activity and cellular PKC isoform expression in monolayers preincubated for 24 h with 50 nM 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA). A: PKC activity in cytosolic (c) and membrane (m) fractions was similar in control monolayers (i). In DOPPA-pretreated monolayers (ii), a 2.5-fold increase in membrane-associated activity and a 50% increase in cytosolic activity were recorded (n = 3; values are means ± SD). B: bar graph of mean total PKC isoform expression levels in the same extracts relative to control as detected by Western blot analysis of Triton X-100-solubilized cell extracts (densitometric values for each immunodetected band normalized to beta-actin). All changes were clearly significant (n = 3, P < 0.01).

 
When the substrate phosphorylating activity was estimated after a 24-h PMA preincubation, a 3.9-fold increase in membrane-bound PKC activity and a 2.2-fold decrease in cytoplasmic activity were recorded (P < 0.001, n = 6; Fig. 2A). When cellular PKC isoform expression levels were quantitated by Western blot analysis, PMA did not significantly change PKC-{alpha}, -{gamma}, and -{epsilon} levels (Fig. 2B), whereas total PKC-beta1 levels decreased significantly (50% of control, P < 0.01) and PKC-beta2 levels increased significantly (144% of control, P < 0.01). Total PKC activity and PKC beta-isoform mass changes were therefore more pronounced but mirrored those recorded for DOPPA (Fig. 1). Similarly, when the membrane translocation status of individual isoforms was assayed (9a), PMA failed to significantly increase the membrane translocation status of either PKC-{alpha} and -{gamma}, slightly increased PKC-beta1 and -{epsilon} membrane translocation, and modestly increased PKC-beta2 membrane association while strikingly promoting large changes in PKC-beta2 cytoplasmic partitioning. Changes in total extractable PKC activity promoted by PMA (Fig. 2A) were therefore also primarily attributed to increases in the activation status of the PKC beta- and {epsilon}-isoforms, with only PKC-beta2 exhibiting postactivation-dependent increases in total cell levels (Fig. 2B).


Figure 2
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  		Fig. 2. Modulation of cellular phorbol ester-sensitive PKC activity and cellular PKC isoform expression in monolayers preincubated for 24 h with 50 nM PMA. A: PKC activity in cytosolic and membrane fractions was evenly distributed in control monolayers (i). In PMA-pretreated monolayers (ii), a dramatic 3-fold increase in membrane-associated activity and a 2-fold decrease in cytosolic activity were recorded (n = 3; values are means ± SD). B: bar graph of mean total PKC isoform expression levels in the same extracts relative to control as detected by Western blot analysis of Triton X-100-solubilized cell extracts (densitometric values for each immunodetected band normalized to beta-actin). All changes were clearly significant (n = 3, P < 0.01).

 
After a 48-h preincubation with a high concentration (500 nM) PMA, substrate phosphorylation activity measured in both cytosolic and membrane fractions was reduced to 17% and 46%, respectively, of carrier-matched controls (n = 3, P < 0.001; Fig. 3A). When the total extractable PKC isoform amount under these conditions was measured by quantitative Western blot analysis (Fig. 3B), PKC-{alpha}, -beta2, and -{gamma} levels remained similar to the control (102 ± 10%, 98 ± 9%, and 101 ± 6%, respectively). A 5-fold decrease in PKC-beta1 (to 17 ± 5% of control) and a 2.4-fold decrease in PKC-{epsilon} levels (to 40 ± 9% of control) were measured. Under these conditions, the reduction in PKC phosphorylation activity did not correlate with the change in PKC-beta2 but correlated with a chronic loss in the cellular PKC beta1-{epsilon}-isoform mass.


Figure 3
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  		Fig. 3. Cellular PKC activity and cellular PKC isoform expression recorded after a 48-h postmonolayer exposure to 500 nM PMA. A: PKC activity in both cytosolic and membrane fractions was similar in control monolayers (i) but decreased after exposure to 500 nM PMA by 4- and 2-fold, respectively (ii). B: bar graph of mean total PKC isoform expression levels in the same extracts relative to control as detected by Western blot analysis of Triton X-100-solubilized cellular proteins (densitometric values for each immunodetected band were normalized to beta-actin). PMA (500 nM) preincubation significantly decreased the total cellular levels of PKC-beta1 and -{epsilon} (P < 0.01), whereas PKC-{alpha}, -{gamma}, and -beta2 expression levels did not change significantly (P > 0.1).

 
Chronic monolayer exposure to low doses of both DOPPA and PMA reduced cellular CFTR mRNA levels but marginally affected cellular CFTR anion channel protein expression. Shown in Fig. 4 are representative Northern and Western blots of CFTR mRNA and protein expression after 24 h in control and 50 nM DOPPA- and 50 nM PMA-treated HT-29 Cl.19A cells.


Figure 4
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  		Fig. 4. Northern blot and protein (immunoprecipitation) analysis of cellular CFTR anion channel expression in 24-h control and DOPPA- and PMA-treated monolayers. A, top left: poly(A)+ mRNA isolated from control [(–) lane; carrier alone], DOPPA-treated [(+d) lane; 50 nM], or PMA-treated [(+p) lane; 50 nM] monolayers probed at high stringency with a CFTR regulatory domain probe. A, bottom left: the same blot was restripped and hybridized with GAPDH probe. A, top and bottom right: corresponding densitometry histograms for each lane plotted at the same vertical scale. Both agonists decreased the CFTR mRNA signal by ~3-fold (normalized to GAPDH, see MATERIALS AND METHODS, n = 3). B: PKA-phosphorylated immunoprecipitates (Genzyme anti-CFTR antibody) obtained from homogenized cell lysates. B, left: denatured protein samples (see MATERIALS AND METHODS) analyzed on a 6% polyacrylamide SDS gel. In vitro phosphorylated CFTR was detected in all lanes as a two-band immunocomplex migrating at ~140 kDa [band A (circled A)] and ~190 kDa [band C (circled C)], respectively. B, right: corresponding densitometry histograms plotted on the same vertical scale. Levels of 32P-labeled CFTR relative to control were 0.80 ± 0.18 and 0.85 ± 0.16 for DOPPA and PMA, respectively. The ratios of 32P-labeled band C to band A for control, DOPPA, and PMA treatment were 1.0, 1.6 and 1.3, respectively (n = 3).

 
Cellular CFTR poly(A)+ mRNA abundance (n = 3) relative to the housekeeping gene GAPDH (n = 3) was observed to decrease after a phorbol ester preincubation (Fig. 4A). The intensity of the 6.5-kb CFTR band in DOPPA- and PMA-preincubated monolayers was 27% and 30% of that encountered in unstimulated conditions, respectively (n = 3, P < 0.001). In the phorbol ester-treated samples, GAPDH migrated with a slightly decreased apparent molecular mass. We attribute this change to reduced polyadenylation of the GAPDH transcript (20) after phorbol ester treatment. Densitometric line histograms for each lane line are shown (Fig. 4). Blots were captured with a 16-bit grayscale camera, and images were analyzed by Universal imaging Metamorph software (see MATERIALS AND METHODS, Densitometric analysis of PKC isoform expression by Western blot analysis). Captured images were not saturated.

To determine whether reductions in cellular mRNA content were reflected at the protein level under identical treatment conditions, CFTR was immunoprecipitated with the Genzyme anti-CFTR regulatory domain antibody and 32P labeled by PKA phosphorylation (see MATERIALS AND METHODS). A 24-h treatment with 100 nM PMA or DOPPA resulted in a modest but not significant reduction in labeled cellular CFTR protein; levels fell to 80 ± 16% and 85 ± 18% of control values, respectively (Fig. 4B; n = 3, P > 0.01). The 32P-labeled immunoprecipitated product running at an apparent molecular mass of ~140 kDa (band A) and second broader immunoprecipitated phosphorylated band running at ~190 kDa (representing complex and mature post-Golgi-processed glycosylation patterns for CFTR, band C) exhibited a similar molecular mass to epithelial CFTR as detected by Western blot analysis with the TAM-18 antibody used previously in isolated colonic crypts (27). Densitometric analysis of the relative intensities for bands A and C within each lane [Fig. 4B, (–), (+d), and (+p) lanes) revealed no glycoprocessing differences. Thus, although low doses of both weak and strong phorbol esters shared the ability to partially downregulate anion channel message levels in confluent HT-29 Cl.19A monolayers, doses of either agonist at twice the concentration used to inhibit CFTR-mediated Cl– section in our functional assays (9a) did not significantly affect cellular CFTR protein content. The effects of sustained PKC-beta2 activation on apical plasma membrane ISC-cAMP therefore must be occurring at another level. We therefore monitored the localization of CFTR in DOPPA- and PMA-treated monolayers.

Both DOPPA and PMA induce the loss of monolayer luminal pole CFTR IMF. Previously, we examined the cellular location of CFTR in HT-29 Cl.19A monolayers (22). Using the same immunostaining protocol and monoclonal antibody against CFTR, we quantified CFTR IMF within two-dimensional (XY) and three-dimensional (XYZ) cellular axial planes after 24 h of incubation with either carrier alone (PBS vehicle), vehicle plus 50 nM DOPPA, or vehicle plus 50 nM PMA (Fig. 5).


Figure 5
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  		Fig. 5. Composite showing the subcellular distribution of CFTR immunostaining in HT-29 Cl.19A monolayers. A: four individual two-dimensional XY planes (PXY) optically sectioned through the brush border (image i), the subapical (sub-Ap) cytoplasm (image ii), the Ap perinuclear (image iii), and the basolateral (Bl) perinuclear region (image iv) of individual cells within the monolayer. B: diagrammatic view of the Z plane sectioning method employed to quantify the subcellular CFTR distribution in three dimensions. Starting below the monolayer (plane = 0), between 160 and 180 Z-axis (0.1 µm) optical sections were collected by progressively focusing upward through and past the Ap pole of cells contained within the monolayer (stack plane of 150 shown). Bar = 5 µm.

 
CFTR staining was first determined in nonphorbol ester-treated control monolayers in individual en face (XY-axes) planes throughout the cellular Z-axis at 1-µm intervals (Fig. 5A; see MATERIALS AND METHODS). The structures exhibiting the clearest labeling were the microvillus brush border and the subapical cytoplasm, which included a circumferential band of IMF within the region of the apical tight junction (Fig. 5A, images i and ii). A lower degree of staining was present in more basal subcellular regions and was absent from the nucleus (Fig. 5A, images iii and iv; n = 24 monolayers). This localization of CFTR was consistent with its geographical role as an apical plasma membrane anion channel involved in apical membrane ISC-cAMP generation. To quantify values from all areas within the 640 (X) x 480 (Y) pixel field of view, we collected individual XY-axes planes at 0.1-µm Z-axis intervals throughout the depth of the monolayer for the whole objective field (~ 45 µm in X and Y planes; Fig. 5B; between 160 and 180 planes encompassing both basal and apical aspects were routinely sampled). To represent the three-dimensional data as a two-dimensional pictograph, we then rendered all 640 "X-axis" pixel values (Formula X1–640) at every Y-axis position (Y1–480) into single average values, PY{sum}X = Y1(Formula X1–640) -> Y480(x X1–640). These ({sum}X) Y-axis slices were then stacked to form a PY{sum}X/Z matrix, where PY{sum}X/Z = (PY{sum}X/PFormula-> PY{sum}X/PFormula, Z0 is the initial Z-axis (Fig. 6). Thus orthogonally constructed images were side-on views looking entirely through ~ 45 µm of the epithelial monolayer containing many cell bodies. For this reason, the resulting image, shown in Fig. 6A, appears more diffuse than would a single orthogonal plane image. However, because 640 pixel planes are averaged for each pixel displayed, this method is quantitatively more accurate for intensity measurement.


Figure 6
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  		Fig. 6. Subcellular distribution of CFTR immunofluorescence (IMF) in 24-h preincubated control and phorbol ester-treated monolayers. A: reconstructed PYZ obtained by X-axis summation of three-dimensional stacks for control (image i), 50 nM DOPPA-treated (image ii), and 50 nM PMA-treated (image iii) monolayers. The resulting images are intensity averaged sideon views through the depth of the monolayer. B: graphical representation of Z-axis intensity distribution averaged along the Y-axis (bullet), control monolayer ({blacktriangledown}), DOPPA ({circ}), and PMA ({triangledown}) nonspecific primaries. Values are means ± SE for 4 separate experiments. DOPPA induced cellular basal pole CFTR IMF accumulation and a reduction in luminal CFTR IMF intensity. This effect was also present in PMA-preincubated monolayers (n = 3).

 
Starting points for the Z0-axis were empirically judged by moving the fine focus in 0.1-µm steps away from the specimen-glass coverslip interface, which was visible as a change in background signal intensity at this boundary. However, when the 25-µm confocal slit width was used to collect sufficient light for immunodetection with minimal laser-induced photobleaching (see MATERIALS AND METHODS), planes close to this interface included an out-of-focus signal [light detection is a function of slit aperture, and wider apertures correspond to widening of the detection sensitivity but lowering of optical resolution (8)]. As a result, starting intensity along the glass-apposed PY{sum}X/Z-axis (Fig. 6, A, plane 0, and B, basolateral membrane) was contaminated by internal reflection between the specimen and glass coverslip. This introduced variability between coverslips. As predicted, background intensity converged at high-PY{sum}X/Z plane values above the luminal membrane of the epithelial cell monolayer (Fig. 6, A, plane 180, and B, apical membrane).

Although this technical limitation prohibited us from measuring quantitatively the area under the curve for each experimental condition, it was clear from PY{sum}X/Z intensity matrixes (Fig. 6A) that control monolayers exhibited a node of IMF signal intensity within the cellular apical pole and that both 50 nM DOPPA and 50 nM PMA decreased the volume of CFTR within this region (n = 4 separate experimental procedures). The effects of DOPPA were subtle in that a higher accumulation of signal was detected in the cellular basolateral pole than within the cellular apical pole, an effect made more apparent when PY{sum}X/Z intensity matrixes were graphically plotted as mean P{sum}YX slice intensity (i.e., x([Y1–480, X1–640] vs. z-axis graphs). The stronger phorbol ester PMA potentiated this effect but reduced IMF intensity throughout the monolayer volume. The reduction in the IMF signal detected when we used PMA-treated samples was unexpected, given that the level of protein remained relatively constant, as determined by CFTR immunoprecipitation studies (Fig. 4B).

Because quantitative intensity measurements using pixel averaging within monolayer planes includes areas where no CFTR fluorescence is found (both within and outside cells) as well as areas containing true CFTR intensity, alterations in the subcellular nature of CFTR protein fluorescence change the resulting averaged signal readout. For instance, CFTR existing in a diffuse cytoplasmic labeling pattern presents the ideal intensity measurement condition. However, when CFTR is volume constrained into structures where the intensity of CFTR fluorescence may be masked (such as when vesicles contain different amounts of protein), then averaged intensity measurements will not remain truly quantitative. Under these conditions, the extent of CFTR intracellular three-dimensional coverage or geography that is no longer intensity encoded but encoded by volume provides another measurement method.

Both DOPPA and PMA induce vesiculation of monolayer apical pole subcellular CFTR IMF into structures bordering the tight junction. To further investigate the changes in CFTR IMF intensity profiles recorded in fixed monolayers (Figs. 5 and 6), the murine monoclonal TAM-18 IgM anti-CFTR antibody used previously for intracellular CFTR fluorescent labeling was employed (27) together with a marker antibody for the cellular tight junction [ZO-1 (18)] and the nuclear stain (TO-PRO-3). The choice of a different primary anti-CFTR antibody made multiple (triple) fluorescent staining in fixed monolayers possible (see MATERIALS AND METHODS). When triple-stained monolayers were visualized at high (x63) magnification on a Zeiss 510 META confocal microscope running in multiscan mode (see MATERIALS AND METHODS), the resulting Cy3-conjugated secondary anti-CFTR pattern exhibited clear diffuse apical localization within the top-most luminal (2 µm) aspect of the monolayer (Fig. 7Ai).


Figure 7
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  		Fig. 7. Ai, Bi, and Ci: triple fluorescently labeled monolayer Z-axis stack and deconvolved YZ and XZ orthogonal planes [CFTR (red), zonula occludens (ZO)-1 (green), and TO-PRO-3 nuclear acid stain (blue)]. CFTR was detected with TAM-18 monoclonal antibody and Cy-3 secondary antibody. ZO-1 detected with Zymed polyclonal antibody and Cy2 secondary antibody. The far red fluorescence emission of TO-PRO-3 dye was pseudocolored blue (see materials and methods). Aii, Bii, and Cii: scattergrams of red/green (CFTR/ZO-1) intensity within the top-most Z-axis plane. A: 24-h pretreatment with carrier alone; B: 24-h pretreatment with 50 nM DOPPA; C: 24-h pretreatment with 50 nM PMA. Monolayers stained with nonspecific primary antibodies or with primary antibodies omitted failed to exhibit Cy2/Cy3 staining (data not shown). DOPPA and PMA reduced CFTR staining within the luminal (top 2 µm) aspect of the monolayer (n = 6, 30 x 0.35-µm Z-axis planes). Bars = 5 µm.

 
Deconvolved orthogonal YZ and XZ planes exhibited brush-border CFTR (red) labeling interspersed with a circumferential ZO-1 (green) staining pattern (Fig. 7). The stained caps of nuclei (blue) were found 4–6 µm below the majority of CFTR IMF (Fig. 7). When a scattergram [two-dimensional histograms mapping all the possible (red) CFTR/(green) ZO-1 pixel colocalization events] was constructed and color encoded by frequency of incidence (blue to red; Fig. 7Aii), no clear colocalization was found between CFTR and ZO-1 (complete XY colocalization produces a straight line through the origin). Thus CFTR staining was constrained above and by the ZO-1-labeled cellular tight junctions. When CFTR staining was visualized in monolayers treated for 24 h with 50 nM DOPPA, the CFTR signal within the monolayer was redistributed from the luminal (top 2 µm) aspect basally into the cell and exhibited areas of high intensity at intracellular structures closely apposed to, but not colocalized with, ZO-1-labeled tight junctions (Fig. 7Bi). Also seen were random blebs of ZO-1 staining within the apical cellular pole not associated with junctions (see also GoGoFig. 10). When scattergrams were constructed, no clear colocalization between CFTR and ZO-1 IMF was found (Fig. 7Bii), although CFTR did appear to surround both tight junctions and the dispersed cytoplasmic blebs of ZO-1 (data not shown). In monolayers exposed to 50 nM PMA for 24 h, the punctate CFTR staining pattern appeared even more eroded and vesiculated. Apical pole CFTR IMF was close to background levels, and the cellular CFTR signal was concentrated into fewer areas (Fig. 7Ci). The scattergram of CFTR versus ZO-1 colocalization (Fig. 7Cii) continued to show no significant degree of colocalization, although, in this instance, there were more coincident red-green pixels. Thus the results confirmed the averaged intensity measurements made in Fig. 6 in that DOPPA and PMA induced subapical pole CFTR relocalization into what appeared to be vesicle-like structures.


Figure 8
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  		Fig. 8. Stacks shown in Fig. 7 viewed "en face" as front plane prioritized (Ap -> Bl) shadow projections (bit-plane mapped, 45° incidence light) at x1 magnification in Ai, Bi, and Ci and x2 magnification in Aii, Bii, and Cii [CFTR (red), ZO-1 (green), and TO-PRO-3 (blue)]. A: control monolayers treated for 24 h with carrier alone exhibited an evenly dense CFTR IMF signal at the Ap cell surface, bordered by ZO-1 staining. B: CFTR staining in monolayers treated for 24 h with 50 nM DOPPA exhibited a punctate staining pattern highest in areas adjacent to ZO-1-stained cellular borders. C: CFTR staining in monolayers pretreated for 24 h with 50 nM PMA exhibited a loss of the diffuse CFTR staining pattern and more eroded punctate staining adjacent to ZO-1-stained cellular borders. Underlying nuclear cap TO-PRO-3 fluorescence (blue) was visible in areas where CFTR IMF levels (and hence, protein accumulation) was low (30 x 0.35-µm Z-axis planes). Bars = 5 µm.

 

Figure 9
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  		Fig. 9. Stacks shown in Fig. 7 viewed "en face" as back plane prioritized (Bl -> Ap) shadow projections (bit-plane mapped, 45° incidence light) at x1 magnification in Ai, Bi, and Ci and x2 magnification in Aii, Bii, and Cii [CFTR (red), ZO-1 (green), and TO-PRO 3 nuclear acid stain (blue)]. A: control monolayers treated for 24 h with carrier alone exhibited nuclear caps and ZO-1 staining in the foreground, and apical CFTR staining was masked in this projection. B: in monolayers treated for 24 h with 50 nM DOPPA, punctate CFTR staining extended into the foreground region bordering nuclear caps and CFTR puncta were now positioned below ZO-1-labeled tight junctions. C: the severely eroded punctate CFTR staining pattern in monolayers pretreated for 24 h with 50 nM PMA shared foreground priority with nuclear caps and was closely opposed to ZO-1-stained cellular borders (30 x 0.35-µm Z-axis planes). Bar = 5 µm.

 

Figure 10
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  		Fig. 10. Ai, Bi, and Ci: high-resolution voxel-based image rendered from Z-axis stack data presented in Fig. 7 showing ZO-1-stained apically oriented circumferential and cellular tight junctions (green) together with nuclear caps (blue). For simplicity, the information encoded by CFTR (red channel) was omitted during rendering. Aii, Bii, and Cii: voxel rendering at x 2 magnification. The resulting images lack pixel intensity encoding but contain three-dimensional pixel (voxel) spatial information. A: 24-h exposure to carrier alone yielded monolayers with irregular ZO-1 lattices structurally defining apically oriented cellular tight junctions. B: 24-h exposure to 50 nM DOPPA resulted in circumferential rings that were larger and more regular with ZO-1 accumulation at tricellular borders. C: 24-h exposure to 50 nM PMA generated monolayers with even larger belts of ZO-1 staining but less ZO-1 accumulation at tricellular borders (30 x 0.35-µm Z-axis planes). Bars = 5 µm.

 
Further confirmation of the compartmentalized nature of the internal CFTR signal was sought by constructing bit plane shadow masks of the triple-labeled Z-axis stacks. This technique encodes (by 4 x 4 binning) each 2,048 x 2,048 bitmap into a 512 x 512 bitmap that is not voxel (three-dimensional pixel volume) rendered but retains the stack plane information. Because of the need for transparency in the top-most layers, the image is no longer rigidly intensity encoded. The shadow mask images shown in Fig. 8 are reconstructed from Fig. 7 with front plane (apical -> basal) priority.

An evenly diffuse apically oriented CFTR IMF signal was recorded in control monolayers treated with carrier alone (Fig. 8A, images i and ii). Shadowing effects revealed that CFTR was positioned uniformly above the plane of the tight junction, and, in some instances, structures resembling CFTR-labeled apical plasma membrane microvilli were visible. Apical priority shadow mask rendering of the 24-h DOPPA-pretreated monolayer demonstrated that less CFTR IMF was found in the top-most (apical) planes and that CFTR IMF accumulated within puncta that resembled cytoplasmic vesicles localized near the tight junctions (Fig. 8B, images i and ii). After 24 h of treatment with 50 nM PMA, CFTR was clearly found at or below the level of the ZO-1-labeled cellular tight junction and was dispersed in less frequently occurring punctate vesicle-like structures. Comparative analysis of the overall size of the vesicular puncta under DOPPA and PMA conditions proved too difficult because of masking due to high levels of small (0.1–0.3 µm) apically oriented puncta in DOPPA-pretreated monolayers. However, when clear isolated vesicles below the ZO-1-labeled tight junction were measured, those found under PMA conditions were larger (>0.3 µm, data not tabulated).

When the same stacks were visualized with back plane shadow masking (basal -> apical priority), important geographical differences in CFTR location were observed after phorbol ester exposure (Fig. 9).

Shadow masking of control Z-axis stacks (Fig. 9A, images i and ii) with back plane priority showed a CFTR signal below the planes of the nuclear (blue) and ZO-1 signal (green) (i.e., little CFTR IMF was detected in back planes), confirming the apical location of the CFTR signal. However, in DOPPA-treated monolayers, the back planes were now populated with red punctate structures. Rendering priority on CFTR now localized in more basal planes caused masking of the green circumferential ZO-1 label (Fig. 9B, images i and ii). Thus CFTR-labeled puncta were present within the central volume of the cell, oriented along basolateral membranes around and below the cellular tight junction. In PMA-treated monolayers viewed from the back plane (Fig. 9C, images i and ii), a dispersed punctate vesicle-like staining pattern similar to that recorded in front plane prioritized stacks (see Fig. 8C, images i and ii) was observed. These findings, repeated in six different experiments, demonstrated that the apparent loss in signal-averaged luminal CFTR intensity after phorbol ester treatment (Fig. 6) was caused by a geographic shift of CFTR protein away from the apical cellular pole into vesicle-like structures found deeper within the cell and associated (DOPPA) with the cellular tight junction. Environmental differences in intracellular CFTR IMF detection may explain the differences between these higher resolution studies (Figs. 7–9) and the earlier intensity-averaged monolayers (Figs. 5 and 6).

The second important finding recorded from the triple-labeling studies related to changes in intracellular tight junction structure observed in DOPPA- and PMA-pretreated monolayers. Shown in Fig. 10 is a volumetric reconstruction of ZO-1-labeled tight junctions from the monolayers shown in Fig. 7.

High-precision volume rendering of control monolayers (Fig. 10A, images i and ii) demonstrated that the circumferential ZO-1-labeled tight junctions exhibited some degree of vertical-axis depth wandering (±0.5 µm) and appeared in most instances as a crinkled circumferential band. Under DOPPA conditions (Fig. 10B, images i and ii), the morphology of the ZO-1-labeled cellular tight junction was changed. The circumferential band appeared larger and more relaxed (reflecting a wider cellular neck region), smoother, and was more vertically constrained (±0.2 µm vertical depth wandering). Also recorded were volume increases in ZO-1 staining at points where adjacent circumferential tight junctions from neighboring cells met (tricellular borders), suggesting that this relatively immobile cytoskeletal element was being redistributed intracellularly within the apical pole. In PMA-treated monolayers (Fig. 10C, images i and ii), the circumferential rings of the ZO-1 labeling were even larger but did not exhibit similar ZO-1 pooling at tricellular borders. Parallel electrical measurements of similarly treated monolayers mounted in Ussing chambers revealed little change in transcellular electrical resistance after DOPPA or PMA preincubation (9a). Thus the chronic inhibitory effects of both DOPPA and PMA on ISC-cAMP (9a) appear related to structural changes in cellular architecture involving PKC-beta2-dependent alterations in subcellular CFTR localization and the cellular tight junction.


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Significance to our recent in vivo findings. We (27, 28) have shown previously that PKC-beta1 activation in vivo leads to elevated colonocyte CFTR mRNA and protein levels. However, only modest increases in transepithelial cAMP-responsive anion secretory current accompanied these changes. We thus hypothesized that this disparity was due to counteracting cellular homeostatic mechanisms. While a picture is emerging for such PKC-dependent effects operating at the level of the basolateral plasma membrane, the findings presented in our companion study (9a) identified an additional focus, incorporating new roles for three individual PKC isoforms (PKC-{alpha}, -beta, and -{epsilon}) at the level of the apical plasma membrane. Using a unique experimental approach that allowed us to monitor the geography of cellular CFTR protein localization and CFTR protein/mRNA expression, we showed that chronic phorbol ester-induced activation and translocation of PKC-beta2 into the cellular cytoplasm, while unrelated to direct apical plasma membrane ISC inhibition, posits a specific role for this isoform in CFTR targeting toward the apical membrane.

By measuring subcellular CFTR IMF in low-dose DOPPA- and PMA-preincubated monolayers, we found that both PKC agonists caused a specific redistribution of CFTR within the subcellular biosynthetic pathway, leading to a loss of CFTR from the cellular apical pole (Fig. 6) and an accumulation of the anion channel in vesicle-like structures within and below the plane of the circumferential tight junction (Figs. 7–9). Because the half-life of CFTR protein was not measured, these effects could have been due to either changes in subcellular CFTR turnover or movement. However, because neither agonist dramatically affected total cellular CFTR protein levels and failed to alter the ratio of high- to low-molecular-weight glycoprocessed forms of CFTR (indicating that the kinetics of immature endoplasmic reticulum/Golgi biosynthetic processing were not changing; Fig. 4), subcellular CFTR movement is predicted. We believe that chronic activation and the subsequent cytoplasmic partitioning of the PKC beta2-isoform was causally associated with subcellular CFTR relocalization within the biosynthetic pathway because the only common correlates between both DOPPA and PMA treatment conditions were enhanced PKC substrate phosphorylation and cytoplasmic PKC-beta2 partitioning.

The tight junction protein ZO-1 has been proposed to be part of a cytoplasmic junctional "waystation" or nexus for membrane trafficking to both apical and basolateral membranes. ZO-1 is known to bind a large variety of cytoskeletal elements (31) and acts as a scaffolding protein for signaling molecules. Coimunoprecipitation studies have shown that ZO-1 associates with G{alpha}-subunits of hetrotrimeric G proteins (19), which are known regulators of targeting within the constitutive biosynthetic pathway (22). It was therefore interesting to note that low (50 nM) concentrations of both DOPPA and PMA affected the shape and appearance of ZO-1-labeled circumferentially oriented apical tight junctions without affecting monolayer electric resistance. High-dose PMA is well characterized as an agent that inhibits cAMP-regulated Cl– secretion as well chronically affecting colonocyte tight junctional integrity, leading to a gradual reduction in transepithelial resistance through PKC-{alpha} activation (26) and the multilayering of cells (13). A more recent study (30) has demonstrated that PKC agonists such as bryostatin-1, a nonphorbol ester activator of conventional and novel PKC isoforms, can increase transepithelial resistance in the colonocyte T84 cell line by specifically activating PKC-{epsilon}, a phenomenon associated with the translocation of ZO-1 and claudin-1 from cytoplasmic to membrane pools. The fact that we did not observe transepithelial resistance changes (9a) reinforces the argument that DOPPA-dependent ZO-1 pooling at tricellular corners, associated with the appearance of CFTR in intracellular vesicles below the apical membrane, supports a structural role for the PKC beta2-isoform in the regulation of CFTR trafficking within a constitutive apical membrane targeting pathway. Figure 11 summarizes our working model for long-term PKC-beta2-dependent inhibition of apical membrane-generated ISC-cAMP common to both DOPPA and PMA monolayer exposure.


Figure 11
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  		Fig. 11. Working model of DOPPA- and PMA-induced CFTR redistribution in HT-29 Cl.19A monolayers. Top: control. The diffuse luminal CFTR staining pattern (red) in untreated monolayers localizes predominantly above the plane of the cellular tight junction (green ovals). Left: DOPPA. After DOPPA treatment, CFTR redistributes into vesicular structures apposed to and found both above and below the plane of the cellular tight junction. Right: PMA. The less specific but more potent phorbol ester induced a more reduced CFTR staining pattern with less spatial distribution within the cell. CFTR was often juxtaposed with both ZO-1 staining at the cellular tight junctions and within cytoplasmic ZO-1-labeled vesicles (Figs. 7–9). The observed coalescence of CFTR into vesicles after PKC-beta activation accounts for the apparent differences in mean overall CFTR IMF intensity X-axis summed YZ planes (Fig. 5) and protein expression levels assayed by 32P radiolabeling (Fig. 4).

 
Evidence supporting a role for PKC-beta2 as a modulator of constitutive CFTR movement within the trans-Golgi network/apical targeting pathway. Although PKC-beta1 and -beta2 are 96% identical [differing in only a small stretch of amino acids within the most terminal region of the catalytic domain (5)], they exhibit homolog-specific subcellular localization. Whereas PKC-beta1 partitions predominantly between the membrane and cytosol, the amino acid differences in the PKC-beta2 COOH-terminal region confer on this molecule the ability to bind to cytoskeletal actin with much greater avidity than PKC-beta1. A number of groups have investigated the signaling aspects of cytoskeletal PKC-beta2 association, and a picture is emerging supporting a role for this isoform in vesicular trafficking to and from the trans-Golgi network (TGN) and at other locals within epithelial cells. A role for PKC in constitutive vesicle release from TGN stacks has been identified in cell-free in vitro systems (10, 29). Furthermore, in polarized MDCK epithelial cells, nonspecific PKC beta-isoform depletion (11) inhibits the appearance of glycoprotein markers at the apical plasma membrane. These inhibitory effects are functionally similar to those recorded for brefeldin A, a fungal antibiotic that prevents antiretrograde vesicle movement within the constitutive biosynthetic pathway by inhibiting guanine nucleotide exchange factor modulation of ADP-ribosylation factor-1-dependent coat protein binding (6). We (21) have previously shown that a 24-h brefeldin A preincubation of HT-29 Cl.19A monolayers downregulates apical membrane-generated ISC-cAMP by preventing CFTR-containing vesicle movement between the TGN and apical plasma membrane. A separate group (2) has reported a temperature sensitivity to this phenomena corresponding to a post-TGN trafficking block, but they, unlike us, only recorded brefeldin A effects on PMA-stimulated ISC. The differences may relate to basal levels of PKC activity between each laboratories' cell lines. More recently, post-TGN apical glycoprotein glucose transporter GLUT2 trafficking in native rat jejunal enterocytes has also been reported as PKC-beta1 sensitive (14). At the molecular level, a receptor for activated PKC-beta (RACK-1) has been shown to accumulate within TGN, move coordinately with PKC-beta2 to the plasma membrane (24), and coimmunoprecipitate with both PKC-beta2 and with the pleckstrin homology domains of phospholipid-binding proteins involved in post-TGN/plasma membrane vesicle transport (23).

On the basis of these observations and our present results, we hypothesize that PKC-beta2 normally facilitates apical plasma membrane CFTR function by promoting channel movement within post-TGN stages of the constitutive biosynthetic pathway. However, during chronic DOPPA and PMA stimulation, PKC-beta2 downregulation (recorded as the cytoplasmic accumulation of kinase; Figs. 1 and 4) inhibits post-TGN vesicle movement and slows the biosynthetic delivery of CFTR into the subcellular apical pole. The corresponding decrease in apical ISC-cAMP (Figs. 1–4 in Ref. 9a) recorded under these conditions is hypothesized to reflect the continuing activity of non-PKC-dependent apical plasma membrane CFTR retrieval mechanisms [endocytosis (7)], thereby providing a cellular basis for apical plasma membrane current inhibition. The DOPPA-induced loss of CFTR in the apical pole associated with increased cytoplasmic partitioning and cellular mass of PKC-beta2 occurred independently of changes in the partitioning/mass of other isoforms. This supports the hypothesis that PKC-beta1/{epsilon} exert their effects on CFTR current generation at or close to the apical plasma membrane, whereas the effects of PKC-beta2 occur more distally within the cellular biosynthetic pathway.


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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-59550 (to A. P. Morris).


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. P. Morris, Div. of Gastroenterology, Depts. of Integrative Biology and Internal Medicine, Univ. of Texas Health Science Center, Rm. 4.236, Medical School Bldg., 6431 Fannin, Houston, TX 77030 (e-mail: Andrew.P.Morris{at}uth.tmc.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.


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 REFERENCES
 

  1. Augeron C and Laboisse CL. Emergence of permanently differentiated cell clones in a human colonic cancer cell line in culture after treatment with sodium butyrate. Cancer Res 44: 3961–3969, 1984.[Abstract/Free Full Text]
  2. Bajnath RB, Dekker K, De Jonge HR, and Groot JA. Chloride secretion induced by phorbol dibutyrate and forskolin in the human colonic carcinoma cell line HT-29Cl.19A is regulated by different mechanisms. Pflügers Arch 430: 705–712, 1995.[CrossRef][Web of Science][Medline]
  3. Bajnath RB, Groot JA, De Jonge HR, Kansen M, and Bijman J. Synergistic activation of non-rectifying small-conductance chloride channels by forskolin and phorbol esters in cell-attached patches of the human colon carcinoma cell line HT-29cl.19A. Pflügers Arch 425: 100–108, 1993.[CrossRef][Web of Science][Medline]
  4. Bargon J, Trapnell BC, Yoshimura K, Dalemans W, Pavirani A, Lecocq JP, and Crystal RG. Expression of the cystic fibrosis transmembrane conductance regulator gene can be regulated by protein kinase C. J Biol Chem 267: 16056–16060, 1992.[Abstract/Free Full Text]
  5. Blobe GC, Stribling DS, Fabbro D, Stabel S, and Hannun YA. Protein kinase C beta II specifically binds to and is activated by F-actin. J Biol Chem 271: 15823–15830, 1996.[Abstract/Free Full Text]
  6. Boman AL, Zhang C, Zhu X, and Kahn RA. A family of ADP-ribosylation factor effectors that can alter membrane transport through the trans-Golgi. Mol Biol Cell 11: 1241–1255, 2000.[Abstract/Free Full Text]
  7. Bradbury NA and Bridges RJ. Endocytosis is regulated by protein kinase A, but not protein kinase C in a secretory epithelial cell line. Biochem Biophys Res Commun 184: 1173–1180, 1992.[CrossRef][Web of Science][Medline]
  8. Brakenhoff GJ, van Spronsen EA, van der Voort HT, and Nanninga N. Three-dimensional confocal fluorescence microscopy. Methods Cell Biol 30:379–98.: 379–398, 1989.[Medline]
  9. Breuer W, Glickstein H, Kartner N, Riordan JR, Ausiello DA, and Cabantchik IZ. Protein kinase C mediates down-regulation of cystic fibrosis transmembrane conductance regulator levels in epithelial cells. J Biol Chem 268: 13935–13939, 1993.[Abstract/Free Full Text]
  10. Broughman JR, Sun L, Umar S, Scott J, Sellin JH, and Morris AP. Chronic PKC-beta activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane current generation. Am J Physiol Gastrointest Liver Physiol 291: G318–G330, 2006.[Abstract/Free Full Text]
  11. Buccione R, Bannykh S, Santone I, Baldassarre M, Facchiano F, Bozzi Y, Di TG, Mironov A, Luini A, and De Matteis MA. Regulation of constitutive exocytic transport by membrane receptors. A biochemical and morphometric study. J Biol Chem 271: 3523–3533, 1996.[Abstract/Free Full Text]
  12. De Matteis MA, Santini G, Kahn RA, Di TG, and Luini A. Receptor and protein kinase C-mediated regulation of ARF binding to the Golgi complex. Nature 364: 818–821, 1993.[CrossRef][Medline]
  13. Hansra G, Garcia-Paramio P, Prevostel C, Whelan RD, Bornancin F, and Parker PJ. Multisite dephosphorylation and desensitization of conventional protein kinase C isotypes. Biochem J 342: 337–344, 1999.
  14. Hecht G, Robinson B, and Koutsouris A. Reversible disassembly of an intestinal epithelial monolayer by prolonged exposure to phorbol ester. Am J Physiol Gastrointest Liver Physiol 266: G214–G221, 1994.[Abstract/Free Full Text]
  15. Helliwell PA, Richardson M, Affleck J, and Kellett GL. Stimulation of fructose transport across the intestinal brush-border membrane by PMA is mediated by GLUT2 and dynamically regulated by protein kinase C. Biochem J 350: 149–154, 2000.
  16. Kiley SC, Olivier AR, Gordge PC, Ryves WJ, Evans FJ, Ways DK, and Parker PJ. 12-Deoxyphorbol-13-O-phenylacetate-20-acetate is not protein kinase C-beta isozyme-selective in vivo. Carcinogenesis 15: 319–324, 1994.[Abstract/Free Full Text]
  17. Liedtke CM and Cole TS. Antisense oligonucleotide to PKC-epsilon alters cAMP-dependent stimulation of CFTR in Calu-3 cells. Am J Physiol Cell Physiol 275: C1357–C1364, 1998.[Abstract/Free Full Text]
  18. MacDonald I, Knox KA, and Gordon J. Stimulation of human B lymphocytes by phorbol esters reported to be selective in the protein kinase C isoforms they activate. Mol Immunol 31: 671–674, 1994.[CrossRef][Web of Science][Medline]
  19. Matter K and Balda MS. Functional analysis of tight junctions. Methods 30: 228–234, 2003.[CrossRef][Web of Science][Medline]
  20. Meyer TN, Schwesinger C, and Denker BM. Zonula occludens-1 is a scaffolding protein for signaling molecules. Galpha(12) directly binds to the Src homology 3 domain and regulates paracellular permeability in epithelial cells. J Biol Chem 277: 24855–24858, 2002.[Abstract/Free Full Text]
  21. Mezquita J, Pau M, and Mezquita C. Several novel transcripts of glyceraldehyde-3-phosphate dehydrogenase expressed in adult chicken testis. J Cell Biochem 71: 127–139, 1998.[CrossRef][Web of Science][Medline]
  22. Morris AP. The regulation of epithelial cell cAMP- and calcium-dependent chloride channels. Adv Pharmacol 46: 209–251, 1999.
  23. Morris AP, Cunningham SA, Tousson A, Benos DJ, and Frizzell RA. Polarization-dependent apical membrane CFTR targeting underlies cAMP-stimulated Cl– secretion in epithelial cells. Am J Physiol Cell Physiol 266: C254–C268, 1994.[Abstract/Free Full Text]
  24. Rodriguez MM, Ron D, Touhara K, Chen CH, and Mochly-Rosen D. RACK1, a protein kinase C anchoring protein, coordinates the binding of activated protein kinase C and select pleckstrin homology domains in vitro. Biochemistry 38: 13787–13794, 1999.[CrossRef][Medline]
  25. Ron D, Jiang Z, Yao L, Vagts A, Diamond I, and Gordon A. Coordinated movement of RACK1 with activated betaIIPKC. J Biol Chem 274: 27039–27046, 1999.[Abstract/Free Full Text]
  26. Shen BQ, Barthelson RA, Skach W, Gruenert DC, Sigal E, Mrsny RJ, and Widdicombe JH. Mechanism of inhibition of cAMP-dependent epithelial chloride secretion by phorbol esters. J Biol Chem 268: 19070–19075, 1993.[Abstract/Free Full Text]
  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. Umar S, Scott J, Sellin JH, Dubinsky WP, and Morris AP. Murine colonic mucosa hyperproliferation. I. Elevated CFTR expression and enhanced cAMP-dependent Cl– secretion. Am J Physiol Gastrointest Liver Physiol 278: G753–G764, 2000.[Abstract/Free Full Text]
  29. Umar S, Sellin JH, and Morris AP. Murine colonic mucosa hyperproliferation. II. PKC-beta activation and cPKC-mediated cellular CFTR overexpression. Am J Physiol Gastrointest Liver Physiol 278: G765–G774, 2000.[Abstract/Free Full Text]
  30. Xu H, Greengard P, and Gandy S. Regulated formation of Golgi secretory vesicles containing Alzheimer beta-amyloid precursor protein. J Biol Chem 270: 23243–23245, 1995.[Abstract/Free Full Text]
  31. Yoo J, Nichols A, Mammen J, Calvo I, Song JC, Worrell RT, Matlin K, and Matthews JB. Bryostatin-1 enhances barrier function in T84 epithelia through PKC-dependent regulation of tight junction proteins. Am J Physiol Cell Physiol 285: C300–C309, 2003.[Abstract/Free Full Text]
  32. Zahraoui A, Louvard D, and Galli T. Tight junction, a platform for trafficking and signaling protein complexes. J Cell Biol 151: F31–F36, 2000.[Abstract/Free Full Text]



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