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CELLULAR AND MOLECULAR

Isoform of Protein Kinase C Alters Barrier Function in Intestinal Epithelium through Modulation of Distinct Claudin Isotypes: A Novel Mechanism for Regulation of Permeability

Departments of Internal Medicine, Pharmacology, and Molecular Physiology, Division of Digestive Diseases, Rush University Medical Center, Chicago, Illinois

Received January 12, 2005; accepted March 2, 2005.

	Abstract

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Using monolayers of intestinal Caco-2 cells, we discovered that the isoform of protein kinase C (PKC), a member of the "novel" subfamily of PKC isoforms, is required for monolayer barrier function. However, the mechanisms underlying this novel effect remain largely unknown. Here, we sought to determine whether the mechanism by which PKC- disrupts monolayer permeability and dynamics in intestinal epithelium involves PKC--induced alterations in claudin isotypes. We used cell clones that we recently developed, clones that were transfected with varying levels of plasmid to either stably suppress endogenous PKC- activity (antisense, dominant-negative constructs) or to ectopically express PKC- activity (sense constructs). We then determined barrier function, claudin isotype integrity, PKC- subcellular activity, claudin isotype subcellular pools, and claudin phosphorylation. Antisense transfection to underexpress the PKC- led to monolayer instability as shown by reduced 1) endogenous PKC- activity, 2) claudin isotypes in the membrane and cytoskeletal pools (claud-1, claud-4 assembly), 3) claudin isotype phosphorylation ( phospho-serine, phospho-threonine), 4) architectural stability of the claudin-1 and claudin-4 rings, and 5) monolayer barrier function. In these antisense clones, PKC- activity was also substantially reduced in the membrane and cytoskeletal cell fractions. In wild-type (WT) cells, PKC- (82 kDa) was both constitutively active and coassociated with claudin-1 (22 kDa) and claudin-4 (25 kDa), forming endogenous PKC-/claudin complexes. In a second series of studies, dominant-negative inhibition of the endogenous PKC- caused similar destabilizing effects on monolayer barrier dynamics, including claudin-1 and -4 hypophosphorylation, disassembly, and architectural instability as well as monolayer disruption. In a third series of studies, sense overexpression of the PKC- caused not only a mostly cytosolic distribution of this isoform (i.e., <12% in the membrane + cytoskeletal fractions, indicating PKC- inactivity) but also led to disruption of claudin assembly and barrier function of the monolayer. The conclusions of this study are that PKC- activity is required for normal claudin assembly and the integrity of the intestinal epithelial barrier. These effects of PKC- are mediated at the molecular level by changes in phosphorylation, membrane assembly, and/or organization of the subunit components of two barrier function proteins: claudin-1 and claudin-4 isotypes. The ability of PKC- to alter the dynamics of permeability protein claudins is a new function not previously ascribed to the novel subfamily of PKC isoforms.

A key discovery in recent years in IBD pathophysiology was the understanding that a leaky intestinal barrier, the so-called hyperpermeable gut, can lead to intestinal inflammation and tissue injury and that maintaining a normal mucosal barrier function is required for intestinal health. For example, transgenic rodents with a hyperpermeable-intestinal-barrier exhibit intestinal mucosal inflammation (Hermiston and Gordon, 1995). Similarly, loss of intestinal barrier function induced by the injection of bacterial derivatives into the mucosa of rodents elicits IBD-like conditions (Yamada et al., 1993). Accordingly, investigating the molecular events underlying the alterations of gut barrier function is of fundamental biological and clinical value.

In our efforts to better understand mechanisms underlying barrier disruption and gut inflammation, we have been investigating molecular mechanisms underlying loss of barrier function in the epithelium of the intestinal mucosa. Using monolayers of intestinal Caco-2 cells, as a well established and widely used model for barrier function, we have reported (Banan et al., 1998a, 1999; 2000a,b, 2001a,b,c) that cytoskeletal assembly and stability is required for epithelial integrity. Moreover, we have shown that changes in protein kinase C (PKC) affect epithelial barrier function (Banan et al., 2001a,b). Although PKC, in general, is known to alter epithelial barrier, the role of specific PKC isoforms in barrier regulation and their mechanisms have remained largely unknown.

PKC consists of a family of serine- and threonine-specific kinases, which includes at least 12 known isoenzymes that can be classified into three subfamilies (Housey et al., 1988; Ponzoni et al., 1993; Maruvada and Levine, 1999; Banan et al., 2001a,b, 2002a, 2003a,b, 2004): conventional (or "classical") PKC isoforms (, 1, 2, and ), "novel" PKC isoenzymes (, , , , and µ), and "atypical" PKC isoforms (, , and ). Intestinal cells (including Caco-2 cells) express at least 10 isoforms of PKC, including PKC-, PKC-1, PKC-2, PKC-, PKC-, PKC-, PKC-, PKC-, PKC-, and PKC- (McKenna et al., 1995; Maruvada and Levine, 1999; Banan et al., 2001a,b, 2002a, 2003a,b, 2004). These isozymes differ in their mechanism of activation, subcellular distribution, substrate type, and expression, suggesting that each of these PKC isoforms can perform unique biological tasks (Persons et al., 1988; Melloni et al., 1990; Mischak et al., 1993; Ponzoni et al., 1993; Banan et al., 2002a, 2003a, 2004). In our previous studies, we demonstrated both damaging and protective mechanisms/pathways affecting gut barrier integrity and permeability. For example, we showed that growth factors (epidermal growth factor or transforming growth factor-) prevent oxidant-induced barrier dysfunction via activation of the classical PKC-1 isoform as well as by the atypical PKC- isoform, leading to monolayer protection (Banan et al., 2001a, 2003a,b). More recently, we reported novel findings that PKC- seems to affect epithelial barrier function (Banan et al., 2004). We showed that activation of the 82-kDa PKC-, a novel PKC isoform, is involved in epithelial barrier alterations. Despite the importance of the isoform of PKC to intestinal barrier permeability, the fundamental mechanisms underlying PKC--mediated alterations in monolayer barrier function still remain largely unexplored.

The ability of the intestinal epithelium to maintain selective barrier permeability depends on a complex array of protein filaments that includes the cytoskeleton and barrier function proteins such as claudins (Banan et al., 1996, 1998a,b, 1999, 2000a,b, 2001a,c; Unno et al., 1997; Furuse et al., 1999; Yoo et al., 2003a). For example, claudins, a crucial family of barrier function proteins, constitute the major components of the junctional strands that delineate apical-lateral membranes as well as form the so-called seal (barrier) in the intercellular space (Furuse et al., 1999; Ishizaki et al., 2003; Yoo et al., 2003a; Fujibe et al., 2004). The claudin family consists of at least 23 different isotypes that are essential in regulation of permeability function of cell monolayers (Furuse et al., 1999). In particular, claudin isotypes such as claudin-1 to -5 are thought not only to form aqueous pores within the cellular junctional strands but also are important to paracellular permeability of the intestinal epithelium (Yoo et al., 2003a). The mechanisms underlying regulation of claudins and intestinal barrier function remain poorly understood.

In the current study, we determined the role of the isoform of PKC in the underlying mechanisms of barrier regulation and investigated possible alterations of key claudin proteins in regulation of the permeability of the intestinal epithelium. Exploring the role of the PKC- in the mechanism of epithelial barrier function is essential because 1) it is of significant biological importance to establish the idea that "specific" isoforms of PKC play fundamental roles in endogenous modulatory mechanisms of cellular proteins required for the maintenance of intestinal barrier function, and 2) a better understanding of effectively modulating (e.g., by PKC-) the permeability function of the intestinal mucosal epithelium could lead to the development of novel therapeutic strategies for inflammatory diseases of the gastrointestinal tract that are related to disruption of barrier function.

To this end, we studied the effects of PKC- on claudin proteins using three complementary molecular approaches. First, using an antisense approach, native (82-kDa) PKC- isoform was reliably underexpressed. Second, using a dominant-negative approach, endogenous PKC- isoform was inactivated. Third, using a sense expression approach, native PKC- was reliably increased. We tested the hypothesis that PKC- isoform is required for changes in the dynamics of claudin assembly and cytoarchitecture and monolayer permeability function in intestinal epithelium. We now report novel mechanisms dependent on the PKC- isoform activity, namely, alterations of the claudin-1 and claudin-4 isotypes phosphorylation, membrane assembly, and distribution as well as permeability function in cell monolayers. The biological ability to change the dynamics of barrier function protein claudins is a new mechanism not previously attributed to the novel subfamily of PKC isoforms in cells.

	Materials and Methods

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Cell Culture. Caco-2 cells were obtained from American Type Culture Collection (Manassas, VA) at passage 15. This widely used colonic cell line was chosen for our studies because they form monolayers that morphologically resemble intestinal cells, with defined apical brush borders and tight junctions, and a highly organized claudin-ring network upon differentiation (Gilbert et al., 1991; Meunier et al., 1995; Banan et al., 1998b). Caco-2 cells are a transformed cell line, and monolayers of tumor cells may respond differently than do nontransformed cells, including enterocytes in native tissue. Nonetheless, there is a wide body of evidence collected on these colonic cells over the past decade that shows their "normal" intestinal epithelial characteristics, which makes them near ideal for gut barrier function studies. For example, Caco-2 cells form monolayers that can be studied for weeks, rather than just days, as is typical of most fresh in vitro preparations (Hurani et al., 1993; Meunier et al., 1995; Unno et al., 1997). This allowed us to measure alterations in intestinal barrier function. In addition, Caco-2 cells closely resemble normal intestinal cells in that they express intestinal hydrolases such as sucrase-isomaltase and alkaline phosphatase. Furthermore, these cells are similar to native intestinal epithelial cells in that they have receptors for prostaglandins, growth factors, vasoactive intestinal peptide, low-density lipoprotein, insulin, and specific substrates such as dipeptides, fructose, glucose, hexoses, and vitamin B₁₂ (Gilbert et al., 1991; Meunier et al., 1995; Banan et al., 1998b). Accordingly, this cell line provides a suitable in vitro model for our studies. To show the general validity of our findings, in select experiments another colonic cell line, namely, HT-29, was used (Balogh et al., 1995; Banan et al., 2002b). Wild-type cells or stably transfected cells (see below) were split at a ratio of 1:6 upon reaching confluence and set up in either six- or 24-well plates for experiments or in T-75 flasks for propagation. Cells were used 7 to 10 days postconfluence. The utility and characterization of these cell lines have been reported previously (Balogh et al., 1995; Meunier et al., 1995; Banan et al., 1996, 1998a,b, 1999, 2000a,b, 2001b,c, 2002ab, 2003a,b, 2004).

Plasmids and Transfection. The sense, antisense, and dominant-negative plasmids of PKC- were constructed as we described previously (Banan et al., 2001a, 2002a, 2004). A unique tetracycline-responsive expression (TRE) system was used to overexpress the native PKC-. cDNA encoding the entire reading frame of PKC- plasmid was subcloned into the TRE vector, creating TRE PKC-. The antisense or dominant-negative PKC- plasmid was also subcloned to create AS-PKC- and dominant-negative PKC-.

Cultures of intestinal cells grown to 50 to 60% confluence were cotransfected with hygromycin resistance plasmid and expression plasmids encoding either sense PKC-, antisense PKC-, or dominant-negative PKC- by Lipofectin (Invitrogen, Carlsbad, CA) as we described previously (Banan et al., 2001a; 2004). Control conditions included vector alone. Briefly, cells were incubated for 16 h at 37°C with the plasmid DNA in serum-free media in the presence of LipofectAMINE (25 µl/25-cm² flask; Invitrogen). Subsequently, the DNA-containing solution was removed and replaced by fresh media containing 10% fetal bovine serum to relieve cells from the shock of exposure to serum-free media. After transfection, cells were subjected to hygromycin selection (1 mg/ml). Resistant cells were maintained in DMEM/fetal bovine serum and 0.2 mg/ml hygromycin (selection medium). For inducible expression of PKC-, cells were transfected with a plasmid expressing the tetracycline-responsive transactivator (tTA) along with a second plasmid conferring resistance to G418 (Geneticin). After selection in 0.6 mg/ml G418 (selection media), one such clone (i.e., parental tTA) was then itself transfected with the TRE PKC- system. Hygromycin resistance plasmid was included to confer resistance to hygromycin (selection marker, 1 mg/ml). Control conditions included vector alone (TRE-z). Multiple clones expressing PKC- or lacking PKC- activity were assessed by immunoblotting and activity assay (Banan et al., 2004) and then used for experiments.

Experimental Design. In the first series of experiments, post-confluent monolayers of wild (naive)-type cells were incubated with vehicle (isotonic saline) for 30 min and then assessed for baseline conditions. Experiments were then repeated using multiple clones of stably transfected cells. In all experiments, we assessed claudin skeletal stability (e.g., subapical ring cytoarchitecture), claudin subcellular distribution and assembly (claudin isotype distribution in membrane/cytoskeletal/cytosolic pools), claudin isotype phosphorylation (phospho-serine and phospho-threonine), PKC- intracellular distribution (membrane, cytoskeletal, and cytosolic fractions), PKC- activity (immunoprecipitation and in vitro kinase assay), and monolayer barrier permeability (clearance of two different sized fluorescent dextran probes, FD70 kDa versus FD4 kDa).

In a second series of experiments, sense-transfected cell clones overexpressing PKC- were used. To this end, multiple sense clones that were stably overexpressing PKC- (i.e., TRE PKC-) were grown as monolayers and then exposed to vehicle. Outcomes measured were as described above. In these overexpression studies, cells were grown for 48 h in the absence of tetracycline before experiments.

In a third series of experiments, monolayers of antisense-transfected cells lacking PKC- activity were treated with vehicle. In all experiments, PKC- activity was determined in immunoprecipitated samples (see below). In a corollary series of experiments, we investigated the effects of a PKC- dominant-negative mutant on the state of monolayer barrier function, claudin isotype subcellular pool/assembly, and claudin isotype phosphorylation and cytoarchitecture. To this end, claudin isotypes (the structural protein subunit of permeability junctions) were isolated and then analyzed by immunoblotting. Claudin integrity was assessed by 1) immunofluorescent labeling and fluorescence microscopy to determine the percentage of cells with normal claudin isotypes, 2) detailed analysis by high-resolution laser scanning confocal microscopy (LSCM), and 3) immunoprecipitation and PAGE analysis of claudins.

Fractionation and Immunoblotting of PKC. Cell monolayers grown in large 75-cm² flasks were processed for the isolation of the cytosolic, membrane and cytoskeletal fractions as we described previously (Banan et al., 2001a, 2004). Protein content of these various fractions was assessed by the Bradford method (Bradford, 1976). Samples (5 µg of protein/lane) were separated by PAGE. The immunoblotted proteins were visualized by ECL (Amersham Biosciences, Inc., Piscataway, NJ) and autoradiography (e.g., 1 h at –20°C). The exposure times were adjusted to ensure linear responses. Under these conditions, the chemiluminescence assay was linear between 1 and 10 µg of total protein. Standard (purified PKC-) loading controls (1 µg/lane) were also run concurrently with each run. To further verify equal loading, blots were routinely stained with 0.1% India ink in TBST buffer.

For the isolation of the cell fractions, after treatments, postconfluent monolayers were scraped and ultrasonically homogenized (GE130, GE Ultrasonic Processor, amplitude 50, 6 pulses/s, duration 20 s) in Tris-HCl buffer (20 mM Tris-HCl, pH 7.5, 0.25 mM sucrose, 2 mM EDTA, 10 mM EGTA, 2 µg/ml aprotinin, 2 µg/ml pepstatin, 2 µg/ml leupeptin, and 2 µg/ml phenylmethylsulfonyl fluoride). The homogenates were then ultracentrifuged (100,000g for 40 min at 4°C), and the supernatant was removed and used as a source of the cytosolic fraction. Next, pellets were washed with 0.2 ml of Tris-HCl buffer and resuspended in 0.8 ml of buffer containing 0.3% Triton X-100 and maintained on ice for 1 h. The samples were then centrifuged (100,000g for 1 h at 4°C), and the supernatant was used as the source of the membrane fraction. To this remaining pellet, we added 0.3 ml of cold (4°C) lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 2 µg/ml aprotinin, 2 µg/ml pepstatin, 2 µg/ml leupeptin, and 2 µg/ml phenylmethylsulfonyl fluoride). The samples were then placed on ice for 1 h and ultracentrifuged as described above. The remainder of the lysate or Triton-insoluble cytoskeletal fraction was then removed. For total extraction, which provides the fraction used to confirm total PKC-, scraped monolayers were placed directly into 1.5 ml of cold lysis buffer and subsequently ultracentrifuged as described above. The supernatant was used for bulk protein determination.

For immunoblotting, samples (5 µg protein/lane) were added to a standard SDS buffer, boiled, and then separated on 7.5% SDS-PAGE (Banan et al., 2004). The immunoblotted proteins were incubated with a primary monoclonal antibody to PKC- [nPKC- (E7), sc-1680 (human reactive), Santa Cruz Biotechnology, Inc., CA] at 1:3000 dilution. A horseradish peroxidase-conjugated antibody (Molecular Probes, Eugene, OR) was used as a secondary antibody at 1:4000 dilution. Proteins were visualized by enhanced chemiluminescence and autoradiography and subsequently analyzed by densitometry. The identity of the PKC- band was assessed by a procedure that we described previously (Banan et al., 2004). 1) Using a PKC- blocking peptide (sc-1680 P; Santa Cruz Biotechnology, Inc.) in combination with the anti-PKC- antibody that prevents the appearance of the corresponding "major" band in Western blots. Additionally, 2) in the absence of the primary antibody to PKC-, no corresponding band for PKC- was observed. 3) The PKC- band ran at the expected molecular mass of 82 kDa as confirmed by a known positive control for PKC- (from rat brain lysates). 4) Prestained molecular weight markers (M_r 67,000 and 93,000) were run in adjacent lanes. In other studies using total PKC extracts, we confirmed our previous findings (Banan et al., 2004) that expression of PKC- or inhibition of PKC- did not affect the relative expression levels of other PKC isoforms.

Immunoprecipitation and PKC- Activity Assay. Immunoprecipitated PKC- was collected and processed for its ability to phosphorylate a synthetic peptide (Banan et al., 2004). Briefly, after treatments, confluent cell monolayers were lysed by incubation for 20 min in 500 µl of cold lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 µg/ml antiprotease cocktail, 10% glycerol, 1 mM sodium orthovanadate, 5 mM NaF, and 1% Triton X-100). The lysates were clarified by centrifugation at 14,000g for 10 min at 4°C. For immunoprecipitation, the lysates were incubated for 90 min at 4°C with anti-PKC- (1:2000 dilution, in excess). The extracts were then incubated with protein A/G plus agarose for 1 h at 4°C. The immunocomplexes were collected by centrifugation (2500g for 5 min) in a microcentrifuge, tube, and washed three times with immunoprecipitation buffer containing 5 mM Tris-HCl, pH 7.4, and 0.2% Triton X-100. They were then washed one time with kinase buffer (20 mM HEPES, pH 7.5, 10 mM MgCl₂, 2 mM MnCl₂, and 20 µM ATP) and resuspended in 20 µl of kinase buffer and 5 µl of 5x reaction buffer (1 mg/ml histone H1 and 0.25 mg/ml L--phosphotidyl-L-serine) plus 5 µCi of [-³²P]ATP and subsequently incubated for 5 min at 30°C. Reactions were then stopped by the addition of 8 µl of 5x sample buffer, and the samples were boiled at 95°C for 5 min before separation by 7.5% PAGE. The extent of histone H1 phosphorylation was determined by scintillation counting of excised Coomassie Blue histone polypeptide bands. Counts for blanks were subtracted from the sample activity. Sample activity was also corrected for protein concentration (Bradford method; Bradford, 1976), and PKC- activity was reported as picomoles per minute per milligram of protein.

Immunofluorescent Staining and High-Resolution Laser Scanning Confocal Microscopy of Claudin Isotypes. Cell monolayers were fixed in a standard cytoskeletal stabilization buffer and then postfixed in 95% ethanol at –20°C as we described previously (Banan et al., 1998a,b, 1999, 2000a,b, 2001a,b,c). Cells were subsequently processed for incubation with an isotype-specific primary antibody, monoclonal anti-claudin isotypes (claudin-1, -2, -3, -4, or -5; Zymed Laboratories, South San Francisco, CA), and then with a secondary antibody (fluorescein isothiocyanate- and/or Texas Red-conjugated; Sigma-Aldrich, St. Louis, MO). After staining of claudins, cells were observed using a 63x oil immersion plan-apochromat objective, numerical aperture 1.4 (Carl Zeiss, Jena, Germany). The claudin elements were examined in a blinded manner for their overall morphology, orientation, and disruption as we have described for other cytoskeletal components previously (Banan et al., 1998a,b, 1999, 2000a,b, 2001a,b,c). At least 1200 cells per group (200 x 6 slides) were examined by a blinded observer in four different subapical fields by laser scanning confocal microscopy, and the percentage of cells displaying normal claudins was determined, reported as mean ± S.E.M. The identity of the treatment groups for all slides was decoded only after examination was complete.

In other studies, immunofluorescent analysis of the subcellular distribution of claudins was performed using monolayers that were 10 days postconfluence. Using laser scanning confocal microscopy, Z-scans of the entire height of cell monolayers were obtained with scans performed in 200-nm steps over a range of 10 µm (25–50 Z-stacks per cell monolayer area). Because claudins such as claudin-1 and claudin-4 exhibited the highest levels of expression at the "subapical" membrane (e.g., 1.8-µm subapical junctional) areas of intestinal cells, we focused on these areas in subsequent immunofluorescent studies.

Fractionation and Immunoblotting of Claudins. Subcellular fractions were isolated using the methods we described for fractionation and immunoblotting of PKC and cytoskeleton (Banan et al., 2003b, 2004). After fractionation, the "claudin skeleton" was recovered by separately incubating (at 37°C for 30 min) the subcellular fractions (membrane, cytoskeletal, and cytosolic) with stabilizing agents, 1 mM MgSO₄ and 1 mM ATP in cytoskeletal stabilization buffer (0.1 M Pipes, pH 6.9, 30% glycerol, 5% dimethyl sulfoxide, 10 µg/ml antiprotease cocktail, 1 mM EGTA, 1 mM MgCl₂, and 1 mM ATP). Claudins were then recovered by centrifugation and resuspended in the above-mentioned stabilization buffer (cytoskeletal stabilization buffer). Fractionated samples were then flash frozen in liquid N₂ and stored at –70°C until immunoblotting. For immunoblotting, samples (5 µg of protein/lane) were placed in a standard SDS sample buffer, boiled, and then subjected to PAGE on 7.5% gels using appropriate (isotype-specific) antibodies. Standard (purified) claudin loading controls (5 µg/lane) were run concurrently with each run. To additionally verify equal loading, blots were routinely stained with 0.1% India ink in TBST buffer. Furthermore, after the blots were stripped, actin (43 kDa) immunoblotting was performed as an internal control for equal loading.

Analysis of Claudin Phosphorylation. Claudins were collected and assessed for phosphorylation by PAGE (Banan et al., 2004). For immunoprecipitation, cell lysates were incubated for 4 h at 4°C with isotype-specific monoclonal anti-claudin antibodies (claudin-1, -2, -3, -4, or -5; 1:50 dilution, in excess). The extracts were then incubated with protein G plus Sepharose 4B (Zymed Laboratories) for 2 h at 4°C. The immunocomplexes were collected by centrifugation (2500g for 5 min) in a microcentrifuge tube, washed three times with immunoprecipitation buffer containing 5 mM Tris-HCl, pH 7.4, and 0.2% Triton X-100. The resultant pellets were resuspended in a standard SDS sample buffer and boiled at 95°C for 5 min before separation by PAGE. Gels were transferred to nitrocellulose membranes, blocked with 1% bovine serum albumin and 0.01% Tween 20 in phosphate-buffered saline for blotting by either anti-phosphoserine or anti-phosphothreonine (1:3000 dilution; BD Transduction Laboratories, Lexington, KY), and for detection of immune complexes by horseradish peroxidase-conjugated secondary antibody, and then incubated with chemiluminescent (ECL) reagents and autoradiographed.

To assess the specificity of claudin phosphorylation, in corollary experiments, blots used to detect the phospho-serine and phosphothreonine states of claudin immunoprecipitates were placed in protein stripping buffer and then reprobed with the corresponding isotype-specific monoclonal claudin antibodies. These protocols were done to confirm that changes in the serine- and threonine-phosphorylation were in fact specifically associated with claudin-1 and claudin-4 proteins (and not due to simple reactivity against nonspecific targets). To further assess phosphorylation specificity, we concurrently coincubated the immunoprecipitated PKC- with the immunoprecipitated claudin (claudin-1 or -4) under in vitro (test tube) conditions. For these protocols, the 1 mg/ml PKC- immunocomplexes were resuspended in 20 µl of kinase buffer (20 mM HEPES, pH 7.5, 10 mM MgCl₂, 2 mM MnCl₂, and 20 µM ATP) and 5 µlof5x reaction buffer (containing 1 mg/ml claudin-1 or -4 and 0.25 mg/ml L--phosphotidyl-L-serine) plus 5 µCi of [-³²P]ATP and subsequently incubated for 5 min at 30°C. Reactions were then stopped by the addition of 8 µlof5x sample buffer, and the samples were boiled at 95°C for 5 min before separation by PAGE. Identical patterns of phosphorylation were seen as that of the protocols using immunoprecipitated claudins alone after PAGE.

In all experiments, positive loading controls were run concurrently with each run. Specifically, for claudin phosphorylation blots, known positive controls (5 µg of corresponding phosphorylated claudins) were run with each gel (purified claudin isotype was phosphorylated by an in vitro kinase reaction via addition of 1 mg/ml PKC- in 20 µl of kinase buffer containing 20 mM HEPES, pH 7.5, 10 mM MgCl₂, 2 mM MnCl₂, and 20 µM ATP). These positive controls also served as additional loading controls (5 µg of loading control per lane) that allowed for the comparison of data derived from different blots (or different days). Moreover, to ensure accuracy, loading controls were routinely run in duplicates or in some cases in triplicates. To further verify equal loading of lanes, blots were routinely stained with 0.1% India ink in TBST buffer (containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20).

Determination of Monolayer Barrier Permeability by Fluorometry. Status of monolayer barrier function was assessed by a widely used and validated technique that measures the apical to basolateral paracellular clearance of fluorescent markers such as fluorescein dextrans (FD70 kDa, 1 mg/ml) as we (Banan et al., 1999, 2000a,b, 20001a,b,c, 2002a) and others (Hurani et al., 1993; Sanders et al., 1995; Unno et al., 1997) described previously. In select experiments, a lower molecular weight fluorescein dextran (FD4 kDa, 1 mg/ml) was used. Briefly, fresh phenol-free DMEM (800 µl) was placed into the lower (basolateral) chamber and phenol-free DMEM (300 µl) containing probe (FD70 or FD4) was placed in the upper (apical) chamber. Aliquots (50 µl) were obtained from the upper and lower chambers at zero time and at subsequent time points and transferred into clear 96-well plates (clear bottom; Costar, Cambridge, MA). Fluorescent signals from samples were quantitated using a fluorescence multiplate reader (FL 600; BIO-TEK Instruments). The excitation and emission spectra for probes were excitation = 485 nm and emission = 530 nm. In exploratory studies assessing barrier function in our PKC--transfected models, we consistently observed picomolar to nanomolar fluxes of FD in a range of sizes (4–70 kDa) across Caco-2 monolayers. Such FD flux was dependent on the transfected clones used (e.g., 1 µg of antisense versus 5 µg of antisense clone) and thus flux (clearance) increased with increasing amounts of DNA transfected. Also, when adding FD at concentrations in the range 0.001 to 1 mg/ml, we observed a graded increase in FD flux. We were also able to consistently observe fluxes of picogram quantities of FD across monolayers. Clearance was then calculated as flux divided by surface area of the monolayer (Hurani et al., 1993; Sanders et al., 1995; Unno et al., 1997). Thus, flux (nanoliters per hour) was calculated and subsequently normalized to surface area of monolayers (0.3 cm²) and reported as a clearance (nanoliters per hour per square centimeter). More specifically, clearance (Cl) was calculated using the following formula: Cl (nL/h/cm²) = Fab/([FD70 or FD4]a x S), where Fab is the apical to basolateral flux of FD70 or FD4 (light units per hour, which is dependent on the fluorescent signals of samples obtained from the lower and upper chambers of Transwell culture inserts), [FD70 or FD4]a is the concentration at baseline (light units per nanoliter), and S is the surface area (i.e., 0.3 cm²). Simultaneous (concurrent) controls were run with each experiment.

Statistical Analysis. Data are presented as mean ± S.E.M. All experiments were carried out with a sample size of at least six observations per treatment group. Statistical analysis comparing treatment groups was performed using analysis of variance followed by Dunnett's multiple range test (Harter, 1960). Correlational analyses were done using the Pearson test for parametric analysis or, when applicable, the Spearman test for nonparametric analysis. p values <0.05 were deemed statistically significant.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Our previous findings (Banan et al., 2004) showed that intestinal cells cotransfected with complementary DNA (cDNA) encoding the hygromycin resistance (for selection) and the PKC- anti-sense (AS-PKC-) stably underexpress the novel , (82-kDa) isoform of PKC (nPKC-). Multiple clones of intestinal cells transfected with 1 to 5 µg of PKC- antisense cDNA demonstrated a dose-dependent underexpression of the PKC- protein (Fig. 1A, immunoblot). The clone transfected with 4 µg of PKC- antisense (-4) led to the largest reduction (99%) in the levels of native PKC- protein while inducing maximum monolayer barrier disruption (increasing clearance of fluorescein sulfonic acid, 0.478 kDa). Transfection of only the empty vector (control vector alone) did not do so. In the present investigation, we have studied the underlying mechanisms by which nPKC- affects barrier function.

View larger version (61K): [in this window] [in a new window]   Fig. 1. A, percentage of intestinal Caco-2 cells with a normal claudin-1 isotype cytoarchitecture from multiple cell clones transfected with a plasmid encoding antisense to native PKC- (AS-PKC-). Uniquely transfected intestinal (Caco-2) cell clones we have recently developed that stably underexpress the novel PKC- were used (see immunoblot). Note the dose-dependent underexpression of native PKC- protein (82 kDa). PKC- antisense underexpression in a dose-dependent manner also deleteriously effects the claudin-1 skeleton as demonstrated by low percentage of cells with normal claudin-1 isotype (paralleling findings on monolayer barrier dysfunction; see Table 1). The 4-µg antisense clone, which effectively suppressed PKC- protein expression, led to the highest number of cells with unstable claudin-1. Wild-type cells (those exhibiting native PKC- protein levels), on the other hand, show a high percentage of cells with stable (normal) claudin-1. Control empty vector clone, as might be expected, did not affect claudin-1. Cell monolayers were processed for immunofluorescent staining with a primary monoclonal anti-claudin-1 isotype antibody and subsequently the claudin-1 architecture was examined in a blinded manner for its overall normal morphology. At least 1200 cells per group were examined in four different subapical fields by laser scanning confocal microscopy and the percentage of cells with normal claudin-1 was determined, reported as mean ± S.E.M. The identity of the treatment groups for all slides was decoded only after examination was complete. *, p < 0.05 versus vehicle in WT cells. +, p < 0.05 versus vector alone-transfected cells. Vector alone, cells transfected with empty vector; AS-PKC-, antisense inhibition of PKC-. n = 6 per group. B, representative immunofluorescent photos showing the intracellular organization of the barrier function protein claudin-1 isotype (an index of monolayer integrity) in intestinal Caco-2 cells of wild-type and transfected origin. Shown are images from both larger fields (top, a–c) and single cells (bottom, a–c). Intestinal monolayers grown on specially coated coverslips were processed for staining and the claudin-1 isotype subapical organization was captured by high-resolution LSCM. a, wild-type monolayers exposed to vehicle. b, PKC- antisense (4-µg) clone exposed to vehicle. c, empty vector clone exposed to vehicle. In wild-type cells (a) claudin-1 look like an intact structure as shown by a smooth and continuous distribution of its ring at areas of cell-to-cell contact. Only in PKC- antisense clones (b), claudin-1 ring looks fragmented, beaded, and disorganized. In empty vector clones (c), as expected, claudin-1 organization is highly maintained and resembles normal (intact) morphology detected in that of wild-type cells. Scale bar, 25 µm. Shown are representative photos. n = 6 per group.

Representative laser scanning confocal microscopy of immunofluorescently labeled claudin-1 corroborates (Fig. 1B, a–c) that the 4-µg clones underexpressing PKC- exhibited a loss of the normal claudin-1 skeletal-ring architecture (b; see arrows). This instability is seen in the intracellular appearance of a torn, beaded, and fragmented claudin-1 "ring" (from the "1.8-µm subapical" area of cell-cell contact). Wild-type cells (a), on the other hand, show an intact caludin-1 ring at these same areas of cell-cell contact. This normal architecture is indistinguishable from the empty vector-transfected clones (c), which were also exposed to vehicle.

View this table: [in this window] [in a new window]   TABLE 1 Effects of transfection of varying amounts of an antisense plasmid for the isoform of PKC on intestinal Caco-2 monolayer barrier function Cells were transfected with varying amounts of an antisense plasmid for PKC- (1, 2, 3, 4, or 5 µg of DNA). The 4-µg antisense clone of PKC- displays the largest loss of monolayer barrier function. As expected, the empty vector alone did not affect monolayer barrier. WT (untransfected) monolayers are also shown. Monolayer barrier function was determined by clearance of two different sized (70 and 4 kDa) FD probes as described under Materials and Methods. Values are means ± S.E.M.

View larger version (64K): [in this window] [in a new window]   Fig. 2. A, antisense transfection against native PKC- isoform (AS-PKC-) induces a dose-dependent instability of monolayer claudin-4 isotype (another barrier function protein) in intestinal Caco-2 cells. Assessment of claudin-4 isotype in multiple PKC- antisense clones demonstrates a graded instability of claudin-4 as shown by reduced percentage of intestinal cells with normal claudin-4 isotype. The 4-µg antisense clone of PKC-, as for claudin-1 isotype, led to the maximum instability of claudin-4. WT cells, in contrast, exhibit a high percentage of cells with normal claudin-4 isotype. The empty vector clone (vector alone), as might be expected, did not affect claudin-4 stability. Intestinal monolayers were processed for staining with a primary monoclonal anti-claudin-4 isotype antibody and then examined in a blinded manner for claudin-4 normal morphology. *, p < 0.05 versus vehicle in WT cells. +, p < 0.05 versus vector alone-transfected cells. Vector alone, cells transfected with empty vector; AS-PKC-, antisense inhibition of PKC-. n = 6 per group. B, architectural organization of the claudin-4 isotype (another index of monolayer integrity) as revealed by LSCM of Caco-2 monolayers of wild-type and transfected origin. Shown are images from both larger fields (top, a–c) and single cells (bottom, panels a-c). Panel a is from wild-type monolayers exposed to vehicle. b, PKC- antisense (4-µg) clone exposed to vehicle. c, empty vector clone exposed to vehicle. In wild-type cells (a), claudin-4 isotype is intact and smoothly distributed at subapical areas of cell-to-cell contact, whereas PKC- antisense clones (b) exhibit clear disruption and instability of claudin-4 ring. In empty vector clones (c), as expected, claudin-4 ring architecture is highly maintained (intact), resembling the wild-type cells. Scale bar, 25 µm. Shown are representative photos. n = 6 per group.

In comparison, other intestinal isotypes of claudin, including claudin-2, claudin-3, and claudin-5, did not seem to be affected by antisense to PKC-. For instance, representative "two-color" (double stain) studies of claudin-1 or -4 compared, for example, with claudin-2 in the same intestinal cells (Fig. 3, A and B, respectively) showed that Caco-2 cells express little or no claudin-2 isotype (compared with either claudin-1 or -4). Similarly, other representative two-color studies of claudin isotypes, including claudin-4 and claudin-2 (Fig. 3C) corroborate the above-mentioned findings. Studies of other claudin isotypes such as claudin-3 and claudin-5 are shown in Fig. 3D.

View larger version (56K): [in this window] [in a new window]   Fig. 3. A to D, representative double stain (two-color) studies of several claudin isotypes in intestinal Caco-2 cells of PKC- antisense-transfected origin. A and B, representative two-color studies of claudin-1 or -4 compared with claudin-2 in the same intestinal cells. Other two color studies of claudin isotypes, including claudin-4 and claudin-2 (C) and claudin-3 and claudin-5 (D) are also shown. Colocalization (yellow merge) is seen for all claudins, except for claudin-2 in Caco-2 cells (i.e., little or no claudin-2 is seen in Caco-2 cells; see B and C). Unlike the disrupted appearance of claudin-1 and -4 isotypes (see A), claudin-3 and -5 isotypes (D) seem intact in PKC- antisense cells. Cells were processed for immunofluorescent staining with the corresponding fluorescein isothiocyanate- and/or Texas Red-conjugated primary antibodies. Shown are representative photos. n = 6 per group.

Furthermore, there was a dose-dependent instability of Caco-2 monolayer barrier function in the above-noted AS-PKC- clones when we used a smaller size permeability probe, namely, 4-kDa fluorescein dextran (FD4) (Table 1). Not surprisingly, the 4-µg clone also exhibited maximum increases in monolayer permeability to FD4 kDa. Indeed, there was a size-dependent increase in monolayer permeability in order of decreasing size for these probes (70 kDa FD < 4 kDa FD), suggesting the dynamic nature of barrier alterations. We observed a similar trend of alterations for disruption of both barrier function and claudin stability in another intestinal epithelial cell line, HT-29 (Table 2). For example, 4 µg of AS-PKC- clone also led to largest instability in claudin (i.e., reduced percentage of normal claudin-1 and claudin-4 isotypes) in HT-29 cells. Because the clone transfected with 4 µg of AS-PKC- cDNA led to maximum levels of monolayer barrier disruption and claudin instability, we used this clone for subsequent mechanistic studies.

View this table: [in this window] [in a new window]   TABLE 2 Effects of PKC- antisense on both monolayer barrier function and claudin integrity in another intestinal cell line, HT-29 Human colonic HT-29 cells were transfected with an antisense plasmid for PKC- (1-5 µg). The 4-µg antisense clone causes the largest instability of both claudin and barrier function. WT cells are also shown. Monolayer barrier function (clearance of FD 70 kDa) and claudin integrity (% normal claudin isotype) were assessed as described under Materials and Methods. Values are means ± S.E.M.

Antisense Reduction of Endogenous nPKC- Leads to Abnormal Alterations in the Membrane and Cytoskeletal Assembly of Claudin Isotypes. We determined the effects of native PKC- underexpression on the cytosolic, membrane, and cytoskeletal pools of claudin-1 and -4 by assessing the 22- and 25-kDa (respectively) structural protein of these isotypes. The various subcellular claudin pools were isolated from the inhibitory (antisense) clones and analyzed after SDS-PAGE fractionation. Tables 3 and 4 show results of analysis for the subcellular distribution of claudin-1 and claudin-4 in the cytosolic, membrane, and cytoskeletal fractions of Caco-2 cells (data presented as a fraction of total claudin isotype). These findings indicate that underexpression of PKC- abnormally decreases claudin-1 (Table 3) and claudin-4 (Table 4) in the membrane and cytoskeletal pools while it concomitantly increases their proportions in cytosolic pools.

For example, PKC--underexpressing clone (4 µg of AS-PKC-) exhibited an abnormal reduction in the particulate (particulate = membrane + cytoskeletal) pool of claudin-1 or claudin-4 as demonstrated by a decrease in its band density (not shown), indicating instability of claudin-1 or -4 assembly. In wild-type cells, we did not find any decreases in the particulate pool of claudin, indicating normal assembly of claudin skeleton. In these wild-type cells, as might be expected, claudin-1 or -4 particulate distribution was comparable with that found in the empty vector clone. Transfection of empty vector, as for its lack of effects on permeability and claudin architecture, had no affect on the particulate pool of claudin. These findings on the subcellular assembly/distribution of claudins parallel the destabilizing effects of antisense, underexpression of native PKC- on intestinal claudin architecture and monolayer barrier function.

Antisense Suppression of nPKC- Causes Alterations in Serine- and Threonine-Phosphorylation States of Claudin Isotypes in Intestinal Epithelium. We subsequently probed molecular processes underlying the observed effects of PKC- on the monolayer claudins and barrier permeability. Accordingly, claudin-1 and claudin-4 isotypes were immunoprecipitated (by isotype-specific antibodies) from the transfected and wild-type cells, and subsequently subjected to PAGE to determine their phosphorylation (Fig. 4, A to D). Immunoprecipitated claudin-1 isotype was largely serine- and threonine-phosphorylated in wild-type cells (Fig. 4, A and B, corresponding lane a), but not in antisense-transfected cells where suppression of native PKC- markedly decreased claudin-1 serine/threonine-phosphorylation (corresponding lane c). We found a similar trend of alterations for the phosphorylation state of claudin-4 isotype in wild-type and antisense clones (Fig. 4, C and D). In the empty vector clones, both claudin isotypes were phosphorylated (corresponding lane b in all blots). The state of claudin serine/threonine-phosphorylation in these vector clones resembled that of the wild-type cells. When the same phosphorylation blots were stripped and reprobed with the corresponding isotype specific monoclonal claudin antibody, we confirmed that changes in phosphorylation were in fact specifically associated with the claudin-1 and -4 proteins (not shown).

View larger version (37K): [in this window] [in a new window]   Fig. 4. A to D, representative immunoblots of alterations in both serine- and threonine-phosphorylation states of immunoprecipitated claudin-1 (A and B) and claudin-4 (C and D) in intestinal Caco-2 cells of transfected origin compared with wild-type cells. Claudin isotypes were immunoprecipitated using the isotype specific monoclonal antibodies and subsequently immunoblotted to determine state of claudin phosphorylation. The bands representing claudin-1 and claudin-4 phospho-serine (A and C, respectively) and claudin-1 and claudin-4 phospho-threonine (B and D, respectively) correspond to a, wild-type cells exposed to vehicle; b, empty vector clone exposed to vehicle; and c, PKC- antisense clone (4 µg) exposed to vehicle. Antisense inhibition of endogenous PKC- isoform decreases claudin phosphorylation (lane c), paralleling its destabilizing effects on claudin subcellular assembly (Tables 3 and 4) and claudin integrity (Figs. 1B and 2B). These abnormally low phosphorylation levels are not seen in wild-type cells (lane a) where claudin isotypes exhibit steady-state serine/threonine-phosphorylation. Empty vector clone (lane b), as expected, did not affect phosphorylation (resembling the wild-type cells). Shown are representative blots. n = 6 per group.

Native nPKC- Isoform Is Complexed with Claudin. To further study the mechanism underlying the unique stabilizing affects of native PKC- on claudins, we used immunoprecipitation analysis (Fig. 5, A–D). In a first series of approaches, cells were initially immunoprecipitated with a monoclonal PKC- antibody and then the immune complexes were analyzed for the presence of claudin-1, assessing whether this PKC isoform physically coprecipitates with claudin-1 isotype. Antisense clones underexpressing endogenous PKC- did not show any complex formation between these two proteins (Fig. 5A, lane c). In contrast, transfection of the empty vector control did not do so, exhibiting coprecipitation of these two proteins (lane d). Similar to empty vector clone, the amount of claudin-1 coprecipitation was markedly enhanced in wild-type (resting) vehicle-treated cells (lane b), indicating likely presence of a PKC-/claudin-1 complex under native conditions. As expected, an irrelevant primary antibody (normal rabbit serum) did not lead to coprecipitation (i.e., no complex formation bands were seen), further suggesting specificity of the coprecipitation seen.

View larger version (20K): [in this window] [in a new window]   Fig. 5. A to D, PKC- antisense leads to a reduction in native coassociation of the novel PKC- isoform and the barrier function protein claudin in intestinal Caco-2 cells. Claudin-1 isotype (A and B) and claudin-4 isotype (C and D). Wild-type cells show the endogenous PKC-/claudin complexes (see corresponding lane b* in A–D). Note the antisense inhibition induced reduction (disappearance) in coimmunoprecipitation of the PKC-/claudin complexes in transfected cells lacking PKC- isoform activity (lane c). For A and C immunoblots, cleared cell lysates were incubated with excess (1:2000 dilution) anti-PKC- bound to protein A beads, and subsequently the immune complexes were resolved by PAGE using the appropriate anti-PKC- or anti-claudin isotypes (claudin-1 or claudin-4) antibodies. Wild-type (vehicle-exposed) resting cells show endogenous PKC-/claudin complexes (see lane b* in A and C). PKC- antisense clone displays no such endogenous PKC-/claudin complexes (lane c). For B and D, blots opposite protocols were done in which the corresponding anti-claudin isotype antibody was used for immunoprecipitation, and then the complexes formed were examined. As before, an almost identical pattern of complex formation (coassociation) is seen between claudin/PKC-. The bands are from a, cell lysates; b, wild-type cells exposed to vehicle; c, PKC- antisense clone (4 µg) exposed to vehicle; and d, empty vector clone exposed to vehicle. WB, Western blot. IP, immunoprecipitated with the shown antibody. Shown are representative immunoblots. n = 6 per group.

In a third series of approaches, we further assessed the specificity of inhibition of formation of the PKC-/claudin-1 complexes in our PKC- antisense clones. Here, we probed lysates from two other PKC isoform antisense clones, the classical PKC-2 and the PKC- AS. As expected, in these other clones, we could not inhibit the formation of PKC-/claudin-1 complexes (not shown).

Using the aforementioned series of approaches, we found a similar trend of coprecipitation and complex formation between PKC- and claudin-4 isotype in intestinal cells (Fig. 5, C and D). As for claudin-1, probing lysates from other PKC isoform antisense clones—the classical PKC-2 AS and the PKC- AS—did not suppress the formation of PKC-/claudin-4 complexes. This again indicates the specificity of PKC-/claudin coprecipitation we observed. Also, incubation with an irrelevant antibody was ineffective (not shown).

Subcellular Activity and Distribution of nPKC-: Native PKC- Isoform Is Constitutively Active and Present Mostly in the Membrane and Cytoskeletal Fractions of Intestinal Cells. Findings from in vitro kinase activity assay of cytosolic, membrane, and cytoskeletal subcellular fractions (Fig. 6A) confirm that the native isoform of PKC is mostly active in the particulate cell fractions (particulate includes membrane and cytoskeletal fractions) with only a small activity in the cytosolic fractions, indicating the constitutive activity of the isoform of PKC under native conditions. Empty vector control was indistinguishable from the wild-type cells. In antisense-transfected clones underexpressing PKC-, on the other hand, there was an almost complete lack of native isoform activity compared with the wild-type cells. Immunoblotting assessment of the subcellular distribution of the PKC- protein (Fig. 6B) additionally shows that the native (82-kDa) isoform of PKC is distributed mostly in the membrane and cytoskeletal fractions of wild-type intestinal cells. Not surprisingly, in antisense clones, we found a near complete absence of PKC- protein in these same subcellular fractions, further indicating PKC- inactivity. Overall, these findings confirm that the endogenous PKC- isoform is "constitutively active" in the membrane and cytoskeletal (particulate) subcellular fractions because it is found to be most active in these fractions under native conditions. These findings on PKC- subcellular activity parallel our findings on the subcellular distribution and coprecipitation of claudin isotypes.

View larger version (26K): [in this window] [in a new window]   Fig. 6. A and B, assessment of PKC- activity (A) and PKC- protein distribution (B) in the subcellular fractions (i.e., cytosolic, membrane, cytoskeletal) of intestinal Caco-2 cells of antisense (AS)-transfected and of wild-type (native) origin. PKC- subcellular activity (by in vitro kinase assay) is shown in A. PKC- subcellular protein distribution (by immunoblotting) is shown in B. Substantial activity/levels of the native PKC- isoform is seen in wild-type cells. In these wild-type cells most of the endogenous PKC- activity and protein is found in the p