Regulation of protein kinase C-δ and -ɛ isoforms by phorbol ester treatment of LLC-PK1 renal epithelia

Kidney International, Vol. 58 (2000), pp. 1004–1015

Regulation of protein kinase C-␦ and -ε isoforms by phorbol ester treatment of LLC-PK1 renal epithelia HILARY CLARKE, NICOLE GINANNI, ALEJANDRO PERALTA SOLER, and JAMES M. MULLIN Lankenau Medical Research Center, Wynnewood, Pennsylvania, USA

Regulation of protein kinase C-␦ and -ε isoforms by phorbol ester treatment of LLC-PK1 renal epithelia. Background. LLC-PK1 renal epithelia are a widely used model for proximal tubular physiology and differentiation. Protein kinase C (PKC) has been observed to play a role in both processes. This study examines the subcellular distribution and down-regulation of PKC-␦ and PKC-ε isoforms in phorbol ester-treated LLC-PK1 epithelia. Methods. Cells were treated with 10⫺7 mol/L 12-O-tetradecanoyl phorbol 13-acetate (TPA) for up to seven days and were extracted as total cell lysates as well as cytosolic, membraneassociated (Triton-X soluble) and a third (Triton-X insoluble) fraction. The expression and cellular localization of PKC-␦ and PKC-ε isoforms were then detected using Western immunoblot and immunofluorescence. Results. Based on the use of an anti-PKC-␦ monoclonal antibody, TPA was observed to cause a rapid decrease in total PKC-␦ content, which then returned to near control levels by seven days of treatment. Immunofluorescence indicated that PKC-␦ had a cytoskeletal localization within the cells, and a subtle cytoskeletal rearrangement occurred upon exposure to TPA. Western immunoblots showed that PKC-␦ did not undergo the expected membrane translocation upon activation by TPA, but simply disappeared immediately from the cytosolic compartment. Conventional cell fractionation procedures such as homogenization and Triton extraction prior to Western immunoblot will, however, fail to evaluate completely PKC-␦ in LLC-PK1 epithelia because of the highly stringent measures necessary to extract PKC-␦ from the cytoskeletal compartment of these cells. Furthermore, we observed that a second (polyclonal) PKC-␦ antibody may recognize phosphorylated forms of PKC-␦, which went unrecognized by the other antibody. PKC-ε was present in the cytosol, membrane, and Triton-X– insoluble fractions of the cells. TPA treatment resulted in a partial translocation of PKC-ε to both the membrane and Triton-X–insoluble fractions of the cell, but total PKC-ε remained essentially unchanged. Conclusions. The present data indicate that the localization of PKC-␦ and subsequent redistribution within the LLC-PK1 cells in response to TPA treatment is highly unique and distinct

Key words: TPA, 12-O-tetradecanoyl phorbol 13-acetate, phosphoprotein, LLC-PK1 cells, isoforms of PKC, cell regulation. Received for publication April 20, 1999 and in revised form February 9, 2000 Accepted for publication April 5, 2000

 2000 by the International Society of Nephrology

from that of PKC-ε and PKC-␣. An important methodological finding is that one given antibody may not recognize all phosphoproteins of a given PKC isoform.

Protein kinase C (PKC) is a family of phospholipiddependent protein kinases consisting of at least 10 different isoforms encoded by separate genes. These isoforms can be classified into three groups—classic (cPKC), new (nPKC), and atypical (aPKC)—based on the structures of their regulatory domains [1, 2]. All show distinct enzymological properties, differential tissue distribution, and specific subcellular localization with distinct modes of cellular regulation [3–5]. These differences in structure, enzymatic properties and intracellular localization strongly suggest specific functions for each of the isoforms of PKC; however, these have not yet been clearly defined, nor have unique target proteins been identified. These isoforms of the PKC family are the major receptors for the tumor-promoting phorbol esters and are therefore thought to play an important role not only in general signal transduction, but also in carcinogenesis. PKC is normally activated by diacylglycerol (DAG) produced by the receptor-coupled hydrolysis of membrane phosphoinositides [6]. Phorbol esters such as 12-O-tetradecanoyl phorbol 13-acetate (TPA) bind to the DAG binding site of the regulatory domain of all but the atypical PKCs and activate their catalytic activity [2]. The fact that phorbol esters are able to replace the endogenous activator DAG in the stimulation of PKC has provided insight into the role of PKC in the regulation of a variety of cellular processes such as exocytosis, gene expression, proliferation, differentiation, as well as tumor promotion [7]. Previous studies from this laboratory have shown that PKC activation in LLC-PK1 cells by phorbol esters causes an increase in tight junction permeability, resulting in an enhanced paracellular flow of solutes between the epithelial cells [8, 9]. The permeability properties of the tight junction such as electrical resistance and cation versus anion selectivity are known to be quite variable in different epithelia, and in a large part determine the

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physiological properties of transepithelial solute transport across the given tissue. The dynamic nature of the tight junctional barrier is evidenced through its regulation by agents as diverse as phorbol esters [10–13], cytokines [14–16], calcium [17, 18], adenosine triphosphate [19], and bacterial toxins [20]. To meet the many physiological and pathological challenges to which epithelial tissues are subjected, the tight junction must be capable of rapid and coordinated responses that require the presence of complex regulatory pathways. We have shown previously that the membrane-associated fraction of PKC-␣ correlated with tight junction permeability [8, 9, 21]. PKC-␦ may also play an important role in this permeability pathway, as cells overexpressing this isoform were seen to have altered permeability properties [22]. Abundant expression of PKC-␦ in several cell types, including ␤ lymphoma cells [23], myeloid cells [24], fibroblasts [25, 26], neural crest-derived PC12 cells [27], and glioma cells [28], suggests that this isoform of PKC is of central importance in intracellular signaling. Although the specific biological functions of PKC-␦ are largely unknown, this enzyme has been implicated as a regulator of cell cycling [29]. It has been shown to have antiproliferative actions upon overexpression in Chinese hamster ovary (CHO) [29] or NIH 3T3 cells [30, 31], and to cause differentiation in myeloid 32D cells [32]. PKC-␦ was also a reasonable choice for study because in response to TPA in polar, gastrointestinal epithelial cell sheets, it regulated in an opposite manner to PKC-␣ [33], which we have previously shown to correlate closely with changes in junctional permeability. PKC-␦ also has an obvious association with cell adhesion [34, 35]. While PKC-ε has also been shown to play a role in cell adhesion [36], this isoform down-regulates very similarly to PKC-␣ in renal epithelia [3] and, moreover, has immunolocalized to the junctional region in gastrointestinal epithelia [37]. Cultured LLC-PK1 renal epithelial cells play an important role in the study of monolayer organization and epithelial barrier function as well as renal function in general [38, 39]. In this work, the established LLC-PK1 renal cell line was used as a model in which phorbol esters perturb epithelial barrier function through PKC activation [8, 9]. The subcellular localization in LLCPK1 epithelia of two calcium-independent PKC isoforms, PKC-␦ and PKC-ε, was determined in the present study in order to add to information that our group has already obtained in this regard about PKC-␣ [9]. The work reported here provides the first information, to our knowledge, on the subcellular distribution and down-regulation of the PKC-␦ and PKC-ε isoforms in phorbol ester-treated LLC-PK1 epithelial cells. METHODS Cell culture LLC-PK1 cells were a gift of Dr. Robert Hull [40] and were used between passages 184 and 202. Cells were cul-

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tured in ␣-minimum essential medium without nuclosides (JRH Biosciences, Lenexa, KS, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) and passaged as described previously [8]. Western blot analysis Seven days after seeding, the differentiated cell sheets cultured in 75 cm2 tissue culture flasks (Falcon, Meylan, France) were refed with fresh culture medium plus the vehicle ethanol (0.01%) or with 10⫺7 mol/L TPA and were incubated for the relevant time period. For chronic studies, cell sheets were refed on days 2 and 4 of the study. Samples taken for total PKC were washed once in ice-cold phosphate-buffered saline (PBS), scraped, and rinsed into 2 mL of lysis buffer [150 mmol/L NaCl, 50 mmol/L Tris HCl, 1 mmol/L ethylene glycol-bis [␤-aminoethylether]-N,N,N⬘,N⬘-tetraacetic acid (EGTA), 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 1% NP-40, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 20 ␮g/mL aprotinin, 10 ␮g/mL leupeptin, and 200 ␮mol/L phenylsulfonyl fluoride] at 4⬚C. The samples were then processed as described previously [8]. To investigate the different cell fractions, the cells were scraped into 2 mL of buffer A (20 mmol/L Tris HCl, pH 7.5, 0.25 mol/L sucrose, 10 mmol/L EGTA, 2 mmol/L EDTA, 20 ␮g/mL leupeptin, 10 ␮g/mL aprotinin, and 200 ␮mol/L phenylsulfonyl fluoride) at 4⬚C and were sonicated and separated into cytosolic, membrane-associated (Triton-X–soluble) and fraction 3 (Triton-X– insoluble) fractions as described previously [8]. The pellet remaining after the third extraction was subjected to a further extraction by boiling for five minutes in 2⫻ sample buffer [0.0625 mol/L Tris, 2.0% SDS, 10% glycerol, 5% mercaptoethanol, and 0.025% (wt/vol) bromophenol blue]. This fraction was then called the fourth fraction. SDS-polyacrylamide gel electrophoresis (PAGE) was performed using 8% polyacrylamide gels on a Protean II electrophoresis apparatus (Bio-Rad, Hercules, CA, USA). Protein transfer to 0.45 ␮m nitrocellulose (Micron Separation, Westborough, MA, USA) was performed overnight at 15 V using a Bio-Rad Transblot cell. After nonspecific binding was blocked with 3% bovine serum albumin (BSA), the immunoblot was incubated with a primary mouse monoclonal anti–PKC-␦ antibody (Transduction Laboratories, Lexington, KY, USA) at a 1:500 dilution in 3% BSA for one hour at room temperature. A rabbit polyclonal anti–PKC-␦ antibody (Santa Cruz, Santa Cruz, CA, USA) was also used in this study at a dilution of 1:100 for two hours at room temperature after nonspecific binding was blocked with 3% milk. A horseradish peroxidase-labeled rabbit anti-mouse IgG secondary antibody or a goat anti-rabbit IgG secondary antibody was then used in conjunction with the Renaissance Western blot chemiluminescence kit (DuPontNEN, Boston, MA, USA). The labeled immunoblot was

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then placed against reflection autoradiography film (Eastman Kodak, Rochester, NY, USA) and developed in a Kodak M35A X-OMAT processor. For PKC-ε detection, the previously mentioned immunoblot was then placed in stripping buffer (100 mmol/L 2-mercaptoethanol, 2% SDS, 62.5 mmol/L Tris-HCl, pH 6.7) for 30 minutes at 37⬚C and processed as before, but now with a mouse monoclonal anti–PKC-ε antibody (Transduction Laboratories) also at a 1:500 dilution. Protein phosphatase treatment Fifty micrograms of cell lysate from either the cytosolic membrane or third fraction was resuspended in 10⫻ phosphatase buffer (10 mmol/L dithiothreitol, 500 mmol/L Tris HCl, 1 mol/L NaCl, and 1 mmol/L EDTA) containing 400 U of lambda phosphatase (Recombinant, Escherichia coli, specific activity 300,000 U/mg protein; Calbiochem, La Jolla, CA, USA). After incubation for 30 minutes at 30⬚C, the reaction was stopped by addition of 75 ␮L of 2⫻ sample buffer. PKC-␦ was analyzed as described previously in this article by 8% SDS-PAGE, and then subjected to Western immunoblot with the Santa Cruz rabbit polyclonal anti–PKC-␦. Immunofluorescence detection of PKC-␦ and PKC-ε One ⫻ 106 cells were seeded into Falcon 3102 filter rings. Three days after seeding, the cell sheets were refed with fresh medium plus the vehicle ethanol (0.01%) or the phorbol ester TPA (10⫺7 mol/L). For the seven-day time point, the cell sheets were refed daily. After the appropriate treatment period, the cell sheets were rinsed in PBS, then fixed in 3.2% paraformaldehyde for 20 minutes, rinsed again, and permeabilized in 0.1% Triton-X for three minutes. After additional rinses in PBS, cell sheets were exposed to 10% normal goat serum for one hour at room temperature and then incubated overnight in either mouse monoclonal antibody to PKC-␦ (1:50), rabbit polyclonal to PKC-␦ (1:50), or PKC-ε (1:50; Transduction Laboratories). After rinses in PBS, cell sheets were incubated with a 1:100 CY3 goat anti-mouse secondary antibody or 1:100 CY3 goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for one hour in the dark. After final rinses in PBS and distilled water, cell sheets were mounted in glycerol and viewed with epifluorescence illumination using rhodamine filters. RESULTS Chronic TPA treatment modulates total cellular expression of PKC-␦ and PKC-ε In accordance with previous reports by Amsler et al, immunoblot analysis demonstrated the presence of both PKC-␦ and PKC-ε isoforms in whole-cell homogenates of

Fig. 1. Western immunoblot of protein kinase C (PKC) -␦ and -ε in a total cell lysate of LLC-PK1 cell sheets exposed to 10⫺7 mol/L TPA for up to seven days. When confluent, cells were either processed immediately, 0 minutes (lane 1), or treated with vehicle for one, four, or seven days (lanes 2, 4, and 6, respectively), or 10⫺7 mol/L TPA for one, four, or seven days (lanes 3, 5, and 7, respectively). Cells were refed on days 2 and 4 of the study. Each lane received 50 ␮g of total protein. A commercially available positive control (⫹) from rat brain lysate (Transduction Laboratories) was run, and the bands observed comigrated with the 78 (␦) and 90 kD (ε) bands shown here. Exclusion of the primary antibody (mouse monoclonal from Transduction Laboratories) resulted in the disappearance of all bands (data not shown).

LLC-PK1 epithelial cells (Fig. 1) [41]. TPA caused a nearly complete disappearance of PKC-␦ after one day, but by day four it reappeared in the treated cell homogenate, but at much lower levels than that seen in the control cells. While PKC-ε underwent a slight decrease after TPA treatment for one day, the cell sheets from four and seven days did not seem to show a change in the total levels of this isoform within the cells. Translocation and down-regulation of PKC-␦ and PKC-ε To investigate the effect of TPA treatment on the subcellular location of the PKC isoform, confluent LLC-PK1 cell monolayers, maintained in the absence or presence of 10⫺7 mol/L TPA for up to seven days, were then extracted and fractionated by ultracentrifugation. Proteins contained within the subcellular fractions were then analyzed by immunoblotting with isozyme-specific antibodies. In control cell populations, PKC-␦ was detected only in the cytoplasmic fraction of the cells with a monoclonal anti–PKC-␦ antibody (Transduction Laboratory). PKC-ε, however, while located predominantly in the cytoplasmic fraction, was also found in the membrane-associated and Triton-X–insoluble (cytoskeletal) fractions (Fig. 2). The basal subcellular distribution of these two isoforms is therefore distinct within LLC-PK1 cells. TPA treatment caused PKC-␦ to disappear completely from the cytoplasmic fraction as early as 15 minutes after its addition to the cell sheet (Fig. 2). PKC-␦ was not detected in the membrane or third fraction after treatment, indicating that either PKC-␦ was not translocated in response to

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Fig. 2. Western immunoblot of PKC-␦ (A) and PKC-ε (B) in cytosolic, membrane and Triton-X–insoluble fractions of LLC-PK1 cell sheets exposed to 10⫺7 mol/L TPA for up to 60 minutes. Confluent, differentiated cell sheets cultured in 75 cm2 tissue culture flasks were refed with control medium for 15, 30, 45, or 60 minutes (lanes 1, 3, 5 and 7, respectively) or TPA for 15, 30, 45, or 60 minutes (lanes 2, 4, 6 and 8, respectively) and extracted at the times indicated as described in the Methods section. Each lane received 50 ␮g of total protein. Exclusion of the primary antibody resulted in the disappearance of all bands (data not shown).

TPA, was down-regulated at a faster rate than translocation, or was not recognized by this particular primary antibody after its translocation. PKC-ε was also seen to depart rapidly but not disappear from the cytoplasmic fraction following the addition of TPA. It underwent a partial translocation to the membrane and Triton-X– insoluble fractions where it remained elevated and manifested a second, possibly phosphoprotein, band. At later time points, up to six hours, treatment of cells with TPA resulted in a continuation of the previously mentioned patterns of translocation and/or degradation of PKC-␦ and PKC-ε (Fig. 3). PKC-␦ continued to be absent from all three fractions after TPA exposure, while PKC-ε was partially translocated from the cytoplasmic to the membrane and third fractions. Continuing administration of TPA to cells for up to seven days resulted in PKC-␦ still being undetectable in the cytoplasmic, membrane and Triton-insoluble fractions (Fig. 4). PKC-ε was now, like PKC-␦, also absent from the cytoplasmic compartment of the TPA-treated cells, but quite unlike PKC-␦, PKC-ε remained detectable in the membrane-associated fraction of these cells. Detection of PKC-␦ in a fourth fraction As a means of explaining the persistence of PKC-␦ in TPA-treated cells as seen in total cell lysates (Fig. 1) and in immunofluorescence microscopy as described later in

this article, a further, more stringent extraction step was carried out with the pellet remaining after the third extraction. This pellet, which contained the remaining PKC-␦, was Triton-X and 0.1% SDS insoluble, and was thus very likely tightly bound to the cytoskeleton. It was able to be further extracted in the sample buffer containing 2% SDS. PKC-␦ was clearly detected in this fourth fraction at the correct molecular weight (Fig. 5). This indicates that there was a further subcellular pool of PKC-␦ that was not successfully extracted by the first three standard fractionation steps. Immunofluorescence detection of PKC-␦ and PKC-ε Using the same antibodies to PKC-␦ and PKC-ε as employed in the immunoblot studies, the cellular localization of these two isoforms in the epithelia was analyzed by immunofluorescence. This procedure was necessary because a chronically TPA-treated cell sheet is a heterogeneous population of both multilayered and monolayered areas of cells [9, 21]. The Western blot techniques involve homogenization of an entire cell sheet, which will average out any differences in PKC isoforms that exist between these two cell populations. Using immunofluorescence microscopy, any heterogeneity in cellular distribution of PKC isoforms can be observed. There appeared to be a paradox between the cellular localization of both PKC-␦ and PKC-ε observed by West-

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Fig. 3. Western immunoblot of PKC-␦ (A) and -ε (B) in cytosolic, membrane and Triton-X–insoluble fractions of LLC-PK1 cell sheets exposed to 10⫺7 mol/L TPA for up to six hours. When confluent, differentiated cell sheets cultured in 75 cm2 tissue culture flasks were either processed immediately, 0 hour (lane 1), or were refed with control medium for two, four, or six hours (lanes 2, 4, and 6, respectively) or TPA for two, four, or six hours (lanes 3, 5 and 7, respectively) and extracted at the times indicated as described in the Methods section. Each lane received 50 ␮g of total protein. Exclusion of the primary antibody resulted in the disappearance of all bands (data not shown).

Fig. 4. Western immunoblot of PKC-␦ (A) and -ε (B) in cytosolic, membrane and Triton-X–insoluble fractions of LLC-PK1 cell sheets exposed to 10⫺7 mol/L TPA for up to seven days. When confluent, differentiated cell sheets cultured in 75 cm2 tissue culture flasks were refed with control medium for one, two, four, or seven days (lanes 1, 3, 5 and 7, respectively) or TPA for one, two, four, or seven days (lanes 2, 4, 6, and 8, respectively). Cells were refed on days 2 and 4 of the study and extracted at the times indicated as described in the Methods section. Each lane received 50 ␮g of total protein. Exclusion of the primary antibody resulted in the disappearance of all bands (data not shown).

Clarke et al: Phorbol ester regulation of PKC-d and PKC-ε

Fig. 5. Western immunoblot of PKC-␦ in a fourth fraction of control cells. The pellet remaining after the third extraction was subjected to a further extraction by boiling for five minutes in 2⫻ sample buffer and was then called the fourth fraction. Exclusion of the primary antibody resulted in the disappearance of all bands (data not shown).

ern immunoblotting and immunofluorescence. This may be explained at least in part by the presence of substantial amounts of PKC-␦ and PKC-ε in a fourth subcellular pool, which was not extractable in the first three cell fractions and only appeared after boiling and high levels of SDS detergent. From Figure 6A, it can be seen that in the control cell sheets, PKC-␦ was mainly localized to cytoskeletal fibers with punctuated staining of the nuclei also present. Treatment of the cell monolayer with TPA for one hour resulted in a more diffuse staining pattern of PKC-␦ throughout the cells (Fig. 6B). Seven days of treatment with TPA caused a pattern of localization that more closely resembles the control cells, with PKC-␦ again being detected in close proximity to the cytoskeleton. This more intense PKC-␦ staining at the day 7 time point is due, at least in part, to increased cell number and multilayering. As seen in Figure 6C, PKC-␦ levels are quite high in areas of multilayering, as was demonstrated earlier for PKC-␣ [9]. PKC-ε does not seem to be uniformly distributed throughout the control epithelium (Fig. 6D). In cells exhibiting staining, PKC-ε appears in a diffuse non-nuclear pattern. Following treatment with TPA for one hour, PKC-ε translocated to the border of the cells (Fig. 6E). In contrast to the continued presence of PKC-ε detected by immunoblot after seven days of TPA treatment, low levels of this isoform were detected by immunofluorescence at this time point. The previously mentioned results for PKC-␦ were somewhat surprising since a disappearance of this isoform from the cells was not observed, and yet was seen in the Western immunoblot. This apparent discrepancy between the immunoblot profile and the immunofluorescence data obtained using the Transduction mouse monoclonal PKC-␦ antibody could be partially explained by our above finding of PKC-␦ in a fourth fraction, but it also caused us to re-examine the expression levels of this PKC isoform with a different anti–PKC-␦ antibody. A rabbit polyclonal anti–PKC-␦ antibody (Santa Cruz) directed against the carboxy terminus of human PKC-␦ was used to detect PKC-␦ within the cells. Surprisingly, as shown in Figure 7, a different pattern of subcellular localization of PKC-␦ was obtained using this antibody. Unlike the profile previously observed using the mono-

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clonal PKC-␦ antibody raised against amino acids 114 to 289 of human PKC-␦ (Fig. 2), the control cells exhibited PKC-␦ in all three cell fractions and in multiple bands as well (Fig. 7). Treatment with TPA for one hour caused a translocation of PKC-␦ from the cytosol with an increase in its expression in both the membrane-associated and third fractions. Several cytoskeletal bands nearly disappeared, while others exhibited marked decreases. Prolonged treatment of the cells with TPA for seven days resulted in a substantial down-regulation of PKC-␦ in both the membrane and third fraction. A third antibody for PKC-␦ (Chemicon, Temecula, CA, USA), which was a sheep polyclonal antibody raised against amino acids 128 to 142 of mouse PKC-␦, did not detect any PKC-␦ in our LLC-PK1 cells (data not shown). It did, however, recognize a positive control LLC-PK1 cell line in which human PKC-␦ was overexpressed. The cellular localization of PKC-␦, as detected by the anti–PKC-␦ rabbit polyclonal antibody, was then further investigated by immunofluorescence. Again, unlike the immunofluorescence profile seen using the monoclonal anti–PKC-␦ (Transduction), the control cells exhibited a diffuse cellular staining (Fig. 8A). One hour of treatment with TPA (Fig. 8B) caused a movement of PKC-␦ to the borders of some of the cells. Treatment of the cell monolayer with TPA for seven days (Fig. 8C) resulted in an overall down-regulation of PKC-␦ expression with a distinct staining persisting at the cell borders, a finding now in closer agreement to the Western immunoblot profile seen with this antibody. Protein phosphatase treatment To investigate whether the phosphorylation state of PKC-␦ may be affecting antibody recognition and therefore antibody binding to the antigen, the different cell fractions were subjected to dephosphorylation by incubation with lambda phosphatase. The Western blot profile for PKC-␦ was then investigated with the Santa Cruz polyclonal antibody. The less phosphorylated form of PKC-␦ (lane 1) was detected as a band that migrated slightly faster (lower arrow, Fig. 9) than the corresponding lane (lane 2) without phosphatase (upper arrow, Fig. 9). TPA treatment resulted in the appearance of additional bands in the membrane-associated and third fractions (lane 3). It can be seen from the TPA-treated cells of both the membrane-associated and third fractions that treatment of these fractions with lambda phosphatase resulted in a different pattern of antibody recognition (lane 3 compared with lane 4, Fig. 9), establishing these additional bands as phosphoproteins. DISCUSSION As PKC is the major receptor for tumor-promoting phorbol esters, it is thought that modulation of this family

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Fig. 6. Immunofluorescence of PKC-␦ and PKC-ε isoforms in control, acutely and chronically TPA-treated LLC-PK1 cell sheets. (A) PKC-␦ in control sheets. (B) PKC-␦ in cell sheet treated for one hour with 10⫺7 mol/L TPA. (C) PKC-␦ in cell sheet treated for seven days with 10⫺7 mol/L TPA. (D) PKC-ε in control sheets. (E) PKC-ε in cell sheet treated for one hour with 10⫺7 mol/L TPA. (F) PKC-ε in cell sheet treated for seven days with 10⫺7 mol/L TPA. For the seven-day time point, the cell sheets were refed daily. After fixation, cells were exposed to an anti–PKC-␦ or anti– PKC-ε monoclonal antibodies and processed for immunofluorescence as described in the Methods section. In controls, PKC-␦ was expressed predominantly in a cytoskeletal stress fiber-like pattern, while PKC-ε seemed to be more diffusely distributed within the LLC-PK1 cells. Exposure time for PKC-␦ with sevenday TPA treatment (C) was half that of A and B because of the high intensity of staining in this cell sheet. Exposure time for PKC-ε was the same for every panel as was the magnification setting of all panels (bar, 25 ␮m).

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Fig. 7. Western immunoblot of PKC-␦ (Santa Cruz rabbit polyclonal antibody) in cytosolic, membrane and Triton-X–insoluble fractions of LLC-PK1 cell sheets exposed to 10⫺7 mol/L TPA for one hour or seven days. Confluent, differentiated cell sheets cultured in 75 cm2 tissue culture flasks were refed with control medium for zero hours, one hour, or seven days (lanes 2, 3, and 5, respectively) or TPA for one hour or seven days (lanes 4 and 6, respectively). A positive control from P␦5 cells overexpressing PKC-␦ was run, and the bands observed comigrated with the 78 kD PKC-␦ (lane 1). Each lane received 50 ␮g of total protein. Exclusion of the primary antibody (rabbit polyclonal from Santa Cruz) resulted in the disappearance of all bands (data not shown).

of PKC isoforms is a key step in tumorigenesis. TPA binding to PKC and resultant activation, translocation, and down-regulation of specific PKC isoforms is thought to play a critical role in phorbol ester-induced cell adhesion changes, including an increase in tight junction leakiness followed after four days by partial recovery of epithelial barrier function [42]. This study follows the phorbol ester-induced changes in the regulation of PKC-␦ and PKC-ε within confluent monolayers of LLC-PK1 renal epithelia. It has been shown previously by this group that the level of PKC activity and the amount of membraneassociated PKC-␣ temporally correlate with the TPAinduced alterations in tight junction permeability [8, 9]. It was also shown that overexpression of PKC-␣ in conjunction with phorbol ester exposure induced transepithelial leakiness and eventual cell detachment [43]. More recently, it has been shown that overexpression of PKC-␦ also causes increased tight junction permeability in LLCPK1 epithelia. This occurs even in the absence of phorbol esters, suggesting a possible role for this isoform in the

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regulation of tight junction permeability [22]. However, the physiological relevance of overexpressing an enzyme within a cell is complex, since the exogenous kinase may be expressed in areas or compartments of the cells where the endogenous form is not found. This led us to investigate further the possible role of PKC-␦ with the more physiologically relevant model of activation of endogenous PKC within the epithelial cell sheet by TPA. The role of another calcium-independent isoform, PKC-ε, in which the overexpression is oncogenic in rat colon epithelial cells [44], was also investigated. Similar to the atypical PKC isoforms, PKC-␭ and PKC-␨, which colocalize to the cell junctional complex in the renal epithelial Madin-Darby canine kidney (MDCK) [45] and LLC-PK1 cells [46], PKC-ε has been seen to stain in the region of the tight junction of intestinal epithelial cells [37], suggesting a putative role in tight junctional regulation. Our investigation of the regulation of these two PKC isoforms by total cell lysates and Western immunoblots revealed that TPA caused a rapid decrease in total PKC-␦ content, which then returned to near control levels, whereas PKC-ε levels remained almost unchanged throughout. Immunofluorescence indicated that PKC-␦ had both cytosolic and cytoskeletal localization within the cells, and a subtle cytoskeletal rearrangement occurred upon TPA treatment. Western immunoblots showed that PKC-␦ did not undergo the expected membrane translocation upon activation by TPA, but merely disappeared immediately from the cytosolic compartment. However, the observed pattern of PKC-␦ distribution and translocation was found to depend strongly on the type of antibody used. This difference stems from the apparent inability of certain antibodies to recognize specific phosphoproteins of PKC-␦. Protein kinase C-ε was localized to the cytosol, membrane, and Triton-X–insoluble fractions of the cells. TPA treatment resulted in a partial translocation of PKC-ε to both the membrane and Triton-X–insoluble fractions of the cell. However, it was the level of total PKC-␦ and not PKC-ε that temporally corresponded to the recovery of epithelial barrier function after chronic TPA treatment [8, 9]. PKC-␦ levels were markedly decreased with 24 hours of TPA treatment. However, at the later time points of four and seven days, there seemed to be a reappearance of PKC-␦ in the treated cells, albeit at lower levels than seen in the controls, a change that would correlate somewhat with the temporal changes in tight junction permeability [42]. Amsler et al noted an increase in PKC-␦ levels after chronic TPA treatment in LLC-PK1 cells, but reported barely detectable levels in control cells [41]. This discrepancy in levels of PKC-␦ detected in that study and those reported here may be due to the different PKC extraction procedures used in the two studies. There seemed to be a slight decrease in PKC-ε levels after one day of TPA treatment, but at the

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Fig. 8. Immunofluorescence of PKC-␦ isoforms in control, acutely and chronically TPA-treated LLC-PK1 cell sheets. (A) PKC-␦ in control sheets. (B) PKC-␦ in a cell sheet treated for one hour with 10⫺7 mol/L TPA. (C) PKC-␦ in a cell sheet treated for seven days with 10⫺7 mol/L TPA. For the seven-day time point, the cell sheets were refed daily. After fixation, cells were exposed to an anti–PKC-␦ polyclonal antibody (Santa Cruz) and processed for immunofluorescence, as described in the Methods section. In controls (A), PKC-␦ exhibited a diffuse cellular staining, while treatment with TPA for one hour (B) resulted in the movement of PKC-␦ to the cell borders. Seven-day treatment with TPA (C) resulted in an overall down-regulation of PKC-␦. Exposure time was the same for every panel, as was the magnification setting of all panels (bar, 25 ␮m).

Fig. 9. Western immunoblot of PKC-␦ (Santa Cruz rabbit polyclonal antibody) in cytosolic, membrane and Triton-X–insoluble fractions of LLC-PK1 cell sheets exposed to 10⫺7 mol/L TPA for one hour and incubated in the presence or absence of lambda phosphatase. Confluent, differentiated cell sheets cultured in 75 cm2 tissue culture flasks were refed with control medium (Lanes 1 and 2) or TPA (lanes 3 and 4) for one hour and extracted as described in the Methods section. Fifty micrograms of protein were incubated with lambda phosphatase (400 U) for 30 minutes at 30⬚C. Each lane received 50 ␮g of total protein. Exclusion of the primary antibody (rabbit polyclonal from Santa Cruz) resulted in the disappearance of all bands (data not shown).

later time points, there did not appear to be a significant effect of TPA on this isoform’s total level of expression within the cells. In addition to total PKC-␦ levels seen in Western immunoblots, the TPA-induced cytoskeletal rearrangement of PKC-␦ seen in immunofluorescence also correlated with the complete transepithelial resistance (Rt) profile, unlike the partial translocation of PKC-ε seen in immunoblots, which correlated with the

initial decrease in Rt but not the later recovery. These present data thus suggest that PKC-␦, but not PKC-ε, may play a role in the regulation of LLC-PK1 epithelial barrier function. A general model for the regulation of PKC activity often includes translocation (tight association) of PKC to the membrane in response to activator binding, accompanied by a conformational change of the enzyme within the lipid environment of the membrane that results in a catalytically active PKC [5]. This is often followed by a rapid inactivation, apparently because of a decrease in activator binding of PKC from the membrane complex. However, in this present study, the usual pattern of redistribution of PKC to the membrane fraction followed by down-regulation does not seem to hold true for PKC-␦ based on results with the monoclonal antibody to PKC-␦ (Transduction). Treatment of cells with TPA appeared to result in a rapid disappearance of PKC-␦ from the cytoplasmic compartment without reappearance in the membrane or Triton-X–insoluble fractions. In T84 human epithelial cells, TPA was observed to cause persistent translocation of PKC-␦ to the membrane compartment, but also caused a 400% increase in PKC-␦ mRNA after 24 hours [33]. Our lack of detected PKC-␦ in the membrane-associated fraction of the LLC-PK1 cells in the present study was first thought not to be an antibody recognition problem, as treatment of the cells with another known PKC activator, bryostatin-1, did result in the detection of PKC-␦ in the membrane-associated compartment by 10 minutes using the same primary antibody (unpublished results). From the immunofluorescence results it would seem that the majority of the PKC-␦ isoform associates tightly with a cytoskeletal fraction within these cells, which was inextractable by the conditions normally used for Western immunoblot anal-

Clarke et al: Phorbol ester regulation of PKC-d and PKC-ε

ysis. To investigate this more fully, the pellet remaining after the third fraction (Methods section) was subjected to a further extraction in 2% SDS followed by boiling, and the obtained sample was analyzed by Western immunoblot. A further pool of PKC-␦ was identified that may belong to a very tightly associated cytoskeleton fraction (Fig. 5). It is interesting to note, however, that in the total cell lysates, a reappearance of PKC-␦ was observed at days 4 and 7, while in the subcellular localization studies, PKC-␦ did not reappear in the cytosol, the fraction from where it originally disappeared. This unexpected disappearance of PKC-␦ from the cytosol without apparent translocation to the membrane compartment of the cell was further investigated by using a second antibody directed to a different epitope of PKC-␦. Surprisingly, as in Figure 7, a different expression pattern of PKC-␦ within the cell compartments was obtained for this isoform of PKC. PKC-␦ was now detected in all three fractions of the control cells with a TPAinduced translocation of this isoform from the cytosolic fraction to the membrane-associated and third fractions after one hour. Protein kinase C-␦ has been shown to be phosphorylated in many different cell types on phorbol ester activation [47, 48]. A possible explanation for the different PKC-␦ binding patterns, which we observed with the two different PKC-␦ antibodies, may be that different antibodies to PKC-␦ recognize different phosphorylated forms of the protein. This was investigated by incubation of the different cell fractions of both control and TPAtreated cell extracts with a lambda phosphatase. The less phosphorylated form of PKC-␦ was observed as a slightly faster migrating PKC-␦ band, which was about 2 kD smaller than the corresponding PKC-␦ band from a cell extract, which had not been incubated with the phosphatase. It can be seen clearly from the cell fractions in lanes 3 and 4 of Figure 9 that at least partial dephosphorylation of PKC-␦ by lambda phosphatase resulted in a greater number of PKC-␦ bands becoming visible when using the Santa Cruz polyclonal antibody. This antibody may be able to recognize the less phosphorylated forms of PKC-␦ because of a slightly different conformational state of the protein and/or the phosphorylation state of the recognition site. Phosphorylation of PKC-␦ may mask the antigenic binding site of the monoclonal antibody (Transduction Labs) and prevent detection of PKC-␦, thus explaining the different subcellular localization patterns obtained. This finding has been reported previously where two different PKC-␮ antibodies were able to selectively detect nonphosphorylated or phosphorylated forms of this PKC isoform [49]. Therefore, such properties of an antibody should be carefully studied before being used to quantitatively determine protein levels. In summary, the phorbol ester TPA seems to be causing a differential regulation of PKC-␦ and PKC-ε in LLC-

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PK1 cells. Although we have temporally correlated the changes in amount and localization of PKC-␦, but not PKC-ε, with the previously observed changes in paracellular permeability, it is difficult to prove an etiological relationship. If PKC-␦ is, in fact, playing a role in the phorbol ester-induced changes in LLC-PK1 epithelial barrier function, it would seem to be doing so in a different subcellular compartment than PKC-␣. Unlike PKC-␣, PKC-␦ is a calcium-insensitive isoform of PKC and might be expected to have a distinct subcellular distribution in these epithelial cells. Unlike the diffuse localization of PKC-␣ in LLC-PK1 cells [9], the colocalization of PKC-␦ with the cytoskeleton detected with the mouse monoclonal anti–PKC-␦ would indicate that this isoform may modulate epithelial permeability through phosphorylation of a target protein localized to the cytoskeleton or the cytoskeleton itself. This compartmental model with different target proteins gains credibility from studies done with PKC-␣ and PKC-␦ overexpressing LLC-PK1 cells [22]. The cells overexpressing PKC-␣ required TPA to induce gross morphological changes and a decrease in Rt, while cells with high levels of the PKC-␦ isoform were constitutively leaky along the paracellular pathway. This indicates that exogenously expressed PKC-␦ in these cells may be located in a DAG-rich compartment, resulting in a basally activated form of this isozyme. A secondary but important methodological finding of this study is that the Western immunoblot data obtained from a single specific antibody to a PKC isoform may reveal only part of the profile of translocation and downregulation of the isoform. We believe that this is so because the two different anti–PKC-␦ antibodies used in our studies appeared to recognize different PKC-␦ phosphoproteins. This explanation seems reasonable because (1) PKC-␦ activation is known to result in changes in the PKC-␦ phosphorylation state [47]; (2) phorbol ester exposure resulted in shifts in the apparent molecular weight of PKC-␦ bands when using one but not the other antibody in our studies; (3) the use of lambda phosphatase shows these different bands to be phosphoproteins; and (4) our two antibodies have distinct antigenic recognition sites on PKC-␦. One should therefore be aware of the possibility that any treatment capable of increasing the phosphorylation state of a PKC isoform may cause a false “disappearance” of one or more bands in a Western immunoblot, depending on the antibody used. The importance of the LLC-PK1 cell line as a model for study of renal apoptosis [50, 51] and of PKC-␦ in the regulation of apoptosis [52, 53] makes our observations here on PKC-␦ in LLC-PK1 epithelia particularly pertinent. ACKNOWLEDGMENTS This work was funded in part by National Institutes of Health grants CA48121 and CA67113. We are grateful to Ms. Jennifer Kampherstein and Dr. Colleen Marano (Lankenau Medical Research Center, Wynne-

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wood, PA, USA) for their insights and comments. We thank Dr. Thomas O’Brien for advice given in the preparation of the manuscript, and the editorial staff of the Lankenau Medical Research Center for their help in the preparation of this manuscript. Reprint requests to Hilary Clarke, Ph.D., Lankenau Medical Research Center, 100 Lancaster Avenue, Wynnewood, Pennsylvania 19096, USA. E-mail: [email protected]

REFERENCES 1. Nishizuka Y: The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334:661–665, 1988 2. Nishizuka Y: Protein kinase C and lipid signaling for sustained cellular responses. FASEB J 9:484–496, 1995 3. Huwiler A, Fabbro D, Pfeilschifter J: Differential recovery of protein kinase C-␣ and -ε isozymes after long-term phorbol ester treatment in rat mesangial cells. Biochem Biophys Res Commun 180:1422–1428, 1991 4. Crabos M, Fabbro D, Stabel S, Erne P: Effect of tumor promoting phorbol ester thrombin and vasopressin on translocation of three distinct protein kinase C isoforms in human platelets and regulation by calcium. Biochem J 288:891–896, 1992 5. Nishizuka Y: Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258:607–614, 1992 6. Nishizuka Y: The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature 308:693–698, 1984 7. Hug H, Sarre TF: Protein kinase C isoenzymes: Divergence in signal transduction. Biochem J 291:329–343, 1993 8. Mullin JM, Soler AP, Laughlin KV, Kampherstein JA, Russo LM, Saladik DT, George K, Shurina RD, O’Brien TG: Chronic exposure of LLC-PK1 epithelia to the phorbol ester TPA produces polyp-like foci with leaky tight junctions and altered protein kinase C-␣ expression and localization. Exp Cell Res 227:12–22, 1996 9. Mullin JM, Marano CW, Laughlin KV, Nuciglio M, Stevenson BR, Soler AP: Different size limitations for increased transepithelial paracellular solute flux across phorbol ester and tumor necrosis factor-treated epithelial cell sheets. J Cell Physiol 171:226–233, 1997 10. Ojakian GK: Tumor promoter-induced changes in the permeability of epithelial cell tight junctions. Cell 23:95–103, 1981 11. Mullin JM, O’Brien TG: Effect of tumor promoters on LLC-PK1 renal epithelial tight junctions and transepithelial fluxes. Am J Physiol 251:C597–C602, 1986 12. Hecht G, Robinson B, Koutsouris A: Reversible disassembly of an intestinal epithelial monolayer by prolonged exposure to phorbol ester. Am J Physiol 266:G214–G221, 1994 13. Stenson WF, Easom RA, Riehl TE, Turk J: Regulation of paracellular permeability in Caco-2 cell monolayers by protein kinase C. Am J Physiol 265:G955–G962, 1993 14. Marano CW, Lewis SA, Garulacan LA, Soler AP, Mullin JM: Tumor necrosis factor-␣ increases sodium and chloride conductance across the tight junction of Caco-2 BBE, a human intestinal epithelial cell line. J Membr Biol 161:263–274, 1998 15. Mullin JM, Snock KV: Effect of tumor necrosis factor on epithelial tight junctions and transepithelial permeability. Cancer Res 50:2172–2176, 1990 16. Madara JL, Stafford J: Interferon-gamma directly affects barrier function of cultured intestinal epithelial monolayers. J Clin Invest 83:724–727, 1989 17. Cereijido M, Robbins ES, Dolan WJ, Rotunno CA, Sabatini DD: Polarized monolayers formed by epithelial cells on a permeable and translucent support. J Cell Biol 77:853–880, 1978 18. Martinez-Palomo A, Meza I, Beaty G, Cereijido M: Experimental modulation of occluding junctions in a cultured transporting epithelium. J Cell Biol 87:736–745, 1980 19. Canfield PE, Geerdes AM, Molitoris BA: Effect of reversible ATP depletion on tight junction integrity in LLC-PK1 cells. Am J Physiol 261:F1038–F1045, 1991 20. Moore R, Pothoulakis C, Lamont JT, Carlson S, Madara JL:

21.

22.

23.

24.

25. 26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

C. difficile toxin A increases intestinal permeability and induces Cl⫺ secretion. Am J Physiol 259:G165–G172, 1990 Mullin JM, Snock KV, Shurina RD, Noe KG, Misner L, Imiazumi S, O’Brien TG: Effects of acute vs. chronic phorbol ester exposure on transepithelial permeability and epithelial morphology. J Cell Physiol 152:35–47, 1992 Mullin JM, Kampherstein JA, Laughlin KV, Clarkin CE, Miller RD, Szallasi Z, Kachar B, Soler AP, Rosson D: Overexpression of protein kinase C-␦ increases tight junction permeability in LLC-PK1 epithelia. Am J Physiol 275:C544–C554, 1998 Shih NY, Floyd-Smith G: Invariant chain (CD74) gene regulation: Enhanced expression associated with activation of protein kinase C delta in a murine B lymphoma cell line. Mol Immunol 32:643–650, 1995 Mischak H, Kolch W, Goodnight J, Davidson WF, Rapp U, Rose-John S, Mushinski JF: Expression of protein kinase C genes in hemopoietic cells is cell-type and B-cell-differentiation stage specific. J Immunol 147:3981–3987, 1991 Mizuno K, Kubo K, Saido TC, Osada S, Kuroko T, Ohno S, Suzuki K: Structure and properties of a ubiquitously expressed protein kinase C, nPKC delta. Eur J Biochem 18:931–940, 1991 Ohno S, Mizuno K, Adachi Y, Hata A, Akita Y, Akimoto K, Osada S, Hirai S, Suzuki K: Activation of novel protein kinase C␦ and C⑀ upon mitogenic stimulation of quiescent rat 3Y1 fibroblasts. J Biol Chem 269:17495–17501, 1994 O’Driscoll KR, Teng KK, Fabbro D, Greene LA, Weinstein IB: Selective translocation of protein kinase C-delta in PC12 cells during nerve growth factor-induced neuritogenesis. Mol Biol Cell 6:449–458, 1995 Chen C-C: Protein kinase C ␣, ␦ and ε and zeta in C6 glioma cells: TPA induces translocation and down-regulation of conventional and new PKC isoforms but not atypical PKC zeta. FEBS Lett 332: 169–173, 1993 Watanabe T, Ono Y, Taniyama Y, Hazama K, Igarashi K, Ogita K, Kikkawa U, Nishizuka Y: Cell division arrest induced by phorbol ester in CHO cells overexpressing protein kinase C-␦ subspecies. Proc Natl Acad Sci USA 89:10159–10163, 1992 Mischak H, Goodnight J, Kolch W, Martiny-Baron G, Schaechtle C, Kazanietz MG, Blumberg PM, Pierce JH, Mushinski JF: Overexpression of protein kinase C-␦ and -ε in NIH 3T3 cells induces opposite effects on growth, morphology, anchorage dependence, and tumorigenicity. J Biol Chem 268:6090–6096, 1993 Hirai S, Izumi Y, Kaibuchi K, Mizuno K, Osada S, Suzuki K, Ohno S: Ras-dependent signal transduction is indispensable but not sufficient for the activation of AP1/Jun by PKC delta. EMBO J 15:2331–1240, 1994 Li W, Yu JC, Michieli P, Beeler JF, Ellmore N, Heidaran MA, Pierce JH: Stimulation of the platelet-derived growth factor beta receptor signaling pathway activates protein kinase C-delta. Mol Cell Biol 14:6727–6735, 1994 Assert R, Schatz H, Pfeiffer A: Upregulation of PKC delta- and downregulation of PKC-alpha mRNA and protein by phorbol ester in human T84 cells. FEBS Lett 388:195–199, 1996 Owen PJ, Johnson GD, Lord JM: Protein kinase C-delta associates with vimentin intermediate filaments in differentiated HL60 cells. Exp Cell Res 225:366–373, 1996 Barry ST, Critchley DR: The RhoA-dependent assembly of focal adhesions in Swiss 3T3 cells is associated with increased tyrosine phosphorylation and the recruitment of both pp125FAK and protein kinase C-delta to focal adhesions. J Cell Sci 107:2033–2045, 1994 Haller H, Lindschau C, Maasch C, Olthoff H, Kurscheid D, Luft FC: Integrin-induced protein kinase C␣ and Cε translocation to focal adhesions mediates vascular smooth muscle cell spreading. Circ Res 82:157–165, 1998 Saxon ML, Zhao X, Black JD: Activation of protein kinase C isozymes is associated with post-mitotic events in intestinal epithelial cells in situ. J Cell Biol 126:747–763, 1994 Gstraunthaler G, Pfaller W, Kotanko P: Biochemical characterization of renal epithelial cell cultures (LLC-PK1 and MDCK). Am J Physiol 248:F536–F544, 1985 Rabito CA, Auseillo DA: Na-dependent sugar transport in a

Clarke et al: Phorbol ester regulation of PKC-d and PKC-ε

40. 41. 42.

43.

44. 45.

46.

cultured epithelial cell line from pig kidney. Am J Physiol 250: F734–F746, 1980 Hull RN, Cherry WR, Weaver GW: The origin and characteristics of a pig kidney cell strain, LLC-PK1. In Vitro 12:670–677, 1976 Amsler K, Murray J, Cruz R, Chen JL: Chronic TPA treatment inhibits the expression of proximal tubule-specific properties by LLC-PK1 cells. Am J Physiol 270:C332–C340, 1996 Mullin JM, Kampherstein JA, Laughlin KV, Saladik DT, Peralta Soler A: Transepithelial paracellular leakiness induced by chronic phorbol ester exposure correlates with polyp-like foci and redistribution of protein kinase C-␣. Carcinogenesis 18:2339–2345, 1997 Rosson D, O’Brien TG, Kampherstein JA, Szallasi Z, Bogi K, Blumberg P, Mullin JM: Protein kinase C-␣ activity modulates transepithelial permeability and cell junctions in the LLC-PK1 epithelial cell line. J Biol Chem 272:14950–14953, 1997 Perletti GP, Folini M, Lin HC, Mischak H, Picinini F, Tashjian AH: Overexpression of protein kinase C epsilon is oncogenic in rat colonic epithelial cells. Oncogene 12:847–854, 1996 Izumi Y, Hirose T, Tamai Y, Hirai S, Nagashima Y, Fujimoto T, Tabuse Y, Kemphues KJ, Ohno S: An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3. J Cell Biol 143:95–106, 1998 Dodane V, Kachar B: Identification of isoforms of G proteins and PKC that colocalize with tight junctions. J Membr Biol 149:199–209, 1996

1015

47. Li W, Zhang J, Bottaro DP, Pierce JH: Identification of serine 643 of protein kinase C-delta as an important autophosphorylation site for its enzymatic activity. J Biol Chem 272:24550–24555, 1997 48. Denning MF, Dlugosz AA, Howett MA, Yuspa SH: Expression of an oncogenic rasHa gene in murine keratinocytes induces tyrosine phosphorylation and reduced activity of protein kinase C-delta. J Biol Chem 268:26079–26081, 1993 49. Rennecke J, Johannes FJ, Richter KH, Kittstein W, Marks F, Gschwendt M: Immunological demonstration of protein kinase C in murine tissues and various cell lines. Differential recognition of phosphorylated forms and lack of down-regulation upon 12-Otetradecanoylphorbol-13-acetate treatment of cells. Eur J Biochem 242:428–432, 1996 50. Zhan Y, Van Der Walter B, Wang Y, Stevens JL: The roles of caspase-3 and bcl-2 in chemically-induced apoptosis but not necrosis or renal epithelial cells. Oncogene 18:6505–6512, 1999 51. Lau AH: Apoptosis induced by cisplatin nephrotic injury. Kidney Int 56:1295–1298, 1999 52. Koriyama H, Kouchi Z, Umeda T, Saido TC, Momoi T, Ishiura S, Suzuki K: Proteolytic activation of protein kinase C delta and epsilon by caspase-3 in U937 cells during chemotherapeutic agentinduced apoptosis. Cell Signal 11:831–838, 1999 53. Pongracz J, Webb P, Wang K, Deacon E, Lunn OJ, Lord JM: Spontaneous neutrophil apoptosis involves caspase 3-mediated activation of protein kinase C-delta. J Biol Chem 274:37329–37334, 1999