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Digestive Diseases and Sciences, Vol. 46, No. 7 (July 2001), pp. 1490 –1499

Phorbol Ester Treatment Increases Paracellular Permeability Across IEC-18 Gastrointestinal Epithelium In Vitro C.W. MARANO, PhD,* L.A. GARULACAN, BS, N. GINANNI, BS, and J.M. MULLIN, PhD

The phorbol ester, TPA, transiently increases the transepithelial permeability across the gastrointestinal epithelium formed by IEC-18. There was a significant decrease in transepithelial resistance (RT) between 0 and 1.5 hr, accompanied by increased flux of polyethylene glycol (4000 MW), suggesting that the increase was across the tight junction. By 2 hr, the decrease in RT reversed and maintained control level. The transepithelial permeability increase was prevented by coincubation with the protein kinase C (PKC) inhibitor bisindolylmaleimide. There was a rapid (within 15 min) translocation of PKC-␣ from the cytosolic to the “membrane-associated” compartment, followed by a down-regulation that was detectable within 60 min of TPA treatment. The down-regulation of PKC-␣ from the membrane was prevented by either calpain inhibitor I or MG-132 and resulted in a sustained permeability increase. The permeability changes were not accompanied by significant effects on the amount or localization of the tight junctional proteins, occludin and ZO-1. However, occludin did show a reversible increase in phosphorylation with TPA treatment. Together these data support a role for PKC-␣-mediated regulation of barrier permeability in an in vitro model of small intestinal epithelium, perhaps through modulation of the phosphorylation state of the tight junctional protein, occludin. KEY WORDS: tight junction; protein kinase C; transepithelial permeability; colon; occludin.

We have documented the regulation of transepithelial permeability by the phorbol ester tumor promoter and PKC activator, TPA, using LLC-PK1 epithelial cell sheets derived from pig kidney cortex and cultured on permeable supports (1, 2). The TPAinduced increase in permeability is rapid, measured as a decrease in transepithelial resistance detectable Manuscript received May 1, 2000; revised manuscript received February 27, 2001; accepted March 2, 2001. From the Lankenau Institute for Medical Research, Wynnewood, Pennsylvania. *Current address: Centocor, Inc., 200 Great Valley Parkway, Malvern, Pennsylvania 19355-1307. We gratefully acknowledge the support of the Lankenau Foundation. Address for reprint requests: J.M. Mullin, Cell Physiology, Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, Pennsylvania 19096.

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within minutes and reaching its nadir of 10% of initial levels within 1–2 hr. It is accompanied by increased transepithelial flux of cell impermeant molecules ranging in size from (182 MW) D-mannitol to (2 ⫻ 106 MW) dextran and the electron dense dye ruthenium red, highlighting TPA’s effect to increase tight junctional permeability (3). Furthermore, the increased permeability extends to epidermal growth factor (EGF) and insulin, which retain their biological activity upon crossing the epithelium (4, 5). Additional studies conducted in our laboratory have shown that the translocation of the PKC isoform, PKC-␣, from the cytosolic to the “membrane-associated” compartment temporally associates with the increase in transepithelial permeability (6 – 8). Furthermore, it associates with a decrease in the phosphorylation Digestive Diseases and Sciences, Vol. 46, No. 7 (July 2001)

0163-2116/01/0700-1490$19.50/0 © 2001 Plenum Publishing Corporation

TPA ALTERS INTESTINAL BARRIER PERMEABILITY

state of the tight junctional protein, occludin, on threonine residues (9). Thus, we have demonstrated that activation of PKC-␣ coincides with an increase in transepithelial permeability to a range of molecules, including growth factors that retain their biological activity on passing through the epithelial barrier formed by the renal LLC-PK1 cell line. One of the ramifications of this observation in vivo is that growth factor receptors either on the basolateral surface of the epithelial cells themselves or on adjacent nonepithelial cells are now accessible to ligands normally sequestered in the luminal compartment. Receptor binding could then lead to disruption in the balance between proliferative and apoptotic signals, resulting in altered cellular turnover. Such dysregulation of cellular turnover could result in further altered permeability properties of the epithelium as well as in tumor formation. The aim of this study was to determine whether this effect of TPA was characteristic of other epithelial barriers, including those lining the gastrointestinal tract. We selected IEC-18, a nontumorigenic cell line derived from rat ileum (10, 11), as a model system to test this hypothesis in nontransformed epithelial cells derived from the gut mucosa. MATERIALS AND METHODS Cell Culture. IEC-18, a nontumorigenic gastrointestinal epithelial cell line derived from rat ileum (10, 11), was obtained from ATCC (CRL-1589) at passage 15. Cells were maintained in Dulbecco’s modified minimal essential medium (DMEM) with 4.5 g/liter glucose, supplemented with 5% fetal bovine serum (Hyclone), 2 mM L-glutamine and 0.1 units/ml insulin (Gibco) and incubated at 37°C and 10% CO2–90% air. IEC-18 were routinely split every seven days and seeded at 1 ⫻ 105 cells/per 75 cm2 tissue culture flask. Confluent flasks were used for the study of cell morphology and western immunoblotting. For permeability and immunofluorescence studies, cells were cultured at 5 ⫻ 105 onto Millicell PCF (4.2 cm2) or Falcon 3102 (4.5 cm2) filter assemblies, respectively. The data presented represent results obtained using confluent cell sheets 7–10 days after seeding. Transepithelial Electrical and Flux Measurements. These methods have been described in detail in earlier publications from our laboratory (3). In both the electrical and flux studies, cell sheets were refed with standard medium prior to the start of the experiments. The cell sheets were returned to 37°C and 10% CO2–90% air for 1 hr. Measurement of the transepithelial resistance (RT) was made at t ⫽ 0 hr, prior to the addition of TPA to the medium, and at 0.5 to 1-hr intervals thereafter. For flux studies, the basolateral medium was supplemented with 0.1 ␮Ci/ml of the respective radiolabeled molecules D-[14C]mannitol (NEN Life Sciences, 51.5 mCi/ mmol); [14C]polyethylene glycol (Amersham, 11.2 mCi/g); and [14C]methyldextran [Sigma, 2.1 mCi/g] ⫾ TPA at t ⫽ 0 Digestive Diseases and Sciences, Vol. 46, No. 7 (July 2001)

hr, and their appearance in the apical medium was monitored by sampling (25–50␮l) at 0.5-to-2 hr, intervals up to 5 hr. These samples were then subjected to liquid scintillation counting to determine the rate of transepithelial flux of the radiolabeled trace as a function of the presence and absence of TPA with or without pharmacological modifiers. The accumulation of PEG, over a fixed time interval, was used to determine the integrity of the IEC-18 epithelial barrier in studies looking at the effect of proteasomal and proteolytic inhibitors on the recovery of permeability during TPA treatment. Briefly, cell sheets were incubated in the presence of TPA ⫾ the indicated inhibitors for 5 hr. At the end of 5 hr, the basolateral media were supplemented with 0.1 ␮Ci/ml [14C]PEG and 0.1 M unlabeled PEG. The incubation was continued for an additional 2 hr, after which the apical media were sampled as described above. Western Immunoblot. At the indicated times of treatment with TPA, the medium was aspirated from confluent cell sheets. The cell sheets were then rinsed with ice cold Ca2⫹/ Mg2⫹-free phosphate-buffered saline (PBS) and scraped into 1 ml of buffer A (20 mM Tris; 0.2 M sucrose; 10 mM EGTA; 2 mM EDTA; 20 ␮g/ml leupeptin; 10 ␮g/ml aprotinin; 50 ␮M phenylmethylsulfonyl fluoride; 50 mM NaF; and 1:100 dilution of phosphatase inhibitor cocktail II (Sigma) pH7.5) at 4°C. The cell suspension was then sonicated (Branson Sonifier 450) on ice for 1 min at an output of 1. It was centrifuged (Beckman L-70 ultracentrifuge) at 4°C and 39,000 rpm for 35 min using a Ti70 rotor. The supernatant (cytosol) was removed, a sample was taken for protein determination, and the remainder was diluted 1:1 with 2X sample buffer (0.0625 M Tris; 2% SDS; 10% glycerol; 5% mercaptoethanol, and 0.05% bromophenol blue, pH 6.8), boiled for 5 min, and stored at ⫺20°C. The remaining pellet (membrane-associated) was suspended in 150␮l of buffer A supplemented with 1% Triton X-100 and the protease and phosphatase inhibitors cited above. The suspension was then incubated with shaking at 4°C for 1 hr. A membrane-associated fraction (supernatant) was then obtained by centrifugation as detailed above, placed in 2X sample buffer, boiled, and stored at ⫺20°C. A third Tritoninsoluble fraction was then generated by incubating the remaining pellet with lysis buffer (150 mM NaCl; 50 mM Tris base; 1 mM EGTA; 1 mM EDTA; 1% IGEPAL; 0.1% sodium deoxycholate; 0.1% SDS) supplemented with the protease and phosphatase inhibitors cited above. The resulting supernatant was derived as described previously. Protein content of the extracted fractions was measured using the Bradford assay with bovine serum albumin as the standard (12). To obtain total protein extracts, TPA-treated IEC-18 epithelial cell sheets were scraped into lysis buffer as described above and centrifuged for 1 hr at 39,000 rpm in a Beckman Ti70 rotor at 4°C. The supernatant was aliquoted for determination of the protein content of the extract and the remaining supernatant was diluted 1:1 with 2X sample buffer, boiled for 5 min, and frozen at ⫺20°C pending western blot analysis. Aliquots (25–50 ␮g protein) of the extracted fractions were separated on 6.5 or 8% SDS polyacrylamide gels depending on the protein of interest. The separated proteins were transferred to nitrocellulose (0.45 ␮m; Osmonics, Westborough, Massachusetts, USA) and incubated with anti-protein kinase C, type III, a monoclonal IgG, against

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MARANO ET AL TABLE 1. TPA INCREASES THE TRANSEPITHELIAL FLUX THROUGH THE PARACELLULAR PATHWAY OF IEC-18 CELL SHEETS* Amount (nmol/3hr/cm2)

14 C]Mannitol [14C]Polyethylene glycol 14 [ C]Methyldextran

D[

Control

TPA (10⫺7 M)

9.2 ⫾ 2.3 (8) 3.3 ⫾ 0.2 (6) 1.3 ⫾ 0.1 e⫺3 (4)

9.3 ⫾ 2.1 (9)a 5.6 ⫾ 0.3 (8)b 1.8 ⫾ 0.1 e⫺3 (4)c

*Data represent the mean ⫾ SEM of (N) individual cell sheets from one or two separate experiments of the rate of appearance of each radiolabeled probe into the apical compartment of IEC-18 epithelial cell sheets cultured on permeable filter ring assemblies for 3 hr. a, NS vs control; b, P ⬍ 0.001 vs control; c, P ⬍ 0.03 vs control.

Fig 1. TPA transiently decreases the transepithelial resistance across IEC-18 cell sheets. Confluent IEC-18 monolayers were incubated with medium ⫾ TPA (10⫺7 M) for 5 hr. The data are expressed as a percent of time matched control at each interval and represent the mean ⫾ SEM of data collected from four separate experiments involving a total of 11–14 individual cell sheets per condition. The mean initial RT was 17.7 ⫾ 2.0 ohms ⫻ cm2.

the catalytic domain (1 ␮g/ml; Upstate Biotechnology Incorporated); rabbit polyclonal anti-occludin (1 ␮g/ml; Zymed); or a rabbit polyclonal anti-ZO-1 (2 ␮g/ml, Zymed) for 1–2 hr at room temperature. The blots were then rinsed 3X with 0.3% Tween/PBS and incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (1:10,000; Southern Biotechnology Associates, Inc.) for 1 hr at room temperature. The blots were then rinsed as described above and the proteins detected using chemiluminescence (NEN Life Sciences, Inc.). ␭ Phosphatase Assay. Fifty micrograms of extracted total protein from TPA-treated IEC-18 epithelial cell sheets was mixed with 7.5 ␮l of 10⫻ phosphatase buffer [DTT (10 mM); Tris HCl (500 mM, pH 7.5); NaCl (1 M); and EDTA (1 mM)], and dH2O ⫾ 1 ␮l of recombinant ␭ protein phosphatase (400 units/ml; Calbiochem) in a final reaction volume of 75 ␮l (9). The tubes were vortexed and incubated at 30°C for 30 min, after which an equal volume of 2X sample buffer was added per tube. The samples were boiled for 5 min and frozen at ⫺20°C pending western immunoblot analysis as described above. Detection of Apoptotic Cells. In separate studies, the morphology and frequency of apoptotic cells during TPA treatment of the IEC-18 epithelium was assessed using the DNA stain, DAPI. The cell sheets were viewed under an epifluorescent microscope and photographs were taken of a 40X field. The frequency of apoptotic cells was determined relative to the total number of nuclei per field as described in an earlier publication (13).

RESULTS Confluent cultures of IEC-18 responded to incubation with TPA (10⫺7 M) with a rapid but small increase in transepithelial permeability, measured inversely as a decrease in RT (transepithelial resistance),

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within 0.5 hr (Figure 1; P ⬍ 0.001 vs time-matched control). This increase in transepithelial permeability was maintained through the 2-hr exposure to the phorbol ester. The RT of TPA-treated IEC-18 cell sheets then recovered and was sustained at the control level for up to 5 hr of observation (Figure 1). The observed increase in permeability was confirmed in a series of transepithelial flux studies utilizing the cellimpermeant radiotracer molecules D-[14C]mannitol (180 MW), D-[14C]polyethylene glycol (4000 MW) and [14C]methyldextran (2 ⫻ 106 MW). In these studies there was no significant effect of TPA on the already high transepithelial flux of mannitol (Table 1), reflecting that IEC-18 is a very leaky epithelium compared to either the CACO-2 BBE (14) or LLCPK1 (3) epithelial cell sheets that we routinely utilize in the laboratory. However, incubation with TPA did increase the transepithelial flux of the larger molecules, polyethylene glycol (P ⬍ 0.0001, Table 1) and methyldextran (P ⬍ 0.03, Table 1), across the IEC-18 cell sheet when compared to control-treated epithelia. In additional experiments using the transepithelial flux of [14C]polyethylene glycol as the probe for changes in transepithelial permeability, the PKC selective inhibitor, bisindolylmaleimide [GF109203X (2 ␮M)], effectively blocked the TPA-induced transepithelial permeability increase (P ⬍ 0.001 vs TPA alone; Table 2). TABLE 2. TRANSEPITHELIAL FLUX OF [14C]POLYETHYLENE GLYCOL ACROSS TPA-TREATED IEC-18 CELL SHEETS IN THE PRESENCE OR ABSENCE OF PROTEIN KINASE C INHIBITION*

Control ⫹2 ␮M GFX TPA (10⫺7 M) ⫹2␮M GFX

([14C]PEG nmol/5hr/cm2)

P

3.99 ⫾ 0.07 (21) 3.98 ⫾ 0.0.08 (9) 5.96 ⫾ 0.07 (21) 4.12 ⫾ 0.07 (9)

NS vs control ⬍0.001 vs control ⬍0.001 vs TPA

*Data represent the means ⫾ SEM of N cell sheets given in paranthesis over multiple experiments. Statistical significance of the data was determined using the Mann-Whitney rank sum test. Digestive Diseases and Sciences, Vol. 46, No. 7 (July 2001)


Fig 2. TPA causes a rapid down-regulation of membraneassociated PKC-␣ in IEC-18. Incubation of IEC-18 with TPA (10⫺7 M) causes a rapid translocation of PKC-␣ from the cytosol (A) to the membrane-associated (B) fraction, reaching a peak within 15–30 min (lanes 3 and 4) after the start of exposure. The PKC-␣ content within the membrane then begins to decrease by 60 min (lane 5) and is barely detectable by 4 hr (lane 7). Lane (⫹) PKC from rabbit brain cytosol (Upstate Biotechnology Inc); lane 1, control, t ⫽ 0 hr; lane 2, TPA, 15 min; lane 3, TPA; 30 min; lane 4, TPA, 60 min; lane 5, TPA, 2 hr; and lane 6, TPA, 4 hr.

Coincident with these changes in permeability, TPA treatment resulted in a translocation of PKC-␣ from the cytosol to the membrane-associated compartment of the IEC-18 epithelial cell sheet. Within 15 min of TPA treatment there was a detectable increase in membrane-associated PKC-␣ accompanied by a corresponding decrease of the kinase in the cytosolic-associated fraction (Figure 2). However, despite the continued presence of the phorbol ester, the PKC-␣ present in the membrane-associated fraction began to down-regulate as early as 1 hr after the start of treatment (Figure 2). Down-regulated PKC-␣ was not returning to the cytosolic compartment, as there was no detectable level of the enzyme in this fraction 4 hr after the start of TPA treatment. The rapid decrease of PKC-␣ in the cytosolic compartment was associated with the translocation of the kinase to the membrane and was not effected by cotreatment with the proteolytic/proteosomal inhibitor, calpain inhibitor I (ALLN; 10 ␮M). However, the presence of ALLN did inhibit down-regulation of PKC-␣ from the membrane-associated fraction at 4 hr following exposure of the IEC-18 epithelium to TPA (Figure 3).

Fig 3. Cotreatment with protease inhibitor blocks the TPA-induced down-regulation of PKC-␣. Preincubation of IEC-18 cell sheets with 10 ␮M calpain inhibitor prevents the down-regulation of PKC-␣ from the membrane-associated fraction normally observed with TPA treatment alone. Lane 1, PKC from rabbit brain cytosol; lane 2, TPA (10⫺7M), 4 hr; lane 3, TPA (10⫺7 M) and calpain inhibitor I (10 ␮M), 4 hr. Digestive Diseases and Sciences, Vol. 46, No. 7 (July 2001)

Fig 4. Transepithelial accumulation of [14C]polyethylene glycol of TPA-treated IEC-18 cell sheets is sustained in the presence of proteosomal or proteolytic inhibition. The data represent the summary of three separate experiments in which IEC-18 cell sheets were incubated with or without TPA (10⫺7 M), ALLN (75 ␮M), and MG-132 (5 ␮M) for 5 hr. At the end of 5 hr, the basolateral media were supplemented with radiolabeled and unlabeled PEG and the incubation was continued for an additional 2 hr as described in Materials and Methods. The data are presented as the mean ⫾ SEM. The statistical significance of the data was analyzed by a t test comparing TPA alone to TPA in the presence of inhibitors (*P ⬍ 0.052; **P ⬍ 0.002).

The proteasomal inhibitor MG-132 also did not affect the TPA-induced translocation of PKC-␣ to the membrane, and its presence led to increased levels of detectable PKC-␣ in the membrane compared to TPA treatment alone at 4 hr (data not shown). The retention of PKC-␣ in the membrane, mediated by either calpain inhibitor I or MG-132 in the presence of TPA, was associated with a sustained increase in transepithelial permeability. This could be measured as an increased accumulation of [14C]PEG in the apical fluid compartment of IEC-18 cell sheets 5–7 hr after treatment with TPA in the presence of the inhibitors when compared to TPA treatment alone, which was not significantly different from its timematched control (Figure 4). This comparison could be made because the reversal of TPA’s effect on PEG permeability between 5 and 7 hr (1.6 ⫾ 0.4 control vs 3.3 ⫾ 0.5 nmol PEG/2 hr/cm2, TPA) contrasted with the significant increase measured during the first 1–3 hr of exposure (1.6 ⫾ 0.4 control vs 3.8 ⫾ 0.6 nmols PEG/2 hr/cm2, TPA; P ⬍ 0.04). Alone, neither inhibitor had a statistically significant effect on baseline [14C]PEG accumulation. The physiological and biochemical changes described above for TPA-treated IEC-18 gastrointestinal epithelial cells were also accompanied by some remarkable changes in the morphology of the epithe-

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Fig 5. Incubation with TPA causes a transient alteration in the morphology of confluent IEC-18 epithelial monolayers. The phase micrographs (40X) are of confluent IEC-18 epithelial monolayers at various times before and after treatment with TPA (10⫺7 M). (A) is representative of cell sheets at t ⫽ 0 hr before TPA treatment. (B) is representative of the cell sheet after 30 – 40 min of incubation with TPA. (C) is typical of a cell sheet that has been in the continuous presence of TPA for 4.5 hr.

TPA-treated IEC-18 epithelium at any time during the course of exposure (Table 3). The increased transepithelial flux of the cell impermeant radiotracer molecules strongly suggested that the measured increase in permeability was paracellular. As the permeability of the paracellular pathway is mediated by the presence of the tight junction, we sought to determine whether TPA treatment was having its effect by altering the characteristics of two proteins localized at the tight junction. We focused on occludin and ZO-1 (15–17) as much work has been done detailing their roles in tight junction permeability regulation and commercially available antibodies exist to both. The total expression of ZO-1 and occludin in IEC-18 epithelial cell sheets was not affected at any time during treatment with TPA (Figures 6A, B, respectively). However, it appears that occludin, but not ZO-1, became phosphorylated within 15 min of TPA treatment. The phosphorylation maximized at

lium when viewed by phase contrast microscopy. At t ⫽ 0 hr and throughout the duration of the study, control-treated IEC-18 cell sheets presented a relatively uniform appearance with the cytoplasm appearing moderately dense (Figure 5A). Within 15–30 min of TPA treatment, two morphologically distinct populations of cells appeared. The first was the moderately dense and flat cell characteristic of the control condition. The second cell type presented a smaller and denser cytoplasm that appeared to rise above the plane of focus (Figure 5B). The appearance of these taller, denser cells became more apparent at 1 hr after treatment. There was then a reversal of the phenotype at 2 hr with a nearly control-type epithelium present 4 –5 hr after TPA (Figure 5C), which associates with the recovery of RT (see Figure 1). These morphological changes were not associated with the induction of apoptosis as there was no significant increase in the frequency of apoptotic cells within the TABLE 3. FREQUENCY

OF

APOPTOSIS

IN

TPA

TREATED

IEC-18 CELL SHEETS*

Time (hr)

Total nuclei Mitotics Apoptotic fragments Apoptotic Fragments as a percent of total nuclei (%)

0

0.25

0.50

1

2

4

185 ⫾ 19 0.7 ⫾ 0.6 2.3 ⫾ 0.9 1.2 ⫾ 0.4

168 ⫾ 11 1.3 ⫾ 0.3 3⫾1 1.8 ⫾ 0.7

165 ⫾ 27 0.7 ⫾ 0.3 2 ⫾ 0.6 1.3 ⫾ 0.4

170 ⫾ 19 1 ⫾ 0.6 1.5 ⫾ 0.5 0.8 ⫾ 0.2

169 ⫾ 32 0.3 ⫾ 0.3 3.3 ⫾ 1.9 1.7 ⫾ 0.7

166 ⫾ 24 3⫾1 1.5 ⫾ 0.5 0.9 ⫾ 0.2

*Data represent the mean ⫾ SE of intact, mitotic and apoptotic nuclei of DAPI-stained IEC-18 epithelial cell sheets before and during treatment with TPA (10⫺7 M) from three individual experiments. The data at 4 hr was collected from two separate experiments.

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Fig 6. Incubation with TPA transiently increases the phosphorylation of occludin without affecting its total cellular expression or expression of ZO-1. The data are representative immunoblots of total cell extracts of TPA-treated IEC-18 cell sheets probed for the tight junctional proteins ZO-1 (A) and occludin (B). Whereas ZO-1 (A) remains a compact band throughout the 4-hr period of TPA treatment, the immunoreactive occludin band (B) shows evidence of a smearing pattern as early as 15 min (lane 2) of TPA, suggestive of phosphorylation. The maximum amount of phosphorylation is detected between 30 (lane 3) and 60 min (lane 4). Thereafter the smearing pattern diminishes at 2 hr (lane 5) and by 4 hr (lane 6) appears similar to that at 0 hr, (lane 1). The experiment was repeated three times with similar results. Lanes: 1, control, t ⫽ 0 hr; 2, TPA, 15 min; 3, TPA, 30 min; 4, TPA, 60 min; 5, TPA, 2 hr; and 6, TPA, 4 hr.

1 hr and began to return to baseline as early as 2 hr after the start of TPA treatment (Figure 6B). This increase in the phosphorylation state of occludin by western immunoblot analysis resulted in a slightly higher molecular weight protein species, which was retarded in the SDS-PAGE gel resulting in a smearing pattern above the main band for occludin at 65 kDa. We confirmed that these upper bands were indeed phosphorylation products by treating extracts with lambda phosphatase, which convincingly removed the upper phosphorylated bands from the occludin blot (Figure 7). There was also no evidence in additional Western immunoblot analysis that TPA was affecting the cellular distribution of occludin or ZO-1, despite its apparent ability to cause phosphorylation of the latter molecule. Immunoreactive occludin could be identified in the cytosolic, membrane, and cytoskeletal compartments of the IEC-18 epithelial cell sheet. The majority of occludin was identified with the membrane-associated compartment, as would be expected for a transmembrane protein, and was the source for the phosphorylation previously observed in total extracts (Figure 8). ZO-1 immunoreactivity was greatest in the cytosolic and cytoskelDigestive Diseases and Sciences, Vol. 46, No. 7 (July 2001)

Fig 7. Treatment with ␭ phosphatase confirms the phosphorylation of occludin in total cell extracts of TPA-treated IEC-18. Total cell extracts of IEC-18 epithelial cells, treated with TPA for 0, 1, and 4 hr, were incubated in the presence (⫹) or absence (⫺) of recombinant ␭ phosphatase (E. coli) as described in Materials and Methods. The resulting mixture was diluted in 2X sample buffer and 25 ␮g of protein was loaded on an 8% SDS PAGE gel for western immunoblot with anti-occludin as the probe. This immunoblot is representative of duplicate experiments yielding similar results.

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Fig 8. TPA treatment does not alter the cellular distribution of occludin in IEC-18 epithelial cell sheets. Cytosolic (A), membrane (B; Triton-soluble), and cytoskeletal (C; Triton-insoluble) fractions were prepared from TPA-treated IEC-18 epithelial cell sheets. From these fractions, 30 ␮g protein was loaded onto 8% SDS PAGE gels and immunoblotted with anti-occludin as described in Materials and Methods. Immunoreactive occludin can be identified in each of the three cellular compartments with the majority of the protein expressed in the membrane-associated fraction (B) followed by the Triton-insoluble cytoskeletal compartment (C). Its distribution among these three cellular compartments is not affected by TPA treatment. Of particular interest is the appearance of phosphorylated species of occludin in the membrane fraction whose intensity increases transiently with TPA treatment. This immunoblot is representative of duplicate experiments yielding similar results. Lanes; 1, control, t ⫽ 0 hr; 2, TPA, 15 min; 3, TPA, 30 min; 4, TPA, 60 min; 5, TPA, 2 hr; and 6, TPA, 4 hr.

etal fractions of the IEC-18 epithelium. Its distribution within these compartments was not remarkably affected by TPA treatment in three separate experiments (data not shown). DISCUSSION In addition to our own published studies with the LLC-PK1, renal epithelial cell line (1–7), TPA has also been reported to increase transepithelial permeability across the renal MDCK (18), the tumorigenic gastrointestinal CACO-2 (19), T84 cell lines (20), Sertoli (21) and thyroid (22) epithelial cell sheets as well as endothelial cell sheets (23, 24). With TPA treatment of the LLC-PK1 cell line, PKC-␣ disappears from the cytosolic compartment and translocates to the membrane-associated fraction as early as 5 min

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after-treatment, with all of the detectable isoform found within the membrane by 60 min (5). Detectable levels of PKC-␣ remained in the membrane fraction of LLC-PK1 cells up to 24 hr after-treatment with none of the enzyme found associated with the cytosolic compartment. This intracellular redistribution of PKC-␣ temporally associates with a rapid and sustained increase in tight junctional and transepithelial permeability across LLC-PK1 cell sheets. Furthermore, overexpression of PKC-␣ in LLC-PK1 results in exaggerated permeability and cell adhesion responses to phorbol esters (25). These accumulated data strongly argued for a role of PKC-␣ in the regulation of at least this epithelial barrier’s permeability. However, when we went to determine the effect of TPA on an accessible epithelial tissue, namely, the rat Digestive Diseases and Sciences, Vol. 46, No. 7 (July 2001)


distal colonic mucosa mounted in an Ussing chamber, TPA had no effect on transepithelial permeability at concentrations as high as 10⫺4 M. In contrast, use of the hydrophilic tumor promoter phorbol dibutyrate (PDBU) in rat and human distal colon resulted in a transient and significant, although relatively minor, increase in transepithelial permeability (measured by a reduction in RT and an increased flux of mannitol) that rapidly and spontaneously reversed (26). It did not mirror the near total loss of barrier function that we have previously observed with TPA treatment of LLC-PK1 renal epithelia in vitro (1, 2), but it was consistent with the general lack of effect of phorbol ester treatment on the permeability of isolated epithelial tissues (27, 28). Even when phorbol esters increased the permeability across epithelial tissues, the effect was transient and relatively weak (29, 30). With this apparent discrepancy in the response to TPA between freshly isolated colonic mucosa and epithelial cell culture systems, we decided to return to the latter and examine the TPA responsiveness of a nontransformed gastrointestinal epithelial cell culture, IEC-18, without the potential influence of other cell types, e.g., fibroblasts, neural or immune cells. Similar to rat and human distal colonic mucosa (26, 29, 30), the phorbol ester-treated IEC-18 epithelium responded with a rapid, albeit small and transient, increase in transepithelial permeability. This increase was measured both electrically, as a decrease in RT, and also as an increased flux of cell impermeant radiolabed molecules consistent with an increase in paracellular permeability. Both the decrease in RT and the increased accumulation of the radiolabeled molecules were reversible despite the continued presence of TPA. This is very different from the dramatic and sustained increase in TJ permeability that phorbol esters have been shown to induce in LLC-PK1 (1, 6), MDCK, CACO-2 and T84 (18 –20). The duration of the changes in transepithelial permeability across IEC-18 as well as LLC-PK1 strongly associate with the length of time that PKC-␣ remains in the membrane fraction (7). In the case of TPAtreated IEC-18, PKC- ␣ is more rapidly downregulated from the membrane fraction than is LLCPK1 and the baseline permeability characteristics of this epithelium are more quickly restored with RT slightly ahead of flux in returning to control levels. This is not the first time that a dissociation between RT and flux have been observed (31, 32). We sought to strengthen this association by preventing the downregulation of membrane-associated PKC-␣ by cotreatment of the IEC-18 epithelium with inhibitors of Digestive Diseases and Sciences, Vol. 46, No. 7 (July 2001)

calpain-mediated proteolysis or proteasomal degradation, as both have been reported to contribute to the down-regulation of PKC-␣ following its TPAinduced redistribution to the membrane (9, 33, 34). Prolonging the presence of PKC-␣ in the membrane of TPA-treated epithelium by blocking its proteolytic degradation with the calpain inhibitor I, ALLN, was associated with a sustained increase in TPA-mediated permeability. Inhibition of the proteasomal degradation with MG-132 had a similar effect on the retention of PKC-␣ in the membrane and also resulted in a sustained increase in transepithelial permeability relative to TPA treatment alone. These results suggest that the proteolytic/proteasomal degradation of PKC-␣ is responsible for the rapid reversal of the TPA-induced permeability increase in IEC-18. The association of PKC-␣ translocation with changes in the permeability characteristics of the paracellular pathway of the IEC-18 epithelium led us to evaluate the effect of TPA on the expression, distribution, and phosphorylation of two tight junctional proteins thought to be important in both the maintenance and regulation of this pathway. We chose to study occludin and ZO-1 because of their potential ability to influence paracellular permeability (15–17) as well the availability of commercially available antibodies that would recognize these proteins in rat tissue. Using LLC-PK1, an earlier study from our laboratory showed that TPA had no effect on the total expression or cellular distribution of occludin, whereas ZO-1 staining patterns were disrupted (8). In IEC-18, TPA treatment had no profound effects on either the total distribution or cellular localization of either occludin or ZO-1, either by western immunoblot or immunofluorescence analysis, despite the observed dramatic changes in cell morphology. However, TPA treatment did result in a transient increase in phosphorylation of occludin as evidenced by the higher molecular weight banding pattern of this protein by western blot analysis. Phosphorylated occludin was detected as early as 15 min after TPA addition and reversed after reaching a maximal level at 1 hr. This is in stark contrast to LLC-PK1 cell sheets in which TPA treatment results in a dephosphorylation of occludin on threonine residues (9). On the other hand, increased occludin phosphorylation and paracellular permeability have been observed in endothelial cells treated with H2O2 (35) or VEGF (36). Alternatively, the phosphorylation and permeability changes have been inhibited by dominant negatives of Rho or inhibition of RhoAactivated kinase (37). It is possible that PKC-␣ is

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actually further upstream in the signaling pathway that leads to the observed changes in paracellular permeability. At this time the data suggest an association between PKC-␣, occludin phosphorylation, and paracellular permeability regulation. However, we have no evidence that PKC-␣ is directly responsible for the observed phosphorylation of occludin. This is despite the coincidence of PKC-␣ translocation to the membrane and its resulting down-regulation with the transient increase in transepithelial permeability and occludin phosphorylation during TPA treatment. It is possible that PKC-␣ in turn phosphorylates/activates a kinase or inactivates a phosphatase whose target is then occludin. Together with the earlier work of Clarke et al (9), these data support a role in general for the phosphorylation state of occludin as a determining factor in tight junctional/paracellular permeability. In summary, the results reported using IEC-18 describe a response to phorbol esters that is more closely characteristic of isolated rat and human colonic mucosa (26, 29, 30). Furthermore, it suggests that there is no single unifying model epithelium in which to study this response. Epithelial cells isolated from various organs, in which they evolved to serve distinct roles in barrier function, apparently will not necessarily respond to stimuli in a similar manner or magnitude. The IEC-18 epithelium demonstrated responses to phorbol esters that were similar to normal, isolated colonic epithelium consistent with its gastrointestinal origins. IEC-18 could, therefore, function as a cell culture model to study the contribution of PKC activation in dysregulation of epithelial barrier function specific to the gastrointestinal tract. ACKNOWLEDGMENTS We are grateful to Dr. Hilary M. Clarke for her helpful comments regarding this manuscript. We thank the Editorial Department of the Lankenau Institute for Medical Research for their help in the preparation of this manuscript.

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