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Departments of Otolaryngology (J.K., N.O., R.K., M.K., M.G., A.H., T.H.) and Pathology (T.K., M.M., M.O., H.C., N.S.), Sapporo Medical University School of Medicine, Sapporo, Japan
Received for publication November 21, 2007.
Accepted for publication May 2, 2008.
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Abstract |
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, PKC-
, and PKC-
. Treatment with TPA increased transepithelial electrical resistance, with tight junction barrier function more than 4-fold that of the control, together with up-regulation of tight junction proteins, occludin, zona occludens (ZO)-1, ZO-2 and claudin-1 at the transcriptional level. Furthermore, it affected the subcellular localization of the tight junction proteins and the numbers of tight junction strands. The up-regulation of barrier function and tight junction proteins was prevented by a pan-PKC inhibitor, and the inhibitors of PKC- and PKC- but not PKC-. In primary human nasal epithelial cells, transcriptional factors GATA-3 and -6 were detected by reverse transcription-polymerase chain reaction. The knockdown of GATA-3 using RNA interference resulted in inhibition of up-regulation of ZO-1 and ZO-2 by treatment with TPA. These results suggest that TPA-induced PKC signaling enhances the barrier function of human nasal epithelial cells via transcriptional up-regulation of tight junction proteins, and the mechanisms may contribute to a drug delivery system.
; van Kempen et al., 2000). The epithelial barrier is regulated in large part by the apical-most intercellular junctions, referred to as tight junctions (Takano et al., 2005; Koizumi et al., 2007; Kurose et al., 2007).
Tight junctions, the most apical component of intercellular junctional complexes, separate the apical from the basolateral cell surface domains to establish cell polarity (performing the function of a fence). Tight junctions also possess a barrier function, inhibiting the flow of solutes and water through the paracellular space (Gumbiner, 1993). They form a particular netlike meshwork of fibrils created by the integral membrane proteins occludin and claudin and members of the Ig superfamilies junctional adhesion molecule (JAM) and Coxsackie adenovirus receptor (CAR) (Tsukita et al., 2001; Sawada et al., 2003; Schneeberger and Lynch, 2004). Several peripheral membrane proteins, ZO-1, ZO-2, ZO-3, 7H6 antigen, cingulin, symplekin, Rab3B, Ras target AF-6, and ASIP, an atypical protein kinase C-interacting protein, have been reported (Tsukita et al., 2001; Sawada et al., 2003; Schneeberger and Lynch, 2004). More recently, tricellulin was identified at tricellular contacts at which there are three epithelial cells and the tricellulin has a barrier function (Ikenouchi et al., 2005). Furthermore, ZO-1 and ZO-2 can independently determine whether and where claudins are polymerized (Umeda et al., 2006).
Protein kinase C (PKC) is a family of serine-threonine kinases known to regulate epithelial barrier function (Tsukamoto and Nigam, 1999; Andreeva et al., 2001; Seth et al., 2007). PKC has been shown to induce both assembly and disassembly of tight junctions depending on the cell type and conditions of activation (Stuart and Nigam, 1995; Andreeva et al., 2001). The activation of PKC causes an increase in permeability in the renal epithelial cell lines LLC-PK1 and Madin-Darby canine kidney (Ellis et al., 1992; Clarke et al., 2000), whereas it causes a decrease in permeability in the human colon carcinoma cell line HT29 (Sjö et al., 2003). Bryostatin enhances tight junction barrier function in T84 cells through a PKC signaling pathway (Yoo et al., 2003). PKC seems to regulate the subcellular localization, phosphorylation states, and transcription of several tight junction-associated proteins (Banan et al., 2005), although the isozyme specificity has not been clearly elucidated. At least 11 different isozymes of PKC are known. These can be subdivided in three classes according to their responsiveness to activators (Newton, 1997). The classic or conventional isozymes (
, βI, βII, and 
) are both Ca2+- and diacylglycerol (DAG)-dependent. The novel (nPKC) isozymes (, , , 
, and µ) are Ca2+-independent but DAG-dependent. The atypical isozymes (
/
and 
) are neither Ca2+-nor DAG-dependent. In the human intestinal epithelial cell lines HT-29 and Caco-2, stimulation with TLR2 ligands leads to activation of specific PKC isoforms PKC- and PKC- and enhances barrier function through translocation of ZO-1 on activation (Cario et al., 2004).
Furthermore, activation of PKC by 12-O-tetradecanoylophorbol-13-acetate (TPA) causes increases in transcription of occludin, ZO-1, and claudin-1 in T84 cells and melanoma cells (Weiler et al., 2005; Leotlela et al., 2007). The claudin-2 promoter is activated by CDX2, HNF-1, and GATA-4 in a cooperative manner (Escaffit et al., 2005). Although activation of PKC exerts its effect directly at the transcriptional level, the responsible transcription factors related to PKC activation remain unknown.
We reported previously that in the epithelium of human nasal mucosa from patients with allergic rhinitis, occludin, JAM-A, ZO-1, and claudin-1, -4, -7, -8, -12, -13, and -14 were detected together with continuous tight junction strands that formed well developed networks (Takano et al., 2005). However, the detailed mechanisms of regulation of tight junctions in human nasal epithelial cells remain unclear. In this study, to investigate the mechanisms of regulation of tight junctions through a PKC signaling pathway, primary cultures of human nasal epithelial cells were treated with TPA as a PKC activator. We found that short treatment with TPA greatly enhanced epithelial barrier function together with an increase in expression of occludin, ZO-1, ZO-2, and claudin-1 at the transcriptional level. When we focused on the transcriptional factor GATA family to investigate the transcriptional mechanisms, up-regulation of ZO-1 and ZO-2 by treatment with TPA was regulated by GATA-3 via a PKC signaling pathway.
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Materials and Methods |
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, anti-PKC-, anti-PKC-, anti-PKC-, anti-PKC-, and mouse monoclonal anti-phosphoserine antibodies were obtained from BD Pharmingen (San Diego, DA). Rabbit polyclonal anti-extracellular signal-regulated kinase 1/2 antibodies were obtained from Promega (Madison, WI.). Rabbit polyclonal anti-actin was obtained from Sigma Chemical Co. (St. Louis, MO). Rabbit polyclonal anti-panPKC and mouse anti-GATA-3 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
PKC inhibitor GF109203X, MAPK inhibitors PD98059 and U0126, p38 MAPK inhibitor SB203580, PI3K inhibitor LY294002, epidermal growth factor receptor (EGFR) inhibitor PD153035, PKC- inhibitor rottlerin, PKC- inhibitor myristoylated PKC- pseudosubstrate peptide inhibitor, and PKC- inhibitor PKC- translocation inhibitor peptide were purchased from Calbiochem-Novabiochem Corporation (San Diego, CA).
Human Nasal Mucosa Tissues. Human nasal mucosa tissues were obtained from patients with hypertrophic rhinitis and chronic sinusitis who underwent inferior nasal turbinectomy. Informed consent was obtained from all patients, and the study was approved by the ethics committee of Sapporo Medical University, the Sapporo Hospital of the Hokkaido Railway Company, and the KKR Sapporo Medical Center Tonan Hospital.
Isolation and Cell Culture. Human nasal mucosa tissues were minced into pieces 2 to 3 mm3 in volume and washed with phosphate-buffered saline (PBS) containing 100 U/ml penicillin and 100 µg/ml streptomycin (Lonza Walkersville, Walkersville, MD) four times. These tissue specimens were suspended in 10 ml of dispersing solution with 0.5 µg/ml DNase I (Sigma) and 0.08 mg/ml Liberase Blenzyme 3 (Roche, Basel, Switzerland) in PBS and then incubated at 37°C for 20 min. The dissociated specimens were subsequently filtrated with 300-µm mesh followed by filtration with 40-µm mesh. After centrifugation at 1000g for 4 min, the cells were cultured in serum-free bronchial epithelial basal medium (Lonza Walkersville) supplemented with 0.5 µg/ml hydrocortisone, 5 µg/ml insulin, 10 µg/ml transferrin, 0.5 µg/ml epinephrine, 6.5 µg/ml triiodothyronine, 50 µg/ml gentamicin, 50 µg/ml amphotericin B, 0.1 ng/ml retinoic acid, and 0.5 ng/ml epidermal growth factor (Lonza Walkersville), bovine pituitary extract [1% (v/v); Pel-Freez Biologicals, Rogers, AR], 100 U/ml penicillin, and 100 µg/ml streptomycin. The isolated human nasal epithelial cells were plated with modified bronchial epithelial basal medium containing 10% fetal bovine serum (PAA Laboratories, Etobicoke, ON, Canada) on 35- or 60-mm culture dishes (Corning Life Sciences, Acton, MA), which were coated with rat tail collagen (500 µg of dried tendon/ml of 0.1% acetic acid) in a humidified, 5% CO2/95% air incubator at 37°C (Koizumi et al., 2007; Kurose et al., 2007). The first-passaged cells using 0.05% trypsin-EDTA (Sigma) were used at day 7 after plating for the experiments.
The cells were treated with 10 or 100 nM TPA (Sigma) for 6 h. Some cells were treated with 2 µM GF109203, 10 µM PD98059, 20 µM U0126, 2 µM PD153035, 10 µM SB203580, 10 µM LY294002, 15 µM rottlerin, 5 µM myristoylated PKC- pseudosubstrate peptide inhibitor, or 10 µM PKC- translocation inhibitor peptide for 2 h before treatment with 100 nM TPA.
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Measurement of Transepithelial Electrical Resistance. The cells were cultured to confluence on inner chambers of 12-mm Transwell inserts with 0.4-µm pore-size filters (Corning Life Sciences). TER was measured using an EVOM voltameter with an ENDOHM-12 (World Precision Instruments, Sarasota, FL) on a heating plate (Fine, Tokyo, Japan) adjusted to 37°C. The values are expressed in standard units of ohms per square centimeter and presented as the mean ± S.D. For calculation, the resistance of blank filters was subtracted from that of filters covered with cells.
Measurement of Permeability. To determine the paracellular flux, the cells were cultured on 12-mm Transwell, 0.4-µm pore-size filters, and then FITC-labeled dextran (MW: 70 kDa)-containing medium was added to the inner chamber. Samples were collected from the outer chamber at 15, 30, 60, and 120 min and were measured with a Wallac 1420 multilabeled counter (PerkinElmer Life and Analytical Sciences, Waltham, MA).
Western Blot Analysis. The cells grown on the inner chambers were scraped in 300 µl of buffer (1 mM NaHCO3 and 2 mM phenylmethylsulfonyl fluoride), collected in microcentrifuge tubes, and then sonicated for 10 s. The protein concentrations of the samples were determined using a BCA Protein Assay Reagent Kit (Pierce Chemical Co, Rockford, IL). Aliquots of 15 µg of protein/lane for each sample were separated by electrophoresis in 4/20% SDS polyacrylamide gels (Daiichi Pure Chemicals Co., Tokyo, Japan). After electrophoretic transfer to a nitrocellulose membrane (Immobilon; Millipore, Billerica, MA), the membrane was saturated for 30 min at room temperature with blocking buffer (25 mM Tris, pH 8.0, 125 mM NaCl, 0.1% Tween 20, and 4% skim milk) and incubated with anti-phospho-pan-PKC, anti-phospho-MARCKs, anti-phospho-MAPK, anti-extracellular signal-regulated kinase 1/2, anti-PKC-, anti-PKC-, anti-PKC-, anti-PKC-, anti-PKC-, anti-occludin, anti-ZO-1, anti-ZO-2, anti-claudin-1, anti-claudin-4, anti-claudin-7, anti-GATA3, and anti-actin antibodies at room temperature for 1 h. The membrane was incubated with horseradish peroxidase-conjugated anti-rabbit or mouse IgG (Dako Denmark A/S, Copenhagen, Denmark) at room temperature for 1 h. The immunoreactive bands were detected using an ECL Western blotting system (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK).
Immunoprecipitation. The dishes were washed with PBS twice, and 300 µl of Nonidet P-40 lysis buffer (50 mM Tris-HCl, 2% Nonidet P-40, 0.25 mM sodium deoxycholate, 150 mM NaCl, 2 mM EGTA, 0.1 mM Na3VO4, 10 mM NaF, and 2 mM phenylmethylsulfonyl fluoride) was added to 60-mm dishes. The cells were scraped and collected in microcentrifuge tubes and then sonicated for 10 s. Cell lysates were incubated with protein A-Sepharose CL-4B (Pharmacia LKB Biotechnology, Inc., Uppsala, Sweden) for 1 h at 4°C and then clarified by centrifugation at 15,000g for 10 min. The supernatants were incubated with the polyclonal anti-occludin antibody bound to protein A-Sepharose CL-4B overnight at 4°C. After incubation, immunoprecipitates were washed extensively with the same lysis buffer and were subjected to Western blot analysis using anti-phosphoserine and anti-phosphothreonine antibodies.
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Immunocytochemistry. The cells grown on inner chambers were fixed with ice-cold absolute acetone or a 1:1 ethanol/acetone mixture at -20°C for 10 min. The cells were stained with anti-occludin, anti-ZO-1, anti-ZO-2 and anti-claudin-1 antibodies overnight at 4°C. Alexa Fluor 488 (green)-conjugated anti-rabbit IgG and Alexa Fluor 592 (red)-conjugated anti-mouse IgG (Invitrogen) were used as secondary antibodies. The specimens were examined and photographed with an Axioskop 2 plus microscope (Carl Zeiss, Jena, Germany) and confocal laser scanning microscope (MRC 1024; Bio-Rad Laboratories, Hercules, CA). Phase-contrast photomicrographs were taken with a Zeiss Axiovert 200 inverted microscope.
Freeze-Fracture Analysis. For freeze-fracture experiments, primary cultured human nasal epithelial cells grown on 60-mm dishes were centrifuged into pellets and then immersed in 40% glycerin solution after fixation in 2.5% glutaraldehyde in 0.1 M PBS, pH 7.3. The specimens were mounted on a copper stage, frozen in liquid nitrogen, fractured at -150°C- -160°C, and replicated by platinum/carbon from an electron beam gun positioned at a 45° angle, followed by carbon applied from overhead in a JFD-7000 freeze-fracture device (JEOL, Tokyo, Japan). After the replicas were thawed, they were floated on filtered 10% sodium hypochlorite solution for 10 min in a Teflon dish. Replicas were washed in distilled water for 30 min, mounted on copper grids, and examined at 80 kV using a JEOL 1200EX transmission electron microscope. Morphometric analysis was performed on freeze-fracture replica images of tight junctions, which were printed at a final magnification of 20,000x. The mean number of tight junction strands was determined by taking numerous counts along a line drawn perpendicular to the junctional axis at 200-nm intervals (Stevenson et al., 1988).
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; Andreeva et al., 2001; Seth et al., 2007). To investigate effects of the PKC activator TPA on tight junction barrier function of human nasal epithelial cells, primary cultures of human nasal epithelial cells at day 7 after plating were treated with 10 or 100 nM TPA and then examined for TER and paracellular flux of FITC-labeled dextran (molecular mass, 70 kDa).
In primary human nasal epithelial cells cultured with 10% fetal bovine serum at day 7 after plating, the maximum value of TER was 200 ± 20 
/cm2. The TER was markedly increased beginning from 1 h after treatment with 10 or 100 nM TPA in a dose-dependent manner (Fig. 1A). The TER values from 2 to 6 h after treatment with 100 nM TPA were more than 4-fold those of the control (Fig. 1A). The TER value at 24 h after treatment with 10 or 100 nM TPA recovered to the level of the control (data not shown).
When the paracellular flux of FITC-labeled dextran (70 kDa) was measured in the cells at 2 h after treatment with 100 nM TPA, in which the TER was more than 4-fold compared with the control, the paracellular flux after treatment with TPA was significantly decreased compared with the control (Fig. 1B). The paracellular flux at 2 h after treatment with 10 nM TPA was also decreased compared with the control and similar to that of 100 nM TPA (Supplemental Fig. 1)
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; Kurose et al., 2007). In primary human nasal epithelial cells at 1 and 2 h after treatment with 100 nM TPA, in which a marked increase of TER was observed (Fig. 1A), we investigated the changes in expression of tight junction proteins occludin, ZO-1, ZO-2, claudin-1, -4, and -7 using Western blot and RT-PCR analyses. In Western blotting, up-regulation in proteins of occludin, ZO-1, ZO-2, and claudin-1, but not claudin-4 and -7, was observed at 1 and 2 h after treatment with TPA (Fig. 2, A to C). In RT-PCR, up-regulation of mRNAs of occludin, ZO-1, ZO-2, claudin-1 and -4, but not claudin-7, was observed at 1 and 2 h after treatment with 100 nM TPA (Fig. 2, B and D). No changes in expression of claudin-2, -8, -9, and -12 and JAM-A, which were detected in primary cultures of human nasal epithelial cells, were observed (data not shown). Changes in Distribution of Tight Junction Proteins by Treatment with TPA. To investigate changes in localization of tight junction proteins in the cells at 2 h after treatment with 100 nM TPA, we performed immunocytochemistry for occludin, ZO-1, ZO-2, and claudin-1. In the control at day 5 after plating, discontinuous lines of occludin, ZO-1, and ZO-2 and weak expression of claudin-1 were observed in some cells (Fig. 3A, C, E, and G). In the cells after treatment with TPA, expression of occludin, ZO-1, ZO-2, and claudin-1 was observed as continuous wide lines at cell borders (Fig. 3, B, D, F, and H).
Changes of Tight Junction Strands Induced by Treatment with TPA. To investigate changes of tight junction strands in the cells at 2 h after treatment with 100 nM TPA, we performed freeze-fracture analysis. In the control, a network composed of several continuous tight junction strands was observed (Fig. 3I). In the cells after treatment with TPA, the mean number of tight junction strands was increased compared with the control and a well developed network of tight junction strands was observed (Fig. 3, J and K).
Changes of PKC- and MAPK-Associated Proteins Induced by Treatment with TPA. There are at least 11 different isozymes of PKC classified into three broad groups (Newton, 1997). To investigate which specific PKC isozymes affect expression and function of tight junction proteins, we performed Western blotting to examine the expression of the five PKC isoforms PKC-, PKC-, PKC-, PKC-, and PKC- and the phosphorylation status of PKC, MARCKs, and MAPK in the cells at 1 and 2 h after treatment with 100 nM TPA. In the cells at 1 and 2 h after treatment with TPA, increases in expression of PKC-, PKC-, and PKC- and activation of phosphorylation of panPKC, MARCKS, and MAPK were observed (Fig. 4, B and D).
PKC Inhibitors but Not MAPK and EGFR Inhibitors Prevent Increase of Barrier Function Induced by Treatment with TPA. TPA is known to induce phosphorylation of MAPK, not only through phosphorylation of PKC but also through EGFR (Barbosa et al., 2003). TPA is involved in multiple signal transduction pathways, including c-Jun NH2-terminal kinase and PI3K (Huang et al., 1997; Yu et al., 2006). To investigate which signaling pathways affected the increase of barrier function measured as TER in the cells after treatment with TPA, we used PKC inhibitor GF109203X, MAPK inhibitors PD98059 and U0126, and EGFR inhibitor PD153035. Treatment with GF109203X but not with PD98059, U0126, or PD153035 completely prevented the marked increase of TER values at 2 h after treatment with 100 nM TPA (Fig. 5A, Supplemental Fig. 1). Although the p38 MAPK inhibitor SB203580 and the PI3K inhibitor LY294002 prevented up-regulation of TER after treatment with TPA, the effect of the inhibition was weak (Fig. 5B).
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PKC Inhibitor Prevents an Increase in Expression of Tight Junction Proteins by Treatment with TPA. We investigated changes in expression of tight junction proteins and PKC isozymes in the cells treated with the PKC inhibitor GF109203X before pretreatment with TPA. Treatment with GF109203X inhibited up-regulation of occludin, ZO-1, ZO-2, and claudin-1 at the levels of protein and mRNA at 2 h after treatment with 100 nM TPA. (Fig. 6, A-D). Furthermore, the increase of claudin-4 mRNA after treatment with TPA was also inhibited by treatment with GF109203X (Fig. 6, B and D). Treatment with GF109203X inhibited increases in expression of PKC-, PKC-, and PKC- at 2 h after treatment with 100 nM TPA (Fig. 7, A and C).
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inhibitor rottlerin, the PKC- inhibitor myristoylated PKC- pseudosubstrate peptide inhibitor, and the PKC- inhibitor PKC- translocation inhibitor peptide before pretreatment with TPA. Treatment with inhibitors of PKC- and PKC- but not PKC- prevented up-regulation of occludin, ZO-1, ZO-2 and claudin-1 at 2 h after treatment with 100 nM TPA. (Fig. 7, B and D).
Transcriptional Factor GATA-3 Up-Regulates Tight Junction Proteins after Treatment with TPA. Tight junction proteins are regulated by various transcription factors in a cooperative manner (Escaffit et al., 2005). Transcriptional factor GATA-3 is associated with differentiation of the luminal cells in the mammary gland (Kouros-Mehr et al., 2006; Asselin-Labat et al., 2007). When we investigated expression of GATA-1, -2, -3, -4, -5, and -6 in primary cultures of human nasal epithelial cells by RT-PCR, mRNAs of GATA-3 and -6 were detected (Fig. 8A). Up-regulation of mRNAs of GATA-3 and -6 was observed at 1 h but not 2 h after treatment with 100 nM TPA (Fig. 8A). Treatment with the PKC inhibitor GF109203X prevented up-regulation of mRNAs of GATA-3 but not GATA-6 at 1 h after treatment with 100 nM TPA (Fig. 8B).
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Discussion |
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; Seth et al., 2007). PKC has been shown to induce both assembly and disassembly of tight junctions depending on the cell type and conditions of activation (Stuart and Nigam, 1995; Andreeva et al., 2001). In this study, TPA-induced nPKC signaling greatly enhanced the barrier function of human nasal epithelial cells in primary culture together with an increase in expression of tight junction-associated proteins, occludin, ZO-1, ZO-2, and claudin-1 at the transcriptional level. The transcriptional up-regulation of ZO-1 and ZO-2 was controlled via transcriptional factor GATA-3.
PKC seems to regulate the subcellular localization, phosphorylation states, and transcription of several tight junction-associated proteins, although the PKC isozyme specificity has not been clearly elucidated (Banan et al., 2005). Conventional PKC- participates in tight junction disassembly, whereas nPKC- plays a role in tight junction formation of kidney epithelial cells (Andreeva et al., 2006). In the human intestinal epithelial cell lines HT-29 and Caco-2, stimulation with TLR2 ligands leads to activation of specific PKC isoforms PKC- and PKC- and enhances barrier function through translocation of ZO-1 on activation (Cario et al., 2004). In the primary human nasal primary cells of the present study, treatment with TPA induced expression of specific nPKC-,-, and - and greatly enhanced tight junction barrier function together with increases in expression of occludin, ZO-1, ZO-2, and claudin-1, whereas the pan-PKC inhibitor GF109230X prevented all the changes. Furthermore, treatment with inhibitors of nPKC- and - but not - prevented the up-regulation of tight junction proteins. Treatment with TPA affected the subcellular localization of the tight junction proteins together with an increase of tight junction strands. These indicate that in human nasal epithelial cells in primary culture, TPA induced nPKC- and - may enhance tight junction barrier function by assembly of tight junctions.
It is reported that activation of PKC by TPA causes increases in transcription of occludin, ZO-1, and claudin-1 in T84 cells and melanoma cells (Weiler et al., 2005; Leotlela et al., 2007). In the primary human nasal epithelial cells used in the present study, activation of PKC by TPA increased expression of occludin, ZO-1, ZO-2, and claudin-1 at the transcriptional level. Although activation of PKC exerts its effect on transcription directly, the responsible transcription factors related to PKC activation remain unknown. The claudin-2 promoter is activated by CDX2, HNF-1, and GATA-4 in a cooperative manner (Escaffit et al., 2005). Tight junctions are regulated via direct repression of the gene expression of claudins/occludin by transcription factor Snail during epithelial to mesenchymal transition (Ikenouchi et al., 2003). In this study, to investigate the transcriptional mechanisms, we focused on the transcriptional factor GATA family. The GATA family, included in zinc finger transcription factors, has been recognized in the differentiation of the endoderm in several evolutionarily diverse organisms (Reiter et al., 2001). They form an ancient family of transcription factors that evolved into six related factors in mammals (Clarke and Berg, 1998). GATA-1, -2, and -3 are predominantly associated with the hematopoietic cell lineage, whereas GATA-4, -5, and -6 are mainly associated with development of endodermally derived organs such as the liver, lung, pancreas, and heart (Lieuw et al., 1997). More recently, it has been reported that GATA-3 is associated with differentiation of the luminal cells in the mammary gland, not only in embryos but also in adults (Kouros-Mehr et al., 2006; Asselin-Labat et al., 2007). In the primary human nasal epithelial cells of the present study, GATA-3 and -6 were detected by RT-PCR and their expression was increased by treatment with TPA. The PKC inhibitor GF109230X prevented up-regulation of GATA-3 but not GATA-6. Furthermore, knockdown of GATA-3 by its RNAi inhibited up-regulation of expression of ZO-1 and ZO-2 via the PKC signaling pathway induced by treatment with TPA. These results suggest that in human nasal epithelial cells, GATA-3 is one of the transcription factors responsible for PKC activation induced by treatment with TPA and may in part regulate ZO-1 and ZO-2. ZO-1 and ZO-2 are membrane-associated guanylate kinase homologs that can bind to transmembrane tight junction proteins, occludin, claudins, and JAMs, the cytoskeleton, and signal transduction molecules (Tsukita et al., 2001; Sawada et al., 2003; Schneeberger and Lynch, 2004). Furthermore, ZO-1 and ZO-2 are closely associated with polymerization of claudins (Umeda et al., 2006). In this study, it is possible that up-regulation of the expression of ZO-1 and ZO-2 might have indirectly affected the expression and localization of occludin and claudin-1.
Although TPA is a typical PKC activator, it induces phosphorylation of MAPK not only through phosphorylation of PKC but also through that of EGFR (Barbosa et al., 2003). Furthermore, TPA is involved in multiple signal transduction pathways, including c-Jun NH2-terminal kinase and PI3K (Huang et al., 1997; Yu et al., 2006). In primary human nasal epithelial cells, to investigate which signaling pathways mainly affect up-regulation of barrier function by treatment with TPA, we used PKC inhibitor GF109203X, MAPK inhibitor PD98059, EGFR inhibitor PD153035, p38MAPK inhibitor SB203580, and PI3K inhibitor LY294002. Treatment with the PKC inhibitor, but not inhibitors of MAPK and EGFR, completely prevented up-regulation of barrier function measured as TER values. Furthermore, inhibitors of p38 MAPK and PI3K also prevented up-regulation of barrier function by treatment with TPA, although the effect was weak. These results suggested that the increase of barrier function induced by treatment with TPA in human nasal epithelial cells was directly regulated by a PKC pathway, not via MAPK pathway, and was indirectly controlled by distinct signaling pathways, including p38 MAPK and PI3K.
TPA induces the rapid phosphorylation of occludin in Madin-Darby canine kidney cells cultured in low extracellular calcium medium with concomitant translocation of occludin to the regions of cell-cell contact (Andreeva et al., 2001). Phosphorylation of claudin-4 via PKC- by treatment with TPA regulates tight junction barrier function in ovarian cancer cells (D'Souza et al., 2007). PKC- alters barrier function through changes in phosphorylation and cellular localization of claudin-1 and -4 in Caco-2 (Banan et al., 2005). In the human nasal epithelial cells used in the present study, treatment with TPA slightly increased activities of serine and threonine phosphorylation of occludin protein (Supplemental Fig. 2). The serine and threonine phosphorylation of occludin leads to up-regulation of barrier function (Andreeva et al., 2001; Seth et al., 2007). In this study, an increase in not only expression of tight junction proteins but also serine and threonine phosphorylation of occludin might have contributed to the enhanced tight junction barrier function of human nasal epithelial cells.
PKC regulates a number of fundamental processes, such as membrane trafficking, cytoskeletal organization, ion transport, cell growth, and differentiation (Venkatachalam et al., 2004). In the human nasal epithelial cells of the present study, treatment with TPA caused phosphorylation of the endogenous PKC substrate MARCKS, which serves as an actin cross-linking protein (Hartwig et al., 1992), potentially participating in modulation of epithelial integrity. It is possible that PKC-mediated human nasal epithelial barrier enhancement results not only from up-regulation of tight junction proteins but also from potential convergence of various signaling interactions and pathways downstream of PKC, which may interact by bridging to the actin cytoskeleton.
In conclusion, the tight junction barrier function of human nasal epithelial cells is up-regulated in part by transcriptional factor GATA-3 via an nPKC signaling pathway, and the PKC-enhanced epithelial tight junction barrier may play a crucial role in innate immunity against a wide variety of exogenous antigens. The up-regulation of human nasal epithelial cells via the nPKC signaling pathway may also be caused by the stimuli such as the cigarette smoke and various dusts (Wyatt et al., 1999; Romberger et al., 2002). Furthermore, the regulation of PKC signaling may contribute to a drug delivery system via human nasal epithelium.
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ABBREVIATIONS: ZO, zona occludens; PKC, protein kinase C; DAG, diacylglycerol; nPKC, novel PKC; TPA, 12-O-tetradecanoylophorbol-13-acetate; MARCKS, myristoylated alanine-rich protein kinase C substrate; MAPK, mitogen-activated protein kinase; GF109203X, 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride; PD98059, 2'-amino-3'-methoxyflavone; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; PI3K, phosphatidylinositol 3-kinase; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; EGFR, epidermal growth factor receptor; PD153035, 4-((3-bromophenyl)amino)-6,7-dimethoxyquinazoline; PBS, phosphate-buffered saline; siRNA, small interfering RNA; RT-PCR, reverse transcription-polymerase chain reaction; TER, transepithelial electrical resistance; FITC, fluorescein isothiocyanate.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Takashi Kojima, Department of Pathology, Sapporo Medical University School of Medicine, S1. W17. Sapporo 060-8556, Japan. E-mail: ktakashi{at}sapmed.ac.jp
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