A novel splice variant of occludin deleted in exon and its role in cell apoptosis and invasion Jin-Mo Gu1, Seung Oe Lim1, Young Min Park2 and Guhung Jung1 Department of Biological Sciences and Seoul National University, Korea Hepatology Center and Laboratory of Hepatocarcinogenesis, Bundang Jesaeng General Hospital, Kyungkido, Korea Keywords apoptosis; calcium; invasion; occludin; splice variant Correspondence G Jung, Department of Biological Sciences and Seoul National University, 56-1 Shillimdong, Kwanak-gu, Seoul 151-747, Korea Fax: +82 872 1993 Tel: +82 880 7773 E-mail: drjung@snu.ac.kr (Received 18 February 2008, revised 11 April 2008, accepted 15 April 2008) doi:10.1111/j.1742-4658.2008.06467.x The tight junction protein occludin participates in cell adhesion and migration and has been shown to possess antitumorigenic properties; however, the exact mechanism underlying these effects is poorly understood In liver cell lines, we identified an occludin splice variant deleted in exon (OccDE9) Furthermore, comparison analysis of wild-type occludin (OccWT) and OccDE9 revealed that exon played important roles in the induction of mitochondria-mediated apoptosis and the inhibition of invasion, along with the downregulation of matrix metalloproteinase expression In addition, by using the calcium indicator X-rhod-1, and the inositol trisphosphate receptor inhibitor 2-aminoethoxydiphenyl borate, we found that OccWT but not OccDE9 increased calcium release from the endoplasmic reticulum In conclusion, our results showed that occludin mediates apoptosis and invasion by elevating the cytoplasmic calcium concentration and that exon of occludin is an important region that mediates these effects Occludin is a tight junction (TJ) protein and the first identified TJ-associated molecule [1] Occludin is the product of a single gene located on human chromosome band 5q13.1 and produces several different mRNAs as a result of alternative splicing [2] Recently, several variants of occludin, such as occludin 1B and a fourth transmembrane domain (TM4)-deleted variant, have been discovered [3,4] Occludin 1B contains a 193 bp insertion corresponding to an alternatively spliced exon in the gene encoding a unique N-terminus Conversely, the TM4deleted variant has a missing fourth TM corresponding to exon In addition to these variants, there is evidence to show that two distinct promoters, P1 and P2, confer separate transcriptional start sites [5] Promoter P2 is located downstream of promoter P1, and both promoter regions are regulated by tumor necrosis factor-a [5] Occludin ( 65 kDa) is composed of two extracellular loops that form four membrane-spanning domains; occludin specifically forms TJ complexes with phosphoproteins such as zonula occludens protein 1, zonula occludens protein 2, and P-130 Other possible signaling molecules include Ga subunits, tyrosine kinase, small GTPases (Rab ⁄ Rho), and junctional adhesion molecules [6] Interactions of these proteins with the actin cytoskeleton are major determinants of the TJ complex and play significant roles not only in cell–cell contact but also in signal transmission [7] Accumulating evidence points to TJ disruption in malignant phenotypes, including local tumor growth, invasion, and metastasis [8] In humans, TJ disruption Abbreviations 2-APB, 2-aminoethoxydiphenyl borate; 5¢-aza-dC, 5¢-aza-2¢-deoxycytidine; BrdUTP, 5-bromo-2¢-deoxyuridine-5¢-triphosphate; ER, endoplasmic reticulum; ERK1 ⁄ 2, extracellular signal-regulated kinase ⁄ 2; HA, hemagglutinin; IP3, inositol trisphosphate; IP3R, inositol trisphosphate receptor; JNK, Jun N-terminal kinase; MMP, matrix metalloproteinase; MSP, methylation-specific PCR; shRNA, short hairpin RNA; TJ, tight junction; TM, transmembrane domain; TSA, trichostatin A; TUNEL, terminal deoxyribonucleotide transferase-mediated nick-end labeling FEBS Journal 275 (2008) 3145–3156 ª 2008 The Authors Journal compilation ª 2008 FEBS 3145 Exon of occludin in apoptosis and invasion J.-M Gu et al is a distinguishing characteristic of epithelial and many other types of cancers, where decreases in TJ expression are associated with cancer stage and metastatic potential [9] Furthermore, the overexpression of TJ proteins such as claudin-1 and claudin-4 induces apoptosis and suppresses metastatic potencies [10,11] Occludin has also been reported to induce apoptosis and apoptotic sensitization, which are regarded as antitumorigenic activities [12] Moreover, various studies have investigated how specific occludin domain deletions affect the localization and function of occludin [13,14] Overexpression of deletion mutants resulted in a lack of capability for membrane localization and induction of apoptosis [12,15] However, the mechanism underlying the specific role of occludin in cancer cell apoptosis remains poorly understood Furthermore, most studies on multiple signal pathways involving the TJ complex have focused on the effects of TJ biogenesis, including occludin [16,17] For example, membrane-localized kinases, calcium, Ga proteins, calmodulin and phospholipase were analyzed in several types of cancer cells from the perspective of TJ formation [18,19] Gaq protein, as a subunit of Ga, induces the formation of inositol trisphosphate (IP3), which binds to the IP3 receptor (IP3R) located on the endoplasmic reticulum (ER), thereby releasing calcium into the cytoplasm [20,21] When calcium concentrations in the cytoplasm increase, calcium accumulation occurs along the inner membrane of mitochondria This destabilizes membrane potentials and facilitates the initiation of apoptosis [22] In addition, elevated calcium levels contribute to cell invasion [23,24] In contrast, in the case of occludin, an ER-mediated increase in intracellular calcium levels is not considered to be the mechanism underlying its effects in cancer cell apoptosis and invasion [21] In this article, we provide evidence of an additional occludin variant deleted in exon (OccDE9) On the basis of a comparative analysis of the involvement of wild-type occludin (OccWT) and variant occludin in apoptosis and invasion, as determined by assay, we revealed that exon played a major role in the induction of mitochondria-mediated apoptosis and the reduction of cell invasiveness, along with the downregulation of matrix metalloproteinase (MMP) expression In addition, on the basis of an analysis using X-rhod-1, a calcium indicator, and 2-aminoethoxydiphenyl borate (2-APB), which inhibits IP3R and thus calcium release from the ER, we suggest that an increase in the intracellular calcium level is a possible explanation for occludin-induced apoptosis and reduced invasiveness 3146 Results Identification of the human occludin splice variant We detected a splice variant of occludin in liver cell lines by using an antibody to the occludin N-terminus, which detected two specific bands, one of which had the expected molecular mass for OccWT ( 65 kDa), and the other of which had a smaller molecular mass (Fig 1A) OccWT was not expressed only in Chang cells On the basis of these observations, we selected Chang cells for the identification of the splice variant The PCR product of Chang cell-derived cDNA was amplified by primers bound to each exon, which showed that exon was deleted in this occludin variant (OccDE9) (Fig 1B) To verify whether this deletion occurred due to the loss of genomic DNA, where exon is located, we amplified genomic DNA with the primers bound to the exon region; the size of the resulting PCR product was as expected (data not shown) To determine the detailed occludin variant sequence, we performed 3¢-RACE and obtained an exon 9-deleted splice variant, which was compatible with the results of PCR (Fig 1C) In a previous report, occludin was shown to involve two different promoters [5] Depending on the promoter, either exon or exon 1a was selected for the alternative splicing process [5] Using primers designed for exon and exon 1a, we compared the promoters involved in Chang and Huh7 cells As shown in Fig 1D, PCR products containing exon were produced in Huh7 cells but not in Chang cells To determine whether the different splice variants were caused by a difference in the boundary sequence deciding the splicing points between exon and exon 9, we compared the sequences of these regions in Huh7 and Chang cells; no difference was found (Fig 1E) Figure 1F depicts the schematic of OccDE9 obtained from Chang cells as compared to OccWT, based on PCR analysis OccWT is epigenetically silenced by promoter hypermethylation in Chang cells On the basis of the different usages of exon and exon 1a in Huh7 and Chang cells, we decided to analyze the relationship between usage of exon 1a and P1 promoter methylation In liver cell lines, we initially performed methylation specific-PCR (MSP) with primers amplifying the CpG island of the occludin P1 promoter We observed methylated DNA only in Chang cells Unmethylated DNA was not detected (Fig 2A) FEBS Journal 275 (2008) 3145–3156 ª 2008 The Authors Journal compilation ª 2008 FEBS J.-M Gu et al Exon of occludin in apoptosis and invasion A C B D E F Fig Identification of a human occludin splice variant in liver cell lines (A) Occludin protein expression was examined with an antibody to the N-terminus of occludin in HepG2, Hep3B, Huh7, Chang and HLE cells (B) cDNA synthesized from Chang cells was amplified with primers matching each exon of occludin (C) 3¢-RACE was performed on the cDNA of Chang cells, and the PCR product was sequenced (D) PCRs for exon and exon 1a were performed in Huh7 and Chang cells (E) The boundary regions of exon and exon in the genomic DNA of Chang cells were sequenced and aligned with OccWT (F) Schematic of mRNA coding sequence for human occludin, showing the comparison of OccWT and OccDE9 E, exon; P, promoter; CpG, CpG island A B C trichostatin A (TSA), an inhibitor of histone deacetylase (Fig 2B) In the same treated samples, OccWT expression was detected by measuring protein and mRNA levels, using primers for exon (Fig 2C) Furthermore, we confirmed the use of exon by using RT-PCR in Chang cells treated with TSA and 5¢-azadC (Fig 2B) Different localizations of OccWT and OccDE9 Fig OccWT expression is epigenetically silenced by methylation in Chang cells (A) The methylation status of the occludin promoter was analyzed in HepG2, Hep3B, Huh7, Chang and HLE cells by MSP (B) The demethylating agents 5¢-aza-dC (5 lM) and TSA (300 nM) were used to treat Chang cells MSP analysis for the occludin promoter and PCR with primers for exon and exon 1a were performed using demethylating agent-treated cells (C) OccWT expression in Chang cells treated with lM 5¢-aza-dC and 300 nM TSA was analyzed using immunoblotting (upper) and RT-PCR (lower) U, unmethylated DNA; M, methylated DNA; T, 5¢-aza-dC and TSA treatment As expected, was induced with lm inhibitor of demethylation of the occludin promoter in Chang cells by combined treatment 5¢-aza-2¢-deoxycytidine (5¢-aza-dC), an DNA methyltransferase 1, and 300 nm To investigate whether or not the occludin variant was localized in the membrane, we cloned the OccWT and OccDE9 genes into pCMV ⁄ HA and performed immunocytochemistry The expression of each construct in Chang, Hep3B and Huh7 cells was analyzed using immunoblotting (Fig 3A) As shown in Hep3B cells transfected with a control plasmid, endogenous occludin was intermittently localized in the membrane and diffused through the cytoplasm Occludin in the membrane of Huh7 cells was localized in a more disconnected fashion than that in Hep3B cells In Chang cells, stained occludin was observed in the cytoplasmic region Additionally, in OccWT-overexpressing Hep3B and Huh7 cells, regions stained with antibody to hemagglutinin (HA), indicating the expression of exogenous occludin, were located in the cell membranes In OccDE9-overexpressing cells, however, no regions stained with antibody to HA were observed in the membrane (Fig 3B) In contrast, neither OccWT nor FEBS Journal 275 (2008) 3145–3156 ª 2008 The Authors Journal compilation ª 2008 FEBS 3147 Exon of occludin in apoptosis and invasion J.-M Gu et al (Fig 4C) Caspase activity was likewise induced by OccWT but not by OccDE9 (Fig 4D) These data implied that OccWT induced mitochondrial apoptosis [25], and that exon played a major role in occludininduced apoptosis In a previous study, the phosphorylation of mitogen-activated protein kinase was shown to be altered in occludin-overexpressing cells [12] To test whether similar modulation occurred in liver cells, we examined the expression and phosphorylation of extracellular signal-regulated kinase ⁄ (ERK1 ⁄ 2), p38, and Jun N-terminal kinase (JNK) The phosphorylation levels of ERK1 ⁄ were high in OccWT-overexpressing cells but not in OccDE9-overexpressing cells (Fig 5) No differences were observed in the phosphorylation levels of p38 and JNK (data not shown) A B Invasiveness is reduced in OccWT-overexpressing cells but not in OccDE9-overexpressing cells Fig Localization of OccWT and OccDE9 (A) OccWT and OccDE9 were cloned into pCMV ⁄ HA, a mammalian overexpression vector (pCMV ⁄ HA-OccWT and pCMV ⁄ HA-OccDE9), and their expression was confirmed by immunoblot analysis (B) Green fluorescence indicates endogenous occludin stained with antibodies to occludin in Chang, Hep3B and Huh7 cells transfected with pCMV ⁄ HA Red fluorescence indicates exogenous occludin stained with antibodies to HA in Chang, Hep3B and Huh7 cells transfected with pCMV ⁄ HAOccWT or pCMV ⁄ HA-OccDE9 Cont, pCMV ⁄ HA-transfected cells; WT, OccWT-overexpressing cells; DE9, OccDE9-overexpressing cells OccDE9 were localized in the membranes of Chang cells (Fig 3B) Mitochondrial apoptosis is induced by OccWT overexpression but not OccDE9 overexpression Consistent with previous observations [12], in OccWToverexpressing cells, cell proliferation decreased, and the number of apoptotic cells increased as compared to findings in control cells These changes, however, were not observed in OccDE9-overexpressing cells (Fig 4A,B) To identify the pathways linked to apoptosis induced by OccWT, we analyzed caspase activity and the expression of several apoptosis-related genes Owing to the overexpression of OccWT, the expression of the apoptotic genes BAX and Apaf-1 increased, and that of the antiapoptotic gene Bcl-2 decreased OccDE9overexpressing cells, on the other hand, did not show any alteration in the expression of these genes 3148 Using a Matrigel invasion assay and analysis of MMP expression, we next determined whether occludin expression in cancer cells was responsible for invasion The Matrigel invasion assay revealed that significantly fewer OccWT-overexpressing Chang and Huh7 cells were invasive as compared to the number of invasive control and OccDE9-overexpressing cells (Fig 6A) Specifically, in Chang and Huh7 cells, OccWT significantly decreased the expression of MMP2, MMP7, MMP9 and MMP14, and of MMP1, MMP2, MMP3, MMP7, MMP9 and MMP14, respectively (Fig 6B) To determine the function of occludin, we reduced occludin expression in Huh7 cells by using occludin short hairpin RNA (shRNA) After determining the reduced expression level of occludin with immunoblotting (Fig 7A), we analyzed the effects of downregulated occludin on apoptotic sensitization for H2O2 treatment and on invasiveness Occludin shRNA-transfected Huh7 cells were more resistant to apoptosisinducing signal (H2O2), and were more invasive than control shRNA-transfected cells (Fig 7B,C) Occludin-mediated increase in calcium concentration increases apoptosis and decreases invasiveness On the basis of the differences between OccWT and OccDE9 with regard to mitochondria-dependent apoptosis and reduction of invasion, along with downregulation of MMP expression, we postulated that increased intracellular calcium might be involved in the effects of occludin In previous reports, the modulation of calcium concentration has been shown to be related to cell apoptosis and invasion [21,22] FEBS Journal 275 (2008) 3145–3156 ª 2008 The Authors Journal compilation ª 2008 FEBS J.-M Gu et al Exon of occludin in apoptosis and invasion A B C D Fig OccWT but not OccDE9 induces mitochondria-mediated apoptosis by the modulation of expression of apoptosis-related genes and caspase activity Chang, Hep3B and Huh7 cells transfected with pCMV ⁄ HA, pCMV ⁄ HA-OccWT or pCMV ⁄ HA-OccDE9 (A) The viability of cells was determined by CCK-8 assay (B) Apoptotic cell numbers were calculated by counting BrdUTP-incorporating cells (C) The levels of BAX, Bcl-2 and Apaf-1 were determined using real-time RT-PCR (D) Caspase activity was determined using the CaspACE colorimetric assay All results in (A), (B), (C) and (D) are expressed as the fold ratio relative to control (Cont) All numerical data represent mean and standard deviation of three independent experiments Cont, pCMV ⁄ HA-transfected cells; WT, OccWT-overexpressing cells; DE9, OccDE9-overexpressing cells; *P < 0.05 Fig Phosphorylation of ERK1 ⁄ in OccWT-overexpressing cells Immunoblot analyses using specific antibodies against occludin, ERK1 ⁄ 2, pERK1 ⁄ and b-actin were performed in Chang, Hep3B and Huh7 cells transfected with pCMV ⁄ HA, pCMV ⁄ HA-OccWT or pCMV ⁄ HA-OccDE9 Cont, pCMV ⁄ HA-transfected cells; WT, OccWToverexpressing cells; DE9, OccDE9-overexpressing cells To confirm the effects of occludin on the cytoplasmic calcium concentration, we analyzed calcium concentrations by using X-rhod-1, a calcium indicator X-rhod-1 has been developed from rhodamine, and emits red fluorescence [26] Therefore, we cotransfected pCMV ⁄ HA, pCMV ⁄ HA-OccWT or pCMV ⁄ HA-OccDE9 with pEGFP-N1 vector As shown in Fig 8A, OccWTtransfected cells showed increased cytoplasmic calcium concentrations, whereas OccDE9-transfected and control-transfected cell did not show any changes in calcium concentration (Fig 8A,B) We tested whether the OccWT-mediated increase in calcium levels was caused by calcium release from the ER – one of the calcium metabolism pathways related to the TJ complex [20] We examined the effects of blocking calcium release from the ER by using 2-APB, an inhibitor of the IP3R, which is located in the ER membrane Chang cells were treated with 20 lm 2-APB 12 h after transfection with pCMV ⁄ HA, pCMV ⁄ HAOccWT or pCMV ⁄ HA-OccDE9 The concentration of 2-APB was determined by a proliferation assay, and 20 lm 2-APB did not induce apoptosis in Chang cells (data not shown) In 2-APB-treated Chang cells, no difference in apoptosis was seen among the control and FEBS Journal 275 (2008) 3145–3156 ª 2008 The Authors Journal compilation ª 2008 FEBS 3149 Exon of occludin in apoptosis and invasion J.-M Gu et al A B Fig Decreased invasiveness of OccWT-overexpressing cells but not of OccDE9-overexpressing cells (A) A Matrigel assay was performed in Chang and Huh7 cells transfected with pCMV ⁄ HA, pCMV ⁄ HA-OccWT or pCMV ⁄ HA-OccDE9 Cells penetrating the Matrigel-coated inserts were stained with crystal violet The dots are stained cells on the bottom of the Matrigel-coated inserts The number of invasive cells was determined by counting the number of stained cells and then normalizing it to the number of control (Cont) cells (B) Real-time RT-PCR analysis was performed to determine the levels of MMP1, MMP2, MMP3, MMP7, MMP9 and MMP14 in Chang and Huh7 cells transfected with pCMV ⁄ HA, pCMV ⁄ HA-OccWT or pCMV ⁄ HA-OccDE9 All numerical data represent the mean and standard deviation of three independent experiments Cont, pCMV ⁄ HA-transfected cells; WT, OccWT-overexpressing cells; DE9, OccDE9-overexpressing cells; *P < 0.05 the OccWT-overexpressing and OccDE9-overexpressing cells (Fig 7C) In addition, 2-APB abrogated the effects of OccWT overexpression, namely, decreased invasiveness, ERK1 ⁄ activation, and modulation of apoptoticrelated gene expression levels (Fig 8D–F) Discussion TJs are involved in cell adhesion and migration; their downregulation can cause epithelial transformation, whereas their upregulation induces apoptosis and inhibits invasion [8] Occludin, one of the TJ proteins, has also been shown to have several types of splice variant and antitumorigenic activity [3,4,27]; however, its effects on and implications for tumorigenesis are poorly understood Here, we present a novel occludin splice variant deleted in exon 9; in Chang cells, this variant acts via the P2 promoter As the P2 promoter was involved in occludin action, and as no wild-type form was observed in Chang cells, the methylation status of the occludin promoter P1 could be examined Therefore, we tested the occludin promoter 3150 P1-containing CpG island region in liver cell lines, and observed that the occludin promoter was strongly methylated only in Chang cells Osanai et al reported that an occludin mutant deleted in region 478–522, which contains exon 9, could not localize in the membrane or induce apoptosis in mammary cell lines [12] In addition, Wang et al described another occludin mutant deleted in the whole cytoplasmic tail This mutant did not exert a potent inhibitory effect on Raf1-mediated tumorigenesis [15] In liver cells, as in mammary cell lines, we observed that OccWT was localized in the membrane, whereas OccDE9 was localized in the cytoplasm In previous reports, several factors, such as the proteins involved in TJ complexes, affected occludin localization [16,17] On the basis of this, the inability of OccWT to associate with the membrane in Chang cells could be possibly explained by cell-type-dependent events In liver cell lines, OccWT but not OccDE9 induced apoptosis and reduced invasiveness (Figs and 6) The modulation of the expression levels of BAX, Bcl-2 and FEBS Journal 275 (2008) 3145–3156 ª 2008 The Authors Journal compilation ª 2008 FEBS J.-M Gu et al Exon of occludin in apoptosis and invasion A B Fig Effects of occludin shRNA on apoptosis and invasiveness Huh7 cells were transfected with five constructs producing predesigned occludin shRNA (A) Immunoblot analysis confirmed the silencing effects of occludin shRNA The degree of occludin downregulation is represented as a bar graph in the right panel (B) Apoptosis of Huh7 cells transfected with occludin shRNA or control shRNA was analyzed using a TUNEL assay in the presence of 400 and 800 lM H2O2 The number of apoptotic cells is expressed as the ratio of these cells to the number of apoptotic control shRNA-transfected cells (Cont shRNA) (C) The invasiveness of Huh7 cells transfected with occludin shRNA or control shRNA was analyzed using a Matrigel assay The number of invasive cells was determined by counting stained cells and then normalizing to the number in the case of control shRNA-transfected cells All numerical data represent the means and standard deviations of three independent experiments –, control shRNA-transfected cells; +, occludin shRNA-transfected cells; *P < 0.05 Apaf-1 and the induction of caspase activity in OccWT-overexpressing cells indicated that occludin affected mitochondria-mediated apoptosis [25] The mitogen-activated protein kinase pathway, which is known to exhibit some correlation with occludin expression, is inactivated by increased expression of this protein in HeLa cells [12] Our data showed that OccWT activated only ERK1 ⁄ and not p38 and JNK On the basis of our data, exon of occludin may play a role in altering cell behavior via the internal cellular signaling pathway Furthermore, we newly confirmed that OccWT located in the cytoplasmic region induced the same effects (Fig 3) In view of mitochondria-mediated apoptosis, decreased invasiveness, and upregulated phosphorylation of ERK1 ⁄ 2, we speculated that the effects of occludin were controlled in one respect by the intracellular concentration of calcium [21,22] Of various signal pathways that involve calcium, those that are mediated by Gaq protein were examined first [20] We observed that the intracellular calcium level increased in OccWT-overexpressing cells, and that 2-APB inhibited the effects of OccWT Therefore, the increase in intracellular calcium may explain the induction of apoptosis and reduction of invasiveness mediated by occludin In previous reports, intracellular calcium concentrations were reported to influence the onset of apoptosis and invasion [22]; however, the relationship between increased calcium levels and the mechanisms underlying the above-mentioned effects of occludin has not been investigated In conclusion, we have discovered a new occludin splice variant deleted in exon in liver cell lines Moreover, the occludin promoter P1 was found to be methylated, and the effects of demethylating agents on the expression of wild-type occludin were examined in Chang cells Furthermore, in a comparative analysis between OccWT and OccDE9, the exon region played a significant role in promoting apoptosis and inhibiting invasion by regulating signaling pathways Calcium release from the ER has been described as one of the mechanisms of this regulation On the basis of the above findings, our research provides new insights into the role of exon in the regulation of apoptosis and invasion in liver cell lines FEBS Journal 275 (2008) 3145–3156 ª 2008 The Authors Journal compilation ª 2008 FEBS 3151 Exon of occludin in apoptosis and invasion J.-M Gu et al A B C D E F Fig Occludin increases the intracellular calcium concentration in Chang cells (A) Representative cell images obtained using a confocal microscope Chang cells were cotransfected with pCMV ⁄ HA, pCMV ⁄ HA-OccWT or pCMV ⁄ HA-OccDE9 and with pEGFP-N1 vector The cells were stained with X-rhod-1, a calcium indicator, and then analyzed Each image shows one cell in the center (B) The relative fluorescence intensity is represented as a bar graph Chang cells were transfected with pCMV ⁄ HA, pCMV ⁄ HA-OccWT or pCMV ⁄ HA-OccDE9, and then treated with 20 lM 2-APB (C) Apoptotic cell numbers were calculated by counting BrdUTP-incorporating cells (D) The levels of BAX, Bcl-2 and Apaf-1 were determined using real-time RT-PCR analysis The relative expression levels are represented as a bar graph (E) Immunoblot analyses using specific antibodies against ERK1 ⁄ 2, pERK1 ⁄ and b-actin were performed (F) Invasiveness of cells was analyzed using a Matrigel assay Cells penetrating the Matrigel-coated inserts were stained with crystal violet The number of invasive cells was determined by counting the number of stained cells All results in (B), (C), (D), (E) and (F) are expressed as the fold ratio relative to control (Cont) All numerical data represent the means and standard deviations of three independent experiments Cont, pCMV ⁄ HA-transfected cells; WT, OccWT-overexpressing cells; DE9, OccDE9-overexpressing cells; +, 2-APB-treated cells; *P < 0.05 were incubated for another 24 h H2O2 was administered in DMEM with 10% fetal bovine serum for 24 h Experimental procedures Cell cultures and treatment Human liver cell lines Huh7, HepG2, Hep3B, and HLE, and Chang cells, were cultured in DMEM with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) To examine the effect of a demethylating agent, the cells were treated with lm 5¢-aza-dC (Sigma Aldrich, St Louis, MO, USA) for 48 h, and then with a histone deacetylase inhibitor, TSA (300 nm) (Sigma Aldrich) for 24 h 2-APB (20 lm) (Sigma Aldrich) was administered in DMEM with 10% fetal bovine serum at 12 h after transfection Cells 3152 Construction of expression vectors and transfection cDNA fragments representing the complete ORFs of occludin (GenBank accession no NM002538) and occludin exon deletion variant were cloned into the eukaryotic expression vector pCMV ⁄ HA (Clontech, Carlsbad, CA, USA) One microgram of each plasmid was transfected using FuGENE (Roche, Indianapolis, IN, USA) transfection reagent in six-well plates MISSION pLKO.1 vector FEBS Journal 275 (2008) 3145–3156 ª 2008 The Authors Journal compilation ª 2008 FEBS J.-M Gu et al constructs expressing occludin shRNA were purchased from Sigma and transfected with FuGENE (Roche) Transfected cells were selected with puromycin (0.5 lgỈmL)1) (Sigma) The pLKO.1 vector that does not contain an shRNA insert was used as a control 3¢-RACE For 3¢-RACE, modified protocols from Scotto-Lavio et al were used [28] First-strand cDNA synthesis was performed using the adaptor primer 5¢-CCAGTGAGCAGAGTGAC GAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTT-3¢, and then two rounds of 3¢-RACE PCR were performed, using forward gene-specific primer (GSP1), 5¢-TGGGAG TGAACCCAACTGCT-3¢ (sense) and 5¢-CCAGTGAGCA GAGTGACG-3¢ (antisense), for the first round, and genespecific primer (GSP2), 5¢-CTCGTTACAGCAGCGGTG GTAACTT-3¢ (sense) and 5¢-GAGGACTCGAGCTCAA GC-3¢ (antisense), for the second round The PCRs were performed using the following conditions: 35 cycles of 95 °C for 30 s, 53 °C for 30 s and 72 °C for for the first round PCR; and 35 cycles of 95 °C for 30 s, 53 °C for 30 s and 72 °C for for the second round Immunocytochemistry For immunocytochemistry, cells were fixed in acetone ⁄ methanol (1 : 1) at )20 °C for 10 and stained using mouse anti-HA IgG (1 : 100) (Sigma Aldrich) and rabbit anti-occludin IgG (1 : 100) (Zymed, San Francisco, CA, USA) The secondary antibodies were anti-(mouse Alexa 594) IgG (1 : 200) and anti-(rabbit Alexa 488) IgG (1 : 200) (Molecular Probes, Carlsbad, CA, USA) Nuclei were stained with 4¢,6-diamidino-2-phenylindole (Molecular Probes) After mounting, cells were visualized using a multiphoton confocal laser scanning microscope equipped with a 40· water immersion lens (Carl Zeiss, Thornwood, NY, USA) RT-PCR Total RNA was isolated with Trizol (Invitrogen) according to the manufacturer’s instructions RNA (1 lg) was reverse transcribed with oligo-dT by using avian myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, WI, USA) For PCR of occludin variants, the primers used were: 5¢-ACTCGACAATGAACAATCCGTCAGAA-3¢ (sense) and 5¢-AGAGTATGCCATGGGACTGTCA-3¢ (antisense) for exon 5; 5¢-TGCAGG-TGCTCTTTTTGAAGGT-3¢ (antisense) for exon 6; 5¢-GC-TCTTGTATTCCTGTAGGC CAG-3¢ (antisense) for exon 7; 5¢-GTATTCATCAGCAG CAGCC-3¢ (antisense) for exon 8; and 5¢-CTGTCTATCA TAGTCTCCAACCAT-CTTC-3¢ (antisense) for exon PCR was carried out at 53 °C for 32 cycles Exon of occludin in apoptosis and invasion Genomic DNA was extracted from the cells using a standard phenol protocol For PCR, the primers used were: 5¢-CAGCAATTGTCACACATCAAGAA-3¢ (sense) and 5¢-T-ACATGTAGGTATGAAGACATCGTC T-3¢ (antisense) for exon 9; 5¢-TCCCTGCTTCCTCTGGC GGA-3¢ (sense) and 5¢-AGCCATAGCCATAGCCACTTC C-3¢ (antisense) for exon 1; 5¢-CCGGAGGGTCGGGCC CAGTT-3¢ (sense) and 5¢-AGCCATAGCCATAGCCACT TCC-3¢ (antisense) for exon 1a; 5¢-TAATAGGCTGCTGC TGATGAATA-3¢ (sense) and 5¢-GGTATGTGGTCACAT TGTGAAAATT-3¢ (antisense) for the exon 8–intron boundary; and 5¢-ACTGCCAGGCACCTTGCGTATTT-3¢ (sense) and 5¢-TATCATAGTCTCCAACCATCTTCTTGA -3¢ (antisense) for the intron–exon boundary PCR was carried out at 58 °C for 30 cycles All PCR products were analyzed on agarose gels and stained with ethidium bromide Real-time RT-PCR analysis Real-time RT-PCR analysis was performed with specifically designed primers using primer express software (Applied Biosystems, Foster City, CA, USA) Primers for b-actin were: 5¢-GCAAAGACCTGTACGCCAACA-3¢ (sense) and 5¢-TGCATCCTGTCGGCAATG-3¢ (antisense) Primers for MMP1 were: 5¢-TGTGGCTCAGTTTGTCCTC ACT-3¢ (sense) and 5¢-TTGGCAAATCTGGCGTGTA A-3¢ (antisense) Primers for MMP2 were: 5¢-TGTGACGC CACGTGACAAG-3¢ (sense) and 5¢-GCCTCGTATACCG CATC-AATC-3¢ (antisense) Primers for MMP3 were: 5¢-TCG-TTGCTGCTCATGAAATTG-3¢ (sense) and 5¢-AC AGGCGGAACCGAGTCA-3¢ (antisense) Primers for MMP7 were: 5¢-TGCTGACATCATGATTGGCTTT-3¢ (sense) and 5¢-TCCTCATCGAAGTGAGCATCTC-3¢ (antisense) Primers for MMP9 were: 5¢-ATGCGTGGAG AGTCGAAATCTC-3¢ (sense) and 5¢-GGTTCGCATG GCCTTCAG-3¢ (antisense) Primers for MMP14 were: 5¢-GACTACCTCCCGGCCTTCTG-3¢ (sense) and 5¢-ATGG CCACGGTGTCAAAGTT-3¢ (antisense) To test expression levels of BAX, Bcl-2 and Apaf-1 with real-time PCR, the primers used were: 5¢-TGGAGCTGCAGAGGATG ATTG-3¢ (sense) and 5¢-CCAGTTGAAGTTGCCGTCA GA-3¢ (antisense) for BAX; 5¢-GGATTGTGGCCTTCTTT GAGTT-3¢ (sense) and 5¢-CGGTTCAGGTACTCAGT CATCCA-3¢ (antisense) for Bcl-2; and 5¢-ACGGGAGAT GACAATGGAGAAAT-3¢ (sense) and 5¢-CATGGGTAG CAGCTCCTTCTTC-3¢ (antisense) for Apaf-1 Total RNA was extracted from cultured cells using Trizol reagent (Invitrogen) according to the manufacturer’s protocol cDNA was synthesized using lg of RNA with AMV reverse transcriptase (Promega) and oligo-dT primers Transcript levels were assessed by quantitative real-time PCR (ABI 7300; Applied Biosystems); all experiments were normalized to b-actin FEBS Journal 275 (2008) 3145–3156 ª 2008 The Authors Journal compilation ª 2008 FEBS 3153 Exon of occludin in apoptosis and invasion J.-M Gu et al Immunoblot analysis Cells were washed three times in NaCl ⁄ Pi and scraped with lysis buffer (ReadyPrep Sequential extraction kit, Reagent 3; Bio-Rad, Hercules, CA, USA) Next, the protein concentration was determined by measuring with Bradford reagent (Bio-Rad) Cell lysates (20 lg) were resolved by SDS ⁄ PAGE and transferred onto poly(vinylidene difluoride) membranes The blots were blocked with 5% nonfat milk in NaCl ⁄ Pi containing 0.1% Tween-20, and probed with antibodies to occludin (Zymed), ERK1 ⁄ (pERK1 ⁄ 2; Cell Signaling Technology, Danvers, MA), pERK1 ⁄ (Cell Signaling Technology), and b-actin (Sigma Aldrich) After being washed with NaCl ⁄ Tris containing 0.1% Tween-20, the membranes were incubated for h with horseradish peroxidase-conjugated secondary antibodies Detection of peroxidase-coupled antibodies was performed using the Western Lightning chemiluminescence kit (Perkin-Elmer, Boston, MA, USA) ium [29] Boyden chambers were incubated for 48 h Following removal of noninvading cells from the upper surface with a cotton swab, invading cells were fixed, counted, and normalized to control sample MSP One microgram of genomic DNA was treated with sodium bisulfate using the One Day MSP Kit (IN2GEN, Seoul, Korea) To analyze the occludin promoter, we designed MSP primers using the Methyl Primer Express system (Applied Biosystems) Primers for methylated DNA were: 5¢-AAGTAGGCGGAGTATCGAAC-3¢ (sense) and 5¢-GAAAAAACGCGATCCTACTT-3¢ (antisense) Primers for unmethylated DNA were: 5¢-GAAGTAGGTGGAGT ATTGAAT-3¢ (sense) and 5¢-CAAAAAAACACAATCCT ACTT-3¢ (antisense) Caspase activity Cell proliferation assay For the cell proliferation assay, cells were seeded on the 96-well plates, transfected using FuGENE according to the manufacturer’s manual, and incubated in 5% CO2 at 37 °C for 48 h Cell proliferation was analyzed using Cell Counting Kit-8 as described by the manufacturer (Dojindo Laboratories, Kumamoto, Japan) Terminal deoxyribonucleotide transferasemediated nick-end labeling (TUNEL) assay Cells subjected to the TUNEL assay were seeded on coverslips and transfected using FuGENE The DeadEND Fluorometric TUNEL assay kit was used as described by the manufacturer (Promega) after 48 h of transfection Cells were fixed in 4% formaldehyde for 25 at °C and permeabilized with 0.2% Triton X-100 for at room temperature After being washed with NaCl ⁄ Pi, coverslips were incubated with fluorescently labeled 5-bromo-2¢-deoxyuridine-5¢-triphosphate (BrdUTP) (Molecular Probes) Nuclei were stained with 4¢,6-diamidino-2-phenylindole After mounting, cells were visualized with a fluorescence microscope (Olympus, Thornwood, NY, USA) Invasion assays Invasion assays were performed as described, with modifications [29–31] An lm cell culture insert (BD Biosciences) was briefly coated with reconstituted Growth Factor Reduced BD Matrigel (10 lgỈcm)2) (BD Biosciences) according to the manufacturer’s instructions Transfected cells (1.0 · 105) were resuspended in serum-free medium and then plated in the upper part of the chamber The lower chamber was filled with NIH ⁄ 3T3 conditioned med- 3154 Cells subjected to the caspase assay were seeded on a 24-well plate and transfected with FuGENE The caspase assay was performed using the CaspACE colorimetric assay kit as described by the manufacturer (Promega) Twentyfour hours after transfection, cells were harvested and lysed with supplied lysis buffer by freeze–thawing Ac-DEVDpNA, caspase substrate, was added to the cell extract and incubated for h Measurement of the caspase activity was done at 405 nm Green fluorescent protein expression construct and fluorescence imaging of calcium Cells were seeded on coverslips coated with poly(l-lysine) (Sigma Aldrich) and cotransfected with pCMV ⁄ HA containing occludin genes and pEGFP-N1 (Clontech) at a ratio of 10 : using FuGENE Coverslips with attached cells were incubated in growth medium supplemented with 10% fetal bovine serum for 24 h After washing with Hepes buffer (120 mm NaCl, 5.5 mm KCl, 1.8 mm CaCl2, mm MgCl2, 25 mm glucose, 20 mm Hepes, pH 7.2), lm X-rhod-1 (Molecular Probes) was added, and cells were incubated for 45 at room temperature After mounting, cells were visualized with a multiphoton confocal laser scanning microscope (Carl Zeiss, Thornwood, NY, USA) Relative fluorescence intensity was determined using imagemaster 2d elite software 4.01 (Amersham Bioscience, Uppsala, Sweden) Statistical analysis Data in bar graphs are expressed as the mean and standard deviation of three independent experiments Student’s t-tests were used in order to compare the differences FEBS Journal 275 (2008) 3145–3156 ª 2008 The Authors Journal compilation ª 2008 FEBS J.-M Gu et al Exon of occludin in apoptosis and invasion between different groups A P-value < 0.05 was considered to be statistically significant 11 Acknowledgements This study was supported by a grant from the National R&D Program for Cancer Control, the Ministry of Health & Welfare, Korea (0520020) Seung Oe Lim and Jin-Mo Gu were supported by a BK21 Research 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Authors Journal compilation ª 2008 FEBS ... occludin variant deleted in exon (OccDE9) On the basis of a comparative analysis of the involvement of wild-type occludin (OccWT) and variant occludin in apoptosis and invasion, as determined by assay,... 5¢-CAGCAATTGTCACACATCAAGAA-3¢ (sense) and 5¢-T-ACATGTAGGTATGAAGACATCGTC T-3¢ (antisense) for exon 9; 5¢-TCCCTGCTTCCTCTGGC GGA-3¢ (sense) and 5¢-AGCCATAGCCATAGCCACTTC C-3¢ (antisense) for exon. .. reverse transcriptase (Promega, Madison, WI, USA) For PCR of occludin variants, the primers used were: 5¢-ACTCGACAATGAACAATCCGTCAGAA-3¢ (sense) and 5¢-AGAGTATGCCATGGGACTGTCA-3¢ (antisense) for exon