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Vol. 19, Issue 5, 1912-1921, May 2008

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Tight Junction Proteins Claudin-2 and -12 Are Critical for Vitamin D-dependent Ca²⁺ Absorption between Enterocytes

Hiroki Fujita^*,,, Kotaro Sugimoto^*,, Shuichiro Inatomi^*,, Toshihiro Maeda^*, Makoto Osanai^*, Yasushi Uchiyama, Yoko Yamamoto^||, Takuro Wada, Takashi Kojima^*, Hiroshi Yokozaki^¶, Toshihiko Yamashita, Shigeaki Kato^||, Norimasa Sawada^*, and Hideki Chiba^*

*Departments of Pathology and Orthopedic Surgery, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan; Chugai Pharmaceutical Co., Ltd., Tokyo 104-8301, Japan; ^||Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan; and ^¶Division of Surgical Pathology, Department of Biomedical Informatics, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan

Submitted September 28, 2007; Revised January 14, 2008; Accepted February 8, 2008
Monitoring Editor: Asma Nusrat

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Ca²⁺ is absorbed across intestinal epithelial monolayers via transcellular and paracellular pathways, and an active form of vitamin D₃, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], is known to promote intestinal Ca²⁺ absorption. However, the molecules driving the paracellular Ca²⁺ absorption and its vitamin D dependency remain obscure. Because the tight junction proteins claudins are suggested to form paracellular channels for selective ions between neighboring cells, we hypothesized that specific intestinal claudins might facilitate paracellular Ca²⁺ transport and that expression of these claudins could be induced by 1,25(OH)₂D₃. Herein, we show, by using RNA interference and overexpression strategies, that claudin-2 and claudin-12 contribute to Ca²⁺ absorption in intestinal epithelial cells. We also provide evidence showing that expression of claudins-2 and -12 is up-regulated in enterocytes in vitro and in vivo by 1,25(OH)₂D₃ through the vitamin D receptor. These findings strongly suggest that claudin-2- and/or claudin-12-based tight junctions form paracellular Ca²⁺ channels in intestinal epithelia, and they highlight a novel mechanism behind vitamin D-dependent calcium homeostasis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Calcium plays a fundamental role in various physiological functions such as bone mineralization, blood coagulation, neuromuscular transmission, and muscle contraction, as well as cell–cell adhesion and intracellular signaling. Ca²⁺ is absorbed in the intestinal mucosa by two distinct routes, the transcellular and paracellular pathways (Bronner et al., 2003; Hoenderop et al., 2005). The transcellular Ca²⁺ transport in the intestine is thought to comprise a multistep process: uptake across the apical plasma membrane via the Ca²⁺ channel protein transient receptor potential vanilloid receptor channel (TRPV)6, intracellular transport by the Ca²⁺-binding proteins calbindins, and extrusion through plasma membrane Ca²⁺-ATPase PMCA_1b (Hoenderop et al., 2005). In contrast, the molecular basis for paracellular Ca²⁺ absorption, which occurs throughout the intestine (Bronner et al., 1986), is largely unknown.

One of the most important hormones to enhance intestinal Ca²⁺ absorption is an active form of vitamin D₃, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] (Norman, 1990; Hoenderop et al., 2005). Most of its actions are mediated by the vitamin D receptor (VDR), a member of the nuclear receptor superfamily (Mangelsdorf et al., 1995). In fact, VDR knockout (VDRKO) mice exhibit decreased Ca²⁺ uptake in the intestine with a concomitant reduction in expression of some of the aforementioned transcellular Ca²⁺ transport proteins (Yoshizawa et al., 1997; Li et al., 2001; Van Cromphaut et al., 2001; Song et al., 2002), indicating the significance of VDR in transcellular Ca²⁺ absorption. It is also suggested that vitamin D could promote paracellular Ca²⁺ transport across intestinal epithelial cells (Wasserman, 2004). However, it remains obscure which molecules driving paracellular Ca²⁺ absorption are targets for the vitamin D signaling.

Tight junctions are the apical-most constituent of the intercellular junctional complex in mammalian epithelial cell sheets, and they act as a semipermeable barrier to the paracellular transport of ions and solutes (Anderson and Cereijido, 2001). Among molecular components of tight junctions, claudins (Cldns) are the major transmembrane proteins, and they consist of 24 members of a gene family (Furuse et al., 1998; Tsukita et al., 2001). In addition, distinct sets of claudins are generally expressed in a cell- and tissue-specific manner. Recent studies have disclosed that claudins are the major determinant of the barrier function of tight junctions. Importantly, the first extracellular loop, in which there is a wide variation in the position and number of charged amino acids depending on each claudin, is shown to create paracellular pores (channels) for cations or anions between neighboring cells (Van Itallie and Anderson, 2006). Along this line, we assumed that the specific species of intestinal claudins might contribute to paracellular Ca²⁺ transport. We also hypothesized that expression of these claudins could be up-regulated by VDR. To examine this assumption, we compared the expression of putative cation-permissive claudins in the intestine of VDRKO mice with that of wild-type (WT) mice, and we used RNA interference (RNAi) and overexpression approaches in the vitamin D-responsive intestinal cell line Caco-2. We report here that expression of claudins-2 and -12 seems to be activated by VDR in vivo and in vitro and that these two claudins are essential for Ca²⁺ absorption between intestinal epithelial cells, providing a novel mechanism underlying vitamin D-dependent intestinal Ca²⁺ transport.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Antibodies
Rabbit polyclonal antibodies (pAbs) against Cldn7, Cldn12, and Cldn15 were generated, and the specificity of these antibodies were verified as described previously (Satohisa et al., 2005; Fujita et al., 2006; Sakai et al., 2007). Preimmune sera from each rabbit before being immunized with antigens were also used as negative controls to confirm the selectivity of these pAbs. A rabbit pAbs against Cldn2 were obtained from Immuno-Biological Laboratories (Minneapolis, MN) and Zymed Laboratories (South San Francisco, CA). Mouse monoclonal antibodies (mAbs) against occludin and VDR and rabbit pAbs against actin, ezrin/radixin/moesin-binding phosphoprotein 50 (EBP50), and TRPV6 were purchased from Zymed Laboratories, Santa Cruz Biotechnology (Santa Cruz, CA), Sigma-Aldrich (St. Louis, MO), Affinity BioReagents (Golden, CO), and Alomone Labs (Jerusalem, Israel), respectively. A rat mAb against ezrin were obtained from Sanko Junyaku (Tokyo, Japan). The secondary antibodies used were horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse immunoglobulin (Ig)G (Dako Denmark, Glostrup, Denmark), Alexa Fluor 488 (green)-labeled anti-rabbit IgG (Invitrogen, Carlsbad, CA), Alexa Fluor 594 (red)-labeled anti-mouse IgG (Invitrogen), and fluorescein isothiocyanate-conjugated anti-rat IgG (DakoCytomation).

Animals and Tissue Preparation
VDRKO mice were generated and maintained as reported previously (Yoshizawa et al., 1997). Twelve-week-old wild-type and VDRKO mice were anesthetized with diethyl ether, and specimens of the intestine were obtained. All aspects of the study were approved by the animal use and care committee of Sapporo Medical University School of Medicine (Sapporo, Japan).

Cell Lines and Cell Culture
The human intestinal cell line Caco-2 (clone BBe; CRL-2102) was obtained from American Type Culture Collection. To establish Caco-2 cell lines expressing Cldn2, Cldn7, Cldn12, and Cldn15 (Caco-2: Cldn2, Caco-2: Cldn7, Caco-2: Cldn12 and Caco-2: Cldn15, respectively), cells were transfected with 10 µg of individual expression vectors containing the corresponding mouse claudin cDNAs (Ishizaki et al., 2003; Fujita et al., 2006) along with 1 µg of the puromycin-resistant gene expression vector pHRLpuro1 (Chiba et al., 1997a) by using Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer's protocols. Puromycin (5 µg/ml)-resistant clones were screened by immunofluorescence staining and immunoblotting.

Cells were plated in DMEM supplemented with 20% fetal bovine serum (heat-inactivated and charcoal-stripped), 100 U/ml penicillin, and 100 µg/ml streptomycin, and they were grown at 37°C in a humidified 5% CO₂ atmosphere. They were refed every 2 d, and they were exposed to the vehicle or 1,25(OH)₂D₃ (Sigma-Aldrich).

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
For analysis of mRNA expression, total RNA was isolated from the intestine and cells using TRIzol reagent (Invitrogen), and RT-PCR was performed as reported previously (Chiba et al., 1997b, 2006). The PCR primers for cDNA were as follows: mouse VDR (GenBank/EMBL/DDBJ accession no. NM_009504), 5'-AACGCTATGACCTGTGAAGGC-3' (nucleotides [nt] 250-270) and 5'-CCTGTACTTACGTCTGCACGA-3' (nt 612-632); human Cldn2 (GenBank/EMBL/DDBJ accession no. NM_020384), 5'-GCTTCTACTGAGAGGTCTG-3' (nt 306-324) and 5'-TTCTTCACACATACCCTG-3' (nt 1006-1023); human Cldn7 (GenBank/EMBL/DDBJ accession no. NM_001307), 5'-AGGCATAATTTTCATCGTGG-3' (nt 792-811) and 5'-GAGTTGGACTTAGGGTAAGAGCG-3' (nt 1021-1043); human Cldn12 (GenBank/EMBL/DDBJ accession no. NM_012129), 5'-CTCCCCATCTATCTGGGTCA-3' (nt 635-654) and 5'-GGTGGATGGGAGTACAATGG-3' (nt 816-835); human Cldn15 (GenBank/EMBL/DDBJ accession no. NM_014343), 5'-AAATACGGCAGAAACGCCTA-3' (nt 915-934) and 5'-CGACTTCCCAAGAGCAGTTC-3' (nt 1109-1128). The primers for mouse claudins and those for the housekeeping gene 36B4 encoding the ribosomal protein were described previously (Kubota et al., 2001; Fujita et al., 2006).

To confirm that amplifications were in the linear range, PCR was performed using three different numbers of cycles between 21 and 36, depending on the gene analyzed. Aliquots of PCR products were loaded onto 2% agarose gel, and then they were analyzed after staining with ethidium bromide. The mRNA signals were quantified using Image 1.62c software (Scion, Frederick, MD).

Gel Electrophoresis and Immunoblotting
The mouse intestinal tissue and Caco-2 cells grown on 60-mm tissue culture plates were washed twice with ice-cold phosphate-buffered saline (PBS), sonicated for 20 s in ice-cold NaHCO₃ buffer (1 mM NaHCO₃ and 1 mM phenylmethylsulfonyl fluoride [PMSF], pH 7.5), and put on ice for 30 min. They were mixed with 2x SDS sample buffer and boiled for 5 min. Total cell lysates were then resolved by one-dimensional SDS-polyacrylamide gel electrophoresis (PAGE) and electrophoretically transferred onto a polyvinylidene difluoride membrane (Immobilon; Millipore, Billerica, MA). The membrane was saturated with PBS containing 4% skim milk, and then it was incubated for 1 h at room temperature with primary antibodies in PBS. After rinsing in PBS containing 0.1% Tween 20, the membrane was incubated for 1 h at room temperature with HRP-conjugated anti-rabbit or anti-mouse IgG (diluted 1:1000) in PBS. It was then rinsed again, and finally it was analyzed using an ECL Western blotting detection system (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). The blots were stripped with Restore Western blot stripping solution (Nacalai Tesque, Kyoto, Japan) according to the manufacturer's instructions, and immunoprobed with an anti-actin antibody. When immunoblotted signals were faint, ECL Advance Western blotting detection system (GE Healthcare) was used according to the manufacturer's recommendations. Signals in immunoblots were quantified using Image 1.62c software.

Immunohistochemistry
Ten-micrometer-thick frozen sections of the mouse intestine were fixed in 95% ethanol for 30 min at 4°C and in 100% acetone for 1 min at room temperature. After being washed three times with PBS, they were incubated for 1 h at room temperature with primary antibodies and rinsed again with PBS, followed by reaction for 1 h at room temperature with appropriate secondary antibodies. For immunohistochemistry of Caco-2 cells, cells grown on coverslips were fixed in 1% formaldehyde in PBS for 10 min. After being washed three times with PBS, they were treated with 0.2% Triton X-100 in PBS for 10 min, rinsed again with PBS, and preincubated in PBS containing 5% skim milk. They were subsequently incubated with primary and secondary antibodies as described above. All samples were examined using a laser-scanning confocal microscope (MRC 1024; Bio-Rad, Hercules, CA) and a PlanApo 60x numerical aperture 1.40 oil immersion objective (Nikon, Tokyo, Japan). Photographs were recorded using a Dell computer (PowerEdge 2200) and OS/2 WARP software (IBM, White Plains, NY), and they were processed with Photoshop 6.0 (Adobe Systems, Mountain View, CA). Observations were made at room temperature.

Measurement of Transepithelial Electrical Resistance (TER) and Calcium Transport Studies
Caco-2 cells were grown on 1.0-cm² polycarbonate Transwell-Clear membranes (0.4-µm pore size; Corning Life Sciences, Acton, MA) coated with rat tail collagen. TER was measured using an EVOM voltohmmeter with ENDOHM-12 (WPI, Sarasota, FL) on a heating plate (FHP-30S; Fine Tokyo, Japan) adjusted to 37°C. The values are expressed in standard units of ohms per square centimeter. For calculation, the resistance of blank filters was subtracted from that of filters covered with cells.

Calcium transport across Caco-2 monolayers was assessed as reported previously (Giuliano and Wood, 1991). In brief, cells were washed twice with PBS, and transport buffer (140 mM NaCl, 5.8 mM KCl, 0.34 mM Na₂HPO₄, 0.44 mM NaH₂PO₄, 1 mM MgCl₂, 1 mM CaCl₂, 25 mM glucose, and 20 mM HEPES, pH 7.4) was poured into the inner and outer chambers. After adding 1 µCi/ml ⁴⁵Ca²⁺ to the insert, cells were incubated at 37°C, and samples were collected from the opposite compartment at 15, 30, 60, and 120 min. Radioactivity of ⁴⁵Ca²⁺ was measured using a scintillation counter (LS6500; Beckman).

Electrophysiological Measurement
For electrophysiological measurements, Caco-2 cells were grown on Snapwell filters (0.4-µm pore size; Corning Life Sciences) coated with rat tail collagen. The permeabilities of Na⁺ and Cl^– across Caco-2 monolayers were determined using an Ussing chamber with an ECV4000 Precision V/I Clamp (WPI) according to the procedure of Hou et al. (2005).

RNAi and Transfection
Stealth small interfering RNA (siRNA) duplex oligonucleotides against human Cldn2 and Cldn12 were synthesized by Invitrogen. The sequences were as follows: Cldn2 RNAi #1, sense (5'-AUUUCAUGCUGUCAGGCACCAGUGG-3') and antisense (5'-CCACUGGUGCCUGACAGCAUGAAAU-3'); Cldn2 RNAi #2, sense (5'-AGAAAUAAUGCCCAAGUAAAGAGCC-3') and antisense (5'-GGCUCUUUACUUGGGCAUUAUUUCU-3'); Cldn2 RNAi #3, sense (5'-AGAGGAUGAUUCCAGCUAUCAGGGA-3') and antisense (5'-UCCCUGAUAGCUGGAAUCAUCCUCU-3'); Cldn7 RNAi #1, sense (5'-AUAGGAGCUCAUCUGCCACUGCGGG-3') and antisense (5'-CCCGCAGUGGCAGAUGAGCUCCUAU-3'); Cldn7 RNAi #2, sense (5'-AUUAGGGCUCGAGUGGCCUGCAAGG-3') and antisense (5'-CCUUGCAGGCCACUCGAGCCCUAAU-3'); Cldn7 RNAi #3, sense (5'-AUACGGGCCUUCUUCACUUUGUCGU-3') and antisense (5'-ACGACAAAGUGAAGAAGGCCCGUAU-3'); Cldn12 RNAi #1, sense (5'-UAUAAGGGAAGUCAUAAACAGGCCC-3') and antisense (5'-GGGCCUGUUUAUGACUUCCCUUAUA-3'), Cldn12 RNAi #2, sense (5'-AUUUCAAUGGCAGAGAGGCGAGAGC-3') and antisense (5'-GCUCUCGCCUCUCUGCCAUUGAAAU-3'); and Cldn12 RNAi #3, sense (5'-UUCUCGUUUCUGUUGAAUGUGAUCA-3') and antisense (5'-UGAUCACAUUCAACAGAAACGAGAA-3'). Cells were transfected with 20 pmol of siRNAs or a Stealth RNAi negative control by using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocols, and they were treated 12 h after transfection followed by exposure to 1,25(OH)₂D₃.

Statistical Analysis
All measured values are presented as the mean ± SD. Statistical significance of differences was evaluated using the unpaired Student's t test.

	RESULTS

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Claudins 2 and 12 Are down-Regulated in the Intestine of VDRKO Mice
Among claudin species that are abundantly expressed in the intestine, Cldn2, Cldn7, Cldn12, and Cldn15 are suggested to act as paracellular channels for cations (Amasheh et al., 2002; Colegio et al., 2002; Van Itallie et al., 2003; Alexandre et al., 2005; Fujita et al., 2006; Hou et al., 2006). Therefore, we first compared, by RT-PCR and Western blot analyses, the mRNA and protein levels of these claudins throughout the intestinal tract in VDRKO mice with those in WT mice. The lack of VDR transcripts and proteins in the intestine of VDRKO mice was confirmed by RT-PCR and immunoblot experiments (Figures 1A and 2, A and C; data not shown). As shown in Figure 1, A and B, expression levels of Cldn3 transcripts were higher in the jejunum in VDRKO mice than in WT mice, in good agreement with the finding that the gene expression of Cldn3 is decreased in the rat small intestine after 1,25(OH)₂D₃ treatment (Kutuzova and Deluca, 2004). In contrast, the mRNA expression of Cldn2 and Cldn12, but not Cldn7 or Cldn15, was reduced in the VDRKO mouse jejunum compared with that of the WT mouse (3- and 4-fold decreases, respectively). Similarly, levels of Cldn2 and Cldn12 proteins, but not Cldn7 or Cldn15 proteins, were much lower in the VDRKO mouse jejunum than in that of WT mice (Figure 1, C and D) (3- and 7-fold less, respectively). Cldn2 and Cldn12 were also down-regulated in the duodenum, ileum, and colon of the VDRKO mouse at both mRNA and protein levels (Figure 2, A–D; data not depicted).

View larger version (64K): [in this window] [in a new window]   Figure 1. Reduced expression of claudins-2 and -12 in the jejunum of VDRKO mice. (A) Gene expression of Cldns in the jejunum of wild-type (+/+; lanes 1–3) and VDRKO mice (–/–; lanes 4–6) at 12 wk of age. One microgram of total RNA from the jejunum was subjected to RT-PCR analysis for the indicated genes. PCR was performed for 20 (36B4), 21 (Cldn3 and Cldn7), 25 (Cldn15), 28 (Cldn2), 32 (Cldn12), or 36 (VDR) cycles. (B) RT-PCR analysis was performed as described in A for at least five independent experiments. The mRNA levels were normalized to the corresponding 36B4 levels and expressed relative to the amount present in the wild-type mice, which was taken as 100. Values represent the mean ± SD (error bars; n = 5 for Cldn7 and n = 6 for other Cldns). (C) Expression of Cldn proteins in the jejunum of each genotype at 12 wk of age. Twenty-five micrograms of whole cell extract from the jejunum was separated by SDS-PAGE and immunoblotted with the corresponding antibodies followed by chemiluminescence detection. Each blot was stripped and reimmunoprobed with an anti-actin antibody. (D) Western blot analysis was performed as in C for at least four independent experiments. The protein levels are normalized to the corresponding actin levels and expressed relative to the amount present in wild-type mice, which was taken as 100. Values represent the mean ± SD (error bars; n = 4 for Cldn15, n = 6 for Cldn2 and Cldn7, and n = 7 for Cldn12).

View larger version (45K): [in this window] [in a new window]   Figure 2. Down-regulation of claudins-2 and -12 in the ileum and colon of VDRKO mice. (A) Gene expression of claudin-2 (Cldn2) and Cldn12 in the colon of wild-type (+/+; lanes 1–3) and VDRKO mice (–/–; lanes 4–6) at 12 wk of age. One microgram of total RNA from the jejunum was subjected to RT-PCR analysis for the indicated genes. PCR was performed for 20 (36B4), 23 (Cldn2), or 36 (Cldn12 and VDR) cycles. (B) RT-PCR analysis was performed as described in A for at least four independent experiments. The mRNA levels are normalized to the corresponding 36B4 levels and expressed relative to the amount present in the wild-type mice, which was taken as 100. Values represent the mean ± SD (error bars; n = 4 for Cldn12 and n = 6 for Cldn2). (C) Expression of Cldn2 and Cldn12 proteins in the ileum of each genotype at 12 wk of age. Twenty-five micrograms of whole cell extract from the ileum was separated by SDS-PAGE and immunoblotted with the corresponding antibodies followed by chemiluminescence detection. Each blot was stripped and reimmunoprobed with an anti-actin antibody. (D) Western blot analysis was performed as described in C for at least five independent experiments. The protein levels are normalized to the corresponding actin levels and expressed relative to the amount present in wild-type mice, which was taken as 100. Values represent the mean ± SD (error bars; n = 5 for Cldn2 and n = 8 for Cldn12).

View larger version (121K): [in this window] [in a new window]   Figure 3. Distribution of claudins in intestinal mucosa of wild-type (+/+) and VDRKO (–/–) mice. Sections of the jejunum (A), ileum (B), and colon (C) of mice at 12 wk of age were subjected to immunostaining with the corresponding antibodies. Bar, 30 µm.

View larger version (72K): [in this window] [in a new window]   Figure 4. Induction of the expression of claudins-2 and -12 by 1,25(OH)2D3 in Caco-2 cells. (A) Expression of Cldn mRNAs in Caco-2 cells treated for 48 h with the vehicle (lane 1) or 100 nM 1,25(OH)2D3 (lane 2). One microgram of total RNA from the cells was subjected to RT-PCR analysis for indicated genes. PCR was performed for 22 (36B4), 30 (Cldn12 and Cldn15) or 32 (Cldn2 and Cldn7) cycles. (B) Expression of Cldn proteins in Caco-2 cells grown for 48 h in the absence (lane 1) or presence of 100 nM 1,25(OH)2D3 (lane 2). Twenty-five micrograms of whole cell extract was separated by SDS-PAGE and immunoblotted with antibodies against the corresponding claudins, followed by chemiluminescence detection. The blots were stripped and immunoprobed with an anti-actin antibody. (C) Western blot analysis was performed as in C for three independent experiments. The protein levels are normalized to the corresponding actin levels and expressed relative to the amount present in the cells cultured without 1,25(OH)2D3, which was taken as 100. Values represent the mean ± SD (error bars; n = 3). (D) Expression of Cldn2 and Cldn12 proteins in Caco-2 cells exposed to 100 nM 1,25(OH)2D3 for 0, 12, 24, and 48 h (lanes 1–4, respectively). Western blot analysis was performed as described in B for three independent experiments, and the protein levels are expressed as described in C (error bars; n = 3). (E) Expression of Cldn2 and Cldn12 proteins in Caco-2 cells treated for 48 h with 0, 1, 10, and 100 nM 1,25(OH)2D3 (lanes 1–4, respectively). Western blot analysis was performed as described in B for three independent experiments, and the protein levels are expressed as described in C (error bars; n = 3). (E) Staining pattern of Cldn2, Cldn7, Cldn12, and Cldn15 in Caco-2 cells grown for 48 h in the absence or presence of 100 nM 1,25(OH)2D3. Cells were subjected to immunostaining with the corresponding antibodies, and they were observed under a laser-scanning confocal microscope. Bars, 20 µm.

Claudins-2 and-12 Are Required for Vitamin D-induced Calcium Absorption in Caco-2 Cells
Treatment of Caco-2 cells with 1,25(OH)₂D₃ is known not only to decrease TER, an indicator of the paracellular barrier to ion conductance but also to increase Ca²⁺ transport (Giuliano and Wood, 1991; Chirayath et al., 1998; Fleet and Wood, 1999; also see Figure 5, C and D). Therefore, we subsequently verified whether expression of endogenous Cldn2 and/or Cldn12 was required for 1,25(OH)₂D₃-induced Ca²⁺ absorption. To this end, we used an RNAi strategy to knock down the expression of Cldn2 and Cldn12. Caco-2 cells were transfected with three distinct siRNAs against human Cldn2, those against human Cldn12 or negative control siRNA, and then they were incubated for 12 h after transfection followed by treatment for 48 h with 10^–7 M 1,25(OH)₂D₃. Western blot and immunofluorescence analyses showed that two different Cldn2 siRNAs (#1 and #3) and a Cldn12 siRNA (#1) effectively diminished the expression of Cldn2 and Cldn12, respectively (Figure 5, A and B; data not shown). Neither Cldn7 nor Cldn15 expression was affected by the Cldn2 or Cldn12 siRNAs, indicating the specific knockdown effects of these siRNAs. Interestingly, when the expression Cldn2 or Cldn12 was suppressed, reduction of TER values by 1,25(OH)₂D₃ was partially but significantly attenuated (Figure 5C and Supplemental Figure S2A). More importantly, the knockdown of Cldn2 or Cldn12 expression resulted in a significant decrease in 1,25(OH)₂D₃-induced ⁴⁵Ca²⁺ transport (Figure 5D and Supplemental Figure S2B). Note that suppression of Cldn2 or Cldn12 expression did not alter expression of the transcellular Ca²⁺ channel protein TRPV6 or distribution of apical markers (ezrin and EBP50) (Supplemental Figure S3, A and C). By contrast, the specific knockdown of Cldn7 expression did not significantly alter the levels of TER or ⁴⁵Ca²⁺ permeability in Caco-2 cells grown in the presence and absence of 1,25(OH)₂D₃ (Supplemental Figure S4; data not shown). Together, these results indicated that 1,25(OH)₂D₃ induced paracellular Ca²⁺ transport at least in part through the upregulation of both Cldn2 and Cldn12 expression.

View larger version (79K): [in this window] [in a new window]   Figure 5. Knockdown of claudins-2 and -12 impairs vitamin D-induced calcium transport across Caco-2 cells. (A) Suppression of claudin-2 (Cldn2) and Cldn12 expression in Caco-2 cells by RNAi. Cells were transfected with negative control siRNA (lanes 1 and 3) or siRNAs against Cldn2 (#1) (lane 2) and Cldn12 (#1) (lane 4), incubated for 12 h after transfection, and then treated for 48 h with 100 nM 1,25(OH)2D3. Twenty-five micrograms of whole cell extract from the cells was separated by SDS-PAGE and immunoblotted with the corresponding antibodies, followed by chemiluminescence detection. The blots were stripped and immunoprobed with an anti-actin antibody. (B) Disappearance of Cldn2 and Cldn12 along cell borders in Caco-2 cells by RNAi. Cells were transfected and treated as described in A, subjected to immunostaining with the corresponding antibodies, and observed under a laser-scanning confocal microscope. Bars, 20 µm. (C and D) Suppression of Cldn2 and Cldn12 expression in Caco-2 cells prevents 1,25(OH)2D3-dependent decrease in TER levels and increase in 45Ca2+ transport. Cells were transfected and treated as described in A, subjected to measurement of TER (C) and calcium transport studies (D). The values of TER and 45Ca2+ permeability are expressed relative to the level in cells transfected with negative control siRNA and grown without 1,25(OH)2D3, which was taken as 1, and they represent the mean ± SD (error bars; n = 4). *p < 0.05 and **p < 0.01 compared with values of cells grown without 1,25(OH)2D3.

View larger version (73K): [in this window] [in a new window]   Figure 6. Overexpression of claudins-2 and -12 promotes calcium transport in Caco-2 cells. (A) Expression of claudin-2 (Cldn2), Cldn7, and Cldn12 proteins in Caco-2 and Caco-2:Cldn transfectants. Twenty-five micrograms of whole cell extract from the cells was separated by SDS-PAGE and immunoblotted with antibodies against the corresponding claudins, followed by chemiluminescence detection. The blots were stripped and reimmunoprobed with an anti-actin antibody. (B) Staining pattern of Cldn2, Cldn7, and Cldn12 in Caco-2 and Caco-2:Cldn transfectants. Cells were subjected to immunostaining with the corresponding claudin antibodies. Bar, 20 µm. (C and D) Relative levels of TER (C) and 45Ca2+ transport (D) in Caco-2 and Caco-2:Cldn transfectants. The values of TER and 45Ca2+ permeability in Caco-2:Cldn transfectants (clones #1 and #2) are expressed relative to the level in the mock-transfected cells, which was taken as 1, and they represent the mean ± SD (error bars; n = 4). *p < 0.05 and **p < 0.01 compared with values of the mock-transfected cells.

View larger version (32K): [in this window] [in a new window]   Figure 7. Weak induction of claudin-2 does not contribute to increased calcium transport in Caco-2 cells overexpressing claudin-12. (A and B) Slight induction of endogenous Cldn2 protein (A) and mRNA (B) in Caco-2:Cldn12 cells (clones #1 and #2). (A) Twenty-five micrograms of whole cell extract from the cells was separated by SDS-PAGE and immunoblotted with antibodies against the corresponding claudins, followed by chemiluminescence detection. The blots were stripped and reimmunoprobed with anti-actin antibody. (B) One microgram of total RNA from the cells was subjected to RT-PCR analysis for indicated genes. PCR was performed for 23 (36B4) or 30 (Cldn2) cycles. (C) Knockdown of endogenous Cldn2 expression in Caco-2:Cldn12 cells (clone #1) by RNAi. Cells were transfected with negative control siRNA (left) or siRNA against Cldn2 (#1; right), incubated for 12 h after transfection, and then they were refed and grown for 48 h. They were subjected to immunostaining with the corresponding antibodies, and they were observed under a laser-scanning confocal microscope. Bars, 20 µm. (D and E) Effects of suppressed endogenous Cldn2 on TER (D) and 45Ca2+ permeability in Caco-2:Cldn12 cells (clones #1 and #2). Cells were transfected and grown as described in C, and then they were subjected to measurement of TER (D) and calcium transport studies (E). The values are expressed relative to the level in Caco-2 cells transfected with negative control siRNA, which was taken as 1, and they represent the mean ± SD (error bars; n = 6 [left] and n = 3 [right]). *p < 0.01 compared with values of the mock-transfected cells.

Slight induction of endogenous Cldn2 expression was observed in two independent Caco-2:Cldn12 clones at both mRNA and protein levels (Figure 7, A and B). Therefore, to verify that weak Cldn2 expression could influence the permeability property in Caco-2:Cldn12 cells, we subsequently suppressed endogenous Cldn2 expression by using RNAi (Figure 7C). As shown in Figure 7, D and E, the values of TER and ⁴⁵Ca²⁺ permeability were not largely altered in Caco-2:Cldn12 cells when Cldn2 expression was knocked down.

Overexpressed Claudin-2, but Not Claudin-12, Promotes Na⁺ Permeability across Caco-2 Cells
Because Cldn2 and Cldn12 seemed to participate in Ca²⁺ transport, we next analyzed the permeability of Na⁺ and Cl^– in Caco-2:Cldn2 and Caco-2:Cldn12 cells (Figure 8, A and B). Overexpression of Cldn2 in Caco-2 cells enhanced Na⁺ permeation (3-fold), but not Cl^– permeation, resulting in a threefold increase of P_Na/P_Cl. In contrast, forced expression of Cldn12 did not basically change the permeability of Na⁺ or Cl^– in Caco-2 cells. Thus, differences in Na⁺ permeability between Caco-2:Cldn2 and Caco-2:Cldn12 cells might contribute to the TER values in these cells (Figure 6C). Nor did suppression of weak induction of endogenous Cldn2 in Caco-2:Cldn12 cells influence Na⁺ or Cl^– conductance (Figure 8, C and D).

View larger version (29K): [in this window] [in a new window]   Figure 8. Overexpression of claudin-2, but not claudin-12, increases Na+ permeability in Caco-2. (A and B) Na+ permeability (A) and relative levels of Na+/Cl– permeability (B; PNa/PCl) in Caco-2, Caco-2:Cldn2 (clone #2) and Caco-2:Cldn12 (clone #1). (C and D) Effects of suppressed endogenous Cldn2 on Na+ permeability (C) and Na+/Cl– (D) in Caco-2:Cldn12 cells (clones #1). Cells were transfected with negative control siRNA (left) or siRNA against Cldn2 (#1, right), incubated for 12 h after transfection, and then they were refed and grown for 48 h. They were then subjected to the measurement. The values are expressed relative to the level in Caco-2 cells transfected with negative control siRNA, which was taken as 1, and they represent the mean ± SD (error bars; n = 4). *p < 0.01 compared with values of the mock-transfected cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We previously demonstrated that two types of nuclear receptors, retinoid receptors and hepatocyte nuclear receptor 4 (HNF4), triggered expression of Cldn6 and Cldn7 in embryonal carcinoma cell line F9 (Kubota et al., 2001; Chiba et al., 2003; Satohisa et al., 2005). Subsequently, Battle et al. (2006) showed that Cldn1 was absent in hepatocytes in mice with conditional KO of the HNF4 gene. More recently, Gareus et al. (2007) found that retinoid receptors and the kinase IB kinase complex 1 cooperatively regulated the expression of retinoid target genes, including Cldn23, in keratinocytes. Furthermore, comparison of gene expression profiling between wild-type and Sertoli cell-specific androgen receptor (AR)-knockout mice revealed that AR mediated activation of the Cldn3 expression in Sertoli cells (Meng et al., 2005). Thus, taken collectively with our present work, expression of different claudin species seems to be up-regulated by distinct members of the nuclear receptor superfamily in a cell-specific manner.

The most important conclusion of the present study is that Cldn2 and Cldn12 facilitate the paracellular conductance and Ca²⁺ transport in intestinal epithelial cells. This conclusion was drawn from results using RNAi and overexpression strategies in Caco-2 cells, in which endogenous Cldn2 and Cldn12 expression was hardly detected along cell interfaces and was induced upon 1,25(OH)₂D₃ treatment. The suppression of either Cldn2 or Cldn12 expression by RNAi in Caco-2 cells hindered 1,25(OH)₂D₃-induced passage of electricity and ⁴⁵Ca²⁺, indicating that these claudin species were at least in part necessary for vitamin D-triggered calcium absorption. Furthermore, overexpression of Cldn2 and Cldn12, but not Cldn7 or Cldn15, led to significant increases in the electrical conductivity and ⁴⁵Ca²⁺ transport across Caco-2 cells. These results highlighted that Cldn2 and Cldn12 were not only required but also sufficient to promote the paracellular conductance and calcium absorption in intestinal epithelial cells. In this sense, it should be noted that, among the different segments of the intestinal tract, Cldn2 and Cldn12 are most abundantly expressed in the ileum (Fujita et al., 2006; Holmes et al., 2006), where the majority (65–88%) of dietary calcium is absorbed (for review, see Wasserman, 2004). It is also noteworthy that, in both mice and humans, Cldn2 has three negatively charged amino acids (positions 53, 65, and 75) within the first extracellular loop (Van Itallie et al., 2003), and that Cldn12 contains four negatively charged residues (positions 62, 66, 71 and 74) in the same domain (Fujita et al., 2006), suggesting that these claudins form paracellular pores for cations. Hence, although a relative contribution of paracellular and transcellular Ca²⁺ transport has not been established (McCormick, 2002; Bronner et al., 2003; Wasserman, 2004; Hoenderop et al., 2005), we propose that Cldn2- and/or Cldn12-based tight junctions create paracellular Ca²⁺ channels in enterocytes to maintain calcium homeostasis together with the transcellular pathway.

Several lines of evidence have suggested that Cldn16/paracellin-1 (PCLN-1) plays a key role in the reabsorption of Mg²⁺ and Ca²⁺. First, positional cloning has identified that familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), an autosomal recessive disease showing severe Mg²⁺ and Ca²⁺ wasting, is caused by mutations in the Cldn16/PCLN-1 gene (Simon et al., 1999). It is also known that Cldn16 is highly expressed at tight junctions of epithelial cells in the thick ascending loop of Henle, where the reabsorption of divalent cations occurs (Simon et al., 1999). Second, disease-associated mutations in patients with FHHNC do indeed affect the intracellular traffic and paracellular Mg²⁺ transport functions of Cldn16 (Kausalya et al., 2006). Third, overexpression of Cldn16 enhances Mg²⁺ and Ca²⁺ transport in Madin-Darby canine kidney (MDCK) cells (Ikari et al., 2004, 2006). Fourth, using the pig renal epithelial cell line LLC-PK1 and transgenic mice, Cldn16, which also seems to cause profound effects on permeability of monovalent cations, is shown to drive the reabsorption of Mg²⁺ and Ca²⁺ (Hou et al., 2005, 2007). However, Cldn16 seems not to be expressed along the intestinal tract (Fujita et al., 2006; Holmes et al., 2006), suggesting that other claudin species may contribute to Ca²⁺ absorption in the intestine. These reports, taken together with our present findings, indicate that distinct claudins, Cldn2 and Cldn12 in the intestine and Cldn16 in the kidney, are most likely involved in paracellular Ca²⁺ transport.

Cldn2 is well known to function as paracellular channel to Na⁺ in renal epithelial cells without altering Cl^– conductance. For example, overexpression of Cldn2 in MDCK strain I cells lacking endogenous Cldn2 increases paracellular Na⁺ permeability and decreases TER (Furuse et al., 2001; Amasheh et al., 2002). Similar effects are observed when Cldn2 is overexpressed in LLC-PK1 cells expressing little endogenous Cldn2 (Van Itallie et al., 2003). Conversely, knockdown of endogenous Cldn2 expression in MDCK strain II cells reduces paracellular Na⁺ permeation and elevates TER (Hou et al., 2006). We have revealed that forced expression of Cldn2 in Caco-2 cells results in enhancement of permeability of Na⁺ and Ca²⁺, indicating that Cldn2 forms paracellular channel for both cations in intestinal epithelial cells. In contrast, overexpression of Cldn12 promoted conductance of Ca²⁺, but not of Na⁺, suggesting that Cldn12, unlike Cldn2 (this study) and Cldn16 (Hou et al., 2005, 2007), may preferentially serve as a paracellular channel to Ca²⁺ in enterocytes.

Another issue that should be discussed is weak induction of endogenous Cldn2 expression by Cldn12. Overexpression of Cldn2, Cldn7, and Cldn15 in Caco-2 cells did not alter the levels of endogenous claudins, in good agreement with previous results using MDCK and LLC-PK1 cells (Furuse et al., 2001; Van Itallie et al., 2003; Alexandre et al., 2005; Hou et al., 2006). In contrast, increased expression of endogenous Cldn2 was detected in two independent Caco-2:Cldn12 clones, but not in Caco-2:Cldn7 and Caco-2:Cldn15 cells, at both transcription and protein levels. In this regard, it is worth noting that forced expression of Cldn8 in MDCK strain II cells results in down-regulation of endogenous Cldn2 protein (Yu et al., 2003). Although it is unclear by which mechanism Cldn12 activates Cldn2 expression, we excluded, by RNAi experiments, the possibility that slight induction of Cldn2 influenced the permeability of both Ca²⁺ and Na⁺ as well as TER in Caco-2:Cldn12 cells.

In conclusion, we have shown that expression of Cldn2 and Cldn12 is up-regulated in intestinal epithelial cells by 1,25(OH)₂D₃ via VDR. We also found that these two claudin species acted as paracellular Ca²⁺ channels in intestinal mucosa. These findings identify essential roles of Cldn2 and Cldn12 in the vitamin D-dependent intestinal Ca²⁺ absorption, providing new insight into Ca²⁺ homeostasis. Further studies will be required to determine whether expression of Cldn2 and Cldn12 genes are directly and/or indirectly regulated by VDR. It will also be important to generate and characterize Cldn2- and/or Cldn12-deficient mice, as well as to elucidate possible contributions of these claudins to pathological conditions such as intestinal malabsorption and impaired Ca²⁺ absorption. Moreover, it will be interesting to investigate whether the luminal Ca²⁺ concentration in the intestine might affect expression of Cldn2 and Cldn12.

	ACKNOWLEDGMENTS

 
We are grateful to Immuno-Biological Laboratories for cooperation in generation of anti-claudin-7, anti-claudin-12, and anti-claudin-15 antibodies; Dr. T. Onaga for technical advice; N. Sakai, K. Takeda, and M. Nakazawa as well as the staff of the animal facility for technical assistance; and K. Barrymore for help with manuscript.

This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Akiyama Foundation, and the Takeda Science Foundation.

	Footnotes

 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-09-0973) on February 20, 2008.

These authors contributed equally to this work.

Address correspondence to: Hideki Chiba (hidchiba{at}sapmed.ac.jp)

Abbreviations used: Cldn, claudin; EBP50, ezrin/radixin/moesin-binding phosphoprotein 50; FHHNC, familial hypomagnesemia with hypercalciuria and nephrocalcinosis; KO, knockout; PCLN-1, paracellin-1; siRNA, small interfering RNA; TER, transepithelial electrical resistance.; VDR, vitamin D receptor; WT, wild type.

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