Heterogenous Gene Expression of Bicellular and Tricellular Tight Junction-Sealing Components in the Human Intestinal Tract

Abstract

A major site for the absorption of orally administered drugs is the intestinal tract, where the mucosal epithelium functions as a barrier separating the inside body from the outer environment. The intercellular spaces between adjacent epithelial cells are sealed by bicellular and tricellular tight junctions (TJs). Although one strategy for enhancing intestinal drug absorption is to modulate these TJs, comprehensive gene (mRNA) expression analysis of the TJs components has never been fully carried out in humans. In this study, we used human biopsy samples of normal-appearing mucosa showing no endoscopically visible inflammation collected from the duodenum, jejunum, ileum, colon, and rectum to examine the mRNA expression profiles of TJ components, including occludin and tricellulin and members of the claudin family, zonula occludens family, junctional adhesion molecule (JAM) family, and angulin family. Levels of claudin-3, -4, -7, -8, and -23 expression became more elevated in each segment along the intestinal tract from the upper segments to the lower segments, as did levels of angulin-1 and -2 expression. In contrast, expression of claudin-2 and -15 was decreased in the large intestine compared to the small intestine. Levels of occludin, tricellulin, and JAM-B and -C expression were unchanged throughout the intestine. Considering their segment specificity, claudin-8, claudin-15, and angulin-2 appear to be targets for the development of permeation enhancers in the rectum, small intestine, and large intestine, respectively. These data on heterogenous expression profiles of intestinal TJ components will be useful for the development of safe and efficient intestinal permeation enhancers.

INTRODUCTION

The oral route of administering drugs is considered superior because it has high compliance, is easy for patients, and is non-invasive. Most orally administered drugs are absorbed into the systemic blood flow in the intestine and then subsequently distributed to tissues. The intestinal mucosa separates the inside body from the outside environment, with the mucosal epithelium acting as a barrier to prevent the free movement of xenobiotics, including drugs, from the outside to the inner body across the epithelial cell sheets. To improve the absorption of orally administered drugs, modulators of this intestinal barrier, called intestinal permeation enhancers, are being developed. The targets for these permeation enhancers are the apical membrane and the intercellular seals, or tight junctions (TJs).

Intestinal permeation enhancers are classified into first-generation absorption enhancers, which have transcellular permeation activity, and second-generation absorption enhancers, which achieve paracellular permeation by loosening TJs.¹⁾ Although the first-generation enhancers salcaprozate sodium and sodium caprate are in clinical use in oral formulations of glucagon-like peptide 1 analogs and octreotide, respectively,²⁾ second-generation enhancers have so far only been investigated in small-animal models, and not in non-human primates.

TJs seal the intercellular spaces between adjacent cells. An electron microscopic analysis revealed that TJs comprise distinct bicellular and tricellular types, the components of which form an anastomosing network consisting of strands of intramembranous particles in the lateral membrane.³⁾ TJs have a complex molecular composition comprising various proteins including claudins, TAMP (tight junction-associated MARVEL proteins, which are occludin, tricellulin and MARVELD3), angulins, junctional adhesion molecules (JAMs), and members of the zonula occludens family (ZOs).^4,5) Occludin, JAMs, claudins, and MARVELD3 are transmembrane proteins in bicellular TJs, and tricellulin and angulins are transmembrane proteins in tricellular TJs.⁵⁾

Of these components, claudins are the most attractive target for developing second-generation absorption enhancers because claudins have solute selectivity, ensuring that only the selected molecules are able to pass through the paracellular barrier, and tissue specificity, which ensures that the barrier functions according to the specific needs of the particular tissue.^1,5–8) In mammals, the claudin family comprises 27 tetra-transmembrane proteins⁸⁾ whose expression profiles differ between tissues. These properties of solute selectivity and tissue specificity mean that claudin-targeted permeation enhancers are gaining attention as a drug delivery platform for safe and effective non-invasive oral administration of biologics.^1,5,6)

In rats, claudin-3 and -4 binders enhance the absorption of macromolecules in the jejunum but not the colon.⁹⁾ Immunoblot analysis has revealed heterophilic expressions of claudin-2, -3, -4, and -5 in the duodenum, jejunum, ileum, and colon of rats.¹⁰⁾ Other research in rats has shown that the expression profiles of claudins correlate with intestinal barrier tightness in the following ways: claudin-1, -3, -4, -5, and -8, which all tighten the TJ seal, are expressed more highly in the colon than in the small intestine (duodenum, jejunum, and ileum) and intestinal barrier integrity is also stronger in the colon than in the small intestine; there is a similar correlation between heterogenous expression of claudins and barrier integrity in the duodenum, jejunum, and ileum.¹¹⁾

In addition to claudins, members of the angulin family (angulin-1, -2, and -3) could be other potential targets for intestinal permeation enhancement. Angulin-1 and -3 binders enhanced jejunal absorption in rats.¹²⁾

A comprehensive understanding of the heterogenous expression of bicellular and tricellular TJ components is important for the development of safe and effective TJ-targeted permeation enhancers. In this study, we used biopsy samples of mucosa showing no endoscopically visible inflammation and conducted a comprehensive investigation of the mRNA expression profiles of TJ components in the human intestine.

MATERIALS AND METHODS

Intestinal Biopsy

This study was approved by the institutional review boards at Sapporo Medical University and Osaka University (Yakuhito 2019-3). We obtained written informed consent from all participants. This study was performed in accordance with the Declaration of Helsinki and the Human Ethical Guidelines of the Ministry of Health, Labor, and Welfare of Japan. Eligibility criteria were (1) a confirmed diagnosis of Crohn’s disease or ulcerative colitis according to Japanese clinical guidelines¹³⁾ and (2) age of 20 years or more with adequate organ function. Patients currently using anticoagulants or antiplatelet agents were excluded. Patients with non-inflammatory bowel diseases (non-IBD) undergoing endoscopy for diagnosis or to investigate or rule out the presence of tumors were also recruited. Intestinal biopsy samples of normal-appearing mucosa showing no endoscopically visible inflammation were collected using cold biopsy forceps from the small intestine (duodenum, jejunum, and ileum) or the large intestine (colon, rectum) during upper gastrointestinal endoscopy, small intestinal endoscopy, or lower gastrointestinal endoscopy. From each participant we collected a single biopsy sample from one of these five regions of the intestinal tract. Each intestinal biopsy sample was immediately placed into a stabilizing agent (RNAlater Reagent; QIAGEN, Düsseldorf, Germany) and stored at −80 °C.

RNA Analysis

Total RNA from 61 fresh-frozen biopsies was isolated by QIAzol (QIAGEN) extraction after removal of the stabilizing reagent. The RNA samples were purified using an RNeasy Mini kit (QIAGEN). To evaluate the RNA quality, a BioAnalyzer (Agilent Technologies, CA, U.S.A.) and RNA 6000 nanochip (Agilent Technologies) were used to derive an RNA integrity number (RIN). Library construction for RNA-seq analysis was performed using a TruSeq Stranded Total RNA Library Prep kit (Illumina, CA, U.S.A.).

For sequencing, a HiSeq 4000 system and NovaSeq 6000 system (Illumina) were used to generate FASTQ files (Wingett and Andrews, 2018); then FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc), FASTX (http://hannonlab.cshl.edu/fastx_toolkit/), and FastQ Screen¹⁴⁾ were used to check the quality of the FASTQ files. Single-end reads (40–100 million per sample) from the FASTQ files were mapped against the Genome Reference Consortium Human Build 38 using the STAR aligner.¹⁵⁾ Quantification and TPM normalization (TPM, transcripts per million¹⁶⁾) were done by using Strand NGS 3.4 (Strand Life Sciences, Karnataka, India) on all sample sets.

The data are available at Gene Expression Omnibus (Accession Number: GSE215285).

Statistical Analysis

Data are expressed as the mean ± standard deviation (S.D.). Statistical analyses were performed by using the Dunn's multiple comparison test (Prism version 9, GraphPad Software, Boston, MA, U.S.A.).

RESULTS

Intestinal Biopsy Samples

We collected duodenum samples from 11 subjects, jejunum samples from 3, ileum samples from 15, colon samples from 27, and rectum samples from 5; the mean age of the subjects in each group was 44, 61, 34, 37, and 54, respectively (Table 1). All subjects were Japanese. The RNA samples extracted from the biopsy samples were confirmed to almost all be of high quality (mean RIN >7; Table 2).

Table 1. Demographics and Baseline Characteristics of Study Subjects

Segment sampled	Number of subjects	Mean age in years (min–max)	Sex [n (%)]		Type of disease
			Female	Male	Non-IBD	Crohn’s disease	Ulcerative colitis
Duodenum	11	44 (20–74)	3 (27)	8 (73)	5	5	1
Jejunum	3	61 (35–78)	0 (0)	3 (100)	3	0	0
Ileum	15	34 (23–62)	2 (13)	13 (87)	0	9	6
Colon	27	37 (21–71)	11 (41)	16 (59)	3	8	16
Rectum	5	54 (31–72)	0 (0)	5 (100)	1	1	3

Non-IBD: Non-inflammatory bowel disease.

Table 2. Quality of RNA Extracted from Biopsy Samples

Segment	Mean RIN (min–max)
Duodenum	7.9 (7.4–8.4)
Jejunum	8.6 (8.5–8.8)
Ileum	8.0 (7.3–8.7)
Colon	8.0 (4.4–9.1)
Rectum	8.1 (7.6–8.8)

RIN, RNA integrity number.

Expression Profiles of Bicellular TJ Components:

Claudin Family

We investigated the expression of claudins-1 to -25 in biopsied samples from each intestinal segment. Claudin-1, -5, -6, -9, -10, -11, -14, -16, -17, -18, -19, -20, -22, -24, and -25 were slightly expressed in all intestinal segments (Supplementary Fig. S1). Claudin-12 showed similar expression levels in all the intestinal segments (Fig. 1A). Expression levels of claudin-3, -4, -7, -8, and -23 each became progressively more elevated from the duodenum in the upper tract to the rectum in the lower tract (Fig. 1A). Although claudin-2 and -15 were expressed in the upper tract (duodenum, jejunum, and ileum), they were decreased in the lower tract (colon and rectum) (Fig. 1A, Supplementary Fig. S1).

Fig. 1. Expression Profiles of the Claudin Family Components of Bicellular Tight Junctions in the Human Intestine

(A) Comparison of mRNA expression levels of the claudin family along the intestinal tract. Expression of mRNA was investigated in biopsy samples of mucosa with no endoscopically visible inflammation collected from the duodenum, jejunum, ileum, colon, and rectum. Data are means ± S.D. Significant difference between the indicated intestinal segments (* p < 0.05, ** p < 0.01, *** p < 0.001). (B) Proportion of mRNA expressed by each member of the claudin family in each intestinal segment.

Heterogeneity in claudin expression also differed between segments (Fig. 1B). The five most highly expressed claudins in each intestinal segment (as a percentage of the expression level of all claudin members in each segment) were: claudin-7 (35.3%), claudin-15 (17.2%), claudin-4 (13.8%), claudin-2 (8.8%), and claudin-12 (6.8%) in the duodenum; claudin-7 (45.2%), claudin-15 (18.0%), claudin-4 (12.1%), claudin-3 (8.5%), and claudin-12 (5.9%) in the jejunum; claudin-7 (38.1%), claudin-4 (15.1%), claudin-15 (13.5%), claudin-3 (8.9%), and claudin-2 and -12 (each 6.6%) in the ileum; claudin-7 (41.6%), claudin-4 (21.1%), claudin-3 (9.9%), claudin-23 (9.3%), and claudin-8 (7.1%) in the colon; and claudin-7 (33.3%), claudin-8 (22.2%), claudin-4 (19.1%), claudin-3 (10.8%), and claudin-23 (6.5%) in the rectum (Fig. 1B).

Occludin, JAM Family, and ZO Family

The other transmembrane proteins composing the bicellular TJ seal are occludin and JAM-A, -B, and -C.⁵⁾ Although expression levels of occludin, JAM-B, and JAM-C were stable throughout the intestinal segments, JAM-A expression was lower in the jejunum than in other segments (Fig. 2A). By proportion, the expression of JAM family members was: JAM-A (83.8%), JAM-B (7.7%), and JAM-C (8.6%) in the duodenum; JAM-A (71.1%), JAM-B (8.7%), and JAM-C (20.2%) in the jejunum; JAM-A (86.8%), JAM-B (5.0%), and JAM-C (8.1%) in the ileum; JAM-A (83.3%), JAM-B (6.6%), and JAM-C (10.1%) in the colon; and JAM-A (85.6%), JAM-B (4.8%), and JAM-C (9.6%) in the rectum (Fig. 2B).

Fig. 2. Expression Profiles of the Bicellular Tight Junction Components Occludin, Zonula Occludens (ZO) Family, and Junctional Adhesion Molecule (JAM) Family in the Human Intestine

(A) Comparison of mRNA expression levels of occludin, ZO family, and JAM family in the duodenum, jejunum, ileum, colon, and rectum. Data are means ± S.D. Significant difference in expression level between the indicated intestinal segments (* p < 0.05, ** p < 0.01). (B, C) Proportion of mRNA expression level in each member of the JAM family (B) or the ZO family (C) in the duodenum, jejunum, ileum, colon, and rectum. Numbers given after each protein name denote the proportion of expression of each bicellular TJ component, calculated as a percentage of the expression level of all bicellular TJ components.

Expression levels of ZO-1 and -2 did not change along the intestinal segments. ZO-3 expression was higher in the lower segments than in the upper segments (Fig. 2A). The proportional expression of ZO family members was: ZO-1 (34.1%), ZO-2 (35.3%), and ZO-3 (30.6%) in the duodenum; ZO-1 (29.6%), ZO-2 (31.7%), and ZO-3 (38.7%) in the jejunum; ZO-1 (33.8%), ZO-2 (33.0%), and ZO-3 (33.2%) in the ileum; ZO-1 (33.8%), ZO-2 (27.4%), and ZO-3 (38.8%) in the colon; and ZO-1 (33.2%), ZO-2 (26.6%), and ZO-3 (40.2%) in the rectum (Fig. 2C).

Expression Profiles of Tricellular TJ Components:

Tricellulin and the Angulin Family

Angulin-3 was very slightly expressed along the intestinal tract (Fig. 3A). The tricellulin expression level was similar in all regions of the intestine. In contrast, expression levels of angulin-1 and -2 were higher in the lower tract than in the upper tract. The proportions for each tricellular TJ component out of all tricellular components were tricellulin (52.1%), angulin-1 (37.9%), angulin-2 (9.1%), and angulin-3 (0.9%) in the duodenum; tricellulin (75.3%), angulin-1 (18.3%), angulin-2 (5.3%), and angulin-3 (1.1%) in the jejunum; tricellulin (53.6%), angulin-1 (37.6%), angulin-2 (8.0%), and angulin-3 (0.8%) in the ileum; tricellulin (39.6%), angulin-1 (40.0%), angulin-2 (19.0%), and angulin-3 (1.4%) in the colon; and tricellulin (31.0%), angulin-1 (48.6%), angulin-2 (20.0%), and angulin-3 (0.5%) in the rectum (Fig. 3B). The proportion of tricellulin expression in the lower intestine was lower than that in the upper intestine. In contrast, the proportions of angulin-1 and -2 expression were increased in the lower intestine compared to those in the upper intestine.

Fig. 3. Expression Profiles of the Tricellular Tight Junction Components Tricellulin and Angulin Family Members in the Human Intestine

(A) Comparison of mRNA expression levels of tricellulin and angulin family members in the segments of the human intestine. Data are means ± S.D. Significant difference between the indicated intestinal segments (* p < 0.05, ** p < 0.01, *** p < 0.001). (B) Proportion of each tricellular component’s mRNA expression level out of all tricellular TJ components, shown as a number after each name.

It is reported that expression of some tight junction proteins is altered in active IBD. Claudin-1, -2, and -18 proteins are reported to be increased in Crohn’s disease and ulcerative colitis, and claudin-3, -4, and -7 proteins are reported to be decreased.¹⁷⁾ Angulin-1 mRNA is downregulated in active Crohn’s disease but is upregulated in controlled Crohn’s disease and in cases in remission.¹⁸⁾ Although all samples in our study were of normal-appearing mucosa with no endoscopically visible inflammation, our samples were collected from patients with non-IBD, Crohn’s disease, and ulcerative colitis, which might possibly influence our findings. To evaluate the validity of our results, we compared the mRNA expression profiles of a number of TJ components grouped by disease type. The levels of claudin-7 mRNA expression in ulcerative colitis were lower than in non-IBD, but there were no significant differences in the levels of claudin-1, -2, -3, -4, and -18, and angulin-1 mRNA expression in the colon between patients with non-IBD, Crohn’s disease, and ulcerative colitis (Figs. 4A, B). These data suggest that the present data on biopsy samples are useful for understanding expression profiles of bicellular and tricellular TJ components in the human intestine, despite them being collected from patients with non-IBD, Crohn’s disease, and ulcerative colitis.

Fig. 4. Comparison of Expression Profiles of Bicellular and Tricellular Components in the Human Intestine of Patients with Non-IBD, Crohn’s Disease, and Ulcerative Colitis

(A) mRNA expression levels of claudin family members and (B) mRNA expression levels of other bicellular and tricellular TJ components: occludin, tricellulin, zonula occludens (ZO) family, junctional adhesion molecules (JAM), and angulin family. Expression of mRNA was investigated in biopsy samples of mucosa showing no endoscopically visible inflammation collected from the duodenum, jejunum, ileum, colon, and rectum. Data are means ± S.D. Significant difference between disease type (* p < 0.05).

DISCUSSION

Claudins were the first of the TJ components to gain attention as promising targets for the development of permeation enhancers for oral administration of chemicals and biologics, because combinations of the 27 members of the claudin family achieve tissue-specific functions in the paracellular barrier and solute-selective paracellular transport.^1,7,19) As mentioned earlier, in rats, claudin-3 and -4 binders enhance the absorption of macromolecules in the jejunum but not in the colon.⁹⁾ Subsequent research in rats showed that a broadly specific claudin binder has more potent jejunal absorption–enhancing activity than the claudin-3 and -4 binders.²⁰⁾ Claudin binders are bicellular TJ permeation-enhancers.

The second component to gain attention was the angulin family, whose members have been focused on as targets for the development of a tricellular TJ-permeation enhancer. Angubindin-1, which was developed as the first tricellular TJ modulator, binds to angulin-1 and -3 to enhance jejunal absorption of a macromolecule in rat.¹²⁾

The expression profiles of some claudins have been investigated in the intestinal tracts of mice, rats, and humans,^{10,11,17,21–25)} and expression profiles of the angulin family were investigated in mice.²⁶⁾ However, there is still no available information on the comprehensive gene expression patterns of bicellular and tricellular TJ components in the human intestinal tract.

In the present study using biopsy samples of mucosa from the human intestine showing no endoscopically visible inflammation, we found heterogenous expressions of claudins and angulins in the human duodenum, jejunum, ileum, colon, and rectum. In this study, comparison of the upper (duodenum, jejunum, and ileum) and the lower (colon and rectum) regions of the intestine showed that the expression levels of claudin-3, -4, -7, -8, and -23 were higher in the lower segments than in the upper segments. Although angulin-1 and -2 were more highly expressed in the lower segments (colon and rectum) than in the upper segments (duodenum, jejunum, and ileum), angulin-2 showed a more specific expression in the colon and rectum compared to angulin-1. Angulin-3 exhibited slight expression in all segments. Previous research has shown that mannitol permeability is higher in the small intestine than in the colon in humans.²⁷⁾ Claudin-3 and -4 are known to be barrier-forming claudins.⁵⁾ A deficiency of claudin-7 in the intestine enhanced the permeation of a small molecule (457 Da) but not a macromolecule (4400 Da) in the colon.²⁸⁾ Claudin-8 prevented paracellular permeation of sodium²⁹⁾ and was also found to be decreased in the intestine of patients with Crohn’s disease showing an impaired intestinal barrier.³⁰⁾ Although claudin-23 is down-regulated in colorectal cancer compared to the levels in healthy colon tissue, it is unclear whether claudin-23 functions as an intestinal barrier.³¹⁾ Taken together, the accumulated knowledge on intestinal heterogenous expression of claudins-3, -4, -7, and -8 shows that these members of the claudin family may contribute to intestinal integrity in the intestine.

Expression of claudin-2 and -15 was lower in the lower segments (colon and rectum) than in the upper segments (duodenum, jejunum, and ileum). The upper and the lower tracts are the major sites for absorption of nutrients and water, respectively. Some intestinal glucose transporters, amino acid transporters, and a bile acid transporter work via the Na⁺ symport system,³²⁾ in which Na⁺ concentration in the intestinal lumen is important for regulation of these nutrient-related transporters. Claudin-2 and -15 function as paracellular transporters of Na⁺ from the submucosa to the intestinal lumen in mice.³³⁾ Our findings suggest that, similarly to the results found in animal experiments, claudin-2 and -15 might be essential for intestinal absorption of glucose, amino acids, and bile acid by paracellular transport of Na⁺ in humans.

Expression of claudin-2 protein is increased in the colon in mice,¹¹⁾ whereas claudin-2 mRNA expression was decreased in the colon in humans. Although claudin-5 protein is also higher in the colon than in the duodenum, jejunum, and ileum in rats,^10,11) the expression of claudin-5 mRNA was low throughout the intestinal tract in humans. Intestinal permeability of a macromolecule (4400 Da) is higher in rats than in humans.²⁷⁾ Mannitol permeability is higher in the small intestine than in the colon in humans but is higher in the ileum than in the jejunum and colon in rats.²⁷⁾ A possible explanation for the difference in expression profiles of some claudins between rats and mice and humans may be species differences.

Expression profiles of other bicellular TJ components, that is, occludin and the JAM family, were almost constant throughout the intestinal tract. Those of the ZO family, which are intracellular scaffold proteins in the TJ, were also similar throughout the tract. Regarding the tricellular TJ components, tricellulin was constantly expressed in all intestinal segments. Angulins are required for epithelial cells to form the intestinal barrier and are determinant factors for subcellular localization of tricellulin at points of tricellular contact.^26,34) Thus, expression profiles of angulin may reflect the involvement of tricellulin in maintaining intestinal barrier integrity at points of tricellular contact. Angulin might be a target for developing a tricellular TJ permeation enhancer.

In this study, we explored the heterogenic expression profiles of bicellular and tricellular TJ components in the human intestinal tract. If TJ components were suitably selected as targets for a drug, segment-specific absorption of the drug may be achieved. For instance, claudin-8, angulin-1, and angulin-2, which are highly expressed in the rectum, might be targets of intestinal permeation enhancers for a suppository. Claudin-2 and -15 could be potential targets for permeation enhancers for upper intestinal drug absorption. Claudin-7, occludin, and tricellulin may be targets for enhancing drug absorption in all segments. Broadly specific claudin-3, -4, and -7 modulators would be the strongest intestinal permeation enhancers. These findings on expression profiles of TJ components will be useful for future development of a safe and effective intestinal permeation enhancer.

Acknowledgments

This study was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan [Grants: 22K19936, 22H03953]; the Japan Agency for Medical Research and Development (AMED) [Grants: JP23ae0121042 and 23mk0121258]; and the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) of AMED [Grants: JP22ama121052 and JP23ama121054]. We are grateful to Drs. Denis Delic and Ramona Schmid (Boehringer Ingelheim Pharma GmbH & Co. KG) for the preparation and sequencing of a cDNA library and for bioinformatics support, including primary processing of the data and QC, respectively.

Conflict of Interest

Drs. Fujioka and Kishimoto are employees of Nippon Boehringer Ingelheim Co., Ltd. The other authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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