Intestinal Epithelial Tight Junction Barrier Regulation by Novel Pathways

Abstract

Intestinal epithelial tight junctions (TJs), a dynamically regulated barrier structure composed of occludin and claudin family of proteins, mediate the interaction between the host and the external environment by allowing selective paracellular permeability between the luminal and serosal compartments of the intestine. TJs are highly dynamic structures and can undergo constant architectural remodeling in response to various external stimuli. This is mediated by an array of intracellular signaling pathways that alters TJ protein expression and localization. Dysfunctional regulation of TJ components compromising the barrier homeostasis is an important pathogenic factor for pathological conditions including inflammatory bowel disease (IBD). Previous studies have elucidated the significance of TJ barrier integrity and key regulatory mechanisms through various in vitro and in vivo models. In recent years, considerable efforts have been made to understand the crosstalk between various signaling pathways that regulate formation and disassembly of TJs. This review provides a comprehensive view on the novel mechanisms that regulate the TJ barrier and permeability. We discuss the latest evidence on how ion transport, cytoskeleton and extracellular matrix proteins, signaling pathways, and cell survival mechanism of autophagy regulate intestinal TJ barrier function. We also provide a perspective on the context-specific outcomes of the TJ barrier modulation. The knowledge on the diverse TJ barrier regulatory mechanisms will provide further insights on the relevance of the TJ barrier defects and potential target molecules/pathways for IBD.

Key Messages.

• Inflammatory bowel disease (IBD) is associated with dysregulated intestinal TJ permeability.

• We reviewed and provided a perspective on the latest findings in the regulation of intestinal TJ barrier.

• The understanding of TJ barrier regulatory mechanisms will help targeting TJ barrier defects in IBD.

Intestinal Epithelial Tight Junction Composition and Function

Among the 3 main types of cell junctions in the intestinal epithelia, tight junctions (TJs), adherens junctions (AJs), and gap junctions (GJs), the most apically located TJs polarize the epithelial cell membrane into apical and basolateral regions (fence function) and regulate passive diffusion of solutes and macromolecules between adjacent cells (gate function).¹ The TJs are formed by an array of transmembrane proteins, such as occludin and claudins, which are linked to the cytoskeleton by cytoplasmic plaque protein zona occludens (ZO-1, -2, and -3). By forming a paracellular barrier, TJs serve as the first line of defense against paracellular permeation of noxious antigens into the epithelium and subepithelial host tissues.^2,3 The TJs consist of barrier-forming proteins, such as occludin, and claudin-1, -4, -8 etc., as well as channel-forming claudins, such as claudin-2 and -15 (Figure 1). Furthermore, the TJs are known to have 2 pathways: a small-size, cation-selective, high-capacity “pore” pathway and a large-size, non-charge-selective “leak” pathway. The pore pathway is largely regulated by the claudin proteins, in a charge- and size-selective manner, while leak pathway may be regulated by barrier-forming occludin.^4-6 The TJ barrier can be modified by distinct complexes formed by different claudins, as recently demonstrated by claudin-23-mediated regulation of claudin-3 and claudin-4 distribution in the TJs.⁷ Similarly, incorporation of an individual claudin family member into the structures and channels formed by other claudins has been shown as a new mechanism of barrier regulation. In this case, claudin-4, which minimally regulates the TJ permeability in the absence of pore-forming claudins, selectively inhibits flux and membrane localization of cation channel-forming claudin-2 and claudin-15.⁸

Figure 1.

The structure of TJs. The TJs consist of an array of transmembrane proteins, including pore-forming claudin-2 and -15; barrier-forming claudins, such as claudin-1, -3, -4, and -8; and barrier-forming occludin. These transmembrane proteins are linked to the actin cytoskeleton via cytoplasmic plaque proteins, such as ZO-1 and ZO-2. The intestinal TJ permeability is defined by 2 distinct pathways. The pore pathway, mediated by claudin-2 and -15, allows movement of cations and small solutes of up to 0.6 nm diameter. The leak pathway is thought to be regulated by occludin and allows non-charge selective passage of solutes of up to 12.5 nm diameter.⁴ This figure was generated using BioRender. Abbreviations: TJ, tight junction; ZO, zona occluden.

The intracellular vesicular membrane transport, a key process in the formation of TJ domains, provides an interactive and dynamic nature to the TJ barrier, which is essential for the physiological functions of TJs.^9,10 The TJ complex adopts to the physiological needs via constant remodeling and intracellular trafficking, where TJ proteins are continuously inserted and internalized from the membrane.¹¹ The selective nature of TJ permeability allows for absorption of nutrients and water while restricting the bulk movement of luminal contents. Under physiological conditions, the paracellular transport of solutes and water is coordinated with transcellular transport, particularly through myosin light chain kinase (MLCK)-mediated rearrangement of perijunctional actin cytoskeleton.^12,13

Intestinal TJ Barrier in Systemic and Intestinal Diseases

In humans, increased intestinal permeability or TJ barrier dysfunction is associated with distinct diseases, including metabolic, autoimmune, neurologic, allergic, and infectious conditions (recently reviewed by Horowitz et al¹⁴), including the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.¹⁵ Furthermore, in severe, life-threatening, multisystem inflammatory syndrome in children (MIS-C), presence of SARS-CoV-2 in the gastrointestinal tract was associated with increased intestinal permeability, detected by systemic presence of zonulin. Interestingly, larazotide, a zonulin antagonist, reduced antigenemia and inflammatory markers and improved the patient’s clinical response,¹⁶ indicating the importance of intestinal TJ barrier in systemic illness. Accordingly, intestinal permeability markers have been reported to be elevated systemically in patients with celiac disease,¹⁷ non-celiac, type I diabetes mellitus,¹⁸ systemic lupus erythematosus,¹⁹ multiple sclerosis,²⁰ Alzheimer’s disease,²¹ Parkinson’s disease,²² schizophrenia,²³ rheumatoid arthritis,²⁴ irritable bowel syndrome,²⁵ necrotizing enterocolitis,²⁶ metabolic dysfunction-associated steatotic liver disease,²⁷ etc. Gut dysbiosis, neurogenic mechanisms, inflammation, stress, infections, and metabolism are some of the major factors postulated to be the mediators of increased intestinal permeability in these various disease conditions.

Loss of intestinal TJ barrier, whether causative or resultant, is a key feature of inflammatory bowel disease (IBD). In a large prospective study, increased intestinal permeability, as early as 3 years before the clinical diagnosis, was found to be associated with the later development of Crohn’s disease (CD).²⁸ In clinical IBD studies, persistent increase in intestinal permeability due to defective TJ barrier was found to be predictive of poor clinical outcome, and normalization of intestinal permeability correlated with long-term clinical remission.^29,30 Similarly, in animal models of IBD, the increase in intestinal permeability preceded intestinal inflammation, whereas therapeutic enhancement of intestinal TJ barrier prevented the intestinal inflammation.^31,32 Experimentally, however, an increase in TJ barrier permeability can be both protective against or promote susceptibility to colitis depending on the context.^33-35 For instance, channel-forming claudin-2 promotes immune-mediated experimental colitis,³⁶ while it is protective against infectious and chemical-induced experimental colitis.^35,37 The physiological need of epithelial paracellular communication for the appropriate gut immune response is also demonstrated by a study where a transient breach in epithelial paracellular permeability promoted adaptive immunity and T-reg development.³⁸ On the other hand, the TJs in follicle-associated epithelium (FAE) covering Payer’s patches are remarkably impermeable on account of increased expression of barrier-forming claudins. This makes an epithelial transcellular route a primary way of antigen presentation to underlying immune cells.³⁹ This selective physiological mechanism is turned into a pathological mechanism when claudin-4 enriched at the apex of FAE TJs becomes a target for Clostridium perfringens enterotoxin.^40,41

TJ Regulation by Ion Transporters

The ion transporters and ion channels are integral components of intestinal epithelial cells, and thus, their role in the TJ permeability has been investigated for a while. Beyond the previously shown role of the transporters, such as chloride channel ClC-2 in the intracellular transport of TJ components,^42-44 sodium hydrogen exchanger 3 (NHE3) and Na⁺-glucose cotransporter 1 (SGLT-1) in the TJ-associated cytoskeletal rearrangement,⁴⁵ and Na⁺, K⁺-ATPase in the scaffolding of TJ-related proteins,⁴⁶ new mechanisms were recently unveiled. In a recent study, genetic deletion of the oxalate-secreting intestinal transporter putative anion transporter 1 (PAT1) in mice (Slc26a6^−/−) impaired intestinal TJ barrier and increased susceptibility to experimental colitis, associated with gut dysbiosis, changes in microbial metabolites, and alteration in TJ gene expression.⁴⁷ It is remarkable that the patients with IBD have increased risk of hyperoxaluria and nephrolithiasis,⁴⁸ emphasizing the connection between gut microbial metabolites and the TJ barrier demonstrated by this study with Slc26a6^−/− mice.⁴⁷ In an in vivo model of alcohol-associated endotoxemia, transient receptor potential cation channel subfamily V member 6 (TRPV6)-mediated Ca2⁺ influx and Src activation induced TJ disruption. Moreover, H185 residue in the ankyrin repeat domain 4 (ARD4) of hTRPV6 was identified as a potential alcohol-binding site in TRPV6, indicating direct targeting of a ion channel in ethanol-induced TJ barrier disruption.⁴⁹ Among the genetic mechanisms that regulate the TJ barrier, RNA-binding proteins CUG triplet repeat, RNA-binding protein 1 (CUGBP1) and HuR competitively bind to occludin mRNA to regulate the expression of occludin.⁵⁰ Such posttranscriptional regulation of TJ barrier was also evident in mice deficient in chloride anion exchanger downregulated in adenoma (DRA) protein (encoded by Slc26a3 gene). The Slc26a3^−/− mice have diminished TJ barrier on account of the increased binding of RNA-binding protein CUGBP1 with occludin and E-cadherin mRNAs, leading to decreased protein levels.⁵¹ Also, interleukin-33 (IL-33)-driven type 2 immune response was found to be altered in Slc26a3^−/− mice colon, indicating an alteration in epithelial-immune cell crosstalk accompanying the TJ barrier dysfunction.⁵²

Genetic Regulation of TJs

In transcriptional regulation of the TJ barrier, GATA-binding protein 6 (GATA6), a transcription factor which regulates epithelial-to-mesenchymal transition (EMT) and tumor dissemination,⁵³ promotes ZO-1 expression by directly binding to ZO-1 promoter.⁵⁴ As a result, epithelial-specific deletion of GATA6 leads to the TJ barrier disruption and exaggerates inflammation in colitis models.⁵⁴ Also, loss of protein tyrosine phosphatase non-receptor type 2 (PTPN2) function, which is known to be associated with increased risk of IBD, affects intestinal TJ barrier. The loss-of-function PTPN2 rs1893217 single nucleotide polymorphism (SNP) was found to be associated with increased intestinal claudin-2 expression in patients with IBD, while the T-cell protein tyrosine phosphatase (TCPTP) encoded by PTPN2 protected against cytokine-induced disruption of both pore and leak TJ pathways.⁵⁵ The TCPTP limited claudin-2 levels by inhibiting Signal Transducer and Activator of Transcription 1 (STAT1)-mediated transcription of claudin-2 gene (CLDN2) and additionally by inhibiting hepatocyte growth factor activator inhibitor-1 (HAI-1)-mediated suppression of transcription of matriptase (ST14), which is involved in the regulation of claudin-2 endocytosis from the membrane for proteasomal degradation.⁵⁵

In recent years, noncoding RNAs have been shown to have several biological functions, including TJ regulation. Micro RNAs (miRNAs) bind to the target mRNAs to delay transcription or degrade the mRNAs. The miRNAs such as miR21, miR212a, miR874, miR122a, and miR200c-3p have been associated with occludin degradation and are upregulated in IBD.^56-63 Studies have also shown that miR122a and miR200c are upregulated in response to tumor necrosis factor-α (TNF-α) and IL-1β signaling, further tying occludin expression to inflammatory stimuli and giving perspective into intestinal TJ barrier loss in IBD.^62,63 On the other hand, miRNA-16 was shown to inhibit the inflammatory TLR4/NF-κB pathway and promote TJ integrity in a mouse model of irritable bowel syndrome with diarrhea.⁶⁴ The miRNAs are in turn regulated by long noncoding RNAs (lncRNAs) in the modulation of target gene expression. The lncRNA uc.173 prevents interaction of miR-29b with CLDN1 mRNA to maintain claudin-1 expression.⁶⁵ In another mechanisms, a RNA-binding protein, HuR, inhibits lncRNA H19-mediated biogenesis of miR-675 and prevents degradation of ZO-1 and E-cadherin mRNA.⁶⁶ HuR also mediates the function of lncRNA SPRY4-IT1 in maintaining the intestinal TJ barrier via promoting translation of claudin-1, claudin-3, occludin, and JAM-1 genes.⁶⁷ Another RNA-binding protein, Lin 28A, however, directly binds to occludin mRNA 3ʹ untranslated region (UTR) and represses the translation of occludin transcripts.⁶⁸ The regulation of TJ barrier by noncoding RNAs, thus, presents an important mode of TJ regulation which affects various barrier organ systems and is relevant for a variety of genetic diseases.

Cytoskeletal and Extracellular Matrix Regulation of TJ Barrier Regulation

The integrity of the epithelial architecture is regulated by the actomyosin cytoskeleton, which also serves as a critical regulator of intercellular junctions and cell-matrix adhesions. The perijunctional belt, made up of actin and nonmuscle myosin, maintains TJ and AJ integrity and the paracellular barrier, while the actin-cytoskeletal-mediated focal adhesions largely manage interactions with extracellular matrix (ECM), cell polarity, and cell migration. The importance of nonmuscle myosin II (NM-II) in epithelial barrier integrity was emphasized by a recent study on UNC-45A, a cytoskeletal chaperone that is essential for proper folding of NM-II heavy chains and myofilament assembly.⁶⁹ Absence of UNC-45A impaired assembly of epithelial AJs and TJs, attenuated cell migration, and increased in vivo intestinal permeability, indicating UNC-45A as a novel regulator of intestinal epithelial barrier integrity and repair. Beside actomyosin, the microtubule cytoskeleton is also involved in the gut barrier dysfunction as evident in the gastrointestinal symptoms and diarrheal illnesses caused by preformed toxins produced by Bacillus cereus. These toxicities are now shown to be caused by a pore-forming toxin, alveolysin, via CD59 and activation of PI3K-AKT signaling which increases cilia- and flagella-associated protein 100 (CFAP100).⁷⁰ CFAP100, a microtubule-interacting protein, disrupts TJs and AJs, explaining the gastrointestinal symptoms as well as the systemic illness due to leakage of bacteria through the intestinal barrier.⁷⁰ The PI3K-AKT signaling also links desmosomes to the TJs. The intestinal desmosomal cadherin, Desmoglein 2 (Dsg2), has been shown to sequester PI3K at the cell membrane, and loss of Dsg2 during inflammation promotes the activation of PI3-kinase and AKT phosphorylation, resulting in the upregulation of claudin-2.⁷¹ RAB11A, a small GTPase involved in intracellular trafficking, was previously known to be part of recycling endosomes.⁷² A recent study shows that RAB11A also regulates associations of Yes-associated protein (YAP), a Hippo-signaling transducer involved in cell-cell contact and growth, with AJs and TJs.⁷³ Rab11A appears to be important for the balance between cellular proliferation and differentiation as its deficiency in mouse colonic epithelia led to increased nuclear localization of YAP, disorganized colonic crypts, and reduced goblet cells during the recovery from dextran sodium sulfate (DSS) colitis.

The studies on MLCK1 has been instrumental in defining the role of the cytoskeleton in the TJ barrier in the physiological^12,74,75 as well as pathological context.^33,76,77 Epithelial-specific long MLCK1 via its perijunctional recruitment leads to actomyosin contraction-dependent endocytosis of occludin and an increase in TJ barrier permeability that models action of several proinflammatory cytokines.^78-81 In a recent study from Dr. Turner’s laboratory, the immunoglobulin-cell adhesion molecule domain 3 (IgCAM3) of MLCK1 was shown to facilitate its recruitment to perijunctional region, and a small molecule that binds to the IgCAM3 region, called divertin, prevents leak pathway permeability by inhibiting MLCK1-mediated MLC phosphorylation.⁸² Moreover, FK506-binding protein 8 (FKBP8) was also identified as a MLCK1-binding partner such that the interactions between MLCK1 and FKBP8 mediates TNF-α-induced perijunctional MLCK1 recruitment and consequent TJ barrier loss.⁸³

Several matrix metalloproteinases (MMPs) by their ability to degrade ECM helps in tissue remodeling and wound healing. However, in the event of excessive tissue damage and persistent inflammation, the MMPs can be aberrantly expressed, cleave cell surface receptors, activate cytokines, and degrade junctional proteins.^84-86 We have previously shown that MMP-9, a gelatinase, increases mouse colonic TJ permeability and severity in DSS colitis. On the other hand, MMP-9 knockout (KO) mice have significant reduction in the disease activity and TJ permeability, along with the reduction in DSS-induced expression and activity of MLCK.⁸⁷ In a recent study, we showed that MMP-12, a macrophage elastase, degrades laminin in the basement membrane and enables transmigration of macrophages while increasing the paracellular permeability. Thus, during experimental colitis in wild-type mice, macrophages were seen across the basement membrane, in the proximity of apical TJs, and the severity of experimental colitis was reduced in MMP-12 KO mice.⁸⁸ Similarly, MMP-7 was shown to target claudin-7, but not claudin-1, -8, or -15, to increase the TJ permeability and the severity of DSS and 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis.⁸⁹ The levels of these proinflammatory MMP-7, -9, and -12 are consistently increased in ulcerative colitis (UC) tissue^87-89; however, further studies are needed to understand their role in wound healing and tissue remodeling vs intestinal inflammation. Just as several MMPs are now known to be produced by epithelial cells, human colonic epithelial cells are also source of a elastase, Elastase 2 (ELA2), whose levels are increased in IBD tissue.⁹⁰ ELA2 increases epithelial permeability via direct cleavage of the TJ protein occludin and the AJ protein E-cadherin. It also promotes inflammation via NFκB-mediated upregulation of a chemokine, C-X-C motif chemokine ligand 8 (CXCL8).⁹⁰ Interestingly, MMP-12 mRNA and protein levels were decreased in CD patients who responded to anti-TNF-α antibody, and MMP-12 downregulation was accompanied by a simultaneous improvement of the histological score.⁹¹ Thus, even though MMPs are needed for clearance of injured tissue, degradation of provisional matrix, and re-epithelization,⁹² excessive levels of MMPs may be harmful during the chronic healing process.

Role of Aryl Hydrocarbon Receptor and Nuclear Factor Erythroid 2-Related Factor 2 Pathways in TJ Regulation

We recently showed that alpha-tocopherylquinone (TQ), a quinone-structured oxidation product of vitamin E, enhances the intestinal TJ barrier by increasing barrier-forming claudin-3 and reducing channel-forming claudin-2 in Caco-2 cell monolayers, mouse models, and also human colonic mucosa.⁹³ TQ reduced colonic permeability and ameliorated colitis symptoms in multiple colitis models including chronic DSS colitis, TNBS colitis, and T-cell transfer-mediated colitis. TQ, as a quinone compound, bi-functionally, activates both aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor 2 (Nrf2) pathways. As an environmental sensor, AhR, binds to a variety of synthetic and natural compounds and acts as a class I, basic helix-loop-helix transcriptional regulator.⁹⁴ Upon activation, AhR translocates into the nucleus and dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein. This AhR-ARNT dimer binds to the xenobiotic response elements (XREs; also known as dioxin response element [DRE]) in gene regulatory regions and regulates the expression of a diverse set of genes.⁹⁵ Earlier studies have shown important functions of AhR in intestinal stem cell differentiation, intestinal homeostasis, and immune regulation in the gut.^96,97 In our studies, TQ-induced AhR activation transcriptionally increased claudin-3 levels via XRE in the CLDN3 promoter. Conversely, TQ suppressed claudin-2 expression via Nrf2-mediated STAT3 inhibition. Nrf2 is a transcription factor that binds the antioxidant response element (ARE) to regulate the expression of genes involved in cellular defense against oxidative or electrophilic stress. In addition, the Keap1 (Kelch-like ECH-associated protein)/Nrf2/ARE signaling pathway is reported to inhibit inflammation by regulating anti-inflammatory gene expression.⁹⁸ STAT3 is reported to regulate CLDN2 expression via a STAT binding site in its proximal promoter region,⁹⁹ while STAT3 activation is controlled by Nrf2 via small heterodimer partner (SHP), which acts as a corepressor of STAT3 transcriptional activity.¹⁰⁰ We showed that TQ reduces phospho-STAT3 levels and decreased nuclear accumulation of STAT3 and its abundance in the CLDN2 promoter. Furthermore, TQ also increased the levels of STAT3 corepressor, SHP, resulting in the reduction in CLDN2 transcript levels. Overall, TQ enhances TJ barrier by differentially reducing channel-forming claudin-2 and increasing barrier-forming claudin-3.⁹³

AhR has been shown to play a critical role in several homeostatic functions including intestinal differentiation, stem cell homeostasis, and maintenance of gut microbiome.^96,101 Thus, AhR activation by various agents has been shown to be protective against intestinal TJ barrier disruption and experimental colitis,^102-104 while AhR deficiency has been shown to cause increased susceptibility to experimental colitis.^105,106 In line with the previous reports of modulation of AhR signaling being ligand- and tissue-specific,¹⁰⁷ we found TQ’s dual effect of claudin-2 reduction and increase in claudin-3 to be unique compared to other established AhR activators.⁹³ Consistent with being a master regulator of oxidative stress, Nrf2 has pro-barrier and anti-inflammatory functions.^98,108 Thus, Nrf2 KO mice have increased susceptibility to oxidative damage and inflammation.¹⁰⁹ The gut barrier protective function of Nrf2 pathway has also been demonstrated in various models such as traumatic brain injury¹¹⁰ and chronic kidney disease models,¹¹¹ and in esophageal epithelium.¹¹² Moreover, compounds such as coffee, prenylated xanthones from mangosteen, and Urolithin A, a microbial metabolite derived from fruit polyphenolics, have been reported to protect intestinal TJ barrier via dual activation of AhR and Nrf2 pathways.^103,113,114

Beyond the effect of TQ on the intestinal TJ barrier, we have also shown that TQ-mediated activation of the AhR in immune cells regulates the production of inflammation-inducing cytokines which are also known to induce TJ barrier disruption.¹¹⁵ Specifically, TQ reduced IL-1β, TNF-α, IL-6, and IL-17A production in macrophages by inhibiting the activation of the NFĸB and STAT3 pathways, in an AhR-dependent manner. TQ also uniquely inhibited the IL-6 signaling axis in T cells and reduced Th17 differentiation.¹¹⁵ Thus, TQ reduces inflammation by congruent effect on intestinal epithelial and immune cells. Overall, our studies suggested that TQ offers a naturally occurring, nontoxic intervention for enhancement of the intestinal TJ barrier. Consistent with these findings, a large number of in vitro, in vivo, animal, and human studies indicate the role of micronutrients in maintaining and healing of intestinal TJ barrier and thus a potential for using these micronutrients as therapeutic adjuvants based on their cell- and tissue-specific beneficial effects on the TJ barrier and cellular health.¹¹⁶ In this regard, vitamin D has been shown to ameliorate disruption of TJ proteins in UC tissue.¹¹⁷ Moreover, the role of vitamin D receptor (VDR) and its ligands in the homeostasis of intestinal barrier has been extensively studied (please see Sun and Zhang¹¹⁸).

Role of Autophagy in TJ Barrier

Autophagy, an intracellular degradation pathway, refers to the engulfment and processing of cellular proteins, including damaged organelles and long-lived and misfolded proteins.¹¹⁹ In a well-coordinated, multistep process of macroautophagy (referred as autophagy hereafter) that includes initiation (formation of the phagophore mediated by 2 key protein kinase complexes, ULK1/2-ATG13-RB1CC1/FIP200 complex and the BECN1/beclin-1-PIK3C3/Vps34-PIK3R4/Vps15- ATG14 complex), expansion of the phagophore (involving ubiquitin-like proteins ATG12 and GABARAP/LC3), autophagosome formation (closure of phagophore which requires lipidation of cytosolic LC3-I to LC3-II), and the fusion of autophagosome with the lysosome to form an autolysosome, the cargo proteins are selectively degraded.¹²⁰ Autophagy has been shown to be associated with preservation of TJ barrier under varied disease conditions, including oxidative stress-induced gastric mucosal damage,¹²¹ oxygen-glucose deprivation/reoxygenation (OGD/R) injury in an ischemia-reperfusion (I/R) model,¹²² and diabetic retinopathy.¹²³ We have previously shown that autophagy profoundly enhances intestinal TJ barrier function.⁵ Induction of autophagy in human intestinal Caco-2 cells by starvation or mammalian target of rapamycin (mTOR) inhibitor rapamycin or PP242 treatment caused reduction in ion permeability, indicating selective autophagy targeting of the TJ pore pathway. Furthermore, the levels of the channel-forming claudin-2 were reduced markedly after starvation or rapamycin treatment and claudin-2 was increasingly targeted for the lysosomal degradation. Pharmacologic as well as genetic inhibition of autophagy prevented the starvation- or rapamycin-induced increase in transepithelial electric resistance (TER), reduction in small solute flux, and degradation of claudin-2.⁵ In our recent study, we demonstrated the molecular mechanism underlying autophagy-induced claudin-2 degradation. We showed that autophagy promotes clathrin-mediated claudin-2 endocytosis; inhibition of clathrin-mediated endocytosis and not micropinocytosis or caveolae-mediated endocytosis prevented starvation-mediated degradation of claudin-2. Moreover, clathrin adaptor protein AP2M1 plays an important role in this process. Activated under autophagic conditions, AP2M1 binds to claudin-2 tyrosine motifs (YXXФ) (67-70 and 148-151) and anchors claudin-2 in clathrin pits. Adaptor protein complex 2 (AP2) also contains LC3 interacting region and acts as a bridge in connecting the endocytosed claudin-2 to autophagy receptor LC3. The deletion of autophagy-related ATG7 gene abrogated claudin-2-AP2M1-LC3 interactions, indicating that ATG7-dependent LC3 lipidation is required for internalization of claudin-2 into autophagosomes.¹²⁴ Furthermore, acute deletion of Atg7 in mice increased colonic claudin-2 levels and the susceptibility to experimental colitis, indicating an important role of autophagy in maintaining the homeostasis of TJ pore pathway.

Autophagy also modulates TJ macromolecular leak pathway.¹²⁵ Recently, we have shown a novel role of autophagy, where it enhances occludin protein levels and retains its membrane localization by reducing its endocytosis. Specifically, autophagy inhibited the constitutive degradation of occludin by preventing its caveolar endocytosis from the membrane and protected against inflammation-induced TJ barrier loss, in ERK1/2-dependent manner. In vivo, autophagy induction by rapamycin enhanced occludin levels in mouse intestines and protected against lipopolysaccharide- and TNF-α-induced TJ macromolecular permeability. Disruption of autophagy with acute Atg7 KO in adult mice decreased intestinal occludin levels, increasing baseline colonic TJ macromolecular permeability and exacerbating the effect of experimental colitis.¹²⁵ As a traditional degradative pathway, autophagy is known to degrade several membrane proteins including amyloid precursor protein, Notch1, focal adhesions, and claudin-2.^124,126-128 Thus, autophagy-mediated shunting of occludin out of the constitutive degradation pathway is a unique function of autophagy and a novel pathway in the regulation of TJ barrier. Interestingly, even though occludin expression is generally known to restrict TJ barrier loss^{44,79,129,130} and the inflamed intestinal mucosa in patients with active IBD has decreased occludin expression,^131-133 occludin is also known to promote apoptosis during inflammation via C-terminal coiled-coil occludin/ELL domain (OCEL) domain or when displaced from the TJs via caspase-3 transcription.^134-136 Thus, though, prima facie, autophagy-mediated increase in occludin levels may seem detrimental to cell survival, the ability of autophagy to restrict occludin to the membrane is consistent with the antiapoptic and cell survival mechanism of autophagy. Overall, autophagy-induced degradation of claudin-2 and upregulation of occludin are highly significant, given the defects in the TJ barrier^131-133 and autophagy^137-139 reported in IBD. However, autophagy regulators and autophagy-related proteins may also have autophagy-independent role in the TJ barrier function. For instance, AMP-activated protein kinase (AMPK) is involved in cytokine-induced TJ barrier modulation, independent of intracellular energy levels,^140,141 while ATG6/beclin-1 regulates endocytosis of occludin in autophagy-independent way.¹⁴² Additionally, the outcomes of autophagy are dependent on the autophagy substrate; autophagy-induced increase in the TER and enhancement of the TJ barrier in claudin-2-rich MDCK II cells is not evident in claudin-2-deficient MDCK I cells.⁵ Numerous studies show that a variety of compounds that have beneficial effects against intestinal inflammation and TJ barrier disruption also induce autophagy. The specific role of autophagy in modulating the TJ barrier and inflammation in the disease ameliorating effects of these compounds needs further investigations.

Environment and TJ Regulation

There have been new investigations into the effect of environmental factors such as drugs, stress, pollution, and dietary constituents on the TJ barrier. One of the most commonly used over-the-counter prescription is the proton pump inhibitors (PPIs). Beyond their efficacy in controlling the gastroesophageal reflux disease (GERD) symptoms, excessive use of PPIs may have potential untoward effects on several organ systems, mostly due to the alterations in gastric pH which can impact host nutrition and microbiome.¹⁴³ In a study examining the effect of long-term PPI administration on intestinal TJ barrier, PPIs administration led to disruption of mouse colonic TJ barrier via activation of MLCK.¹⁴⁴ The PPI administration for up to 4 weeks increased MLCK activity and increased constitutive TJ permeability, which ultimately predisposed for experimental colitis. Moreover, the use of a PPI over a 5-year period in patients with IBD was found to be associated with an significantly increased risk of hospitalization.¹⁴⁴ In another study, alcohol ethoxylates present in the rinse aid and a component of dish detergent led to disruption of TJ barrier and also altered the expression of genes involved in cell survival, epithelial barrier, cytokine signaling, and metabolism. Importantly, significant and toxic levels of rinse aid were found to be present on washed and ready-to-use dishware cleaned through professional dishwashers.¹⁴⁵ Among other environmental toxins, benzo[a]pyrene (B[a]P)-loaded polystyrene microplastics (PSMPs) have been shown to induce mouse colonic TJ barrier disruption via oxidative stress-induced notch signaling,¹⁴⁶ while a neonicotinoid insecticide, imidacloprid, increased paracellular permeability in vitro by disrupting the TJs through NF-κB-MLCK activation.¹⁴⁷ Psychological stress is also an important component of our environment. A study found that a mother’s psychological mood correlated with miR-148a expression in breast milk. The T-score on negative mood subscales was negatively correlated with miR-148a levels, while the T-score on positive mood subscales was positively correlated with miR-148a levels in the breast milk. Additionally, the nursing mice exposed to restrain stress had reduced expression of miR-148a in breast milk, and the neonatal mice nursed by stressed animals had decreased intestinal ZO-1 levels.¹⁴⁸ Though functional assessment of the intestinal TJ permeability was not carried out in this study, the reduction in ZO-1 level was shown to be due to the increase in DNA methyltransferase 1 (DNMT1) in the intestine of infant mice, indicating a potential for stress to alter intestinal TJ barrier. The TJ barrier also adopts to the environment. In a study of intestinal biopsies from environmental enteric dysfunction (EED), which is associated with deficits in physical and intellectual growth, barrier-forming claudin-4 was found to be upregulated, along with upregulation of nutrient and water transporters.¹⁴⁹

Nonjunctional Functions of TJ Proteins

In recent years, several TJ proteins have been shown to have non-barrier functions. Various claudins play a role in EMT, cell migration, invasion, and tumorigenesis (reviewed by Hagen¹⁵⁰). These nonjunctional functions of claudins are related to their non-TJ localizations such as distinct cytoplasmic locations, nuclei, or basolateral membrane. For example, nuclear claudin-1 regulates cell transformation and metastasis in colon cancer cells,¹⁵¹ while claudin-7, a basolaterally expressed TJ protein, suppresses cell proliferation and detachment via interactions with integrin β.¹⁵² Beyond the dispensable role of occludin in establishing the TJ barrier, occludin on epithelial and γδ T cells contributes to the intraepithelial lymphocyte migration, which is necessary to maintain epithelial barrier against pathogen invasion.^153,154 Also, limitation of epithelial apoptosis in the absence of occludin¹⁵⁵ is now proposed to be an adaptive response to limit tissue injury.¹³⁴ Absence of occludin during murine intestinal inflammation limits both intrinsic and extrinsic apoptotic pathways, along with reduced caspase-3 expression, providing a context for reduced occludin expression in IBD.^134,156 Similarly, cytoplasmic plaque protein ZO-1 has potentially TJ barrier-independent role in epithelial wound healing and proliferation on account of its function in WNT-β-catenin signaling and mitotic spindle orientation.^156-158

Perspective

In summary, several novel pathways that can regulate the intestinal epithelial TJ barrier have been investigated. Besides the traditional cell lines, cell-specific mouse genetic models and human intestinal organoids have been used to gain more nuanced and physiologically relevant knowledge of the intestinal epithelial TJ barrier. Beyond the somatic stem cell-derived intestinal organoids that contain only epithelial cells, inducible pluripotent stem cell (iPSC)-derived organoids containing the both epithelial and mesenchymal compartments can recapitulate the complexity of gut tissue as well as the patient-specific TJ barrier defects.¹⁵⁹

The importance of autophagy-mediated TJ barrier modulation is underscored by the IBD-associated defects in intestinal TJ barrier,^4,72,73 identification of mutations in autophagy-related genes as substantiated risk factors for CD,^137-139 the role of autophagy in gut immune responses,^160-164 and autophagy-mediated amelioration of intestinal inflammation in animal models.^164-166 Similarly, the AhR and Nrf2 pathways, considering their essential role in the homeostasis of intestinal function and integrity, are proposed to be the promising targets in IBD therapy.^98,167 The modulation of the TJ barrier-targeting pathways may have context-dependent outcomes and not uniform responses across the various models and diseases. Thus, the future studies should consider cell-type specific, physiologic vs pathologic, tissue vs organ-level outcomes in understanding the role of TJs in health and disease (Figure 2). The impact of various therapies on the TJ barrier under disease conditions is also important during regaining the homeostasis. For instance, the intestinal TJ barrier is targeted by the dysregulated cytokine responses, downstream of pattern recognition receptors (PRRs) pathway activated by microbial products. However, the TJ barrier is also benefitted by several cytokines such as IL-17 or IL-23, possibly explaining inefficiency of anti-IL-17 therapies in IBD.^168,169 Thus, considering the importance of the TJ barrier in mucosal healing and remission, enhancement of the TJ barrier should be a part of strategy involving prevention of excessive inflammatory responses.¹⁷⁰ The advances in the understanding of the intricate functions of the TJ barrier will help improve the therapeutic efforts against IBD and many more autoimmune and systemic diseases.

Figure 2.

The regulation of TJs. The cytoskeletal rearrangement (eg, MLCK or Src activation), proteostasis (regulation of a protein from synthesis to degradation, eg, by autophagy), intracellular trafficking (endocytic vs exocytic processes affecting the membrane localization of TJ proteins), and genetic regulation (by transcriptional factors as well as noncoding RNAs) are some of the major regulatory modes involved in the TJ barrier modulation. The outcome of the TJ barrier modulation, in terms of TJ homeostasis (selective paracellular permeability) or dysregulation (unrestricted paracellular permeability), is influenced by several factors including cell- or tissue-type specific TJ targets (barrier-forming claudins vs channel-forming claudins), the context of a model or disease condition (eg, immune vs chemical-induced experimental colitis), temporal effects of TJ signaling pathways (acute vs chronic conditions), physiologic reserve (the ability of tissue to respond to the TJ barrier modulation), non-barrier roles of TJ proteins (eg, EMT, cell migration), etc. Abbreviations: EMT, epithelial-to-mesenchymal transition; MLCK, myosin light chain kinase; TJ, tight junction; ZO, zona occluden.

Author Contributions

P.A. and K.S. conducted background research, designed experiments, generated figures, and wrote the manuscript. P.N. generated figures, conceived the study, and wrote the manuscript.

Funding

This research work was supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK114024. The sponsors had no role in the study design, collection, analysis, and interpretation of data, manuscript writing, or decision to submit the article for publication.

Conflicts of Interest

None declared.

Data Availability

The authors confirm that the sources of the data are either included in this article or will be made available upon a request.