Relationship between G proteins coupled receptors and tight junctions

ABSTRACT

Tight junctions (TJs) are sites of cell-cell adhesion, constituted by a cytoplasmic plaque of molecules linked to integral proteins that form a network of strands around epithelial and endothelial cells at the uppermost portion of the lateral membrane. TJs maintain plasma membrane polarity and form channels and barriers that regulate the transit of ions and molecules through the paracellular pathway. This structure that regulates traffic between the external milieu and the organism is affected in numerous pathological conditions and constitutes an important target for therapeutic intervention. Here, we describe how a wide array of G protein-coupled receptors that are activated by diverse stimuli including light, ions, hormones, peptides, lipids, nucleotides and proteases, signal through heterotrimeric G proteins, arrestins and kinases to regulate TJs present in the blood-brain barrier, the blood-retinal barrier, renal tubular cells, keratinocytes, lung and colon, and the slit diaphragm of the glomerulus.

KEYWORDS:

• blood brain barrier
• blood retinal barrier
• GPCR
• G protein
• slit diaphragm
• tight junctions

Introduction

In multicellular organisms the exchange of ions and molecules with the external milieu occurs through epithelial cell layers. Epithelial cells have two defining characteristics. The first is the polarization of their plasma membrane in two domains: apical and basolateral, with different biochemical composition, morphology and function [for review see.¹] The apical domain is in contact with the external environment or the content present in the lumen of ducts (e.g. intestine and renal tubules) and cavities (e.g. stomach, uterus and prostate), and displays microvilli and cilia, while the basolateral domain faces the underlying parenchyma and the interior of the organism, and exhibits in the basal and lateral regions, cell to substrate and cell to cell adhesion structures, respectively.

The second canonical characteristic of epithelia is the presence of tight junctions (TJs) at the uppermost portion of the lateral membrane. These cell-to-cell adhesion structures block the free diffusion of lipids and proteins within the membrane between the apical and the basolateral domains.^2–4 and hence are fundamental to maintain the polarized state of the plasma membrane in epithelial cells that guarantees the vectorial transport of ion and substances across epithelial cells. This notion however has been recently challenged by the observation that complete polarization of epithelial cells has been achieved in single cells that lack junctional cell-cell contacts upon activation of LKB1, a serine/threonine kinase whose gene is homologous to Par-4 polarity gene in C. elegans and D. melanogaster.⁵

By thin section transmission electron microscopy TJs appear as points of contact between the outer leaflets of the plasma membrane of adjacent cells. At these points originally referred to as “kisses”, the intercellular space disappears blocking the free transit of molecules and ions through the paracellular route.⁶ By freeze-fracture electron microscopy the TJ is seen as a network of strands in the protoplasmic face of the membrane and of grooves in the E face that surround the cells bellow the apical microvilli.⁷ [for review see.⁸] These strands are formed by the polymerization of claudins, a family of tetraspan proteins that interact between neighboring cells forming channels and seals in the paracellular space that define the ionic selectivity and paracellular permeability of epithelia [for reviews see.^9,10] Besides claudins, other tetraspan proteins, members of the Marvel family named occludin and marvelD3 are found in TJ strands between two cells,¹¹ while tricellulin concentrates at junctions formed between three cells.¹² In addition, integral proteins that belong to the immunoglobulin superfamily like JAMs [for review see.¹³] and angulins establish cell-cell interactions at TJs and mark the site where these structures are to be established in bicellular and tricellular [for review see.¹⁴] junctions, respectively. In the submembranous region of TJs a scaffold of proteins is found that links integral TJ proteins to the actomyosin cytoskeleton. Among these proteins are cingulin and paracingulin, as well as a wide variety of proteins with PDZ domains, including the MAGUK proteins ZO-1, -2, and -3 and Pals1; the inverted MAGUKs also known as MAGI -1, -2 and -3, and proteins containing a single PDZ domain like Par-6 and AF-6 or multiple PDZ domains like MUPP1 and PATJ [for reviews see.^15,16]

TJ integrity and barrier function is regulated by several mechanisms including: 1) expression of TJ proteins, 2) integrity and dynamics of TJ-associated actomyosin cytoskeleton, 3) localization of TJ proteins at the plasma membrane and 4) posttranslational modification of TJ proteins that affects protein-protein interactions. These mechanisms in turn, are modulated by various signaling pathways and molecules like kinases, phosphatases, Rho proteins and G protein-coupled receptors (GPCRs) [for review see.¹⁷] GPCRs constitute the largest protein family in the human proteome, with more than 800 members that share as common characteristic the presence of seven transmembrane helices. GPCRs are targeted by ≈ 30-40% of drugs in the market highlighting their crucial participation in a wide spectrum of physiological processes and diseases.¹⁸ GPCRs are usually located in the plasma membrane, although some concentrate at the membrane of the endoplasmic reticulum. They recognize a wide variety of stimuli including light, ions, hormones, glucocorticoids, peptides, lipids, proteases and neurotransmitters. GPCRs convert these stimuli into intracellular responses through their coupling to intracellular proteins like heterotrimeric guanine-nucleotide-binding proteins (G proteins), arrestins and kinases. Arrestins are adapter proteins that uncouple GPCRs from G proteins and target them to clathrin-mediated endocytosis. Since arrestins form complexes with several kinases, they can recruit them to agonist occupied GPCRs and confer a distinct signaling pathway for these receptors [for review see.¹⁹]

GPCR signal transduction through G proteins starts with the coupling of the receptor to a membrane associated heterotrimeric complex constituted by a GTP hydrolysing Gα subunit and a Gβγ dimeric partner. Upon activation by an agonist, GPCRs undergo a conformational change that promotes their guanine-nucleotide-exchange activity. This leads to the release of GDP and subsequent binding of GTP by the Gα subunit, triggering the dissociation of Gα subunit from Gβγ and the subsequent association of different effectors to specific subtypes of Gα subunits (Fig. 1). Around 20 mammalian G protein α subunits have been identified and organized into four families based on their sequence similarity: Gα_s, Gα_i, Gα_q/11, and Gα_12/13. When the G protein complex is stimulatory, Gα_s activates adenylyl cyclase that converts ATP into cAMP, which can directly activate protein kinase A (PKA). Instead when the complex is inhibitory, Gα_i blocks adenylyl cyclase. Pertussis toxin from Bacillus pertussis catalyses the ADP-ribosylation of Gα_i leaving it locked in a GDP bound state, leading to an increase in the cellular concentration of cAMP. Gα_q/11 subunit activates phospholipase C (PLC), which in turn hydrolyzes phosphatidylinositol 4,5-biphosphate (PIP2) to diacyl glycerol (DAG) and inositol trisphosphate (IP3). The former is a second messenger that activates protein kinase C (PKC), while IP3 induces the release of calcium from the endoplasmic reticulum. Gα_12/13 activates guanine nucleotide exchange factors (GEFs) for the small G protein RhoA [for review see.²⁰]. Table 1 indicates the type of G protein α subunit involved in signaling by GPCRs regulating TJ barrier function.

Figure 1. Schematic representation of GPCR signal transduction through G proteins. Signal transduction starts with the association of the receptor to a G protein heterotrimeric complex formed by a Gα subunit and a Gβγ dimeric partner. GPCR activation leads to the release of GDP by the Gα subunit and subsequent binding of GTP, triggering the dissociation of Gβγ subunit from Gα. The latter then interacts with different effectors. Gαs activates adenylyl cyclase that converts ATP into cAMP, which can directly activate cAMP-dependent protein kinase (PKA) and through the conversion of cAMP to AMP activates AMP dependent kinase (AMPK). Instead, Gαi blocks adenylyl cyclase. Gαq/11 subunit activates activates phospholipase C (PLC) that hydrolyzes phosphatidylinositol 4,5-biphosphate (PIP2) to diacyl glycerol (DAG) and inositol trisphosphate (IP3). DAG activates protein kinase C (PKC) and IP3 induces the release of calcium from the endoplasmic reticulum. While Gα12/13 activates guanine nucleotide exchange factors (GEFs) for RhoA.

Table 1. G protein-coupled receptors regulating tight junction barriers.

*As reported in https://www.ncbi.nlm.nih.gov/gene in the subsection pathways Biosystems

**Mechanisms involved and signaling pathway: 1) Expression of TJ proteins; 2) integrity and dynamics of the TJ-associated actomyosin cytoskeleton; 3) trafficking of TJ proteins, 4) posttranslational modification of TJ proteins that affects protein-protein interactions and 5) signaling pathway.

Symbols: ↑, increase; ↓, decrease; →induces; ↔, delocalization; ┤, inhibition

Abbreviations: ADAM, disintegrin and metalloenzyme; AKT, protein kinase B; AMPK, AMP-activated protein kinase; Ang, angiopoietin; cAMP, cyclic adenosine monophosphate; Ang, angiopoietin; AP, alkaline phosphatase; APC, activated protein C; aPKC, atypical protein kinase C; AQP4, aquaporin-4; cav-1, caveolin-1; Dvl-2, dishevelled-2; EGFR; epidermal growth factor receptor; eNOS, endothelial nitric oxide synthase; EPCR, endothelial protein C receptor; ERK, extracellular signal-regulated protein kinase; FoXO1, forhead box protein O1; FXa, factor Xa; Gab-1, Grb2-associated binding protein 1; GSK-3, glycogen synthase kinase; IL-1β, interleukin-1β; iNOS, inducible nitric oxide synthase; IP3, inositol triphosphate; LRP, low density lipoprotein receptor-related protein; MCP-1, monocyte chemoattractant protein-1; MEK, MAPK/ERK kinase; miR, microRNA; MLCK, Myosin light-chain kinase; MMP, Matrix metalloproteinase; MPO, myeloperoxidase; mTOR, target of rapamycin; MyD88, myeloid differentiation primary response 88; MYPT1, myosin phosphatase target subunit; NFAT, nuclear factor of activated T cell; NFκB, nuclear factor kappa B; p, phosphorylated; Per, permeability; p, phosphorylated; PIP2; Phosphatidylinositol 4,5-bisphosphate; PKA, protein kinase A; PKC, protein kinase C; PLC, Phospolipase C; Rac1, Ras-related C3 botulinum toxin substrate 1; RAFTK, related adhesion focal tyrosine kinase; Rap1, Ras-related protein 1; RhoA, Ras homolog gene family, member A; ROCK, Rho-associated protein kinase; Sox-17, SRY-Box 17 transcription factor; Src, protein-tyrosine kinase; SRF, serum response factor; STAT3, Signal transducer and activator of transcription 3; TER, transepithelial electrical resistance; Tie2, endothelial receptor tyrosine kinase 2; TNF-α, tumor necrosis factor α; ZO-2, zonula occludens 2; ZONAB, ZO-1-associated nucleic acid-binding protein.

Barriers: BBB, blood brain barrier; BBTB, blood brain tumor barrier; BRB, blood retinal barrier; BSCB, blood spinal cord barrier; iBRB, inner blood retinal barrier; oBRB, outer blood retinal barrier; oLMR, outer limiting membrane of the retina.

Receptors: ALX/FPR, lipoxin receptor; AMR/GPR182, adrenomedulin receptor; AT1, angiotensin II receptor 1; A1/ADOR, adenosine receptor; BLT2/LTB4R2, leukotriene B4 receptor type 2; BR2/BKR2/BDKRB2, bradykinin receptor B2; Calcrl, calcitonin receptor-like receptor; CaSR, calcium sensing receptor; CBR, cannabinoid receptor; CCR, C-C motif chemokine receptor; CRHR, corticotropin releasing hormone receptor; CTR, calcitonin receptor; CXCR, C-X-C motif chemokine receptor; C5aR, complement C5a receptor; DOR, opioid receptor delta 1; DRD, dopamine receptor D; EP, E-type prostanoid receptor; ET, endothelin receptor; FSHR; follicle stimulating hormone receptor; FZD, frizzled class receptor; GLPR1, glucagon like peptide 1 receptor; GPR, G protein-coupled receptor; H₁, histamine receptor 1; mAChR, muscarinic Ach receptor; MC, melanocortin receptor; MOR, opioid receptor mu 1; OGR1, ovarian cancer G protein-coupled receptor 1; PAFR/PTAFR, platelet-activating factor receptor; PAR, protease-activating receptor; P2Y, purinergic receptor; PKR2, prokineticin receptor 2; RHO, rhodopsin receptor; SSTR, somatostatin receptor; S1PR, sphingosine-1 phosphate receptor; TGR5/GPBAR1, Takeda G-protein coupled receptor 5; 5-HT, 5-Hydroxytryptamine (serotonin) receptor.

Cell lines: BAEC, bovine aortic endothelial cells; BBB-EC, blood brain barrier-endothelial cell; BEC, blood vascular endothelial cells; Bend.3, mouse brain endothelial cell line; BMVEC, primary human brain microvascular endothelial cells; Caco-2, colonic epithelial cell line; Calu-3, bronchial epithelial cells; CMT93, colon murine intestinal epithelial cells; CuFi, cystic fibrosis bronchial epithelial cell line; EA.hy926, human umbilical vein cell line; ECV304, derivate of T24 human bladder carcinoma; HaCaT, human keratinocyte cell line; HBMEC, human brain microvascular endothelial cells; HCAEC, human coronary artery endothelial cells; HCLE, human corneal epithelial cells; HEEC, normal primary human esophageal epithelial cells; HEK-293, human embryonic kidney cells 293; HMEC-1, human microvascular endothelial cell line; HMVEC-dLys, human dermal lymphatic microvascular endothelial cells; HNEC, human nasal epithelial cells; HPAEC, human pulmonary artery endothelial cells; HPMVEC, human pulmonary microvascular endothelial cells; HRMEC, human retina microvascular endothelial cells; HT-29, human colon adenocarcinoma cell line; HuH-7, hepatocyte-derived carcinoma cell line; HUVEC, human umbilical vein endothelial cells; IEC-6, normal rat intestinal cell line; LSEC, liver sinusoidal endothelial cell; LLC-PK1, kidney proximal tubule cell line; mBMEC, mouse brain microvascular endothelial cells; MCF10A, human mammary epithelial cells; MDCK, Madin Darby canine kidney cel line; MCF-7, breast cancer cell line; MDA-MB-231, breast cancer cell line; MEC, primary mouse mammary epithelial cells; MLEC, murine lung endothelial cells; NSCLC, human non-small cell lung cancer cells; NuLi-1, non-cystic fibrosis bronchial epithelial cell line; PAEC, pulmonary artery endothelial cells; PC-3, human prostate cancer cell line; RCEC, rat coronary microvascular endothelial cells; RBEC, rat brain capillary endothelial cells; RBMEC; rat brain microvascular endothelial cells; SH-SY5Y, Human neuroblastoma cell line; SMG-C6, rat salivary epithelial cells; SVEC4-10, SV40 transformed endothelial cell line; TAL, thick ascending limb of Henle; TM-1, trabecular meshwork cell line; T-84, colonic epithelial cells; 16HBE14o-, human bronchial epithelia.

Since 1991, Balda demonstrated that G proteins are implicated in the regulation of TJ assembly and sealing,²¹ and afterwards, Gα subunits were found to localize at epithelial TJs. Thus, constitutive activation of Gα_12/13, which binds to the SH3 domain of ZO-1, increased the paracellular permeability of epithelial cells.²² TJs were disrupted since Gα_12/13 stimulated Src-mediated phosphorylation of ZO-1 and ZO-2, which in turn caused dissociation of occludin and claudin-1 from the TJ complex. In addition, recruitment of heat shock protein 90 was necessary for Src activation by Gα_12/13. Instead, endogenous Gα_12/13 was activated upon TJ assembly triggered by switching the monolayers from low to normal calcium.²³ Gα_i2 subunit also localizes at TJs and immunoprecipitates with ZO-1, and its activation accelerates TJ biogenesis in epithelia.^24,25

According to sequence similarities within their seven-transmembrane region, GPCRs are classified into six major families: class A also known as rhodopsin receptor family, that includes receptors for odorants and small ligands; class B or secretin receptor family with ligands that include high molecular weight hormones; class C or metabotropic glutamate receptor family that includes GABA, taste, olfactory and pheromone receptors; class D or fungus pheromone receptor family; class E or cAMP receptors found only in Dictyostelium discoideum; and class F or frizzled/smoothened receptor family involved in cell polarity and segmentation [for review see.²⁶]

In this review we focus on GPCRs that influence TJ assembly and sealing evaluated by changes in transepithelial electrical resistance (TER) and paracellular permeability, as well as alterations in the expression of TJ proteins. In the case of claudins, we also report modifications in the isoforms of claudins being expressed. However, in many cases these changes in isoforms have not been correlated to variations in permeability and moreover, in the case of cancers, differences in claudin expression could be the result of a dedifferentiation of cells to a more stem-cell-like state.

Table 1 shows that of the 52 GPCR receptors that regulate the TJ barrier, 43 belong to class A and 5 to class B, while only 2 GPCRs belong to classes C and F, respectively. The two GPCRs of class C promote TJ formation and sealing, whereas the rest of the classes have some GPCRs that induce TJ sealing and others that open the barrier. These effects also vary according to the receptor ligand and tissue.

Here, for clarity purposes, we have organized the information based on the effect of GPCRs on TJs, and have subdivided the data according to the type of agonists that activate the receptors. Schematic summaries of the effect of different GPCRs in TJs of the intestine, kidney, blood-brain barrier (BBB), blood-retinal barrier (BRB) and other endothelia are presented in figs 2–6.

Figure 2. Regulation of TJs in intestinal epithelia by different G protein coupled receptors. Left, schematic representation of colon epithelia, showing a list of GPCRs the stimulate TJ formation (blue arrow) or favor TJ disassembly (red arrow). Right, signaling pathways known to be activated in the colon by GPCRs to promote TJ opening or closure. References for these studies are shown in Table 1. Receptors: A2B, adenosine receptor B; BLT2/LTB4R2, leukotriene B4 receptor type 2; BR2/BKR2/BDKRB2, bradykinin receptor B2; Calcrl, calcitonin receptor-like receptor; CaSR, calcium sensing receptor; CBR, cannabinoid receptor; CRHR, corticotropin releasing hormone receptor; CXCR, C-X-C motif chemokine receptor; EP, E-type prostanoid receptor; GPR, G protein-coupled receptor; OGR1, ovarian cancer G protein-coupled receptor 1; PAR-2, protease-activating receptor 2; SSTR, somatostatin receptor; S1PR, sphingosine-1 phosphate receptor. Other abbreviations: AMPK, AMP-activated protein kinase; cAMP, cyclic adenosine monophosphate; ERK, extracellular signal-regulated protein kinase; IP3, inositol triphosphate; MEK, MAPK/ERK kinase; MLC, Myosin light-chain; MLCK, myosin light-chain kinase; MMP2, matrix metalloproteinase 2; mTOR, target of rapamycin; NFκB, nuclear factor kappa B; PKA, protein kinase A; PKC, protein kinase C; PLC, Phospolipase C; SRF, serum response factor; STAT, Signal transducer and activator of transcription; TNFα, tumor necrosis factor α; ZO-2, zonula occludens 2.

Figure 3. GPCR regulation of TJs in the thick ascending limb of Henle, and of the slit diaphragm in the glomerulus. A) Left, schematic representation of a nephron and the slit diaphragm between podocytes in the glomerulus. The GPCRs that open (red arrow) or tighten (blue arrow) the podocytes slit diaphragms are indicated. Right, signaling pathways activated by GPCRs that regulate the slit diaphragm B) Schematic representation of epithelial cells lining the thick ascending limb of Henle illustrating how activation of CaSR favors claudin-14 expression, blocking in consequence cation reabsorption through the claudin-16/claudin-19 paracellular heteromeric channel. CaSR promotes claudin-14 expression blocking the transcription of miR-9 and miR-374 genes that induce the decay of claudin-14 mRNA. Receptors: AT1, angiotensin II receptor 1; BR2/BKR2/BDKRB2, bradykinin receptor B2; CaSR, calcium sensing receptor; CBR, cannabinoid receptor. Other abbreviations: ADAM, disintegrin and metalloenzyme; EGFR; epidermal growth factor receptor; ERK, extracellular signal-regulated protein kinase; miR, microRNA; PIP2, Phosphatidylinositol 4,5-bisphosphate; PLC, Phospolipase C; Src, protein-tyrosine kinase.

Figure 4. BBB regulation by different G protein coupled receptors. Left, schematic representation of the BBB, showing a list of GPCRs the stimulate TJ formation (blue arrow) or favor TJ disassembly (red arrow). Right, signaling pathways activated in the BBB by GPCRs to promote BBB opening or closure. References for these studies are shown in Table 1. Receptors: AT1, angiotensin II receptor 1; A1/ADOR, adenosine receptor; BLT2/LTB4R2, leukotriene B4 receptor type 2; BR2/BKR2/BDKRB2, bradykinin receptor B2; Calcrl, calcitonin receptor-like receptor; CBR, cannabinoid receptor; CCR, C-C motif chemokine receptor; CXCR, C-X-C motif chemokine receptor; C5aR, complement C5a receptor; EP, E-type prostanoid receptor; ETB, endothelin receptor; FZD, frizzled class receptor; GLPR1, glucagon like peptide 1 receptor; GPR, G protein-coupled receptor; PAR, protease-activating receptor; SSTR, somatostatin receptor; S1PR, sphingosine-1 phosphate receptor. Other abbreviations: cAMP, cyclic adenosine monophosphate; CREB, cAMP response element-binding; eNOS, endothelial nitric oxide synthase; ERK, extracellular signal-regulated protein kinase; iNOS, inducible nitric oxide synthase; LRP, low density lipoprotein receptor-related protein; MLC, Myosin light-chain; MMP, Matrix metalloproteinase; NFκB, nuclear factor kappa B; p, phosphorylated; PKA, protein kinase A; PLC, Phospolipase C; ROCK; Rho-associated protein kinase; Sox-17, SRY-Box 17 transcription factor; ZONAB, ZO-1-associated nucleic acid-binding protein.

Figure 5. Schematic representation of the retina showing GPCRs that regulate the retinal barriers and the known mechanisms involved. Left, schematic representation of the retina showing a list of GPCRs the stimulate TJ formation (blue arrow) or favor TJ disassembly (red arrow) in the iBRB, oBRB and the TJ-like barrier between the photoreceptors and Müller glial cells. Right, signaling pathways activated by GPCRs to promote iBRB closure. Receptors: Calcrl, calcitonin receptor-like receptor; GLPR1, glucagon like peptide 1 receptor; MC, melanocortin receptor; S1PR, sphingosine-1 phosphate receptor. Other abbreviations: AKT, protein kinase B; ERK, extracellular signal-regulated protein kinase; iNOS, inducible nitric oxide synthase; NFκB, nuclear factor kappa B; p, phosphorylated; STAT3, Signal transducer and activator of transcription 3; TNFα, tumor necrosis factor α.

Figure 6. Regulation of endothelial TJs present in tissues different to the retina or the brain by different G protein coupled receptors. Left, schematic representation of TJs between endothelial cells with a list of GPCRs the stimulate TJ formation (blue arrow) or favor TJ disassembly (red arrow). Right, signaling pathways activated in endothelial cells by GPCRs to promote TJ opening or closure. References for these studies are shown in Table 1. Receptors: Calcrl, calcitonin receptor-like receptor; CCR, C-C motif chemokine receptor; H1, histamine receptor 1; PAR, protease-activating receptor; S1PR, sphingosine-1 phosphate receptor.Other abbreviations: AKT, protein kinase B; Ang, angiopoietin; cAMP, cyclic adenosine monophosphate; ERK, extracellular signal-regulated protein kinase; IP3, inositol triphosphate; MLC, Myosin light-chain; NFκB, nuclear factor kappa B; p, phosphorylated; PKA, protein kinase A; PLC, Phospolipase C; Rac1, Ras-related C3 botulinum toxin substrate 1; Rap1, Ras-related protein 1; RhoA, Ras homolog gene family, member A; ROCK, Rho-associated Coiled-coil Kinase; STAT3, Signal transducer and activator of transcription 3; Tie2, endothelial receptor tyrosine kinase 2.

G protein-coupled receptors that induce TJ formation

Ion sensing receptors

Proton-sensing receptor OGR1/GPR68

The ovarian cancer G protein-coupled receptor OGR1/GPR68 also known as a receptor for sphingosylphosphorylcholine is a proton-sensor receptor of mild to moderate extracellular acidification below the physiological set point of pH 7.4. The receptor signals via Gα_q/11 and activates PKC, ERK and gene promoters that depend on the transcription factor serum response factor (SRF), which is a master regulator of actin cytoskeleton.²⁷ OGR1/GPR68 is expressed in osteoblasts,²⁸ kidney and the intestinal epithelium where it is up-regulated during inflammation. In intestinal Caco-2 cells stably over-expressing OGR1/GPR68, an acidic pH shift from pH 7.8 to 6.6 leads to an acute TJ barrier enhancement explained by augmented expression of occludin and ZO-1 and a decrease of claudin-2. This change is accompanied by increased formation of actin stress fibers and reduced cell mobility.²⁹ Hence, OGR1/GPR68 could become a target for innovative therapies dealing with the pathogenesis and progression of inflammatory bowel disease, which is commonly associated to a local pH decrease, TJ barrier disfunction and claudin-2 over-expression. However, chronic activation of OGR1/GPR68 may also exacerbate tissue damage since it also induces genes associated with tissue inflammation like those for interleukin 8 (IL-8),³⁰ chemokines CXCL1 and CXCL2, and prostaglandin-endoperoxide synthase,²⁹ explaining why OGR1/GPR68 deficiency protects from inflammation in the IL-10 knock-out (KO) mice model.³⁰

Zinc-sensing receptor GPR39

Zinc is an essential micronutrient important for human health. Zinc deficiency alters the epidermal, digestive, immune, reproductive and neuronal systems [for review see.³¹] Zinc is a cofactor of several enzymes and binds to zinc fingers domains in transcription factors and other proteins including transporters, ion channels and membrane receptors. G protein-coupled receptor GPR39 that senses extracellular zinc and signals through Gα_q/11, is present in neurons in the hippocampus,³² the thyroid³³ and the gastrointestinal system, including the stomach, small intestine, colon and pancreas.^34,33

GPR39 belongs to a family of receptors that includes those for gherlin, motilin and neurotensin.³⁵ Gherlin is the ligand of growth hormone secretagogue receptor and is known to stimulate appetite. Both gherlin and motilin, stimulate gastric emptying and interdigestive motility. Obestatin, a peptide derived from gherlin precursor peptide is the natural ligand of GPR39 and opposes gherlin's effect on food intake.³⁶ GPR39 KO mice shows symptoms of zinc deficiency like accelerated gastric emptying and increased fecal secretion,³³ accompanied by a decreased expression of ZO-1 and occludin in the colon.³⁷

Activation in colon of zinc/GPR39 signaling regulates proliferation and differentiation of the epithelia and induces TJ formation.³⁷ Hence, GPR39 silencing attenuated the activation of ERK1/2, AKT and mTOR/p70S6K pathways that promote proliferation, but at the same time inhibited alkaline phosphatase activity, a marker of colon cell differentiation. These changes were accompanied by a decrease in TER and a reduced expression of the apical junctional complex proteins occludin, ZO-1 and E-cadherin. Therefore, it is not surprising to observe that in the dextran sulfate sodium (DSS) model of ulcerative colitis, the loss of GPR39 increased inflammation susceptibility due to a lower expression of occludin.³⁸ and that zinc supplementation through GPR39 activation enhanced the amount of ZO-1 and occludin and improved epithelial integrity in Salmonella typhimurium infected colonic cells.³⁹

Zinc activation of GPR39 also leads to epithelial repair. Thus, in keratinocytes zinc/GPR39 signaling up-regulates the activity of the sodium proton exchanger NHE1 and enhances scratches closure.⁴⁰ Interestingly, extracellular zinc can derive from the injured cells in the tissue, revealing a mechanism through which the damaged cells induce the repair of the wound.

Calcium-sensing receptor CaSR

In the kidney, claudins -14, -16 and -19 regulate paracellular reabsorption of calcium. In the thick ascending limb of Henle (TAL), where a major percentage of Ca²⁺ and Mg²⁺ is reabsorbed via the paracellular route [for review see,⁴¹] claudins -16 and -19 form a paracellular heteromeric cationic channel that permeates Ca²⁺ and Mg²⁺.^42,43 Claudin-14 interacts with claudin-16, diminishing the permeability of the paracellular claudin-16/claudin-19 cation channel.⁴⁴

Calcium sensing receptor (CaSR) which is critical for the homeostasis of divalent ions and is upregulated by extracellular Ca²⁺, decreases the phosphorylation in serine residues of claudin-16 and triggers its dissociation from ZO-1 and translocation to the lysosome.⁴⁵ CaSR also favors claudin-14 expression, blocking in consequence Ca²⁺ reabsorption through the claudin-16/claudin-19 channel.⁴⁴ Accordingly, a deficiency of CaSR, down-regulates in kidney the expression of claudin-14, up-regulates claudin-16 and lowers Ca²⁺ urinary excretion⁴⁶ (Fig. 3).

CaSR promotes claudin-14 expression blocking binding of the nuclear factor of activated T cells to the proximal promoter region of miR-9 and miR-374 genes.⁴⁷ These micro RNAs target claudin-14 mRNA and induce its decay and translational repression.^44,47 Inhibitors of histone deacetylase stimulate the transcription of miR-9 and miR-374 and in consequence increased paracellular cation conductance in the TAL and rescued the phenotype of cells and animal models of autosomal dominant hypocalcemia, characterized by a gain of function mutation in CaSR.⁴⁸ Altogether, these observations highlight the importance of CaSR as a novel therapeutic target to treat renal calcium handling pathologies.

CaSR promotes TJ assembly and sealing in diverse tissues. Hence, the over-expression of CaSR in the basal cells of mice epidermis accelerates the differentiation of embryonic epidermal cells and the formation of the epidermal permeability barrier by claudins.⁴⁹ In MDCK cells, transfection of a CaSR gain of function mutant increased TER, and the activation of CaSR, relocated ZO-1 and occludin to the cell borders in cells cultured in low Ca²⁺ media, in a process that promoted the interaction of ZO-1 with I-afadin mediated by AMP-activated kinase (AMPK).⁵⁰ This effect seems surprising since CaSR signals through G_αi that inhibits adenylyl cyclase and lowers AMPK activation. However, CaSR also transmits information through G_αq/11 that via PLC and IP3 releases calcium from the endoplasmic reticulum and activates AMPK. This signaling cascade was demonstrated in intestinal cells where chitosan, an oligosaccharide derived from chitin found in the cytoskeleton of shrimp, crab and insects, activated CaSR and induced TJ assembly.⁵¹

Receptors activated by hormones

Adrenomedullin and intermedin receptor Calcrl

Adrenomedullin and intermedin also known as adrenomedullin2, are peptide hormones of the same family that bind to the G protein-coupled calcitonin receptor-like receptor (Calcrl) when the latter is associated with a receptor activity modifying protein (RAMP). The latter constitutes a family of three members: RAMP-1, -2 and -3, which translocate the Calcrl to the plasma membrane. Adrenomedullin binds to Calcrl/RAMP-2 and Calcrl/RAMP-3 and with less affinity than calcitonin gene-related peptide, to Calcrl/RAMP-1. Intermedin also binds Calcrl/RAMP-1 to -3 but with lower affinity than adrenomedullin [for review see.⁵²]

Adrenomedullin and intermedin are secreted from a variety of organs and tissues including endothelial cells, and plasma levels of adrenomedullin are elevated in patients with hypertension, congestive heart failure and chronic kidney disease.

Endothelial cell specific KO mice of RAMP-2 die perinatally and the surviving adults are afflicted with spontaneous vasculitis. Moreover, in drug inducible adult KO mice, the deletion of endothelial RAMP-2 provoked pronounced edema and vascular leakage. These deleterious effects on endothelial cells barrier function were triggered by a decrease in Rac1-GTP accompanied by an increase in RhoA-GTP that produced fragmentation of the cortical actin ring.⁵³ Adrenomedullin counteracts actomyosin contractility by activating Rap1, a small GTPase similar in structure to Ras, which inhibits RhoA. Accordingly, loss of the actin binding protein cortactin, causes endothelial barrier dysfunction due to actomyosin contractility mediated by a reduced adrenomedullin secretion.⁵⁴

In human umbilical vein endothelial cells (HUVEC), adrenomedullin and intermedin decreased the paracellular hyperpermeability induced by thrombin, through a mechanism involving cAMP accumulation. Adrenomedullin and intermedin reduce stress fibers formation.^55,56 and in the case of adrenomedullin this was found to be accompanied by a decreased phosphorylation of myosin light chain, while the intermedin study reported an increase in Rac1 activation together with a reduced RhoA activity and a consequential diminished actomyosin contraction. Intermedin however, was less potent than adrenomedullin. It is however noteworthy, that in rat coronary microvascular endothelial cells, intermedin increased permeability.⁵⁷ This effect, opposite to that observed in HUVEC is due to the fact that although intermedin inactivated the RhoA/ROCK pathway in both cell types, it inactivated Rac1 in coronary microvascular endothelial cells but not in HUVEC, highlighting the importance of the Rac1-GTP/RhoA-GTP ratio to preserve the endothelial barrier stability.

Intermedin increased TER of human pulmonary microvascular endothelial cells and attenuated ventilator-induced lung hyperpermeability in mice. These results highlight the potential therapeutical use of intermedin to prevent or ameliorate ventilator induced lung injury in patients receiving mechanical ventilation due to respiratory failure, surgery or trauma.⁵⁸

In lymphatic cells, adrenomedullin tightened the endothelial barrier by concentrating ZO-1 and VE-cadherin at the plasma membrane.⁵⁹ while the temporal deletion of Calcrl in adult mice induced a disorganized expression of ZO-1 and VE-cadherin at lymphatic junctions triggering a pathologic dilation of lymph vessels known as lymphangiectasia.⁶⁰

In the BBB, adrenomedullin increased TER and decreased paracellular permeability, and these changes were accompanied by enhanced claudin-5 expression,⁶¹ although others reported that the improval of BBB function did not affect the expression of claudin-1, occludin and ZO-1.^62,63

In Kimba mice that over-express vascular endothelial growth factor (VEGF) in the retina and display characteristics of diabetic retinopathy, the administration of adrenomedullin ameliorated the capillary dropout and vascular leakage, and in retinal capillary endothelial cells in culture treated with VEGF, adrenomedullin suppressed the enhanced permeability and reduced TER by reducing the level of molecules related to NFκB signaling and inflammation like MCP-1, IL-1β, VCAM-1, ICAM-1 and TNF-α, and by inducing TJ formation.⁶⁴ A somewhat similar effect was observed in HUVEC cells, where intermedin reduced the irregular and over sprouted vasculature caused by VEGF by preventing junctional VE-cadherin dissociation.⁶⁵

Adrenomedullin has a potential therapeutic value for the treatment of inflammatory bowel disease, as it can maintain the intestinal epithelial barrier function in rodent models of colitis induced by 2,4,6-trinitro-benzene-sulfonic acid or DSS. The protective effect that allowed the maintenance of TJ proteins was exerted via down-regulation of myosin light chain kinase (MLCK) and phosphorylated myosin light chain, and suppressed phosphorylation of STAT1 and STAT3 that triggered the decreased expression of inflammatory cytokines TNF-α, IL-6 and IFN-ϒ.^66,67 Adrenomedullin also reduced intestinal permeability in rats with Staphylococcus aureus α-toxin induced septic shock and in Caco-2 cells treated with the α-toxin or H₂O_{2 .}⁶⁸

Somatostatin receptor SSTR

Somatostatin, also known as growth hormone is a peptide hormone that inhibits insulin and glucagon secretion. Somatostatin interacts with five receptors named SSTR 1 to 5 that are coupled to inhibitory, pertussis toxin sensitive G proteins.

Somatostatin maintains the integrity of the BBB and restores the organization of ZO-1 in human brain endothelial cells treated with cytokines and lipopolysaccharide (LPS) to disrupt TJs. Hence, the decreased level of somatostatin found in the cerebrospinal fluid of patients with multiple sclerosis appears to contribute to the leaky BBB present in this disease.⁶⁹

In intestinal Caco-2 cells, somatostatin ameliorates LPS induced TJ damage, by decreasing ERK1/2 phosphorylation and increasing the expression of occludin and ZO-1 and inhibiting their redistribution.⁷⁰ Interestingly, SSTR3 interacts with MUPP1 TJ protein, and as a result of this SSTR3 is targeted to TJs. The interaction with MUPP1 gives the receptor the capacity to increase TER in a pertussis sensitive manner.⁷¹ and in cultured human keratinocytes, treatment with somatostatin increased the expression of claudin-4.⁷² that functions as a cationic barrier,⁷³ hence explaining the increase in TER observed.

Glucagon-like peptide receptor GLPR1

Glucagon-like peptide (GLP-1) is a 30-amino acid long peptide hormone secreted by intestinal enteroendocrine L-cells and certain neurons upon food consumption that signals through GLP-1 receptor (GLPR1). GLP-1 decreases blood sugar level by enhancing the secretion of insulin, and hence has the potential of being used for the treatment of type-2 diabetes.

Treatment of brain capillary endothelial cells with GLP-1 increased TER and the amount of occludin and claudin-5, and decreased paracellular permeability via cAMP/PKA signaling.⁷⁴ An agonist of GLP-1 named exendin-4, protects the inner endothelial blood-retinal barrier (iBRB) from vascular leakage induced by diabetic insult, by up-regulating the expression of GLPR1, claudin-5 and occludin.⁷⁵

Melanocortin receptors MC1 and MC5

Melanocortins are peptide hormones with a wide range of biological activities. Melanocortin receptors MC1, MC3, MC4 and MC5 are ubiquitously expressed, while MC2 is localized at adrenal glands. Intravitreal injection of MC1 and MC5 agonist in streptozotocin-induced diabetic mice augmented the expression of occludin in the retina, attenuating diabetic retinal damage. The mechanism involved is not yet clear, but appears to be related to a decrease to nondiabetic values, of the main mediators of the inflammatory response including cytokines IL-1α, IL-1β and IL-6, and chemokines MIP-1α, MIP-2α and MIP-3α, and of the main markers of vessel proliferation such as VEGF and ki-67.⁷⁶ The ability of melanocortins to reduce production of proinflammatory cytokines relies on their capacity to inhibit NFκB translocation to the nucleus.⁷⁷

G protein-coupled receptors activated by lipids

Receptors activated by metabolites of arachidonic acid

BLT2 a leukotriene B4 receptor type 2 activated by 12-HHT

12-Hydroxyheptadecatrenoic acid (12-HHT) is a 17-carbon metabolite of arachidonic acid that for many years was thought be an inactive byproduct of prostaglandin synthesis. However, recent research demonstrates that it protects epithelial barriers through the activation of G protein-coupled leukotriene B4 (LTB₄) receptor type 2 (BLT2), for which it has even a higher affinity than LTB₄. In mice lacking BLT2 an increased susceptibility to DSS-induced colitis was found, while transfection of BLT2 into MDCK cells decreased paracellular permeability.⁷⁸ and induced a faster recovery of TER after a calcium switch.⁷⁹ 12-HHT treatment of human keratinocytes activated BLT2 and enhanced TJ sealing by inducing claudin-4 expression through a process mediated by Gα_i and MAPK activation.⁷⁹ 12-HHT increased TER even in cell grown under low calcium condition that mimic the basal layer of cells in the epidermis.⁸⁰ This suggests that BLT2 agonist could be employed for the treatment of barrier-disrupted skin diseases.

ALX/FPR2 receptor activated by lipoxin A4

Lipoxins are arachidonic acid metabolites with potent anti-inflammatory and immunoregulatory properties. Lipoxin A₄ that binds to ALX/FPR2 G protein-coupled receptor plays an anti-inflammatory role in human airways and its production is decreased in cystic fibrosis and severe asthma. Lipoxin A₄ increased TER and stimulated ZO-1, claudin-1 and occludin expression in human bronchial epithelia,⁸¹ and delayed Pseudomonas aeruginosa invasion and transepithelial migration in cystic fibrosis and normal bronchial epithelial cell cultures by reducing TJ disruption.⁸²

Cannabinoid receptors

Cannabinoids include endocannabinoids that are produced naturally in the body of animals; phytocannabinoids found in plants like marijuana [e.g the psychoactive compound Δ⁹-tetrahydrocannabinol (THC) and cannabidiol (CBD)], and synthetic cannabinoids. Endocannabinoids are lipids derived from arachidonic acid that undergo rapid degradation by fatty acid amide hydrolase. There are two cannabinoid receptors: CBR1, which is associated with the psychoactive effect of cannabinoids, and CBR2 that mediates anti-inflammatory actions.

Activation of cannabinoid receptors produces potent neuroprotective and anti-inflammatory effects. Thus, CBR1 synthetic agonist CP55940 inhibited HIV-1 glycoprotein 120-induced increase of permeability of human brain microvascular endothelial cells in co-culture with astrocytes, and prevented the down-regulation of ZO-1, claudin-5 and JAM-1, and in vivo blocked BBB permeability and the transmigration of human monocytes into the brain.⁸³ Agonists of CBR2 protect the blood-spinal cord barrier from ischemia reperfusion injury,⁸⁴ and the BBB dysfunction after LPS-induced encephalitis.⁸⁵ or subarachnoid hemorrhage.⁸⁶ by increasing TJ protein expression and reducing barrier leakiness. The mechanism of action has been described on the blood-spinal cord barrier during ischemia reperfusion injury, where CBR2 agonist JWH-015 down-regulates the expression of caveolin-1 and up-regulates in consequence TJ protein expression, and in an in vitro BBB model where this agonist increased TER of brain microvascular endothelial cells by inducing the phosphorylation of phosphoinositide-3 kinase (PI3K) and of transcription factor FoxO1 that binds to the promoter region of caveolin-1 gene and in turn decreased the expression of caveolin-1 protein.⁸⁴

In intestinal and pulmonary epithelia cannabinoids also exert an anti-inflammatory effect coupled with reinforcement of the TJ barrier. So, in mice with DSS-induced colitis, WIN55-212-2, an agonist of CBR1 and CBR2, through the inhibition of p38MAPK, decreased the plasma levels of TNF-α and IL-6, and enhanced the expression of claudin-1.⁸⁷ Interestingly, apical or basolateral treatment of intestinal Caco-2 cells with THC or CBD enhanced through CBR1 the speed of recovery of EDTA-induced permeability, while endocannabinoids exerted this effect only when applied basolaterally. All cannabinoids augmented the mRNA of ZO-1, but endocanabinoids also decreased the mRNA of claudin-1.⁸⁸ In rats with cirrhosis and ascites, activation of CBR2 decreased intestinal oxidative stress, inflammatory cytokines, intestinal mucosal damage, bacterial translocation and spontaneous bacterial peritonitis. These changes respond to a down-regulation by CBR2 agonist JWH133 of systemic TNF-α/NFκB/cytokine signaling cascade that increases epithelial permeability by decreasing TJ proteins.⁸⁹ Similarly, in airway epithelia, THC through CBR2 activation reversed TNF-α induced decrease in TER and increase in permeability due to a decreased expression of occludin and ZO-1,⁹⁰ and in pulmonary edema induced after subarachnoid hemorrhage, JWH133 an agonist of CBR2 inhibit the infiltration of neutrophils reducing pulmonary edema ⁹¹

In kidney in contrast, antagonizing cannabinoids signaling reinforces the slit diaphragm barrier. Thus, AM251, the antagonist of CBR1 prevented diabetes-induced down-regulation of nephrin, podocin and ZO-1 in podocytes, ameliorating albuminuria.⁹²

Receptor GPR40 activated by a gut microbial metabolite of polyunsaturated fatty acids

A gut microbial metabolite of linoleic acid named 10-hydroxy-cis-12-octadecenoic acid (HYA) ameliorated in mice the TNF-α and DSS-induced colitis, suppressing the reduction in intestinal occludin and the increased expression of MLCK. HYA blocked the expression of TNF receptor 2 in intestinal epithelial cells through the activation of GPR40 G-coupled free fatty acid receptor 1 and MEK/ERK signaling pathway.⁹³

Sphingosine-1 phosphate receptors S1PR

Sphingosine-1 phosphate (S1P) is a bioactive lipid generated from sphingomyelin by sphingosine kinases (SphK). S1P is the natural ligand of five different GPCRs named S1PR1-5. S1PR1 is also transactivated as a result of the interaction of the protease activated protein C (APC) with its receptor endothelial protein C receptor (EPCR), resulting in activation of protease activated receptor 1 (PAR-1) and Rac and endothelial barrier enhancement.^94,95 (see also section 3.1.2). S1P analogues have potent barrier protective effects in lung endothelia.⁹⁶ and the BRB in diabetic rats.⁹⁷ and increased expression of SphK1 induced by the anesthetic isoflurane, suppresses BBB disruption after subarachnoid hemorrhage.⁹⁸ In addition, SEW2871, a S1PR1 agonist, improves intestinal barrier function, protecting IL-10 deficient mice from experimental colitis.⁹⁹

In endothelia, a physiological concentration of S1P enhances endothelial integrity through activation of S1PR1-PLC-IP3R-Ca²⁺-Rac pathway, while excessive S1P causes barrier dysfunction by activating S1PR2-calcium influx-RhoA/ROCK ¹⁰⁰ Instead, S1P-mediated activation of S1PR2 up-regulates the expression of E-cadherin and ZO-1 in intestinal epithelial cells.¹⁰¹

Delta opioid receptors activated by flavonoids

Flavonoids are plant or fungus secondary metabolites. Epicatechin, the predominant flavonoid present in dark chocolate, through the activation of δ opioid receptor diminishes the detrimental effect of high glucose, on TJs of the retinal pigment epithelia. Exposure to high glucose that mimics the milieu found under a diabetic condition increases the levels of TNF-α and inducible nitric oxide synthase (iNOS), creating nitrosative stress that results in S-nitrosylation and increased endocytosis of caveolin-1. This condition increases the interaction of caveolin-1 with claudin-1 and occludin and decreases their expression. This cascade is inhibited by treatment with high–polyphenol cocoa or epicatechin that through activation of δ opioid receptor, decrease TNF-α-induced iNOS upregulation and caveolin-1 endocytosis and restore TER in ARPE-19 monolayers.¹⁰²

Glucocorticoid-stimulated receptor GPR158

Trabecular meshwork cells are located in the eye at the base of the cornea and form a spongy tissue that allows draining of the aqueous humor from the anterior chamber of the eye into the tubes of the Schlemm´s canal that flows into the blood system. GPR158 is a glucocorticoid stimulated G protein-coupled receptor, whose over-expression enhances the barrier function of cultured trabecular meshwork cells, by increasing the expression of ZO-1 and occludin. This mechanism is responsible for glucocorticoid induced ocular hypertension that leads to glaucoma.¹⁰³

CCR6 receptor activated by β-defensins

β-defensins are antimicrobial peptides produced by epithelial cells lining various organs. Human β-defensins increased TER and reduced paracellular permeability of keratinocytes increasing the expression and localization of claudins. These effects were reversed with the blockade of G protein-coupled chemokine receptor CCR6.¹⁰⁴

Activation of frizzled-4 receptor by norrin

Norrin is a protein unrelated to Wnt ligands that signals through activation of frizzled-4 G protein-coupled receptor and LRP co-receptor (see also chapter 2.7). The NDP gene, whose mutations in humans cause Norrie disease, an X-linked disorder characterized by retinal hypovascularization and a severe loss of visual function, encodes Norrin. This protein promotes angiogenesis in the retina and is essential for BBB formation. Thus, after subarachnoid hemorrhage, norrin through frizzled-4 activation, protected BBB integrity enhancing the expression of occludin, VE-cadherin and ZO-1.¹⁰⁵

Precocious norrin production in engineered mice leads to premature retinal vascular invasion and delayed norrin production leads to defects in intra-retinal vascular architecture. In the adult retina and cerebellum, gain or loss of norrin/Fzd4 signaling resulted in gain or loss, respectively, of both the BRB and the BBB function,^106,107 while the administration of a monoclonal antibody that neutralized frizzled-4 function in vivo inhibited physiological and pathologic sprouting angiogenesis within the retina and allowed the extravasation of molecules through the BRB.¹⁰⁸ Transcription factor Sox17, up-regulated by norrin/frizzled-4 signaling, appears to plays a central role in the angiogenesis induced by norrin 107).

Rhodopsin, a G protein-coupled receptor activated by light

Rhodopsin is a light-sensitive G protein-coupled receptor found in the eye in the rods of the retina that enables vision in low-light conditions. Retinitis pigmentosa is an inherited disease characterized by primary degeneration of rod photoreceptors and secondary degeneration of cones in the retina. In a mouse model of retinitis pigmentosa, induced by the targeted disruption of rhodopsin gene, the expression of ZO-1, β-catenin and p120-catenin diminishes at the outer limiting membrane of the retina.¹⁰⁹ The latter forms a TJ-like barrier between the photoreceptor cells and Müller glial cells, containing occludin, JAM and ZO-1, at the apical site of the outer nuclear layer of the photoreceptors.¹¹⁰

G protein-coupled receptors that open TJs

Hormone activated receptors

Calcitonin activated receptor

Calcitonin (CT) is a 32-amino acid linear polypeptide hormone that in humans is produced by the parafollicular cells of the thyroid. It reduces blood calcium, in opposition to the effect of parathyroid hormone. Calcitonin is formed by the proteolytic cleavage of the product of CALC1 gene. Calcitonin receptor (CTR) signals through Gα_s, adenylyl cylase and cAMP in target cells.

Expression of CT and CTR is frequently elevated in prostate cancers and activation of CT-CTR axis in non-invasive prostate cancer cells induces an invasive phenotype, while their silencing in highly metastasic prostate cancer cells reduces their tumorigenicity and ability to form distant metastases.¹¹¹ The activation of CTR induces the phosphorylation and inactivation of glycogen synthase kinase-3β (GSK-3β), a key enzyme needed for the cytosolic degradation of β-catenin in the Wnt signaling pathway. This results in β-catenin translocation into the nucleus and transcription of Wnt target genes involved in epithelial to mesenchymal transformation.¹¹² These changes are accompanied by increased secretion of matrix metalloproteinase (MMP) a switch from E-cadherin to N-cadherin, ZO-1 delocalization and loss of cell-cell adhesion.¹¹² The PDZ binding motif present at the carboxy-terminus of CTR binds to the PDZ3 domain of ZO-1,¹¹³ bringing CTR-activated PKA in close proximity to TJs facilitating the phosphorylation of ZO-1 and claudin-3 that is responsible for TJ disassembly ¹¹⁴ These results hence explain why CTR-ZO-1 interaction is required for CTR to destabilize TJs and form metastases of prostate cancer.¹¹³

Neurotransmitter activated receptors

Serotonin activated receptor 5-HT₇

Serotonin is a monoamine neurotransmitter derived from tryptophan. In esophageal squamous epithelial cells, serotonin disrupts the barrier function through serotonin receptor 5-HT₇ that activated p38MAPK and decreased the expression of ZO-1, and by activation of ERK that decreased occludin level. Serotonin also reduced claudin-1 expression but through a mechanism independent of ERK or MAPK activation.¹¹⁵ In mammary epithelium, treatment with 5-HT₇ receptor antagonists.¹¹⁶ or a low concentration of serotonin increased TER, while at higher serotonin concentration and later time points, TER decreased and the TJs were disrupted through 5-HT₇ activation of Gα_s/cAMP and p38, while inhibition of PKA prevented the increase in TER in response to the low serotonin dose.¹¹⁷ Serotonin opening of TJs could be a negative-feedback mechanism in lactation that might serve to dissipate the transepithelial gradients necessary for milk secretion.¹¹⁶

Acetylcholine receptor mAChR

Acetylcholine (ACh) is an ester of acetic acid and choline that functions as a neurotransmitter at the neuromuscular junction. Activation of muscarinic Ach receptors (mAChRs) by carbachol and cevimeline reduced the TER and increased the permeability of rat salivary epithelial cells through a mechanism involving phosphorylation of claudin-4 by ERK1/2, which facilitated the interaction of claudin-4 with β-arrestin2, triggered claudin-4 internalization via clathrin and its subsequent ubiquitin-mediated degradation.¹¹⁸

Histamine receptors

Histamine is an amine formed by the removal of a carboxyl group of the amino acid histidine. Histamine regulates a variety of physiological functions in the gut and acts as a neurotransmitter for the brain, spinal cord and uterus. It participates in the inflammatory response and plays a central role as a mediator of itching. For this purpose, histamine is produced by basophils and mast cells present in the connective tissue. Histamine signals via four G protein-coupled receptors named H1 to H4.

In cultured human nasal epithelial cells, histamine down-regulated ZO-1 mRNA through a process mediated by histamine receptor H1.¹¹⁹ This receptor signals through Gα_q/11 and promotes the expression of transcription factor NFκB that regulates the inflammatory process [for review see.¹²⁰]

In epithelial cells, histamine increased paracellular permeability and stimulated stress fiber formation, myosin light chain phosphorylation and occludin phosphorylation. However, only the cytoskeletal events were mediated by RhoA/ROCK activation.¹²¹ Similarly, in endothelial HUVEC cells and in microlymphatic endothelial cells of dermal origin, histamine decreased TER through a process independent of myosin light chain kinase.¹²²

In a microfluidic model of microcirculation containing both blood and lymphatic vessels, treatment with histamine decreased the junctional localization of claudin-5 and VE-cadherin and increased the permeability of both type of vessels,¹²³ and in a blood-tumor barrier model, histamine increased paracellular permeability, decreased TER, disrupted the integrity of TJs and lowered the expression of ZO-1, occludin and claudin-5.¹²⁴

In endothelial cells histamine also alters the adherens junctions and their link to vimentin. Thus, in HUVEC cells histamine caused a transient and reversible disruption of binding between β-catenin and VE-cadherin,¹²⁵ induced serine/threonine phosphorylation of catenin p120 and its splice variant p100,¹²⁶ and decreased the amount of vimentin that immunoprecipitates with VE-cadherin.¹²⁷

MOR an opioid activated receptor

The activation of μ opioid receptor (MOR) with opioids in human non-small cell lung cancer cells increased the expression of snail, slug and vimentin and decreased the protein level of ZO-1 and claudin-1, consistent with an epithelial to mesenchymal transition.¹²⁸ This observation might explain why epidemiological studies have recently shown a diminished incidence of cancer recurrences following regional anesthesia with lower doses of opioids following surgeries for prostate and colon cancer.^129,130

Chemokines activated receptors

Chemokines are chemotactic cytokines that control the migration and localization of immune cells. There are approximately 50 endogenous chemokines that bind to 20 different G protein-coupled receptors.

Receptors for CXC chemokines

CXC chemokines or α-chemokines have two N-terminal cysteines separated by one amino acid. In the central nervous system, the microglia functions as the immune system and accordingly, in brains of Alzheimer's disease microglia accumulates and surrounds the senile plaques of beta-amyloid. Since bone marrow-derived microglia originates from monocytes, interest has arisen in discovering how these monocytes cross the BBB. In this respect, it has been found that monocytes that over-express chemokine CXCL1, like those derived from Alzheimer's disease patients, decrease TER and increase the permeability of brain microvascular endothelial cells altering the distribution of ZO-1 and occludin, through a mechanism involving chemokine receptor CXCR2 and ROCK.¹³¹ Moreover, in mice an antagonist to chemokine receptor CXCR4 named AMD3100, protects BBB integrity and reduces the inflammatory response after ischemia induced by middle cerebral artery occlusion,¹³² and protects intestinal barrier function from experimental colitis preventing the decreased expression of claudins -1, -3, -5, -7 and -8 and the increase in claudin-2 expression triggered by DSS.¹³³ Interestingly, gliadin the toxic component of gluten that triggers celiac disease, binds to chemokine receptor CXCR3 in intestinal epithelial cells, inducing the recruitment of the adaptor protein MyD88 and zonulin release. The latter is an endogenous regulator of TJs that increases intestinal permeability enabling the paracellular passage of gliadin and other antigens from the lumen to the gut mucosa leading to the pathology associated with celiac disease.¹³⁴

Receptors for CC chemokines

CC chemokines or β-chemokines have two adjacent cysteines near their amino terminal. The monocyte chemoattractant protein-1 (MCP-1) also known as chemokine CCL2 besides its main function of recruiting leukocytes at sites of inflammation opens the BBB. Thus, studies performed in vitro with a co-culture of brain endothelial cells and astrocytes, and under in vivo conditions, demonstrate that MCP-1/CCL2 through the CCR2 receptor present in endothelial cells and signaling via RhoA and ROCK, increases BBB permeability altering the distribution and expression of occludin, claudin-5, ZO-1 and ZO-2,^135,136 Accordingly, during ischemia-reperfusion injury, BBB permeability increased together with MCP-1/CCL2 secretion from both astrocytes and brain endothelial cells, while CCL2 blockade or the absence of CCR2 from endothelial cells decreased BBB permeability.¹³⁷

Another chemokine named eotaxin or CCL11 increased the permeability of coronary artery endothelial cells through receptor CCR3, down-regulating the expression of ZO-1, occludin and claudin-1, producing oxidative stress and the activation of p38MAPK, STAT3 and NFκB pathways.¹³⁸

Receptors activated by complement peptides

Anaphylatoxins or complement peptides C3a, C4a and C5a are peptide fragments produced by the activation of the complement system. C3, C4 and C5 are large complement glycoproteins, which are proteolytically activated by cleavage at specific sites, forming a- and b-fragments. Anaphylatoxins are synthesized in the liver and found in the blood and are known to cause muscle contraction, histamine release from mast cells and enhanced vascular permeability.

Exposure of an in vitro model of BBB to C5a or to serum from patients with systemic lupus, where complement activation occurs, decreased TER and the expression of claudin-5 and ZO-1, through the activation of G protein-coupled receptor C5aR/CD88.¹³⁹ The mechanism through which C5a disrupts BBB integrity involves NFκB signaling, since treatment with a C5aR/CD88 antagonist that preserved ZO-1 expression in mouse brain endothelial cells treated with lupus serum, reduced the translocation of NFκB into the nucleus.¹⁴⁰ These results help to explain why impaired BBB integrity and neurological complications are frequently associated to systemic lupus.

In epithelial cells as well, C5aR destabilizes cell-cell adhesion. Thus, in hepatocellular carcinoma cells, activation of C5aR/CD88 by C5a promoted cell invasion and migration through down-regulation of E-cadherin and claudin-1 and up-regulation of snail accompanied by the activation of ERK1/2 pathway.¹⁴¹

Endothelin receptors activated by vasoconstricting peptides

Endothelins are 21-amino acid peptides that constrict blood vessels and raise blood pressure. In rat, status epilepticus induced the activation of endothelin B receptor, leading to nitric oxide synthase (eNOS) mediated activation of MMP-9 and degradation of claudin-2, occludin and ZO-1 in endothelia from brain cortex.¹⁴² Moreover, transgenic mice with endothelial endothelin-1 over-expression show more severe vascular permeability and BBB breakdown after transient middle cerebral artery occlusion. The brain of these animals displayed oxidative stress, edema, increased MMP-2 expression, BBB leakage and a decreased occludin level. Interestingly, endothelin-1 antagonist A-147627 partially normalized the infarct volume and neurological deficit in these mice, highlighting the contribution of endothelin receptor activation to BBB breakdown under ischemia.¹⁴³

Frizzled receptors activated by Wnt ligands

The canonical Wnt pathway that promotes epithelial to mesenchymal transition and is involved in the development and progression of a wide variety of carcinomas, is activated by Wnt ligands that bind to the G protein-coupled receptor frizzled and the co-receptor LRP5/6. Frizzled receptors constitute a family that in humans has 11 members.¹⁴⁴ In the absence of a Wnt ligand, a complex of scaffold proteins APC and axin, and the kinases GSK3β, and casein kinase 1, is located in the cytosol. β-catenin is phosphorylated by these kinases and is then recognized and ubiquitinated by a multiprotein complex containing β-TrCP, leading to the degradation of β-catenin in the proteosome. However when Wnt ligands activate the pathway, LRP5/6 is phosphorylated by casein kinase 1 and GSK3β, triggering the translocation of the complex from the cytosol to the plasma membrane and the recruitment of dishevelled, another scaffold protein. In this membrane complex, where dishevelled binds to frizzled and axin binds to phosphorylated LRP5/6, β-catenin ubiquitination is suppressed, leading to saturation by accumulation of phosphorylated β-catenin.¹⁴⁵ As a result, newly synthesized β-catenin accumulates in the cytoplasm and enters into the nucleus where it binds to LEF/TCF transcription factor, promoting as a result the expression of a vast group of genes involved in EMT [for review see.¹⁴⁶]

Snail transcription factor that represses the transcription of E-cadherin, claudins and occludin, promoting TJ and adherens junctions disassembly and EMT,¹⁴⁷ is a target of GSK3β-dependent phosphorylation and β-TrCP-directed ubiquitination and proteosomal degradation.¹⁴⁸ Wnt signaling inhibits snail phosphorylation and increases in consequence snail level and activity.¹⁴⁹ This process is mediated by axin2. Thus, β-catenin at the nucleus upon association to LEF/TCF induces the expression of axin2,¹⁵⁰ which functions as a chaperone that promotes the exportation of GSK3β from the nucleus into the cytoplasm, inhibiting in consequence the turnover of snail.¹⁵¹ Therefore activation of frizzled receptors by Wnt ligands disrupts TJ stability.

The placenta regulates the transfer of materials between the mother and the developing fetus. One of the earliest steps of placental development is branching of the sheet of trophoblast cells. These cells are present at the outer layer of the blastocyst, and branching increases the surface of the epithelium of the chorionic villi that exchanges nutrients, gases and wastes between the fetus and the mother's blood present in the intervillous space. Wnt ligands via frizzled-5 receptor and the activation of the Wnt signaling pathway, regulate chorionic branching. Thus, the up-regulation of frizzled 5 triggered by transcription factor Gcm 1 induces the dissociation of cell junctions needed for branching initiation through the down-regulation of ZO-1, claudin-4 and claudin-7 in trophoblast cells, at the base of the chorion.¹⁵²

Receptors activated by lipids

Receptors activated by the phospholipid platelet-activating factor

Platelet-activating factor (PAF) is a phospholipid involved in inflammation and thrombosis that activates a G protein-coupled receptor. Treatment with PAF increased endothelial permeability and triggered the formation of intercellular filopodia and lamellipodia through a process involving Rac1 activation and the association of Rac1 and its GEF, Tiam1, with PAF receptor (PAFR). This process is independent of myosin light chain phosphorylation and MLCK activation,^153,154 Accordingly, serum from patients with severe dengue virus infection with evidence of vascular leakage, displayed high PAF levels and reduced TER and ZO-1 expression when tested on an endothelial cell line.¹⁵⁵ In caecum and distal colon PAF has also been reported to increase paracellular permeability.¹⁵⁶

Takeda G protein-coupled receptor 5 activated by bile acids

The transformation of normal esophageal squamous epithelium to reflux esophagitis, followed by Barrett's esophagus and dysplasia, can finally lead to esophageal adenocarcinoma. Chronic exposure to bile salts in gastro esophageal reflux disease induces inflammation mediated through the bile acid receptors, including the Takeda G protein-coupled receptor 5 (TGR5). Interestingly, in esophageal carcinoma and precancerous lesion, expression of TGR5 is associated with high expression of claudin-2,¹⁵⁷ which is a pore forming claudin whose overexpression has also been reported in colorectal and esophagus carcinomas.^158–160 and inflammatory bowel disease.¹⁶¹

Receptors activated by extracellular nucleotides and nucleosides

Nucleotides are organic molecules constituted by three distinctive chemical units: a five-carbon sugar molecule and a nitrogenous base, which together are called a nucleoside, and one phosphate group. Therefore a nucleotide is also named a nucleoside monophosphate. However common usage has extended the definition in order to include as nucleotides the molecules with two or three phosphates also known respectively as nucleoside diphosphate and nucleoside triphosphate [for review see.¹⁶²]

Nucleotides contain either a purine (adenine or guanine) or a pyrimidine (thymine, uracil or cytosine) nitrogenous base, and are termed ribonucleotides if the sugar is ribose or deoxyribonucleotides if the sugar is deoxyribose. Nucleotides have several functions: 1) as monomer units for forming the nucleic acid polymers DNA and RNA, 2) as packets of chemical energy in the form of the nucleoside triphosphates ATP, GTP, CTP and UTP, 3) as signaling molecules in the form of cyclic nucleotides cGMP and cAMP, and 4) as cofactors of enzymatic reactions.

P2Y₂ receptor activated by nucleotides

The P2Y family of G protein-coupled receptors, is activated by a wide range of extracellular mono and dinucleotides. P2Y₂ receptor, which is activated by ATP, promotes cell invasion and metastasis in prostate cancer cells, triggering the expression of snail and inhibiting E-cadherin and claudin-1 expression.¹⁶³ The ability of P2Y₂ receptor to disrupt epithelial TJ has been employed to improve ocular drug delivery. Thus, in human corneal epithelial cells, treatment with the dinucleotide P¹,P⁴-Di (adenosine-5’) tetraphosphate (Ap₄A) activated ERK and reduced TER and TJ protein levels through a process dependent on P2Y₂ receptor. In addition, the topical application of Ap₄A to rabbit eyes, disrupted ZO-1 membrane distribution in the cornea and improved the delivery of a hypotensive compound that reduces intraocular pressure, into the aqueous humour.¹⁶⁴

Adenosine activated receptors A₁, A_2A and A_2B

Adenosine is a purine nucleoside that besides its role in the metabolism exerts physiological functions by interacting with four receptors: A₁, A_2A, A_2B and A₃. Adenosine is produced inside and outside of cells. Extracellular adenosine is generated by the conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and adenosine monophosphate (AMP) by the extracellular enzyme CD39. AMP it then converted to adenosine by CD73, another extracellular enzyme.

Activation of adenosine receptors A₁ and A_2A increases BBB permeability, facilitating the entry of intravenously administered macromolecules into the brain, including compounds of therapeutic value like chemotherapeutic drugs and antibodies against β-amyloid. Opening of the BBB is reversible and mediated by a reorganization of the actin cytoskeleton induced by RhoA, and involves actomyosin stress fibers formation and a diminished expression of ZO-1, occludin, claudin-5 and VE-cadherin,^165,166 These observations have led to the development of adenosine receptor agonists that have a longer circulation lifetime and hence exert a broader BBB opening time window that can be allowed to match with the pharmacokinetics of the therapeutic agent.¹⁶⁷

Adenosine receptor signaling exerts conflicting effects on the intestinal barrier. Thus, while some reported that inhibition of A_2B adenosine receptors attenuated the decrease in TER and diminished claudin-1, occludin and ZO-1 expression, induced by ischemia/reperfusion injury or acute hypoxia,¹⁶⁸ others showed that adenosine receptor signaling induced by AMP cleavage, had a protective role against Clostridium difficile toxins TcdA and TcdB, reversing the reduced TER and increased paracellular permeability of intestinal cells.¹⁶⁹

G protein-coupled receptors with dual effect on TJs

Protease-activated receptors

PAR-2

Proteinase-activated receptor-2 (PAR-2) is a G protein-coupled receptor activated by a proteolytic cleavage on the N-terminal extracellular region that unmasks amino terminal residues that serve as tethered ligands that activate the receptor. PAR-2 is activated by trypsin, chymase and mast cell tryptase, which are highly expressed in the intestine. The colonic administration of PAR-2 agonist up-regulates PAR-2 expression and induces an inflammatory reaction that decreases transepithelial resistance.¹⁷⁰ and increases paracellular permeability,¹⁷¹ and that is accompanied by the redistribution of perijunctional F-actin, ZO-1 and occludin.¹⁷² and the reduction of claudin-5 expression.¹⁷⁰ The mechanism through which mast cells induce an inflammatory reaction in the colon following degranulation and the activation of PAR-2 involves association of the receptor to the multiadaptor protein β-arrestin that mediates activation of kinases ERK1/ERK2 which in turn re-organize the perijunctional ring of F-actin to increase epithelial permeability.¹⁷² In Caco-2 cells, PAR-2 activation with chymase also involves MMP-2 expression and activation.

PAR-2 activation explains why infiltration of mast cells that are replete with proteases including tryptase, delocalizes TJ proteins and increases the permeability of the intestine that is inflamed due to chronic stress, cytokines, allergens and bacterial products. Moreover, the role of PAR-2 is critical to understand TJ disruption in patients with inflammatory bowel disease where luminal trypsin and tryptase are elevated,^173,174 In this respect, it was found that mucosal application in mice of faecal supernatants with increased serine protease activity from diarrhea-predominant irritable bowel syndrome patients, increased colonic paracellular permeability in a manner dependent of PAR-2 expression.¹⁷⁵

Activation of PAR-2 by specific peptides also increases colon permeability. Hence, PAR-2 activation with the peptide SLIGRL increases colonic permeability and alters ZO-1 localization even without causing inflammation, via calmodulin that binds and activates MLCK.¹⁷⁶ In addition, the amino terminal portion of Vibrio cholerae-derived Zonula occludens toxin, has a PAR-2 activating motif (FCIGRL), that augments the phosphorylation by PKCα of ZO-1 and myosin. These modifications induce the dissociation of ZO-1 from occludin, claudin and myosin and open the TJ.¹⁷⁷ Nitric oxide and capsaicin-sensitive afferent neurons are also involved in PAR-2 mediated colonic inflammation and paracellular permeability increase.¹⁷⁸

Activation of PAR-2 by mast cell tryptase is also causative of stress-induced epithelial barrier dysfunction in the esophagus.¹⁷⁹

In epithelial MDCK cells, PAR-2 localizes at the lateral surface explaining, why the application of a PAR-2 activating peptide to the apical side of monolayers exerts no effect on TER (180). In these cells PAR-2 leads to TJ disruption through the activation of p38MAPK.

In mice placenta instead, the expression of PAR-2 is critical for placental barrier function. Thus, mice double deficient in the serine protease matriptase and PAR-2 are embryonic lethal and die at midgestation despite normal development. The defect lies in the sealing of the placental epithelium in direct contact with the maternal blood, named the laberynth and composed of an external layer of syncytiotrophoblast and an underlying layer of cytotrophoblast that surround the mesenchyma with the fetal vessels. Thus, in matriptase and PAR-2 double deficient embryos, the placental epithelium is leaky to the paracellular marker inulin and has a significantly reduced expression of claudin-1.¹⁸¹

PAR-1 and PAR-3

PAR-1 is the major thrombin activated receptor and plays a major role in platelet aggregation, inflammatory responses and neurodegeneration [for review see.¹⁸²] Thrombin promotes coupling of PAR-1 to Gα_12/13 and Gα_q and activation of RhoA.¹⁸³

Upon breakdown of the BBB thrombin enters the brain and activates PAR-1 receptors. Therefore, intracerebral injection of autologous blood produce damage to the brain parenchyma that is significantly reduced in PAR-1 KO mice or by the addition of the thrombin inhibitor, hirudin.¹⁸⁴ Similarly, intracerebral injection of thrombin in mice causes massive brain lesions and brain hemorrhages by BBB disruption, that are markedly reduced in PAR-1 KO mice,¹⁸⁵ and hypothermia, reduces brain water content in thrombin–induced brain edema by lowering the expression of PAR-1.¹⁸⁶ In addition, in an experimental model of multiple sclerosis known as experimental autoimmune encephalomyelitis, inactivation of PAR-1 with KC-A0590 preserved the expression of occludin and ZO-1 in brain endothelial cells and down-regulated the secretion of MMP-9 in astrocytes,¹⁸⁷ and under oscillatory sheer stress that accelerates atherogenesis, endothelial TJ are impaired through a mechanism that involves activation of PAR-1 followed by increased expression of Dickkopf 1, a protein that inhibits the Wnt signaling pathway.

These observations in principle indicate that inhibition of PAR-1 could be employed as a therapeutic strategy for decreasing BBB breakdown in a wide variety of pathological conditions. However, activation of PARs by other serine proteases like APC or factor Xa (FXa), can have a protective effect on endothelial barrier function. APC is an anticoagulant also known as blood coagulation factor XIV, that through association to its receptor EPCR allows activation of PAR-1.¹⁸⁸ Signaling of APC-activated PAR-1 is not mediated by heterotrimeric G proteins signaling, but through β-arrestin and dishevelled-2 scaffolds and Rac1 activation.¹⁸⁹ While thrombin activation of PAR-1 involves a canonical cleavage at Arg41, activation of PAR-1 by APC generates a non-canonical proteolysis at Arg46.¹⁹⁰ FXa, a protease critical in the blood coagulation cascade, can cleave PAR-3 at non-canonical Arg41, similar to APC.¹⁹¹

Activation of PAR-1 or PAR-3 by APC or FXa requires the presence of EPCR and involves activation of Tie2, a transmembrane tyrosine kinase receptor for angiopoietin, and results in up-regulation and translocation of ZO-1 to cell borders and stabilization of endothelial TJs.^191,192 This mechanism explains why recombinant human APC has been approved for patients with severe sepsis, where increased mortality is associated with bleeding risk.¹⁹³

In epithelial cells a similar mechanism promotes TJ barrier integrity. Thus in keratinocytes APC activates Tie2 through a mechanism that requires the sequential activation of EPCR, PAR-1 and EGFR that in turn enhances PI3K/AKT signaling, increases the expression of ZO-1 and claudin-1 and reduces paracellular permeability.¹⁹⁴

Hormone receptors

Angiotensin II activated receptor AT1

Angiotensin II (Ang II) is a peptide hormone that causes vasoconstriction and subsequently increases blood pressure. It also stimulates the release of aldosterone from the adrenal cortex, which promotes sodium retention and augments blood pressure. These effects of Ang II are mediated by G-coupled receptor AT1. In the kidney, podocytes are the cells responsible of the glomerular filtration barrier. Podocytes instead of exhibiting TJ between their neighboring foot processes, form a structure named slit diaphragm that allows the passage of molecules up to 70 ^oA, in contrast to TJ that prevent the passage of molecules >8^oA. Slit diaphragms are constituted by some proteins common to TJs like JAM-A, occludin, cingulin and ZO-1 and other unique membrane proteins like nephrin and podocin¹⁹⁵ (Fig 3).

In podocytes in culture, Ang II changes the pattern of organization of F-actin and ZO-1. Thus, the transcytoplasmic F-actin filaments disappear and ZO-1 distribution at cell-cell contacts becomes fragmented through a process mediated by the activation of Src kinase and phospholipase C.¹⁹⁶ Accordingly, in male MWF rats that develop spontaneous proteinuria with age, treatment with lisinopril, an inhibitor of angiotensin converting enzyme, preserves the distribution of ZO-1 at the slit diaphragm in the glomeruli, and in consequence prevents proteinuria and increases glomerular ultrafiltration coefficient.¹⁹⁷ Moreover, blockade of AT1 with ZD7155 attenuates the reduced expression and cytoplasmic redistribution of ZO-1, observed in rat glomerular epithelial cells exposed to high glucose and in diabetic rats.¹⁹⁸ Interestingly, a combined therapy with an AT1 blocker and a vitamin D analog that suppresses renin biosynthesis, which hydrolyses angiotensinogen to angiotensin I, prevents renal injury in diabetic nephropathy by reversing the decline of slit diaphragm proteins nephrin-1, ZO-1 and α-actinin-4.¹⁹⁹

Ang II also regulates endothelial TJs. Thus, Ang II promotes diabetic retinopathy by inducing VEGF expression and loss of TJ proteins in retinal endothelial cells. In accordance, blockade of Ang II with an inhibitor of angiotensin converting enzyme blocked these effects and attenuated the increased vascular permeability of diabetic retina.²⁰⁰ The effect of Ang II on the BBB remains controversial. Thus, some have reported that in brain microvessel endothelial cells Ang II induced through AT1 activation an increase in albumin permeability and a decrease in TER that is not accompanied by changes in the expression of occludin and claudin-5.²⁰¹ Others instead, observed that angiotensin KO animals have a disorganized pattern of occludin at the BBB, and that activation of AT1 diminishes the permeability of BBB endothelial cells and mobilizes occludin to lipid rafts membrane microdomains.²⁰² Accordingly, multiple sclerosis lesions with a leaky BBB have a reduced number of perivascular astrocytes positive for angiotensinogen.²⁰²

Corticotropin-releasing hormone receptors CRHR1 and CRHR2

Corticotropin-releasing hormone (CRH) is a 41-amino acid peptide secreted by the hypothalamus in response to stress, whose main function is the stimulation of the synthesis of adrenocorticotropic hormone by the anterior pituitary gland. In breast cancer cells, CRH inhibits TGFβ1-mediated transformation mainly via G-coupled CRH receptor 2 (CRHR2), inducing the expression of E-cadherin and occludin and down-regulating that of snail1 and twist1.²⁰³ Instead, activation of CRHR1 exerts a negative effect in TJs of the intestine. Thus, in the Wistar Kyoto rat model of irritable bowel syndrome, the administration of anti IL-6 and CRHR1 antagonist antalarmin, normalized stress-induced defecation and visceral pain sensitivity through a mechanism that involves a reduction in the overexpression of claudin-2.²⁰⁴ In a similar sense, fish oil enhanced intestinal barrier function by increasing the expression of claudin-1, via inhibition of CRHR1 signaling in weaned pigs after lipopolysaccharide challenge.²⁰⁵

Membrane bound G protein-coupled estrogen receptor GPR30

Estrogen is the primary female sex hormone responsible for the development and regulation of the female reproductive system and secondary sex characteristics. In women, estrone, estradiol and estriol constitute the major naturally occurring forms of estrogen, being estradiol the most potent and prevalent estrogen. The canonical mechanism of estrogen action is through nuclear receptors ERα and ERβ, which stimulate gene expression by acting as transcription factors. The non-classical mechanism instead, involves action through GPR30, a G protein-coupled estrogen receptor found in the membrane of the endoplasmic reticulum that promotes signaling through Zn²⁺-dependent matrix metalloproteinases, EGFR, PI3K, Ca²⁺ mobilization and nitric oxide production. In human hepatoma cells, activation of GPR30 leads to cleavage of occludin by MMP-9. Since occludin is a receptor for hepatitis C virus, these results contribute to explain why hepatocellular carcinoma and cirrhosis, in response to hepatitis C virus, is more associated with men and postmenopausal women than with premenopausal women and women receiving hormone replacement therapy.²⁰⁶ Likewise, 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47) increases migration and invasion of neuroblastoma cells, by down-regulating E-cadherin and ZO-1 expression and upregulating MMP-9 via the GPR30/PI3K/AKT signaling pathway.²⁰⁷ In contrast, in the brain GPR30 activation reduces occludin and claudin-5 disruption in the BBB, via inhibition of VEGF expression after ischemic injury.²⁰⁸

Prostaglandin E₂ receptors

Prostaglandins PGE₂, PGF_2α and PGI₂ are lipid autacoids, meaning that these prostanoids, product of cyclooxygenase mediated metabolism of arachidonic acid, are locally produced and metabolized and usually act near their site of synthesis.

During the differentiation of Caco-2 intestinal epithelial cells, a decrease in cyclooxygenase-2 expression and PGE₂ synthesis was observed, accompanied by a reduction in paracellular permeability, while the addition of PGE₂ to differentiated cells disrupted the epithelial barrier function,²⁰⁹ through activation of EP₁ and EP₄ G-coupled PGE₂ receptors. This process that involved redistribution of occludin, ZO-1 and perijunctional actin, and increased MLCK activity, was mediated through the PLC/IP3/ Ca²⁺ /PKC and cAMP-PKA signaling pathways.²¹⁰ Another study revealed that PGE₂ receptor EP₂ was also involved in TJ disassembly in Caco-2 cells by PGE₂ through a mechanism mediated by PKC and not PKA.²¹¹ However, the presence of PGE₂ receptor EP₂ appears to be crucial for maintaining TJ barrier integrity, since EP₂ receptor antagonism or siRNA based silencing decreased claudin-4 expression by proteosomal degradation decreasing in consequence the TER of Caco-2 cells.²¹²

In rat brain, post-ischemic treatment with EP₁ antagonist, SC-51089 or EP₁ genetic ablation, reduces MMP-9/3 activity and preserves ZO-1 and occludin at the BBB, highlighting the possibility of targeting EP₁ receptor to diminish stroke induced neurovascular damage.²¹³

Bradykinin activated receptor BR2

Bradykinin is a 9-amino acid peptide generated by the proteolytic cleavage of high-molecular weight kininogen by the enzymes kallikreins, and is degraded by kininases including angiotensin-converting enzyme. Bradykinin dilates blood vessels causing a fall in blood pressure. Bradykinin and its long-lasting analogue labradimil/RPM-7, selective for the G-coupled bradykinin receptor 2 (BR2), induce a higher increase in permeability in the blood-brain tumor barrier than in the BBB. This feature is of utmost therapeutic importance as it allows the delivery of hydrophilic chemotherapeutics,^214,215 and virus vectors with killing-genes.²¹⁶ to brain tumors and not to healthy brain tissue. In brain microvascular endothelial cells BR2 through Gα_i inhibits adenylyl cyclase decreasing the expression of cAMP and PKA and through Gα_q stimulates PLC to increase intracellular calcium. These changes alter the perijunctional actin cytoskeleton and down-regulates the expression of ZO-1, occludin and claudin-5.²¹⁷ The increase in blood-brain tumor barrier permeability is also mediated by ROCK activation that induces TJ protein relocalization, stress fibers formation, and MLC and cofilin phosphorylation,^218,219 and by a NOS dependent mechanism that induces the translocation to the nucleus of ZONAB transcription factor, which inhibits the promoters of claudin-5 and occludin genes, resulting in a reduced expression of the corresponding TJ proteins.²²⁰

In BR1 KO mice, the associated compensatory over-expression of BR2 in the intestine exacerbates the DSS-induced colitis, decreasing occludin gene expression.²²¹ In contrast, bradykinin decreased podocyte permeability and increased ZO-1 staining at the cell borders, through activation of BR2 that transactivates EGFR by the disintegrin and metalloenzyme ADAM-17.²²²

Regulation of colonic, renal, endothelial, blood-retinal and blood-brain barriers by different G protein-coupled receptors

In this section we summarize the interplay between different G protein-coupled receptors in epithelial and endothelial barriers, highlighting the studies where a clear mechanistic link to TJs has been found.

Colon

Fig. 2 shows that opening and closure of TJ in colon epithelia is regulated by a wide variety of G protein-coupled receptors. Signaling through Gα_s and Gα_q/11 modulates both TJ closure and opening, whereas signaling via Gα_i promotes TJ closure.

TJ formation and closure is obtained upon activation of Calcrl receptor that leads to a decreased phosphorylation of MLC and the STAT transcription factors,^66,67; the activation of CBR1 and CBR2 receptors that lower the expression of NFκB and cytokines^88,89 and upon activation of AMPK⁵¹ and p70S6K³⁷ by receptors CaSR and GPR39, respectively. A decrease in TNF-α that leads to TJ closure appears to be a common step employed by the activation of several GPCRs including CBR1,⁶⁶ CBR2,⁶⁷ and S1PR1⁹⁹ receptors.

The impact of ERK on intestinal TJs is difficult to understand due to contradictory observations. Thus for example, TJ closure has been reported in colon due to a decrease in ERK phosphorylation induced by SSTR,⁷⁰ while others have reported the same effect owed to ERK activation by receptors GPR40⁹³ or OGR1.²⁹ Moreover, receptor CRHR1 activation of Gαs promotes ERK1/2 activation that opens intestinal TJs.²⁰⁴

TJ opening in intestine is also achieved by activation of EP₁ receptor through Gα_q/11 that activates PKC and MLCK, inducing the contraction of prerijunctional actin and the formation of stress fibers.²¹⁰ In colon, G-protein coupled receptors also open TJs signaling through routes independent of Gα. Thus, receptor CXCR3 forms a complex with the adaptor protein MyD88 and gliadin, the toxic compound of gluten, which increases the expression zonulin that opens TJs,¹³⁴ and PAR-2 through β-arrestin activates ERK1/2 and induces TJ opening.¹⁷²

Slit diaphragm in the glomerulus

Closure of the slit diaphragm between podocytes in the glomerulus, is induced by BR2 receptor via Gα_i through activation of ERK,²²² while the opening is triggered by AT1 receptor via Gα_q/11 and a decrease in actin bundle formation.¹⁹⁶ (Fig. 3).

Blood brain barrier

A wide diversity of GPCRs regulate BBB opening and closure (Fig. 4) and while signaling through Gα_s and Gα_i is implicated in both processes, only Gα_q/11 is involved in BBB opening. The mechanisms triggered by GPCRs that promote BBB closure include the participation of Sox-17 transcription factor induced by the activation of frizzled-4 receptor by norrin protein, a non-Wnt ligand,¹⁰⁷ and a decrease in iNOS accompanied by an augmentation of eNOS due to SSTR1 receptor activation.⁶⁹ The latter point appears to be controversial since other studies have shown that an increase in eNOS by activation of receptors ETB¹⁴² and BR2²²⁰ lead to BBB opening.

The BBB appears to be very sensitive to PKA levels, as activation of PKA by receptor GLPR1 tightens the barrier,⁷⁴ while reducing cAMP and PKA levels through BR2 activation opens the BBB ²¹⁷ In addition, activation of MMPs by EP1 receptor,²¹³ of NFκB by C5aR¹⁴⁰ or RhoA by BR2 receptor²¹⁸ also promote BBB opening.

Inner blood-retinal barrier

In the retina it is surprising to observe that both Gα_s and Gα_i, which exert opposite effects on adenyly cyclase, mediate closure of the iBRB (Fig. 5). However, upon closer examination it becomes clear that activation of MC1, MC5⁷⁶ and Calcrl⁶⁴ receptors that signal through Gα_s, and S1PR1 and S1PR3 ⁹⁷ receptors that signal via Gα_i, leads to a decrease in NFκB that induces closure of the iBRB. Other mechanisms that lead to iBRB closure include activation of Gα_s by GLPR1 that induces a decrease in AKT phosphorylation, which diminishes the phosphorylation and activity of STAT3,⁷⁵ as well as the activation of Gα_i by δ opioid receptor that triggers a decrease in TNF-α, iNOS and caveolin endocytosis.¹⁰²

Endothelia in tissues different to the retina or the brain

In endothelia present in tissues different to the retina or the brain, the four Gα subunits are involved in both TJ opening and closure (Fig. 6). It is noteworthy that the outcome of Calcrl activation varies depending on the kind of endothelia. Thus, in rat coronary microvascular endothelial cells (RCECs) signaling through Gα_s leads to an increase in cAMP and PKA activation that blocks RhoA and Rac1 activation and in consequence produces barrier failure.⁵⁷ Instead, in HUVEC Gα_s activation by Calcrl decreases RhoA activity as in RCEC but increases Rac1 activity leading to endothelial barrier enhancement,^56,57 The reason why Calcrl exerts opposites effects on Rac1 in these endothelia remains unknown.

The appearance of stress fibers is deleterious for endothelial TJ closure. Hence, an increase in these fibers triggered by S1PR3 debilitates the barrier,²²³ while a decrease in stress fibers induced by Calcrl promotes TJ closure.^53–57 In endothelia, stimulation of Gα_i by CCR3 augments the expression of NFκB and promotes TJ opening,¹³⁸ in agreement to what has been observed in colon⁹³ and retina.⁹⁷ Other GPCRs that induce TJ closure include PAR-1 activation of Gα_12/13 that through β-arrestin stimulates PI3K and AKT¹⁹⁰; and PAR-3 that through Gα_q/11 leads to the activation of angiopoietin receptor Tie2.¹⁹¹

Concluding remarks

GPCRs activated by diverse stimuli like ions, hormones, lipids, chemokines, nucleotides, proteases and light, signal through heterotrimeric G proteins, arrestins and kinases, to modulate the degree of sealing of TJs in epithelial and endothelial cells throughout the organism. Activation of these receptors regulates TJ proteins by changing their level of expression, localization at the cell borders or degree of phosphorylation. Another mechanism triggered by some GPCRs to induce TJ opening is contraction of the cortical ring of actomyosin. In the BBB, BRB, keratinocytes, colon epithelia, renal tubular cells and the glomerulus, numerous studies have been done revealing the participation of a wide variety of GPCRs in TJ modulation. Nevertheless, the relationship between GPCRs and TJs has not always been studied in depth and deeper knowledge of the mechanisms involved is needed. In addition, it would be helpful if future studies provide insight on the interplay between different GPCRs that act on the same epithelial and endothelial barriers.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Additional information

Funding

This work was supported by grant 237241 to L.G.M of the Mexican National Council of Science and Technology (Conacyt). A.R.S., L.G.G. and C.H.G. were recipients of doctoral fellowships from Conacyt (233193, 340209, 407499).