Tripathi, A. et al. Identification of human zonulin, a physiological modulator of tight junctions, as prehaptoglobin-2. Proc. Natl Acad. Sci. USA 106, 16799-16804

Mucosal Biology Research Center, Center for Vascular and Inflammatory Diseases and Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 20201, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 09/2009; 106(39):16799-804. DOI: 10.1073/pnas.0906773106
Source: PubMed


Increased intestinal permeability (IP) has emerged recently as a common underlying mechanism in the pathogenesis of allergic, inflammatory, and autoimmune diseases. The characterization of zonulin, the only physiological mediator known to regulate IP reversibly, has remained elusive. Through proteomic analysis of human sera, we have now identified human zonulin as the precursor for haptoglobin-2 (pre-HP2). Although mature HP is known to scavenge free hemoglobin (Hb) to inhibit its oxidative activity, no function has ever been ascribed to its uncleaved precursor form. We found that the single-chain zonulin contains an EGF-like motif that leads to transactivation of EGF receptor (EGFR) via proteinase-activated receptor 2 (PAR(2)) activation. Activation of these 2 receptors was coupled to increased IP. The siRNA-induced silencing of PAR(2) or the use of PAR(2)(-/-) mice prevented loss of barrier integrity. Proteolytic cleavage of zonulin into its alpha(2)- and beta-subunits neutralized its ability to both activate EGFR and increase IP. Quantitative gene expression revealed that zonulin is overexpressed in the intestinal mucosa of subjects with celiac disease. To our knowledge, this is the initial example of a molecule that exerts a biological activity in its precursor form that is distinct from the function of its mature form. Our results therefore characterize zonulin as a previously undescribed ligand that engages a key signalosome involved in the pathogenesis of human immune-mediated diseases that can be targeted for therapeutic interventions.

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Available from: Alessio Fasano
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    • "5. Biomarkers mainly used in humans to assess intestinal inflammation and permeability Zonulin is a 47 kDa protein which modulates intestinal permeability by disassembling intercellular tight junctions between epithelial cells in the digestive tract (Wang et al., 2000; Fasano, 2001; Vanuytsel et al., 2013). The effect of zonulin on increased intestinal permeability is mediated through activation of EGF receptor (EGFR) via proteinaseactivated receptor 2 (PAR2) activation (Tripathi et al., 2009). Patients with type 1 diabetes, an autoimmune disease in which the finely tuned regulation of intestinal tight junctions is lost, have increased serum zonulin levels. "
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    ABSTRACT: Liver disease is often times associated with increased intestinal permeability. A disruption of the gut barrier allows microbial products and viable bacteria to translocate from the intestinal lumen to extraintestinal organs. The majority of the venous blood from the intestinal tract is drained into the portal circulation, which is part of the dual hepatic blood supply. The liver is therefore the first organ in the body to encounter not only absorbed nutrients, but also gut-derived bacteria and pathogen associated molecular patterns (PAMPs). Chronic exposure to increased levels of PAMPs has been linked to disease progression during early stages and to infectious complications during late stages of liver disease (cirrhosis). It is therefore important to assess and monitor gut barrier dysfunction during hepatic disease. We review methods to assess intestinal barrier disruption and discuss advantages and disadvantages. We will in particular focus on methods that we have used to measure increased intestinal permeability and bacterial translocation during experimental liver disease models. Copyright © 2015. Published by Elsevier B.V.
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    • "The sTNFR1 is a sensitive marker of low-grade inflammation in the obese [20]. Thus, these results confirm that circulating zonulin is an inflammatory marker, as its precursor, haptoglobin (Hp) [12], a liver acute-phase response protein. "
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    ABSTRACT: The association between gut microbiota and circulating zonulin level, a marker of intestinal permeability, has not been studied yet. The aim of the study is the assessment of plasma zonulin, haptoglobin and proinflammatory cytokines (TNF- α and IL-6) levels in relation to composition of gut microbiota in obese and normal weight subjects. Circulating inflammation markers, such as TNF- α , sTNFR1, sTNFR2, IL-6, zonulin, and haptoglobin levels were measured and semiquantitative analysis of gut microbiota composition was carried out in 50 obese and 30 normal weight subjects without concomitant diseases. Higher circulating zonulin, TNF- α , sTNFR1, sTNFR2, and IL-6 levels were found in the obese subjects. Plasma zonulin level correlated positively with age (r = 0.43, P < 0.001), body mass (r = 0.30, P < 0.01), BMI (r = 0.33, P < 0.01), fat mass and fat percentage (r = 0.31, P < 0.01 and r = 0.23, P < 0.05, resp.). Positive correlations between bacterial colony count and sTNFR1 (r = 0.33, P < 0.01) and plasma zonulin (r = 0.26, P < 0.05) but not haptoglobin levels were found. Additionally, plasma zonulin level was proportional to daily energy intake (r = 0.27, P < 0.05) and serum glucose concentration (r = 0.18, P < 0.05) and inversely proportional to diet protein percentage (r = -0.23, P < 0.05). Gut microbiota-related systemic microinflammation in the obese is reflected by circulating zonulin level, a potential marker of interstitial permeability.
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    • "Zonulin recently identified as preheptaglobin-2 [19], is the endogenous eukaryotic analogue of zonula occludens toxin (Zot) from Vibrio cholera. Zonulin/preheptaglobin-2 opens the paracellular route by binding to a specific surface receptor that triggers the activation of a signaling pathway that involves phospholipase C and protein kinase C activation, actin polymerization, and contraction of the perijunctional action-myosin ring [for review see [20]. "
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    ABSTRACT: Transit of human neural stem cells, ReNcell CX, through the blood brain barrier (BBB) was evaluated in an in vitro model of BBB and in nude mice. The BBB model was based on rat brain microvascular endothelial cells (RBMECs) cultured on Millicell inserts bathed from the basolateral side with conditioned media (CM) from astrocytes or glioma C6 cells. Glioma C6 CM induced a significant transendothelial migration of ReNcells CX in comparison to astrocyte CM. The presence in glioma C6 CM of high amounts of HGF, VEGF, zonulin and PGE2, together with the low abundance of EGF, promoted ReNcells CX transmigration. In contrast cytokines IFN-α, TNF-α, IL-12p70, IL-1β, IL-6, IL-8 and IL-10, as well as metalloproteinases -2 and -9 were present in equal amounts in glioma C6 and astrocyte CMs. ReNcells expressed the tight junction proteins occludin and claudins 1, 3 and 4, and the cell adhesion molecule CRTAM, while RBMECs expressed occludin, claudins 1 and 5 and CRTAM. Competing CRTAM mediated adhesion with soluble CRTAM, inhibited ReNcells CX transmigration, and at the sites of transmigration, the expression of occludin and claudin-5 diminished in RBMECs. In nude mice we found that ReNcells CX injected into systemic circulation passed the BBB and reached intracranial gliomas, which overexpressed HGF, VEGF and zonulin/prehaptoglobin 2.
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