The Integral Role of Tight Junction Proteins in the Repair of Injured Intestinal Epithelium

The intestinal epithelial monolayer forms a transcellular and paracellular barrier that separates luminal contents from the interstitium. The paracellular barrier consists of a highly organized complex of intercellular junctions that is primarily regulated by apical tight junction proteins and tight junction-associated proteins. This homeostatic barrier can be lost through a multitude of injurious events that cause the disruption of the tight junction complex. Acute repair after injury leading to the reestablishment of the tight junction barrier is crucial for the return of both barrier function as well as other cellular functions, including water regulation and nutrient absorption. This review provides an overview of the tight junction complex components and how they link to other plasmalemmal proteins, such as ion channels and transporters, to induce tight junction closure during repair of acute injury. Understanding the components of interepithelial tight junctions and the mechanisms of tight junction regulation after injury is crucial for developing future therapeutic targets for patients experiencing dysregulated intestinal permeability.

MTOR-DRIVEN ALTERATIONS IN THE BRAIN MICROVASCULAR PROTEOME IN ALZHEIMER’S DISEASE

Therapeutic interventions for Alzheimer’s disease (AD) remain limited due to an incomplete understanding of the molecular mechanisms of its onset and progression. Cerebrovascular dysfunction occurs early during disease development. Attenuation of mTOR, a key regulator of aging, attenuates and reverses cerebrovascular deficits by restoring cerebral blood flow, brain vascular density, neurovascular coupling, and vascular amyloid-β clearance in hAPP(J20) mice expressing human amyloid precursor protein carrying two FAD-associated mutations. The mechanisms by which mTOR attenuation alleviates AD pathology are poorly understood. This study defined changes in the microvascular proteome of hAPP(J20) mice arising from mTOR attenuation. At 7 months of age, hAPP(J20) mice were fed vehicle- or rapamycin-supplemented diet (2.24 mg/kg/day) for 4 months. Mass spectrometry of collected brain microvasculature identified significant changes in 840 of 3361 proteins (p<0.05). mTOR attenuation led to significant changes in 26 of these proteins, some of which are involved in pathways including tight junction regulation, calcium signaling, and actin cytoskeleton regulation. Candidate mediators of mTOR-driven cerebrovascular dysfunction were identified by selecting proteins that were aberrantly altered in hAPP(J20) microvasculature and normalized by rapamycin. Examples include members of the heterogeneous nuclear ribonucleoprotein family (hnRNPA/B, hnRNPD) which regulate the mRNA stability of genes related to cellular cycle arrest and inflammatory cytokines as well as localization of crucial mRNA involved in nitric oxide signaling. Also included are nucleoporin 54 and vacuolar ATPase assembly factor, both of which are altered in aging and neurodegeneration. Subsequent studies will elucidate the role of these proteins in mTOR-driven cerebrovascular dysfunction in AD.

Tight junction regulation through vesicle trafficking: bringing cells together

Epithelial layers are integral for many physiological processes and are maintained by intercellular adhesive structures. During disease, these structures can disassemble, leading to breakdown of epithelia. TJs (tight junctions) are one type of intercellular adhesion. Loss of TJs has been linked to the pathogenesis of many diseases. The present review focuses on the role of vesicle trafficking in regulation of TJs, in particular trafficking of the TJ protein occludin. We examine how endocytosis and endosomal recycling modulate occludin localization under steady-state conditions and during stimulated TJ disassembly.

Protein kinase Cζ phosphorylates occludin and promotes assembly of epithelial tight junctions

Protein kinases play an important role in the regulation of epithelial tight junctions. In the present study, we investigated the role of PKCζ (protein kinase Cζ) in tight junction regulation in Caco-2 and MDCK (Madin–Darby canine kidney) cell monolayers. Inhibition of PKCζ by a specific PKCζ pseudosubstrate peptide results in redistribution of occludin and ZO-1 (zona occludens 1) from the intercellular junctions and disruption of barrier function without affecting cell viability. Reduced expression of PKCζ by antisense oligonucleotide or shRNA (short hairpin RNA) also results in compromised tight junction integrity. Inhibition or knockdown of PKCζ delays calcium-induced assembly of tight junctions. Tight junction disruption by PKCζ pseudosubstrate is associated with the dephosphorylation of occludin and ZO-1 on serine and threonine residues. PKCζ directly binds to the C-terminal domain of occludin and phosphorylates it on threonine residues. Thr403, Thr404, Thr424 and Thr438 in the occludin C-terminal domain are the predominant sites of PKCζ-dependent phosphorylation. A T424A or T438A mutation in full-length occludin delays its assembly into the tight junctions. Inhibition of PKCζ also induces redistribution of occludin and ZO-1 from the tight junctions and dissociates these proteins from the detergent-insoluble fractions in mouse ileum. The present study demonstrates that PKCζ phosphorylates occludin on specific threonine residues and promotes assembly of epithelial tight junctions.