David R. Raleigh

Tight Junction Pore and Leak Pathways: A Dynamic Duo

Tissue barriers that restrict passage of liquids, ions, and larger solutes are essential for the development of multicellular organisms. In simple organisms this allows distinct cell types to interface with the external environment. In more complex species, the diversity of cell types capable of forming barriers increases dramatically. Although the plasma membranes of these barrier-forming cells prevent flux of most hydrophilic solutes, the paracellular, or shunt, pathway between cells must also be sealed. This function is accomplished in vertebrates by the zonula occludens, or tight junction. The tight junction barrier is not absolute but is selectively permeable and is able to discriminate between solutes on the basis of size and charge. Many tight junction components have been identified over the past 20 years, and recent progress has provided new insights into the proteins and interactions that regulate structure and function. This review presents these data in a historical context and proposes an integrated model in which dynamic regulation of tight junction protein interactions determines barrier function.

Caveolin-1–dependent occludin endocytosis is required for TNF-induced tight junction regulation in vivo

Although tight junction morphology is not obviously affected by TNF, this proinflammatory cytokine promotes internalization of occludin, resulting in disrupted barrier function within the intestine.

Tight Junction–associated MARVEL Proteins MarvelD3, Tricellulin, and Occludin Have Distinct but Overlapping Functions

This study identifies and characterizes marvelD3, a novel tight junction protein that contains a conserved MARVEL domain. Analyses using phylogenetic, expression profiling, microscopic, and functional approaches show that marvelD3, occludin, and tricellulin are related and have distinct but overlapping functions at the tight junction.

Epithelial Myosin Light Chain Kinase Activation Induces Mucosal Interleukin-13 Expression to Alter Tight Junction Ion Selectivity

Intestinal barrier function is reduced in inflammatory bowel disease (IBD). Tumor necrosis factor (TNF) and interleukin (IL)-13, which are up-regulated in IBD, induce barrier defects that are associated with myosin light chain kinase (MLCK) activation and increased claudin-2 expression, respectively, in cultured intestinal epithelial monolayers. Here we report that these independent signaling pathways have distinct effects on tight junction barrier properties and interact in vivo. MLCK activation alters size selectivity to enhance paracellular flux of uncharged macromolecules without affecting charge selectivity and can be rapidly reversed by MLCK inhibition. In contrast, IL-13-dependent claudin-2 expression increases paracellular cation flux in vitro and in vivo without altering tight junction size selectivity but is unaffected by MLCK inhibition in vitro. In vivo, MLCK activation increases paracellular flux of uncharged macromolecules and also triggers IL-13 expression, claudin-2 synthesis, and increased paracellular cation flux. We conclude that reversible, MLCK-dependent permeability increases cause mucosal immune activation that, in turn, feeds back on the tight junction to establish long-lasting barrier defects. Interactions between these otherwise distinct tight junction regulatory pathways may contribute to IBD pathogenesis.

Occludin S408 phosphorylation regulates tight junction protein interactions and barrier function

Occludin S408 phosphorylation regulates interactions between occludin, ZO-1, and select claudins to define tight junction molecular structure and barrier function.

MLCK-dependent exchange and actin binding region-dependent anchoring of ZO-1 regulate tight junction barrier function

The perijunctional actomyosin ring contributes to myosin light chain kinase (MLCK)-dependent tight junction regulation. However, the specific protein interactions involved in this process are unknown. To test the hypothesis that molecular remodeling contributes to barrier regulation, tight junction protein dynamic behavior was assessed by fluorescence recovery after photobleaching (FRAP). MLCK inhibition increased barrier function and stabilized ZO-1 at the tight junction but did not affect claudin-1, occludin, or actin exchange in vitro. Pharmacologic MLCK inhibition also blocked in vivo ZO-1 exchange in wild-type, but not long MLCK−/−, mice. Conversely, ZO-1 exchange was accelerated in transgenic mice expressing constitutively active MLCK. In vitro, ZO-1 lacking the actin binding region (ABR) was not stabilized by MLCK inhibition, either in the presence or absence of endogenous ZO-1. Moreover, the free ABR interfered with full-length ZO-1 exchange and reduced basal barrier function. The free ABR also prevented increases in barrier function following MLCK inhibition in a manner that required endogenous ZO-1 expression. In silico modeling of the FRAP data suggests that tight junction-associated ZO-1 exists in three pools, two of which exchange with cytosolic ZO-1. Transport of the ABR-anchored exchangeable pool is regulated by MLCK. These data demonstrate a critical role for the ZO-1 ABR in barrier function and suggest that MLCK-dependent ZO-1 exchange is essential to this mechanism of barrier regulation.

Occludin OCEL-domain interactions are required for maintenance and regulation of the tight junction barrier to macromolecular flux.

Occludin loss enhances paracellular macromolecular permeability (radius up to ∼62.5 Å) and is necessary for TNF-induced barrier loss. The latter requires the C-terminal OCEL domain, which stabilizes tight junction–associated occludin and regulates trafficking. Thus OCEL-mediated interactions are critical regulators of macromolecular paracellular flux.

Targeted next-generation sequencing of pediatric neuro-oncology patients improves diagnosis, identifies pathogenic germline mutations, and directs targeted therapy

Background Molecular profiling is revolutionizing cancer diagnostics and leading to personalized therapeutic approaches. Herein we describe our clinical experience performing targeted sequencing for 31 pediatric neuro-oncology patients. Methods We sequenced 510 cancer-associated genes from tumor and peripheral blood to identify germline and somatic mutations, structural variants, and copy number changes. Results Genomic profiling was performed on 31 patients with tumors including 11 high-grade gliomas, 8 medulloblastomas, 6 low-grade gliomas, 1 embryonal tumor with multilayered rosettes, 1 pineoblastoma, 1 uveal ganglioneuroma, 1 choroid plexus carcinoma, 1 chordoma, and 1 high-grade neuroepithelial tumor. In 25 cases (81%), results impacted patient management by: (i) clarifying diagnosis, (ii) identifying pathogenic germline mutations, or (iii) detecting potentially targetable alterations. The pathologic diagnosis was amended after genomic profiling for 6 patients (19%), including a high-grade glioma to pilocytic astrocytoma, medulloblastoma to pineoblastoma, ependymoma to high-grade glioma, and medulloblastoma to CNS high-grade neuroepithelial tumor with BCOR alteration. Multiple patients had pathogenic germline mutations, many of which were previously unsuspected. Potentially targetable alterations were identified in 19 patients (61%). Additionally, novel likely pathogenic alterations were identified in 3 cases: an in-frame RAF1 fusion in a BRAF wild-type pleomorphic xanthoastrocytoma, an inactivating ASXL1 mutation in a histone H3 wild-type diffuse pontine glioma, and an in-frame deletion within exon 2 of MAP2K1 in a low-grade astrocytic neoplasm. Conclusions Our experience demonstrates the significant impact of molecular profiling on diagnosis and treatment of pediatric brain tumors and confirms its feasibility for use at the time of diagnosis or recurrence.

Molecular targets and mechanisms of radiosensitization using DNA damage response pathways

The cellular reaction to genomic instability includes a network of signal transduction pathways collectively referred to as the DNA damage response (DDR). Activated by a variety of DNA lesions, the DDR orchestrates cell cycle arrest and DNA repair, and initiates apoptosis in instances where damage cannot be repaired. As such, disruption of the DDR increases the prevalence of DNA damage secondary to incomplete repair, and in doing so, enhances radiation-induced cytotoxicity. This article describes the molecular agents and their targets within DDR pathways that sensitize cells to radiation. Moreover, it reviews the therapeutic implications of these compounds, provides an overview of relevant clinical trials and offers a viewpoint on the evolution of the field in the years to come.

p50 (NF-κB1) Is an Effector Protein in the Cytotoxic Response to DNA Methylation Damage

The functional significance of the signaling pathway induced by O(6)-methylguanine (O(6)-MeG) lesions is poorly understood. Here, we identify the p50 subunit of NF-κB as a central target in the response to O(6)-MeG and demonstrate that p50 is required for S(N)1-methylator-induced cytotoxicity. In response to S(N)1-methylation, p50 facilitates the inhibition of NF-κB-regulated antiapoptotic gene expression. Inhibition of NF-κB activity is noted to be an S phase-specific phenomenon that requires the formation of O(6)-MeG:T mismatches. Chk1 associates with p50 following S(N)1-methylation, and phosphorylation of p50 by Chk1 results in the inhibition of NF-κB DNA binding. Expression of an unphosphorylatable p50 mutant blocks inhibition of NF-κB-regulated antiapoptotic gene expression and attenuates S(N)1-methylator-induced cytotoxicity. While O(6)-MeG:T-induced, p50-dependent signaling is not sufficient to induce cell death, this pathway sensitizes cells to the cytotoxic effects of DNA breaks.