Analysis of the 'angulin' proteins LSR, ILDR1 and ILDR2--tricellulin recruitment, epithelial barrier function and implication in deafness pathogenesis.

Tricellular tight junctions (tTJs) seal the extracellular space at tricellular contacts (TCs), where the corners of three epithelial cells meet. To date, the transmembrane proteins tricellulin and lipolysis-stimulated lipoprotein receptor (LSR) are known to be molecular components of tTJs. LSR recruits tricellulin to tTJs, and both proteins are required for the full barrier function of epithelial cellular sheets. In the present study, we show that two LSR-related proteins, immunoglobulin-like domain-containing receptor (ILDR) 1 and ILDR2, are also localized at TCs and recruit tricellulin. At least one of LSR, ILDR1 and ILDR2 was expressed in most of the epithelial tissues in mice. The expressions of LSR, ILDR1 and ILDR2 were generally complementary to each other, although LSR and ILDR1 were co-expressed in some epithelia. ILDR1 was required for the establishment of a strong barrier of the epithelium, similar to LSR, when introduced into cultured epithelial cells, whereas ILDR2 provided a much weaker barrier. We further analyzed human ILDR1, mutations in which cause a familial deafness, DFNB42, and found that most DFNB42-associated ILDR1 mutant proteins were defective in recruitment of tricellulin. We also found that tricellulin mutant proteins associated with another familial deafness, DFNB49, were not recruited to TCs by ILDR1. These findings show the heterogeneity of the molecular organization of tTJs in terms of the content of LSR, ILDR1 or ILDR2, and suggest that ILDR1-mediated recruitment of tricellulin to TCs is required for hearing. Given their common localization at epithelial cell corners and recruitment of tricellulin, we propose to designate LSR, ILDR1 and ILDR2 as angulin family proteins.

LSR defines cell corners for tricellular tight junction formation in epithelial cells.

Epithelial cell contacts consist of not only bicellular contacts but also tricellular contacts, where the corners of three cells meet. At tricellular contacts, tight junctions (TJs) generate specialized structures termed tricellular TJs (tTJs) to seal the intercellular space. Tricellulin is the only known molecular component of tTJs and is involved in the formation of tTJs, as well as in the normal epithelial barrier function. However, the detailed molecular mechanism of how tTJs are formed and maintained remains elusive. Using a localization-based expression cloning method, we identified a novel tTJ-associated protein known as lipolysis-stimulated lipoprotein receptor (LSR). Upon LSR knockdown in epithelial cells, tTJ formation was affected and the epithelial barrier function was diminished. Tricellulin accumulation at the tricellular contacts was also diminished in these cells. By contrast, LSR still accumulated at the tricellular contacts upon tricellulin knockdown. Analyses of deletion mutants revealed that the cytoplasmic domain of LSR was responsible for the recruitment of tricellulin. On the basis of these observations, we propose that LSR defines tricellular contacts in epithelial cellular sheets by acting as a landmark to recruit tricellulin for tTJ formation.

A coronary artery disease-associated gene product, JCAD/KIAA1462, is a novel component of endothelial cell-cell junctions.

Cell-cell junctions play crucial roles in the organization and function of epithelial and endothelial cellular sheets. Here, we have identified the protein product for KIAA1462 gene, whose single nucleotide polymorphisms (SNPs) have recently reported to be associated with coronary artery disease, as a novel component of cell-cell junctions. We propose the name of KIAA1462 protein junctional protein associated with coronary artery disease (JCAD). JCAD is a ∼145 kDa protein without any known domains but contains a proline-rich region. Immunolocalization studies revealed that JCAD is specifically localized at cell-cell junctions in endothelial cells but not in epithelial cells. The accumulation of JCAD at cell-cell junctions in cultured endothelial cells was impaired by RNAi-mediated suppression of VE-cadherin expression. In cell adhesion-deficient mouse L fibroblasts, JCAD was recruited to cell-cell contacts when cadherin-mediated cell-cell adhesion was induced. These results indicate that JCAD is a component of VE-cadherin-based cell-cell junctions in endothelial cells. This study also suggests the implication of endothelial cell-cell adhesion in coronary artery disease.

Suppression of STING Associated with LKB1 Loss in KRAS-Driven Lung Cancer.

KRAS-driven lung cancers frequently inactivate TP53 and/or STK11/LKB1, defining tumor subclasses with emerging clinical relevance. Specifically, KRAS-LKB1 (KL)-mutant lung cancers are particularly aggressive, lack PD-L1, and respond poorly to immune checkpoint blockade (ICB). The mechanistic basis for this impaired immunogenicity, despite the overall high mutational load of KRAS-mutant lung cancers, remains obscure. Here, we report that LKB1 loss results in marked silencing of stimulator of interferon genes (STING) expression and insensitivity to cytoplasmic double-strand DNA (dsDNA) sensing. This effect is mediated at least in part by hyperactivation of DNMT1 and EZH2 activity related to elevated S-adenylmethionine levels and reinforced by DNMT1 upregulation. Ectopic expression of STING in KL cells engages IRF3 and STAT1 signaling downstream of TBK1 and impairs cellular fitness, due to the pathologic accumulation of cytoplasmic mitochondrial dsDNA associated with mitochondrial dysfunction. Thus, silencing of STING avoids these negative consequences of LKB1 inactivation, while facilitating immune escape. SIGNIFICANCE: Oncogenic KRAS-mutant lung cancers remain treatment-refractory and are resistant to ICB in the setting of LKB1 loss. These results begin to uncover the key underlying mechanism and identify strategies to restore STING expression, with important therapeutic implications because mitochondrial dysfunction is an obligate component of this tumor subtype.See related commentary by Corte and Byers, p. 16.This article is highlighted in the In This Issue feature, p. 1.

Retinoblastoma Inactivation Induces a Protumoral Microenvironment via Enhanced CCL2 Secretion.

Cancer cell-intrinsic properties caused by oncogenic mutations have been well characterized; however, how specific oncogenes and tumor suppressors impact the tumor microenvironment (TME) is not well understood. Here, we present a novel non-cell-autonomous function of the retinoblastoma (RB) tumor suppressor in controlling the TME. RB inactivation stimulated tumor growth and neoangiogenesis in a syngeneic and orthotropic murine soft-tissue sarcoma model, which was associated with recruitment of tumor-associated macrophages (TAM) and immunosuppressive cells such as Gr1+CD11b+ myeloid-derived suppressor cells (MDSC) or Foxp3+ regulatory T cells (Treg). Gene expression profiling and analysis of genetically engineered mouse models revealed that RB inactivation increased secretion of the chemoattractant CCL2. Furthermore, activation of the CCL2-CCR2 axis in the TME promoted tumor angiogenesis and recruitment of TAMs and MDSCs into the TME in several tumor types including sarcoma and breast cancer. Loss of RB increased fatty acid oxidation (FAO) by activating AMP-activated protein kinase that led to inactivation of acetyl-CoA carboxylase, which suppresses FAO. This promoted mitochondrial superoxide production and JNK activation, which enhanced CCL2 expression. These findings indicate that the CCL2-CCR2 axis could be an effective therapeutic target in RB-deficient tumors. SIGNIFICANCE: These findings demonstrate the cell-nonautonomous role of the tumor suppressor retinoblastoma in the tumor microenvironment, linking retinoblastoma loss to immunosuppression.

Lipolysis-stimulated lipoprotein receptor: a novel membrane protein of tricellular tight junctions.

Tricellular tight junctions (tTJs) are specialized structural variants of tight junctions that restrict the free diffusion of solutes at the extracellular space of tricellular contacts. Their presence at cell corners, situated in the angles between three adjacent epithelial cells, was identified early by electron microscopy, but despite their potential importance, tTJs have been generally ignored in epithelial cell biology. Tricellulin was the first molecular component of tTJs shown to be involved in their formation and in epithelial barrier function. However, the precise molecular organization and function of tTJs are still largely unknown. Recently, we identified the lipolysis-stimulated lipoprotein receptor (LSR) as a tTJ-associated membrane protein. LSR recruits tricellulin to tTJs, suggesting that the LSR-tricellulin system plays a key role in tTJ formation. In this paper, we summarize the identification and characterization of LSR as a molecular component of tTJs.

Identification of adherens junction-associated GTPase activating proteins by the fluorescence localization-based expression cloning.

The junctional complex, including tight junctions (TJs), adherens junctions (AJs), and desmosomes, plays crucial roles in the structure and functions of epithelial cellular sheets. In this study, we evaluated the fluorescence localization-based retrovirus-mediated expression cloning (FL-REX) method as an approach to identify novel molecular components of TJs and AJs. Using an expression library of cDNA-GFP-fusions derived from mRNA of a mouse epithelial cell line, we confirmed that cDNAs for various known TJ- and AJ-components could be cloned in the FL-REX. Furthermore, cDNAs for ARHGAP12 and SPAL3, two putative GTPase activating proteins (GAPs) for small G proteins, were cloned as novel components of the junctional complex. Immunofluorescence staining using antibodies generated in-house demonstrated that these GAPs were localized at epithelial cell-cell junctions in various mouse tissues, and were specific to AJs when observed under confocal laser-scanning microscopy. These data suggest that FL-REX is a powerful tool to identify novel proteins localized at TJs and AJs.

Enhanced neutrophil motility by granulocyte colony-stimulating factor: the role of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase.

The effect of granulocyte colony-stimulating factor (G-CSF) on human neutrophil motility was studied using videomicroscopy. Stimulation of neutrophils with G-CSF resulted in enhanced motility with morphological change and increased adherence. Enhanced neutrophil motility was detected within 3-5 min after G-CSF stimulation, reached a maximum at 10 min, and was sustained for approximately 35 min. The maximum migration rate was 84.4 +/- 2.9 microm/5 min. A study using the Boyden chamber method revealed that G-CSF-stimulated neutrophils exhibited random migration but not chemotaxis. Enhanced neutrophil motility and morphological change were inhibited by MEK [mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase] inhibitors (PD98059 and U0126), and a phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin), but not by a p38 MAPK inhibitor (SB203580). These findings are consistent with the fact that G-CSF selectively activates MEK/ERK and PI3K, but not p38, in neutrophils. MEK/ERK activation was associated with G-CSF-induced redistribution of F-actin and phosphorylated myosin light chain. Enhanced neutrophil motility was observed even in the presence of neutralizing anti-CD18 antibody, which prevented cell adherence. These findings indicate that G-CSF induces human neutrophil migration via activation of MEK/ERK and PI3K.