Stricturing Crohn's Disease Single-Cell RNA Sequencing Reveals Fibroblast Heterogeneity and Intercellular Interactions.

BACKGROUND & AIMS: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding its pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full-thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single-cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. METHODS: We performed scRNAseq of 13 fresh full-thickness CD resections containing noninvolved, inflamed nonstrictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next-generation sequencing, proteomics, and animal models. RESULTS: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and up-regulated, and its profibrotic function was validated using NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and knock-out and antibody-mediated CDH11 blockade in experimental colitis. CONCLUSIONS: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and open potential therapeutic developments. This work has been posted as a preprint on Biorxiv under doi: 10.1101/2023.04.03.534781.

Understanding disruption of the gut barrier during inflammation: Should we abandon traditional epithelial cell lines and switch to intestinal organoids?

Disruption of the intestinal epithelial barrier is a hallmark of mucosal inflammation. It increases exposure of the immune system to luminal microbes, triggering a perpetuating inflammatory response. For several decades, the inflammatory stimuli-induced breakdown of the human gut barrier was studied in vitro by using colon cancer derived epithelial cell lines. While providing a wealth of important data, these cell lines do not completely mimic the morphology and function of normal human intestinal epithelial cells (IEC) due to cancer-related chromosomal abnormalities and oncogenic mutations. The development of human intestinal organoids provided a physiologically-relevant experimental platform to study homeostatic regulation and disease-dependent dysfunctions of the intestinal epithelial barrier. There is need to align and integrate the emerging data obtained with intestinal organoids and classical studies that utilized colon cancer cell lines. This review discusses the utilization of human intestinal organoids to dissect the roles and mechanisms of gut barrier disruption during mucosal inflammation. We summarize available data generated with two major types of organoids derived from either intestinal crypts or induced pluripotent stem cells and compare them to the results of earlier studies with conventional cell lines. We identify research areas where the complementary use of colon cancer-derived cell lines and organoids advance our understanding of epithelial barrier dysfunctions in the inflamed gut and identify unique questions that could be addressed only by using the intestinal organoid platforms.

Unique and redundant functions of cytoplasmic actins and nonmuscle myosin II isoforms at epithelial junctions.

The integrity and functions of epithelial barriers depend on the formation of adherens junctions (AJs) and tight junctions (TJs). A characteristic feature of AJs and TJs is their association with the cortical cytoskeleton composed of actin filaments and nonmuscle myosin II (NM-II) motors. Mechanical forces generated by the actomyosin cytoskeleton are essential for junctional assembly, stability, and remodeling. Epithelial cells express two different actin proteins and three NM-II isoforms, all known to be associated with AJs and TJs. Despite their structural similarity, different actin and NM-II isoforms have distinct biochemical properties, cellular distribution, and functions. The diversity of epithelial actins and myosin motors could be essential for the regulation of different steps of junctional formation, maturation, and disassembly. This review focuses on the roles of actin and NM-II isoforms in controlling the integrity and barrier properties of various epithelia. We discuss the effects of the depletion of individual actin isoforms and NM-II motors on the assembly and barrier function of AJs and TJs in model epithelial monolayers in vitro. We also describe the functional consequences of either total or tissue-specific gene knockout of different actins and NM-II motors, with a focus on the development and integrity of different epithelia in vivo.

P-Cadherin Regulates Intestinal Epithelial Cell Migration and Mucosal Repair, but Is Dispensable for Colitis Associated Colon Cancer.

Recurrent chronic mucosal inflammation, a characteristic of inflammatory bowel diseases (IBD), perturbs the intestinal epithelial homeostasis resulting in formation of mucosal wounds and, in most severe cases, leads to colitis-associated colon cancer (CAC). The altered structure of epithelial cell-cell adhesions is a hallmark of intestinal inflammation contributing to epithelial injury, repair, and tumorigenesis. P-cadherin is an important adhesion protein, poorly expressed in normal intestinal epithelial cells (IEC) but upregulated in inflamed and injured mucosa. The goal of this study was to investigate the roles of P-cadherin in regulating intestinal inflammation and CAC. P-cadherin expression was markedly induced in the colonic epithelium of human IBD patients and CAC tissues. The roles of P-cadherin were investigated in P-cadherin null mice using dextran sulfate sodium (DSS)-induced colitis and an azoxymethane (AOM)/DSS induced CAC. Although P-cadherin knockout did not affect the severity of acute DSS colitis, P-cadherin null mice exhibited faster recovery after colitis. No significant differences in the number of colonic tumors were observed in P-cadherin null and control mice. Consistently, the CRISPR/Cas9-mediated knockout of P-cadherin in human IEC accelerated epithelial wound healing without affecting cell proliferation. The accelerated migration of P-cadherin depleted IEC was driven by activation of Src kinases, Rac1 GTPase and myosin II motors and was accompanied by transcriptional reprogramming of the cells. Our findings highlight P-cadherin as a negative regulator of IEC motility in vitro and mucosal repair in vivo. In contrast, this protein is dispensable for IEC proliferation and CAC development.

Myosin Motors: Novel Regulators and Therapeutic Targets in Colorectal Cancer.

Colorectal cancer (CRC) remains the third most common cause of cancer and the second most common cause of cancer deaths worldwide. Clinicians are largely faced with advanced and metastatic disease for which few interventions are available. One poorly understood aspect of CRC involves altered organization of the actin cytoskeleton, especially at the metastatic stage of the disease. Myosin motors are crucial regulators of actin cytoskeletal architecture and remodeling. They act as mechanosensors of the tumor environments and control key cellular processes linked to oncogenesis, including cell division, extracellular matrix adhesion and tissue invasion. Different myosins play either oncogenic or tumor suppressor roles in breast, lung and prostate cancer; however, little is known about their functions in CRC. This review focuses on the functional roles of myosins in colon cancer development. We discuss the most studied class of myosins, class II (conventional) myosins, as well as several classes (I, V, VI, X and XVIII) of unconventional myosins that have been linked to CRC development. Altered expression and mutations of these motors in clinical tumor samples and their roles in CRC growth and metastasis are described. We also evaluate the potential of using small molecular modulators of myosin activity to develop novel anticancer therapies.

Anillin is an emerging regulator of tumorigenesis, acting as a cortical cytoskeletal scaffold and a nuclear modulator of cancer cell differentiation.

Remodeling of the intracellular cytoskeleton plays a key role in accelerating tumor growth and metastasis. Targeting different cytoskeletal elements is important for existing and future anticancer therapies. Anillin is a unique scaffolding protein that interacts with major cytoskeletal structures, e.g., actin filaments, microtubules and septin polymers. A well-studied function of this scaffolding protein is the regulation of cytokinesis at the completion of cell division. Emerging evidence suggest that anillin has other important activities in non-dividing cells, including control of intercellular adhesions and cell motility. Anillin is markedly overexpressed in different solid cancers and its high expression is commonly associated with poor prognosis of patient survival. This review article summarizes rapidly accumulating evidence that implicates anillin in the regulation of tumor growth and metastasis. We focus on molecular and cellular mechanisms of anillin-dependent tumorigenesis that include both canonical control of cytokinesis and novel poorly understood functions as a nuclear regulator of the transcriptional reprogramming and phenotypic plasticity of cancer cells.

Loss of ß-Cytoplasmic Actin in the Intestinal Epithelium Increases Gut Barrier Permeability in vivo and Exaggerates the Severity of Experimental Colitis.

Intestinal epithelial barrier is critical for the maintenance of normal gut homeostasis and disruption of this barrier may trigger or exaggerate mucosal inflammation. The actin cytoskeleton is a key regulator of barrier structure and function, controlling the assembly and permeability of epithelial adherens and tight junctions. Epithelial cells express two actin isoforms: a ß-cytoplasmic actin and γ-cytoplasmic actin. Our previous in vitro studies demonstrated that these actin isoforms play distinctive roles in establishing the intestinal epithelial barrier, by controlling the organization of different junctional complexes. It remains unknown, whether ß-actin and γ-actin have unique or redundant functions in regulating the gut barrier in vivo. To address this question, we selectively knocked out ß-actin expression in mouse intestinal epithelium. Mice with intestinal epithelial knockout of ß-actin do not display gastrointestinal abnormalities or gross alterations of colonic mucosal architecture. This could be due to compensatory upregulation of γ-actin expression. Despite such compensation, ß-actin knockout mice demonstrate increased intestinal permeability. Furthermore, these animals show more severe clinical symptoms during dextran sodium sulfate induced colitis, compared to control littermates. Such exaggerated colitis is associated with the higher expression of inflammatory cytokines, increased macrophage infiltration in the gut, and accelerated mucosal cell death. Consistently, intestinal organoids generated from ß-actin knockout mice are more sensitive to tumor necrosis factor induced cell death, ex vivo. Overall, our data suggests that ß-actin functions as an essential regulator of gut barrier integrity in vivo, and plays a tissue protective role during mucosal injury and inflammation.

A Novel Pharmacological Approach to Enhance the Integrity and Accelerate Restitution of the Intestinal Epithelial Barrier.

BACKGROUND: Disruption of the gut barrier is an essential mechanism of inflammatory bowel diseases (IBDs) contributing to the development of mucosal inflammation. A hallmark of barrier disruption is the disassembly of epithelial adherens junctions (AJs) driven by decreased expression of a major AJ protein, E-cadherin. A group of isoxazole compounds, such as E-cadherin-upregulator (ECU) and ML327, were previously shown to stimulate E-cadherin expression in poorly differentiated human cancer cells. This study was designed to examine whether these isoxazole compounds can enhance and protect model intestinal epithelial barriers in vitro. METHODS: The study was conducted using T84, SK-CO15, and HT-29 human colonic epithelial cell monolayers. Disruption of the epithelial barrier was induced by pro-inflammatory cytokines, tumor necrosis factor-α, and interferon-γ. Barrier integrity and epithelial junction assembly was examined using different permeability assays, immunofluorescence labeling, and confocal microscopy. Epithelial restitution was analyzed using a scratch wound healing assay. RESULTS: E-cadherin-upregulator and ML327 treatment of intestinal epithelial cell monolayers resulted in several barrier-protective effects, including reduced steady-state epithelial permeability, inhibition of cytokine-induced barrier disruption and junction disassembly, and acceleration of epithelial wound healing. Surprisingly, these effects were not due to upregulation of E-cadherin expression but were mediated by multiple mechanisms including inhibition of junction protein endocytosis, attenuation of cytokine-induced apoptosis, and activation of promigratory Src and AKT signaling. CONCLUSIONS: Our data highlight ECU and ML327 as promising compounds for developing new therapeutic strategies to protect the integrity and accelerate the restitution of the intestinal epithelial barrier in IBD and other inflammatory disorders.

Anillin regulates breast cancer cell migration, growth, and metastasis by non-canonical mechanisms involving control of cell stemness and differentiation.

BACKGROUND: Breast cancer metastasis is driven by a profound remodeling of the cytoskeleton that enables efficient cell migration and invasion. Anillin is a unique scaffolding protein regulating major cytoskeletal structures, such as actin filaments, microtubules, and septin polymers. It is markedly overexpressed in breast cancer, and high anillin expression is associated with poor prognosis. The aim of this study was to investigate the role of anillin in breast cancer cell migration, growth, and metastasis. METHODS: CRISPR/Cas9 technology was used to deplete anillin in highly metastatic MDA-MB-231 and BT549 cells and to overexpress it in poorly invasive MCF10AneoT cells. The effects of anillin depletion and overexpression on breast cancer cell motility in vitro were examined by wound healing and Matrigel invasion assays. Assembly of the actin cytoskeleton and matrix adhesion were evaluated by immunofluorescence labeling and confocal microscopy. In vitro tumor development was monitored by soft agar growth assays, whereas cancer stem cells were examined using a mammosphere formation assay and flow cytometry. The effects of anillin knockout on tumor growth and metastasis in vivo were determined by injecting control and anillin-depleted breast cancer cells into NSG mice. RESULTS: Loss-of-function and gain-of-function studies demonstrated that anillin is necessary and sufficient to accelerate migration, invasion, and anchorage-independent growth of breast cancer cells in vitro. Furthermore, loss of anillin markedly attenuated primary tumor growth and metastasis of breast cancer in vivo. In breast cancer cells, anillin was localized in the nucleus; however, knockout of this protein affected the cytoplasmic/cortical events, e.g., the organization of actin cytoskeleton and cell-matrix adhesions. Furthermore, we observed a global transcriptional reprogramming of anillin-depleted breast cancer cells that resulted in suppression of their stemness and induction of the mesenchymal to epithelial trans-differentiation. Such trans-differentiation was manifested by the upregulation of basal keratins along with the increased expression of E-cadherin and P-cadherin. Knockdown of E-cadherin restored the impaired migration and invasion of anillin-deficient breast cancer cells. CONCLUSION: Our study demonstrates that anillin plays essential roles in promoting breast cancer growth and metastatic dissemination in vitro and in vivo and unravels novel functions of anillin in regulating breast cancer stemness and differentiation.

A Septin Cytoskeleton-Targeting Small Molecule, Forchlorfenuron, Inhibits Epithelial Migration via Septin-Independent Perturbation of Cellular Signaling.

Septins are GTP-binding proteins that self-assemble into high-order cytoskeletal structures, filaments, and rings. The septin cytoskeleton has a number of cellular functions, including regulation of cytokinesis, cell migration, vesicle trafficking, and receptor signaling. A plant cytokinin, forchlorfenuron (FCF), interacts with septin subunits, resulting in the altered organization of the septin cytoskeleton. Although FCF has been extensively used to examine the roles of septins in various cellular processes, its specificity, and possible off-target effects in vertebrate systems, has not been investigated. In the present study, we demonstrate that FCF inhibits spontaneous, as well as hepatocyte growth factor-induced, migration of HT-29 and DU145 human epithelial cells. Additionally, FCF increases paracellular permeability of HT-29 cell monolayers. These inhibitory effects of FCF persist in epithelial cells where the septin cytoskeleton has been disassembled by either CRISPR/Cas9-mediated knockout or siRNA-mediated knockdown of septin 7, insinuating off-target effects of FCF. Biochemical analysis reveals that FCF-dependent inhibition of the motility of control and septin-depleted cells is accompanied by decreased expression of the c-Jun transcription factor and inhibited ERK activity. The described off-target effects of FCF strongly suggests that caution is warranted while using this compound to examine the biological functions of septins in cellular systems and model organisms.

A membrane fusion protein, Ykt6, regulates epithelial cell migration via microRNA-mediated suppression of Junctional Adhesion Molecule A.

Vesicle trafficking regulates epithelial cell migration by remodeling matrix adhesions and delivering signaling molecules to the migrating leading edge. Membrane fusion, which is driven by soluble N-ethylmaleimide-sensitive factor associated receptor (SNARE) proteins, is an essential step of vesicle trafficking. Mammalian SNAREs represent a large group of proteins, but few have been implicated in the regulation of cell migration. Ykt6 is a unique SNARE existing in equilibrium between active membrane-bound and inactive cytoplasmic pools, and mediating vesicle trafficking between different intracellular compartments. The biological functions of this protein remain poorly understood. In the present study, we found that Ykt6 acts as a negative regulator of migration and invasion of human prostate epithelial cells. Furthermore, Ykt6 regulates the integrity of epithelial adherens and tight junctions. The observed anti-migratory activity of Ykt6 is mediated by a unique mechanism involving the expressional upregulation of microRNA 145, which selectively decreases the cellular level of Junctional Adhesion Molecule (JAM) A. This decreased JAM-A expression limits the activity of Rap1 and Rac1 small GTPases, thereby attenuating cell spreading and motility. The described novel functions of Ykt6 could be essential for the regulation of epithelial barriers, epithelial repair, and metastatic dissemination of cancer cells.

Actin-Depolymerizing Factor and Cofilin-1 Have Unique and Overlapping Functions in Regulating Intestinal Epithelial Junctions and Mucosal Inflammation.

The actin cytoskeleton is a crucial regulator of the intestinal mucosal barrier, controlling the assembly and function of epithelial adherens and tight junctions (AJs and TJs). Junction-associated actin filaments are dynamic structures that undergo constant turnover. Members of the actin-depolymerizing factor (ADF) and cofilin protein family play key roles in actin dynamics by mediating filament severing and polymerization. We examined the roles of ADF and cofilin-1 in regulating the structure and functions of AJs and TJs in the intestinal epithelium. Knockdown of either ADF or cofilin-1 by RNA interference increased the paracellular permeability of human colonic epithelial cell monolayers to small ions. Additionally, cofilin-1, but not ADF, depletion increased epithelial permeability to large molecules. Loss of either ADF or cofilin-1 did not affect the steady-state morphology of AJs and TJs but attenuated de novo junctional assembly. The observed defects in AJ and TJ formation were accompanied by delayed assembly of the perijunctional filamentous actin belt. A total loss of ADF expression in mice did not result in a defective mucosal barrier or in spontaneous gut inflammation. However, ADF-null mice demonstrated increased intestinal permeability and exaggerated inflammation during dextran sodium sulfate-induced colitis. Our findings demonstrate novel roles for ADF and cofilin-1 in regulating the remodeling and permeability of epithelial junctions, as well as the role of ADF in limiting the severity of intestinal inflammation.

Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells.

Transdifferentiation of epithelial cells into mesenchymal cells and myofibroblasts plays an important role in tumor progression and tissue fibrosis. Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood. In the present study, we observed that selective siRNA-mediated knockdown of γ-cytoplasmic actin (γ-CYA), but not ß-cytoplasmic actin, induced epithelial-to-myofibroblast transition (EMyT) of different epithelial cells. The EMyT manifested by increased expression of α-smooth muscle actin and other contractile proteins, along with inhibition of genes responsible for cell proliferation. Induction of EMyT in γ-CYA-depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B. Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction. Our findings demonstrate a previously unanticipated, unique role of γ-CYA in regulating epithelial phenotype and suppression of EMyT that may be essential for cell differentiation and tissue fibrosis.

N-ethylmaleimide-sensitive factor attachment protein α (αSNAP) regulates matrix adhesion and integrin processing in human epithelial cells.

Integrin-based adhesion to the extracellular matrix (ECM) plays critical roles in controlling differentiation, survival, and motility of epithelial cells. Cells attach to the ECM via dynamic structures called focal adhesions (FA). FA undergo constant remodeling mediated by vesicle trafficking and fusion. A soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein α (αSNAP) is an essential mediator of membrane fusion; however, its roles in regulating ECM adhesion and cell motility remain unexplored. In this study, we found that siRNA-mediated knockdown of αSNAP induced detachment of intestinal epithelial cells, whereas overexpression of αSNAP increased ECM adhesion and inhibited cell invasion. Loss of αSNAP impaired Golgi-dependent glycosylation and trafficking of ß1 integrin and decreased phosphorylation of focal adhesion kinase (FAK) and paxillin resulting in FA disassembly. These effects of αSNAP depletion on ECM adhesion were independent of apoptosis and NSF. In agreement with our previous reports that Golgi fragmentation mediates cellular effects of αSNAP knockdown, we found that either pharmacologic or genetic disruption of the Golgi recapitulated all the effects of αSNAP depletion on ECM adhesion. Furthermore, our data implicates ß1 integrin, FAK, and paxillin in mediating the observed pro-adhesive effects of αSNAP. These results reveal novel roles for αSNAP in regulating ECM adhesion and motility of epithelial cells.

Dynamics and regulation of epithelial adherens junctions: recent discoveries and controversies.

Adherens junctions (AJs) are evolutionarily conserved plasma-membrane structures that mediate cell-cell adhesions in multicellular organisms. They are organized by several types of adhesive integral membrane proteins, most notably cadherins and nectins that are clustered and stabilized by a number of cytoplasmic scaffolds. AJs are key regulators of tissue architecture and dynamics via control of cell proliferation, polarity, shape, motility, and survival. They are absolutely critical for normal tissue morphogenesis and their disruption results in pathological abnormalities in different tissues. Although the field of adherens-junction research dramatically progressed in recent years, a number of important questions remain controversial and poorly understood. This review outlines basic principles that regulate organization of AJs in mammalian epithelia and discusses recent advances and standing controversies in the field. A special attention is paid to the regulation of AJs by vesicle trafficking and the intracellular cytoskeleton as well as roles and mechanisms of adherens-junction disruption during tumor progression and tissue inflammation.

Novel mechanism of cytokine-induced disruption of epithelial barriers: Janus kinase and protein kinase D-dependent downregulation of junction protein expression.

The ductal epithelium plays a key role in physiological secretion of pancreatic enzymes into the digestive system. Loss of barrier properties of the pancreatic duct may contribute to the development of pancreatitis and metastatic dissemination of pancreatic tumors. Proinflammatory cytokines are essential mediators of pancreatic inflammation and tumor progression; however, their effects on the integrity and barrier properties of the ductal epithelium have not been previously addressed. In the present study, we investigate mechanisms of cytokine-induced disassembly of tight junctions (TJs) and adherens junctions (AJs) in a model pancreatic epithelium. Exposure of HPAF-II human pancreatic epithelial cell monolayers to interferon (IFN)γ disrupted integrity and function of apical junctions as manifested by increased epithelial permeability and cytosolic translocation of AJ and TJ proteins. Tumor necrosis factor (TNF)α potentiated the effects of IFNγ on pancreatic epithelial junctions. The cytokine-induced increase in epithelial permeability and AJ/TJ disassembly was attenuated by pharmacological inhibition of Janus kinase (JAK) and protein kinase D (PKD). Loss of apical junctions in IFNγ/TNFα-treated HPAF-II cells was accompanied by JAK and PKD dependent decrease in expression of AJ (E-cadherin, p120 catenin) and TJ (occludin, ZO-1) proteins. Depletion of E-cadherin or p120 catenin recapitulated the effects of cytokines on HPAF-II cell permeability and junctions. Our data suggests that proinflammatory cytokines disrupt pancreatic epithelial barrier via expressional downregulation of key structural components of AJs and TJs. This mechanism is likely to be important for pancreatic inflammatory injury and tumorigenesis.

Loss of a membrane trafficking protein αSNAP induces non-canonical autophagy in human epithelia.

Autophagy is a catabolic process that sequesters intracellular proteins and organelles within membrane vesicles called autophagosomes with their subsequent delivery to lyzosomes for degradation. This process involves multiple fusions of autophagosomal membranes with different vesicular compartments; however, the role of vesicle fusion in autophagosomal biogenesis remains poorly understood. This study addresses the role of a key vesicle fusion regulator, soluble N-ethylmaleimide-sensitive factor attachment protein α (αSNAP), in autophagy. Small interfering RNA-mediated downregulation of αSNAP expression in cultured epithelial cells stimulated the autophagic flux, which was manifested by increased conjugation of microtubule-associated protein light chain 3 (LC3-II) and accumulation of LC3-positive autophagosomes. This enhanced autophagy developed via a non-canonical mechanism that did not require beclin1-p150-dependent nucleation, but involved Atg5 and Atg7-mediated elongation of autophagosomal membranes. Induction of autophagy in αSNAP-depleted cells was accompanied by decreased mTOR signaling but appeared to be independent of αSNAP-binding partners, N-ethylmaleimide-sensitive factor and BNIP1. Loss of αSNAP caused fragmentation of the Golgi and downregulation of the Golgi-specific GTP exchange factors, GBF1, BIG1 and BIG2. Pharmacological disruption of the Golgi and genetic inhibition of GBF1 recreated the effects of αSNAP depletion on the autophagic flux. Our study revealed a novel role for αSNAP as a negative regulator of autophagy that acts by enhancing mTOR signaling and regulating the integrity of the Golgi complex.

Nonredundant roles of cytoplasmic ß- and γ-actin isoforms in regulation of epithelial apical junctions.

Association with the actin cytoskeleton is critical for normal architecture and dynamics of epithelial tight junctions (TJs) and adherens junctions (AJs). Epithelial cells express ß-cytoplasmic (ß-CYA) and γ-cytoplasmic (γ-CYA) actins, which have different cellular localization and functions. This study elucidates the roles of cytoplasmic actins in regulating structure and remodeling of AJs and TJs in model intestinal epithelia. Immunofluorescence labeling and latrunculin B treatment reveal affiliation of dynamic ß-CYA filaments with newly assembled and mature AJs, whereas an apical γ-CYA pool is composed of stable perijunctional bundles and rapidly turning-over nonjunctional filaments. The functional effects of cytoplasmic actins on epithelial junctions are examined by using isoform-specific small interfering RNAs and cell-permeable inhibitory peptides. These experiments demonstrate unique roles of ß-CYA and γ-CYA in regulating the steady-state integrity of AJs and TJs, respectively. Furthermore, ß-CYA is selectively involved in establishment of apicobasal cell polarity. Both actin isoforms are essential for normal barrier function of epithelial monolayers, rapid AJ/TJ reassembly, and formation of three-dimensional cysts. Cytoplasmic actin isoforms play unique roles in regulating structure and permeability of epithelial junctions.

A membrane fusion protein αSNAP is a novel regulator of epithelial apical junctions.

Tight junctions (TJs) and adherens junctions (AJs) are key determinants of the structure and permeability of epithelial barriers. Although exocytic delivery to the cell surface is crucial for junctional assembly, little is known about the mechanisms controlling TJ and AJ exocytosis. This study was aimed at investigating whether a key mediator of exocytosis, soluble N-ethylmaleimide sensitive factor (NSF) attachment protein alpha (αSNAP), regulates epithelial junctions. αSNAP was enriched at apical junctions in SK-CO15 and T84 colonic epithelial cells and in normal human intestinal mucosa. siRNA-mediated knockdown of αSNAP inhibited AJ/TJ assembly and establishment of the paracellular barrier in SK-CO15 cells, which was accompanied by a significant down-regulation of p120-catenin and E-cadherin expression. A selective depletion of p120 catenin effectively disrupted AJ and TJ structure and compromised the epithelial barrier. However, overexpression of p120 catenin did not rescue the defects of junctional structure and permeability caused by αSNAP knockdown thereby suggesting the involvement of additional mechanisms. Such mechanisms did not depend on NSF functions or induction of cell death, but were associated with disruption of the Golgi complex and down-regulation of a Golgi-associated guanidine nucleotide exchange factor, GBF1. These findings suggest novel roles for αSNAP in promoting the formation of epithelial AJs and TJs by controlling Golgi-dependent expression and trafficking of junctional proteins.

Loss of soluble N-ethylmaleimide-sensitive factor attachment protein α (αSNAP) induces epithelial cell apoptosis via down-regulation of Bcl-2 expression and disruption of the Golgi.

Intracellular trafficking represents a key mechanism that regulates cell fate by participating in either prodeath or prosurvival signaling. Soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein α (αSNAP) is a well known component of vesicle trafficking machinery that mediates intermembrane fusion. αSNAP increases cell resistance to cytotoxic stimuli, although mechanisms of its prosurvival function are poorly understood. In this study, we found that either siRNA-mediated knockdown of αSNAP or expression of its dominant negative mutant induced epithelial cell apoptosis. Apoptosis was not caused by activation of the major prodeath regulators Bax and p53 and was independent of a key αSNAP binding partner, NSF. Instead, death of αSNAP-depleted cells was accompanied by down-regulation of the antiapoptotic Bcl-2 protein; it was mimicked by inhibition and attenuated by overexpression of Bcl-2. Knockdown of αSNAP resulted in impairment of Golgi to endoplasmic reticulum (ER) trafficking and fragmentation of the Golgi. Moreover, pharmacological disruption of ER-Golgi transport by brefeldin A and eeyarestatin 1 or siRNA-mediated depletion of an ER/Golgi-associated p97 ATPase recapitulated the effects of αSNAP inhibition by decreasing Bcl-2 level and triggering apoptosis. These results reveal a novel role for αSNAP in promoting epithelial cell survival by unique mechanisms involving regulation of Bcl-2 expression and Golgi biogenesis.