Cytoskeletal mechanisms regulating attaching/effacing bacteria interactions with host cells: It takes a village to build the pedestal.

The actin cytoskeleton is a key cellular structure subverted by pathogens to infect and survive in or on host cells. Several pathogenic strains of Escherichia coli, such as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), developed a unique mechanism to remodel the actin cytoskeleton that involves the assembly of actin filament-rich pedestals beneath the bacterial attachment sites. Actin pedestal assembly is driven by bacterial effectors injected into the host cells, and this structure is important for EPEC and EHEC colonization. While the interplay between bacterial effectors and the actin polymerization machinery of host cells is well-understood, how other mechanisms of actin filament remodelling regulate pedestal assembly and bacterial attachment are poorly investigated. This review discusses the gaps in our understanding of the complexity of the actin cytoskeletal remodelling during EPEC and EHEC infection. We describe possible roles of actin depolymerizing, crosslinking and motor proteins in pedestal dynamics, and bacterial interactions with the host cells. We also discuss the biological significance of pedestal assembly for bacterial infection.

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.

Novel Functions of the Septin Cytoskeleton: Shaping Up Tissue Inflammation and Fibrosis.

Chronic inflammatory diseases cause profound alterations in tissue homeostasis, including unchecked activation of immune and nonimmune cells leading to disease complications such as aberrant tissue repair and fibrosis. Current anti-inflammatory therapies are often insufficient in preventing or reversing these complications. Remodeling of the intracellular cytoskeleton is critical for cell activation in inflamed and fibrotic tissues; however, the cytoskeleton has not been adequately explored as a therapeutic target in inflammation. Septins are GTP-binding proteins that self-assemble into higher order cytoskeletal structures. The septin cytoskeleton exhibits a number of critical cellular functions, including regulation of cell shape and polarity, cytokinesis, cell migration, vesicle trafficking, and receptor signaling. Surprisingly, little is known about the role of the septin cytoskeleton in inflammation. This article reviews emerging evidence implicating different septins in the regulation of host-pathogen interactions, immune cell functions, and tissue fibrosis. Targeting of the septin cytoskeleton as a potential future therapeutic intervention in human inflammatory and fibrotic diseases is also discussed.

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.

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 vesicle trafficking protein αSNAP regulates Paneth cell differentiation in vivo.

A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo.

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.

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.

Regulation of Epithelial and Endothelial Barriers by Molecular Chaperones.

The integrity and permeability of epithelial and endothelial barriers depend on the formation of tight junctions, adherens junctions, and a junction-associated cytoskeleton. The establishment of this junction-cytoskeletal module relies on the correct folding and oligomerization of its protein components. Molecular chaperones are known regulators of protein folding and complex formation in different cellular compartments. Mammalian cells possess an elaborate chaperone network consisting of several hundred chaperones and co-chaperones. Only a small part of this network has been linked, however, to the regulation of intercellular adhesions, and the systematic analysis of chaperone functions at epithelial and endothelial barriers is lacking. This review describes the functions and mechanisms of the chaperone-assisted regulation of intercellular junctions. The major focus of this review is on heat shock protein chaperones, their co-chaperones, and chaperonins since these molecules are the focus of the majority of the articles published on the chaperone-mediated control of tissue barriers. This review discusses the roles of chaperones in the regulation of the steady-state integrity of epithelial and vascular barriers as well as the disruption of these barriers by pathogenic factors and extracellular stressors. Since cytoskeletal coupling is essential for junctional integrity and remodeling, chaperone-assisted assembly of the actomyosin cytoskeleton is also discussed.

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.

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.

Stricturing Crohn's disease single-cell RNA sequencing reveals fibroblast heterogeneity and intercellular interactions.

Background: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding it's 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 non-involved, inflamed non-strictured, 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 upregulated, and its pro-fibrotic function was validated by NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and two animal models of experimental colitis. Conclusion: 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 opens potential therapeutic developments.

Polyinosinic:polycytidylic acid induces protein kinase D-dependent disassembly of apical junctions and barrier dysfunction in airway epithelial cells.

BACKGROUND: Disruption of the epithelial barrier might be a risk factor for allergen sensitization and asthma. Viral respiratory tract infections are strongly associated with asthma exacerbation, but the effects of respiratory viruses on airway epithelial barrier function are not well understood. Many viruses generate double-stranded RNA, which can lead to airway inflammation and initiate an antiviral immune response. OBJECTIVES: We investigated the effects of the synthetic double-stranded RNA polyinosinic:polycytidylic acid (polyI:C) on the structure and function of the airway epithelial barrier in vitro. METHODS: 16HBE14o- human bronchial epithelial cells and primary airway epithelial cells at an air-liquid interface were grown to confluence on Transwell inserts and exposed to polyI:C. We studied epithelial barrier function by measuring transepithelial electrical resistance and paracellular flux of fluorescent markers and structure of epithelial apical junctions by means of immunofluorescence microscopy. RESULTS: PolyI:C induced a profound decrease in transepithelial electrical resistance and increase in paracellular permeability. Immunofluorescence microscopy revealed markedly reduced junctional localization of zonula occludens-1, occludin, E-cadherin, ß-catenin, and disorganization of junction-associated actin filaments. PolyI:C induced protein kinase D (PKD) phosphorylation, and a PKD antagonist attenuated polyI:C-induced disassembly of apical junctions and barrier dysfunction. CONCLUSIONS: PolyI:C has a powerful and previously unsuspected disruptive effect on the airway epithelial barrier. PolyI:C-dependent barrier disruption is mediated by disassembly of epithelial apical junctions, which is dependent on PKD signaling. These findings suggest a new mechanism potentially underlying the associations between viral respiratory tract infections, airway inflammation, and allergen sensitization.

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.

Profiling of mitochondrial transcriptome in germinating wheat embryos and seedlings subjected to cold, salinity and osmotic stresses.

Mitochondrial functions are potential targets of abiotic stresses that are major environmental factors limiting plant development and productivity. To evaluate mitochondrial responses to abiotic stresses we studied mitochondrial transcriptome profiles at the early stages of wheat development after imbibition under normal and induced stress conditions. Three stresses given were low temperature (4 degrees C), high salinity (0.2 M NaCl) and high osmotic potential (0.3 M mannitol). All these stresses greatly reduced growth but dramatically increased respiration both via the cytochrome and alternative pathways. Macroarray analysis of the mitochondrial transcriptome revealed that most of the changes in transcript levels were stress specific but groups of genes responded commonly to different stresses. Under 3-days continuous stresses, 13 genes showed low temperature specific responses with either up- or down-regulation, while 14 and 23 genes showed responses specific to high salinity and high osmotic potential, respectively. On the other hand, 13 genes showed common responses, among which cob and ccmFn increased their transcript levels while transcripts of the other genes including nad6, atp4 and atp9 decreased. The differential profiles of mitochondrial transcriptome revealed by the macroarray analysis were verified by the quantitative reverse transcriptase PCR analysis. Taken together, three among five nuclear-encoded mitochondria-targeted genes included in the array showed decreases under the stresses, while MnSOD and AOX increased their transcript amounts. Our results indicated the existence of common and different regulatory mechanisms that can sense different abiotic stresses and modulate both nuclear and mitochondrial gene expression in germinating wheat embryos and seedlings.

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.

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 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.