in-vitro-vivo-approaches-to-determine-intestinal-epithelial-cell

<em>In Vitro</em> and <em>In Vivo </em>Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

quantification-of-proliferative-and-dead-cells-in-enteroids

Quantification of Proliferative and Dead Cells in Enteroids

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. ...

Soochow University

The First Affiliated Hospital of Soochow University

Smooth muscle sphincters exhibit basal tone and control passage of contents through organs such as the gastrointestinal tract; loss of this tone leads to disorders such as faecal incontinence. However, the molecular mechanisms underlying this tone remain unknown. Here, we show that deletion of myosin light-chain kinases (MLCK) in the smooth muscle cells from internal anal sphincter (IAS-SMCs) abolishes basal tone, impairing defecation. Pharmacological regulation of ryanodine receptors (RyRs), L-type voltage-dependent Ca(2+) channels (VDCCs) or TMEM16A Ca(2+)-activated Cl(-) channels significantly changes global cytosolic Ca(2+) concentration ([Ca(2+)]i) and the tone. TMEM16A deletion in IAS-SMCs abolishes the effects of modulators for TMEM16A or VDCCs on a RyR-mediated rise in global [Ca(2+)]i and impairs the tone and defecation. Hence, MLCK activation in IAS-SMCs caused by a global rise in [Ca(2+)]i via a RyR-TMEM16A-VDCC signalling module sets the basal tone. Targeting this module may lead to new treatments for diseases like faecal incontinence. 

The molecular basis of the genesis of basal tone in internal anal sphincter.

The regulation of intestinal epithelial permeability requires phosphorylation of myosin regulatory light chain (MLC). The phosphorylation status of MLC is regulated by myosin light chain phosphatase (MLCP) activities. The activity of the catalytic subunit of MLCP (PP1cδ) toward MLC depends on its regulatory subunit (MYPT1). In this study, we revealed the presence of two MYPT1 isoforms, full length and variant 2 in human intestinal (Caco-2) epithelial cells and isolated intestinal epithelial cells (IECs) from mice. In confluent Caco-2 cells, MYPT1 was distributed at cell-cell contacts and colocalized with F-actin. These results suggest that MYPT1 isoforms are expressed in intestinal epithelial cells and MYPT1 may be involved in the regulation of intestinal epithelial barrier function. 

Characterization of isoform expression and subcellular distribution of MYPT1 in intestinal epithelial cells.

Sphingosine kinase 1 (SphK1) is over-expressed in many cancers and therefore serves as a biomarker for cancer prognosis. SKI-5C is a new SphK1 inhibitor, and until now its molecular function in Wilms' tumor cells remained unknown. Here, using CCK-8 and nude mice experiments we assessed cell growth in Wilms' tumor cell lines (SK-NEP-1 and G401)  and . We demonstrated that SphK1 is highly expressed in SK-NEP-1 and G401 cells, and through annexin V/propidium iodide staining and flow cytometry analysis, we detected cell apoptosis. Treatment with SKI-5C inhibited proliferation and induced apoptosis of SK-NEP-1 and G401 cells in a dose-dependent manner. Moreover, SKI-5C treatment inhibited the growth of SK-NEP-1 xenograft tumors in nude mice, with few side effects. Our microarray analysis revealed that SKI-5C-treated SK-NEP-1 cells mostly downregulated PRKACA and significantly inhibited phosphorylation of ERK1/2 and NF-κB p65. These results imply that SKI-5C induces apoptosis of SK-NEP-1 cells through the PRKACA/MAPK/NF-κB pathway. While, further research is required to determine the underlying details, these results provide new clues for the molecular mechanism of cell death induced by SKI-5C and suggest that SKI-5C may act as new candidate drug for Wilms' tumor.

A novel sphingosine kinase 1 inhibitor (SKI-5C) induces cell death of Wilms' tumor cells  and .

Decreased autophagy is accompanied by the development of a myeloproliferative state or acute myeloid leukemia (AML). AML cells are often sensitive to autophagy‑inducing stimuli, prompting the idea that targeting autophagy can be useful in AML cytotoxic therapy. AML NB4 cells overexpressing microtubule-associated protein 1 light chain 3-green fluorescent protein were screened with 69 inhibitors to analyze autophagy activity. AML cells were treated with the polo-like kinase 1 (PLK1) inhibitors RO3280 and BI2536 before autophagy analysis. Cleaved LC3 (LC3-II) and the phosphorylation of mammalian target of rapamycin (mTOR), adenosine monophosphate-activated protein kinase, and Unc-51-like kinase 1 during autophagy was detected with western blotting. Autophagosomes were detected using transmission electron microscopy. Several inhibitors had promising autophagy inducer effects: BI2536, MLN0905, SK1-I, SBE13 HCL and RO3280. Moreover, these inhibitors all targeted PLK1. Autophagy activity was increased in the NB4 cells treated with RO3280 and BI2536. Inhibition of PLK1 expression in NB4, K562 and HL-60 leukemia cells with RNA interference increased LC3-II and autophagy activity. The phosphorylation of mTOR was reduced significantly in NB4 cells treated with RO3280 and BI2536, and was also reduced significantly when PLK1 expression was downregulated in the NB4, K562 and HL-60 cells. We demonstrate that PLK1 inhibition induces AML cell autophagy and that it results in mTOR dephosphorylation. These results may provide new insights into the molecular mechanism of PLK1 in regulating autophagy.

Inhibiting PLK1 induces autophagy of acute myeloid leukemia cells via mammalian target of rapamycin pathway dephosphorylation.

Overexpression of cyclin D1 dependent kinases 4 and 6 (CDK4/6) is a common feature of many human cancers including leukemia. LEE011 is a novel inhibitor of both CDK4 and 6. To date, the molecular function of LEE011 in leukemia remains unclear.

Molecular mechanism of G arrest and cellular senescence induced by LEE011, a novel CDK4/CDK6 inhibitor, in leukemia cells.

Diarrhea is a host response to enteric pathogens, but its impact on pathogenesis remains poorly defined. By infecting mice with the attaching and effacing bacteria Citrobacter rodentium, we defined the mechanisms and contributions of diarrhea and intestinal barrier loss to host defense. Increased permeability occurred within 2 days of infection and coincided with IL-22-dependent upregulation of the epithelial tight junction protein claudin-2. Permeability increases were limited to small molecules, as expected for the paracellular water and Na channel formed by claudin-2. Relative to wild-type, claudin-2-deficient mice experienced severe disease, including increased mucosal colonization by C. rodentium, prolonged pathogen shedding, exaggerated cytokine responses, and greater tissue injury. Conversely, transgenic claudin-2 overexpression reduced disease severity. Chemically induced osmotic diarrhea reduced colitis severity and C. rodentium burden in claudin-2-deficient, but not transgenic, mice, demonstrating that claudin-2-mediated protection is the result of enhanced water efflux. Thus, IL-22-induced claudin-2 upregulation drives diarrhea and pathogen clearance.

IL-22 Upregulates Epithelial Claudin-2 to Drive Diarrhea and Enteric Pathogen Clearance.

Leiomyosarcoma is a rare malignant smooth muscle tumor which can be very unpredictable. Myosin II is involved in many functions, including cell contraction, migration, and adhesion. The phosphorylation of myosin regulatory light chain (MLC) by myosin light chain kinase (MLCK) determines the activity of Myosin II. However, it is still unclear whether MLC phosphorylation is involved in cell proliferation in leiomyosarcoma. In this study, we aimed to explore the role of MLCK-dependent MLC phosphorylation in leiomyosarcoma development. We found that the expression of MLCK, phosphorylated MLC, and Ki67 in leiomyosarcoma was significantly higher than in leiomyoma and adjacent normal smooth muscle cells. MLCK expression was significantly correlated with phosphorylated MLC level. Kaplan-Meier survival analysis revealed that patients with high expression of MLCK or phosphorylated MLC had shorter overall survival times compared with the patients with low expression of MLCK or phosphorylated MLC. In vitro studies revealed a causative link between MLC phosphorylation and cellular proliferation as expression of phosphomimetic MLC (T19D, S20D) increased cellular proliferation as assessed by Ki67 staining. In contrast, MLCK specific inhibitor reduced cellular proliferation. We concluded that MLCK, phosphorylated MLC and Ki67 were overexpressed in leiomyosarcoma. MLCK dependent MLC phosphorylation might be responsible for the high proliferative state in leiomyosarcoma. MLCK and phosphorylated MLC are potential prognostic indicators of leiomyosarcoma.

Myosin regulatory light chain phosphorylation is associated with leiomyosarcoma development.

Epithelial regeneration is essential for homeostasis and repair of the mucosal barrier. In the context of infectious and immune-mediated intestinal disease, interleukin (IL) 22 is thought to augment these processes. We sought to define the mechanisms by which IL22 promotes mucosal healing.

Wei-Qi He

Jiangsu Key Laboratory of Neuropsychiatric Diseases and Cambridge-Suda (CAM-SU) Genome Resource Center

Soochow University

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Quantification of Proliferative and Dead Cells in Enteroids

Wei-Qi He

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Soochow University

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Quantification of Proliferative and Dead Cells in Enteroids

Jiangsu Key Laboratory of Neuropsychiatric Diseases and Cambridge-Suda (CAM-SU) Genome Resource Center