Contributions of Myosin Light Chain Kinase to Regulation of Epithelial Paracellular Permeability and Mucosal Homeostasis.

Intestinal barrier function is required for the maintenance of mucosal homeostasis. Barrier dysfunction is thought to promote progression of both intestinal and systemic diseases. In many cases, this barrier loss reflects increased permeability of the paracellular tight junction as a consequence of myosin light chain kinase (MLCK) activation and myosin II regulatory light chain (MLC) phosphorylation. Although some details about MLCK activation remain to be defined, it is clear that this triggers perijunctional actomyosin ring (PAMR) contraction that leads to molecular reorganization of tight junction structure and composition, including occludin endocytosis. In disease states, this process can be triggered by pro-inflammatory cytokines including tumor necrosis factor-α (TNF), interleukin-1ß (IL-1ß), and several related molecules. Of these, TNF has been studied in the greatest detail and is known to activate long MLCK transcription, expression, enzymatic activity, and recruitment to the PAMR. Unfortunately, toxicities associated with inhibition of MLCK expression or enzymatic activity make these unsuitable as therapeutic targets. Recent work has, however, identified a small molecule that prevents MLCK1 recruitment to the PAMR without inhibiting enzymatic function. This small molecule, termed Divertin, restores barrier function after TNF-induced barrier loss and prevents disease progression in experimental chronic inflammatory bowel disease.

[Hypoxia promotes the growth and invasiveness of prostate cancer cells by down-regulating miR-132 in vitro].

OBJECTIVE: To explore the expression of miR-132 in prostate cancer and its effects on the growth and invasiveness of prostate cancer cells and the influence of hypoxia on the level of miR-132 and biological behavior of prostate cancer cells. METHODS: Real time PCR was used to measure the expression level of miR-132 in the prostate cancer tissue, analyze its relationship with the clinical stage and Gleason score of prostate cancer, and determine the influence of hypoxia on the miR-132 level in the human prostate cancer PC3 cell line in vitro. Sulfor-hodamine B chromatometry and Matrigel invasion assay were employed to detect the effects of hypoxia and miR-132 mimic plasmid transfection on the viability and invasiveness of PC3 cells in vitro. RESULTS: The miR-132 level in the prostate cancer was significantly declined to 52.38% (in T1－T2 stages) and 21.59% (in T3－T4 stages) of that in the cancer-adjacent tissue (both P<0.01). In hypoxia, the expression of miR-132 was significantly decreased in the PC3 cells (P<0.01). After 48 and 72 hours of transfection with miR-132 mimic plasmid, the viability of the PC3 cells was markedly reduced (P<0.05 or P<0.01), and their invasiveness decreased by 57.5% after 48 hours (P<0.01). However, there was no significant difference in the viability or invasiveness of the PC3 cells transfected with miR-132 mimic plasmid between normoxia and hypoxia. CONCLUSIONS: The reduced expression of miR-132 is closely related to the clinical stage and Gleason score of prostate cancer. Hypoxia increases the viability and invasiveness of prostate cancer cells in vitro by down-regulating the expression of miR-132 and consequently may promote the growth and metastasis of prostate cancer.

Altered contractile phenotypes of intestinal smooth muscle in mice deficient in myosin phosphatase target subunit 1.

BACKGROUND & AIMS: The regulatory subunit of myosin light chain phosphatase, MYPT1, has been proposed to control smooth muscle contractility by regulating phosphorylation of the Ca(2+)-dependent myosin regulatory light chain. We generated mice with a smooth muscle-specific deletion of MYPT1 to investigate its physiologic role in intestinal smooth muscle contraction. METHODS: We used the Cre-loxP system to establish Mypt1-floxed mice, with the promoter region and exon 1 of Mypt1 flanked by 2 loxP sites. These mice were crossed with SMA-Cre transgenic mice to generate mice with smooth muscle-specific deletion of MYPT1 (Mypt1(SMKO) mice). The phenotype was assessed by histologic, biochemical, molecular, and physiologic analyses. RESULTS: Young adult Mypt1(SMKO) mice had normal intestinal motility in vivo, with no histologic abnormalities. On stimulation with KCl or acetylcholine, intestinal smooth muscles isolated from Mypt1(SMKO) mice produced robust and increased sustained force due to increased phosphorylation of the myosin regulatory light chain compared with muscle from control mice. Additional analyses of contractile properties showed reduced rates of force development and relaxation, and decreased shortening velocity, compared with muscle from control mice. Permeable smooth muscle fibers from Mypt1(SMKO) mice had increased sensitivity and contraction in response to Ca(2+). CONCLUSIONS: MYPT1 is not essential for smooth muscle function in mice but regulates the Ca(2+) sensitivity of force development and contributes to intestinal phasic contractile phenotype. Altered contractile responses in isolated tissues could be compensated by adaptive physiologic responses in vivo, where gut motility is affected by lower intensities of smooth muscle stimulation for myosin phosphorylation and force development.

Association of Overweight and Inflammatory Indicators with Breast Cancer: A Cross-Sectional Study in Chinese Women.

Objective: This cross-sectional study aimed to explore the association of overweight and inflammatory indicators with breast cancer risk in Chinese patients. Methods: Weight, height, and peripheral blood inflammatory indicators, including white blood cell count (WBC), neutrophil count (NE), lymphocyte count (LY), platelet count (PLT) and the concentration of hypersensitivity C-reactive protein (hsCRP), were collected in 383 patients with benign breast lumps (non-cancer) and 358 patients with malignant breast tumors (cancer) at the First Affiliated Hospital of Soochow University, China, from March 2018 to July 2020. Body mass index (BMI), neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR) and systemic immune-inflammation index (SII) were determined according to the ratio equation. The correlations among overweight, inflammatory indicators, and the proportion of non-cancer or cancer cases were analyzed. Results: BMI is associated with an increased breast cancer risk. Compared with non-cancer patients, the average WBC count, NE count, NLR, and level of hsCRP were significantly higher in cancer patients. The level of hsCRP was closely associated with the size of malignant breast tumors. Conclusion: We conclude that overweight and high levels of hsCRP may serve as putative risk factors for malignant breast tumors in Chinese women.

Current Applications of Colorectal Cancer Organoids: A Review.

Colorectal cancer is a prevalent malignancy, with advanced and metastatic forms exhibiting poor treatment outcomes and high relapse rates. To enhance patient outcomes, a comprehensive understanding of the pathophysiological processes and the development of targeted therapies are imperative. The high heterogeneity of colorectal cancer demands precise and personalized treatment strategies. Colorectal cancer organoids, a three-dimensional in vitro model, have emerged as a valuable tool for replicating tumor biology and exhibit promise in scientific research, disease modeling, drug screening, and personalized medicine. In this review, we present an overview of colorectal cancer organoids and explore their applications in research and personalized medicine, while also discussing potential future developments in this field.

Nutraceuticals for the Treatment of IBD: Current Progress and Future Directions.

Inflammatory bowel disease (IBD) is a chronic relapsing-remitting inflammatory disease of the gastrointestinal tract. Patients are usually diagnosed in adolescence and early adulthood and need lifelong treatment. In recent years, it has been found that diet plays an important role in the pathogenesis of IBD. Diet can change intestinal barrier function, affect the structure and function of intestinal flora, and promote immune disorder, thus promoting inflammation. Many patients believe that diet plays a role in the onset and treatment of the disease and changes their diet spontaneously. This review provides some insights into how nutraceuticals regulate intestinal immune homeostasis and improve intestinal barrier function. We reviewed the research results of dietary fiber, polyphenols, bioactive peptides, and other nutraceuticals in the prevention and treatment of IBD and sought better alternative or supplementary treatment methods for IBD patients.

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. Epithelial barrier function requires the integrity of intestinal epithelial cells. While epithelial cell proliferation maintains a continuous layer of cells that forms a barrier, epithelial damage leads to barrier dysfunction. As a result, luminal contents can across the intestinal barrier via an unrestricted pathway. Dysfunction of intestinal barrier has been associated with many intestinal diseases, such as inflammatory bowel disease. Isolated mouse intestinal crypts can be cultured and maintained as crypt-villus-like structures, which are termed intestinal organoids or "enteroids". Enteroids are ideal to study the proliferation and cell death of intestinal epithelial cells in vitro. In this protocol, we describe a simple method to quantify the number of proliferative and dead cells in cultured enteroids. 5-ethynyl-2'-deoxyuridine (EdU) and propidium iodide are used to label proliferating and dead cells in enteroids, and the proportion of proliferating and dead cells are then analyzed by flow cytometry. This is a useful tool to test the effects of drug treatment on intestinal epithelial cell proliferation and cell survival.

Comparison of gene transcription between subcutaneous and visceral adipose tissue in Chinese adults.

Obese individuals with fat stored in visceral adipose tissue (VAT) generally suffer greater adverse metabolic consequences than those with fat stored predominantly in subcutaneous adipose tissue (SAT), but its molecular basis is not completely understood. We isolated paired samples of SAT and VAT from 15 lean and 15 obese subjects and systematically compared the transcription level of genes that may determine fat distribution and metabolic sequelae between SAT and VAT using quantitative real-time PCR. We found that, leptin levels were lower in VAT than SAT, for both lean and obese subjects. In lean subjects, tumor necrosis factor-alpha (TNF-alpha) was expressed equally in both fat depots, while toll-like receptor 4 (TLR4) and glucocorticoid receptor (GR) showed significantly lower expression in VAT than SAT. In obese subjects, TNF-alpha and TLR4 expression were significantly higher in VAT than SAT, yet GR expression did not differ in these areas. For all subjects, VAT 11beta-hydroxysteroid dehydrogenate type 1 (11beta-HSD1) level was significantly correlated with BMI. GR expression level was significantly correlated with TLR4 expression level. Cultured adipocytes showed higher TLR4 mRNA level after differentiation, and higher TNF-alpha level after treatment with free fatty acids. These results suggest that there are depot-specific differences in leptin, TNF-alpha, TLR4 and GR transcriptions in humans. TLR4 signaling and higher 11beta-HSD1 and GR levels in VAT may contribute predominantly to inflammatory factor production and subsequent metabolic sequelae in obese human.

Interleukin 22 Expands Transit-Amplifying Cells While Depleting Lgr5+ Stem Cells via Inhibition of Wnt and Notch Signaling.

BACKGROUND & AIMS: 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. METHODS: Intestinal stem cell cultures and mice were treated with recombinant IL22. Cell proliferation, death, and differentiation were assessed in vitro and in vivo by morphometric analysis, quantitative reverse transcriptase polymerase chain reaction, and immunohistochemistry. RESULTS: IL22 increased the size and number of proliferating cells within enteroids but decreased the total number of enteroids. Enteroid size increases required IL22-dependent up-regulation of the tight junction cation and water channel claudin-2, indicating that enteroid enlargement reflected paracellular flux-induced swelling. However, claudin-2 did not contribute to IL22-dependent enteroid loss, depletion of Lgr5+ stem cells, or increased epithelial proliferation. IL22 induced stem cell apoptosis but, conversely, enhanced proliferation within and expanded numbers of transit-amplifying cells. These changes were associated with reduced wnt and notch signaling, both in vitro and in vivo, as well as skewing of epithelial differentiation, with increases in Paneth cells and reduced numbers of enteroendocrine cells. CONCLUSIONS: IL22 promotes transit-amplifying cell proliferation but reduces Lgr5+ stem cell survival by inhibiting notch and wnt signaling. IL22 can therefore promote or inhibit mucosal repair, depending on whether effects on transit-amplifying or stem cells predominate. These data may explain why mucosal healing is difficult to achieve in some inflammatory bowel disease patients despite markedly elevated IL22 production.

In Vitro and In Vivo 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 epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

MiR-155 up-regulated by TGF-ß promotes epithelial-mesenchymal transition, invasion and metastasis of human hepatocellular carcinoma cells in vitro.

It has previously been reported that microRNA (miR)-155 is linked to the recurrence and prognosis of hepatocellular carcinoma (HCC) following liver transplantation. However, the role of miR-155 in the invasion and metastasis of HCC cells remains largely unclear. The aim of this study was to investigate the expression of miR-155 in HCC cells and its role in the invasion and migration of HCC cells in vitro. We found that the level of expression of miR-155 in HCC tissues and cells was significantly increased compared with non-tumorous adjacent tissues. Further study revealed that recombinant human transforming growth factor-ß (TGF-ß1) up-regulated the expression of miR-155 in HCC cells in vitro. Further, the overexpression of miR-155 in HCC cell line Huh-7 led to increased levels of cell invasion and migration compared with untreated control Huh-7 cells. MiR-155-overexpressed Huh-7 cells also exhibited altered levels of expression of certain cellular adhesion molecules related to epithelial-mesenchymal transition (EMT), including low levels of CDH1 and higher levels of FN1, SNAI1 and ZEB1, compared with control Huh-7 cells. Moreover, it was found that the overexpression of miR-155 and of TGF-ß1 protein decreased the expression of E-Cadherin and increased the expression of Vimentin in Huh-7 cells. These results indicate that an increased level of miR-155 in HCC cells, possibly due to stimulation by TGF-ß1, accelerates the process of EMT, promotes cellular invasion and migration in vitro, and thereby further promotes the progression of HCC.

Myosin regulatory light chain phosphorylation is associated with leiomyosarcoma development.

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.

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

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.

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

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.