Effect of inflammatory cytokines and high fat diet on inositol-1,4,5-trisphosphate (IP3) receptors binding protein released with IP3 (IRBIT) expression in intestinal cells

IP3, upon binding to the IP3 receptor (IP3R), causes the release of intracellular calcium from the endoplasmic reticulum, which drives many cellular responses (e.g., cell spreading, exocytosis). In addition to releasing calcium, IP3 also causes the release of IRBIT from the IP3R. Over the past decade, IRBIT has been described as a protein that regulates calcium release, due to interaction with the IP3R, the activity of the Na-HCO3 cotransporter, the cystic fibrosis transmembrane regulator, and the Na/H exchanger (NHE3). Lack of reabsorption of Na+ by NHE3 in the intestine is responsible for diarrhea. Recently it was shown that IRBIT and NHE3 expression was decreased in a mouse model of diabetes, and the loss of NHE3 expression induced diarrhea in this model. Insulin treatment restored IRBIT and NHE3 expression, resulting in a decrease of diarrhea. Besides insulin, very little is known about factors regulating IRBIT expression in intestinal epithelial cells. In this work, we set to study the effect of inflammatory cytokines and high calorie diet on IRBIT expression, due to the fact that diabetes is associated with chronic inflammation and high caloric intake. To test the effect of inflammatory cytokines, we used the human colonic crypt cells T84. Exposing T84 cells to interleukin 13 or tumor necrosis factor alpha for 72 hours decreased IRBIT expression by 36% (P \u3c 0.001, n = 5) and 44% (P \u3c 0.001, n = 3) respectively. Finally, we compared the expression of IRBIT in mice fed with low fat milk (control) versus high milk fat (37%). We found that in the duodenum of 3 mice with a high fat diet, a substantial increase of IRBIT expression compared to the control. Our work is the first to demonstrate that inflammatory cytokines and dietary fat can alter IRBIT expression

Elucidating internalization mechanism of the Na-K-2Cl cotransporter 1 and its fate in the endocytotic pathway during protein kinase C activation in epithelial cells

Gut clearance (i.e., fluid secretion) is an important mechanism for host defense. Fluid secretion flushes luminal toxins and prevents bacterial attachment to intestinal epithelial cells, which otherwise would harm the host. In the colon, transepithelial chloride fluid secretion drives fluid secretion. The basolateral Na-K-2Cl cotransporter 1 (NKCC1) is the main protein pumping chloride inside the cell for its secretion by apical chloride channels. Previous studies have demonstrated that activation of the protein kinase C (PKC) causes a rapid internalization of NKCC1, thus decreasing chloride secretion. To date, the protein kinase C downstream targets involved in NKCC1 internalization and the fate of NKCC1 in the endocytic pathway is unknown. Using the human colonic crypt cells T84, we demonstrate that T84 cells express α-adducin and Myristoylated, Alanine-Rich C Kinase Substrate, two substrates of the PKC involved in protein internalization in other cells. In presence of phorbol 12-myristate 13-acetate (PMA), an activator of the conventional and novel PKC, we demonstrate that α-adducin is strongly phosphorylated in T84 cells. Next, we hypothesized that upon activation by PKC, α-adducin binds to NKCC1. In T84 cells subjected to PMA, we show that phospho α-adducin co-immunoprecipitates with NKCC1. Next, we used Mardin Darby Canine Kidney (MDCK) cells stably expressing eGFP-NKCC1. In this model, using immunocytochemistry, we show that NKCC1 colocalizes with α-adducin at the plasma membrane during PKC activation. Finally, we tested the fate of NKCC1 in the endocytic pathway. In MDCK cells exposed to PMA, we found that NKCC1 colocalizes with LAMP1, a marker of the lysosome. In conclusion, our data suggest that α-adducin participates to NKCC1 internalization during PKC activation and NKCC1 is targeted for degradation

Detailed analysis of the genetic and epigenetic signatures of iPSC-derived mesodiencephalic dopaminergic neurons.

Induced pluripotent stem cells (iPSCs) hold great promise for in vitro generation of disease-relevant cell types, such as mesodiencephalic dopaminergic (mdDA) neurons involved in Parkinson's disease. Although iPSC-derived midbrain DA neurons have been generated, detailed genetic and epigenetic characterizations of such neurons are lacking. The goal of this study was to examine the authenticity of iPSC-derived DA neurons obtained by established protocols. We FACS purified mdDA (Pitx3 (Gfp/+) ) neurons derived from mouse iPSCs and primary mdDA (Pitx3 (Gfp/+) ) neurons to analyze and compare their genetic and epigenetic features. Although iPSC-derived DA neurons largely adopted characteristics of their in vivo counterparts, relevant deviations in global gene expression and DNA methylation were found. Hypermethylated genes, mainly involved in neurodevelopment and basic neuronal functions, consequently showed reduced expression levels. Such abnormalities should be addressed because they might affect unambiguous long-term functionality and hamper the potential of iPSC-derived DA neurons for in vitro disease modeling or cell-based therapy

Defining Protein Kinase C Substrates Involved in the Na-K-2Cl Cotransporter 1 Endocytosis in Colonic Epithelial Cells

Fluid secretion in the lungs and intestine lubricates and protects cell lining in these organs’ cavities and defect in secretion is observed in situations such as cystic fibrosis or Sjögren’s syndrome. In colonic epithelial cells, the driving force for fluid secretion is an active transcellular chloride secretion, and the basolateral Na-K-2Cl cotransporter 1 (NKCC1) represents the main transporter pumping chloride inside the cell for its secretion by apical chloride channels. Human colonic T84 cells are used to characterize cellular and molecular mechanisms regulating chloride secretion. Previously it was shown that activated protein kinase C (PKC) epsilon and delta, cause internalization of NKCC1, thus blunting chloride secretion. Today, PKC-mediated internalization of NKCC1 is not fully understood. Myristoylated, alanine-rich C kinase substrate (MARCKS) and α-adducin are known PKC substrates participating in PKC-mediated endocytosis in other cells, but their potential role in NKCC1 endocytosis has not been tested. Here, we show by western blot and immunocytochemistry that T84 cells express both MARCKS and α-adducin and display cytosolic and some plasma membrane localization. Also, activation of PKCs by phorbol 12-myristate 13-acetate (PMA) induced α-adducin and MARCKS phosphorylation in T84 cells. We have performed co-immunoprecipitation experiment to determine whether MARCKS and/or α-adducin participate in NKCC1 internalization during PKC activation. T84 cells exposed to PMA revealed that immunoprecipitating NKCC1 pulled down α-adducin and MARCKS. Finally, using T84 cells and Mardin Darby Canine Kidney cells expressing eGFP-NKCC1 we found that PMA causes co-localization of α-adducin with NKCC1. In conclusion, our data support the hypothesis that α-adducin and MARCKS may participate in NKCC1 endocytosis during PKC activation

Elucidating protein kinase C substrates involved in internalization of the Na+-K+-2Cl− cotransporter in the human colonic crypt cells T84

Elucidating protein kinase C substrates involved in internalization of the Na+-K+-2Cl− cotransporter in the human colonic crypt cells T84 Jesse Smallwood, Natasa Petreska, Alexander Ahlgrim and Patrice G Bouyer, Valparaiso University Fluid secretion in the lungs and colon lubricates and protects cells lining the cavity of those organs. Dysregulation of fluid secretion is the hallmark of diseases such as: cystic fibrosis (defective secretion) or secretory diarrhea (excessive secretion). In epithelial cells, the driving force for fluid secretion is active transcellular chloride secretion, with the basolateral Na-K-2Cl cotransporter 1 (NKCC1) pumping chloride inside the cell for its secretion by apical chloride channels. Previous studies have highlighted the critical role of NKCC1 in the regulation of chloride secretion in the colon. We have demonstrated that activation of the protein kinase C (PKC) causes a rapid internalization of NKCC1, hence, blunting chloride secretion. Nonetheless, the cellular and molecular details of PKC-mediated NKCC1 internalization remain unclear. Myristoylated, alanine-rich C kinase substrate (MARCKS) and α-adducin are two known PKC substrates participating in PKC-mediated endocytosis in other cells, but their potential role in NKCC1 endocytosis has not been tested. In the present study, we showed by Western blot that α-adducin and MARCKS are expressed in the human colonic crypt cells T84. In addition, using phorbol 12-myristate 13-acetate (PMA), an activator of the conventional and novel PKC caused both α-adducin and MARCKS to be phosphorylated and represent therefore PKC substrates in T84 cells. To test whether α-adducin or MARCKS binds to NKCC1 during PKC activation we performed immunoprecipitation experiments. After PKC activation by PMA, we found that immunoprecipitating NKCC1 pulled down MARCKS and α-adducin. In conclusion, our preliminary data strongly suggest that α-adducin and MARCKS are involved in NKCC1 internalization during PKC activation

Chromatin architecture reorganization during stem cell differentiation

Higher-order chromatin structure is emerging as an important regulator of gene expression. Although dynamic chromatin structures have been identified in the genome, the full scope of chromatin dynamics during mammalian development and lineage specification remains to be determined. By mapping genome-wide chromatin interactions in human embryonic stem(ES) cells and four human ES-cell-derived lineages, we uncover extensive chromatin reorganization during lineage specification. We observe that although self-associating chromatin domains are stable during differentiation, chromatin interactions both within and between domains change in a striking manner, altering 36% of active and inactive chromosomal compartments throughout the genome. By integrating chromatin interaction maps with haplotype-resolved epigenome and transcriptome data sets, we find widespread allelic bias in gene expression correlated with allele-biased chromatin states of linked promoters and distal enhancers. Our results therefore provide a global view of chromatin dynamics and a resource for studying long-range control of gene expression in distinct human cell lineages.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000349547400029&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701Multidisciplinary SciencesSCI(E)[email protected]