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TNFα/IFNγ Mediated Intestinal Epithelial Barrier Dysfunction Is Attenuated by MicroRNA-93 Downregulation of PTK6 in Mouse Colonic Epithelial Cells.

ABSTRACT: Since inflammatory bowel diseases (IBD) represent significant morbidity and mortality in the US, the need for defining novel drug targets and inflammatory mechanisms would be of considerable benefit. Although protein tyrosine kinase 6 (PTK6, also known as breast tumor kinase BRK) has been primarily studied in an oncogenic context, it was noted that PTK6 null mice exhibited significantly enhanced colonic epithelial barrier function. Considering that the inflammatory functions of PTK6 have not yet been explored, we hypothesized that cytokines responsible for mediating IBD, such as TNFα/IFNγ, may solicit the action of PTK6 to alter barrier function. After first assessing critical mediators of TNFα/IFNγ driven epithelial barrier dysfunction, we further explored the possibility of PTK6 in this inflammatory context. In this report, we showed that PTK6 siRNA and PTK6 null young adult mouse colonic epithelial cells (YAMC) exhibited significant attenuation of TNFα/IFNγ induced barrier dysfunction as measured by electric cell-substrate impedance sensing (ECIS) assay and permeability assays. In addition, PTK6 null cells transfected with PTK6 cDNA displayed restored barrier dysfunction in response to TNFα/IFNγ, while the cells transfected with vector alone showed similar attenuation of barrier dysfunction. Furthermore, using subcellular fractionation and immunocytochemistry experiments, we found that PTK6 plays a role in FoxO1 nuclear accumulation leading to down-regulation of claudin-3, a tight junction protein. Moreover, we searched for relevant miRNA candidates putative for targeting PTK6 in order to identify and assess the impact of microRNA that target PTK6 with respect to TNFα/IFNγ induced barrier dysfunction. Subsequently, we assayed likely targets and determined their effectiveness in attenuating PTK6 expression as well as cytokine induced barrier dysfunction. Results showed that miR-93 reduced PTK6 expression and attenuated TNFα/IFNγ imposed decrease in transepithelial electrical resistance (TER), as well as excluded FoxO1 from the nucleus. Our results indicate that PTK6 may act as a novel mediator of intestinal epithelial permeability during inflammatory injury, and miR-93 may protect intestinal epithelial barrier function, at least in part, by targeting PTK6.

SUBMITTER: Haines RJ

PROVIDER: S-EPMC4847919 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Hyperglycemia is thought to increase production of inflammatory cytokines and permeability of the large intestine. Resulting intestinal inflammation is then often characterized by excess secretion of tumor necrosis factor alpha (TNFα). Thus, hyperglycemia in hospitalized patients suffering from severe trauma or disease is frequently accompanied by TNFα secretion, and the combined impact of these insults on the intestinal epithelium is poorly understood. This study utilized a simple yet elegant model of the intestinal epithelium, comprised of primary human intestinal stem cells and their differentiated progeny, to investigate the impact of hyperglycemia and inflammatory factors on the colonic epithelium. When compared to epithelium cultured under conditions of physiologic glucose, cells under hyperglycemic conditions displayed decreased mucin-2 (MUC2), as well as diminished alkaline phosphatase (ALP) activity. Conditions of 60 mM glucose potentiated secretion of the cytokine IL-8 suggesting that cytokine secretion during hyperglycemia may be a source of tissue inflammation. TNFα measurably increased secretion of IL-8 and IL-1β, which was enhanced at 60 mM glucose. Surprisingly, intestinal permeability and paracellular transport were not altered by even extreme levels of hyperglycemia. The presence of TNFα increased MUC2 presence, decreased ALP activity, and negatively impacted monolayer barrier function. When TNFα hyperglycemia and ≤30 mM glucose and were combined, MUC2 and ALP activity remained similar to that of TNFα alone, although synergistic effects were seen at 60 mM glucose. An automated image analysis pipeline was developed to assay changes in properties of the zonula occludens-1 (ZO-1)-demarcated cell boundaries. While hyperglycemia alone had little impact on cell shape and size, cell morphologic properties were extraordinarily sensitive to soluble TNFα. These results suggest that TNFα acted as the dominant modulator of the epithelium relative to glucose, and that control of inflammation rather than glucose may be key to maintaining intestinal homeostasis.

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TNFα/IFNγ Mediated Intestinal Epithelial Barrier Dysfunction Is Attenuated by MicroRNA-93 Downregulation of PTK6 in Mouse Colonic Epithelial Cells.

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