TNFα/IFNγ Mediated Intestinal Epithelial Barrier Dysfunction Is Attenuated by MicroRNA-93 Downregulation of PTK6 in Mouse Colonic Epithelial Cells.
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.
University of South Florida, Morsani College of Medicine, Department of Surgery, Tampa, Florida, United States of America.
Ricci J
0000-0002-1706-4268
RJ
Haines
University of South Florida, Morsani College of Medicine, Department of Molecular Pharmacology and Physiology, Tampa, Florida, United States of America.
Richard S
RS
Beard
University of South Florida, Morsani College of Medicine, Department of Surgery, Tampa, Florida, United States of America.
Rebecca A
RA
Eitner
University of South Florida, Morsani College of Medicine, Department of Surgery, Tampa, Florida, United States of America.
Liwei
L
Chen
James A. Haley Veterans' Hospital, Tampa, Florida, United States of America.|University of South Florida, Morsani College of Medicine, Department of Surgery, Tampa, Florida, United States of America.
Mack H
MH
Wu
D000818:Animals; D062465:Claudin-3 / Q000378:metabolism; D003106:Colon / Q000166:cytology; D018076:DNA, Complementary / Q000378:metabolism; D017097:Electric Impedance; D004847:Epithelial Cells / Q000378:metabolism*; D000071161:Forkhead Box Protein O1 / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D007249:Inflammation; D007371:Interferon-gamma / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D035683:MicroRNAs / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D010539:Permeability; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D019108:Tight Junctions / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D062465:Claudin-3; C568123:Cldn3 protein, mouse; D018076:DNA, Complementary; D000071161:Forkhead Box Protein O1; C467200:Foxo1 protein, mouse; C543350:MIRN93 microRNA, human; D035683:MicroRNAs; C551470:Mirn93 microRNA, mouse; D009363:Neoplasm Proteins; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D011505:Protein-Tyrosine Kinases; C088375:PTK6 protein, human; C000598269:Ptk6 protein, mouse; D048670:MAP Kinase Kinase 4