Monochloramine induces reorganization of nuclear speckles and phosphorylation of SRp30 in human colonic epithelial cells: role of protein kinase C.
Intestinal epithelial cells are constantly stimulated by reactive oxidant metabolites (ROMs) in inflamed mucosa. Monochloramine (NH2Cl), a cell-permeant ROM, is particularly relevant to the pathogenesis of inflammation in the gastrointestinal tract. Nuclear speckles, a unique nuclear subcompartment, accumulate a family of proteins, namely, serine- and arginine-rich (SR) proteins. They play important roles in regulation of pre-mRNA splicing. Currently, little is known about the link between inflammatory stimulation and the pre-mRNA splicing process, although gene expression is changed in inflamed tissues. The present study was designed to investigate whether stimulation of human colonic epithelial cells (HT-29 and Caco-2 cell lines) with NH2Cl affects nuclear speckles and their components. By indirect immunofluorescence, nuclear speckles have been shown to undergo rapid aggregation after NH2Cl stimulation. By utilizing Western blotting, SRp30 (a subset of SR proteins) in intestinal epithelial cells was found to be phosphorylated after NH2Cl treatment, whereas other SR proteins were not responsive to NH2Cl stimulation. The cytotoxic effect of NH2Cl was excluded by both negative lactate dehydrogenase assay and propidium iodide staining. Therefore, NH2Cl-induced morphological changes on nuclear speckles and phosphorylated SRp30 do not result from intestinal epithelial injury. Furthermore, the effect of NH2Cl on nuclear speckles and SRp30 was blocked by bisindolylmaleimide I, a selective PKC inhibitor. Together, the available data suggest that stimulation of intestinal epithelial cells with NH2Cl results in a consequent change on pre-mRNA splicing machinery via a distinctive signal pathway involving activation of PKC. This effect may contribute to oxidant-induced pathophysiological changes in the gastrointestinal tract.
Zhu
Ya-Qin
YQ
Disease Pathogenesis Program, Box 217, Children's Memorial Institute for Education and Research, Children's Memorial Hospital, 2300 Children's Plaza, Chicago, IL 60614, USA.
Lu
Yu
Y
Tan
Xiao-Di
XD
D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000494:pharmacology; D022003:Cell Nucleus Structures / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D002700:Chloramines / Q000633:toxicity*; D003106:Colon / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D019073:HT29 Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors
D002352:Carrier Proteins; D002700:Chloramines; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; C015482:protein kinase modulator; D000068103:Serine-Arginine Splicing Factors; D011493:Protein Kinase C; C030816:chloramine