Influence of NFkappaB inhibitors on IL-1beta-induced chemokine CXCL8 and -10 expression levels in intestinal epithelial cell lines: glucocorticoid ineffectiveness and paradoxical effect of PDTC.
OBJECTIVE
Activation of intestinal epithelial cell (IEC) nuclear factor kappaB (NFkappaB) and the consequent chemokine upregulation are crucial events in inflammatory bowel disease (IBD) pathogenesis. Not much is known about the consequences of NFkappaB inhibition in terms of chemokine expression in intestinal cells. Therefore, we aimed to evaluate the efficacy of compounds known to disrupt the NFkappaB pathway on NFkappaB transcriptional activity and CXCL8 and CXCL10 gene expression in intestinal cell lines.


METHODS
The influence of NFkappaB inhibitors (dexamethasone, pyrrolidine dithiocarbamate (PDTC) and BAY 11-7082) on IL-1beta-induced NFkappaB transcriptional activity was investigated by transient transfection of Caco-2 cells with an NFkappaB-secreted alkaline phosphatase reporter plasmid. Il-1beta stimulated CXCL8 and CXCL10 mRNA and protein expression and was studied in Caco-2 and HT29 cells in the presence and absence of the NFkappaB inhibitors by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent serologic assay, respectively. To reveal alternative signalling cascades, experiments were also performed in the presence of the p38MAPK inhibitor SB 203580 and the ERK inhibitor PD 98059.


RESULTS
Dexamethasone did not downregulate chemokine expression sufficiently, probably due to a lack of glucocorticoid receptors in these cells. While BAY11-7082 inhibited chemokine expression, PDTC led to a paradoxical upregulation of CXCL8 in Caco-2 cells, which could be prevented by inhibition of p38MAPK.


CONCLUSIONS
These data explain the frequent unresponsiveness of IBD to glucocorticoid treatment and suggest that alternative NFkappaB inhibition in IECs might be of use in IBD therapy. Drug development based on measuring anti-NFkappaB activity might be misleading and should therefore also include studies on relevant gene products.
Sunil
Yeruva
Y
Centre for Internal Medicine, Department of Gastroenterology and Endocrinology, University of Goettingen, Goettingen, Germany.
Ramadori
Giuliano
G
Raddatzc
Dirk
D
D018938:Caco-2 Cells; D054357:Chemokine CXCL10 / Q000235:genetics* / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D005419:Flavonoids / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D005938:Glucocorticoids / Q000494:pharmacology; D019073:HT29 Cells; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D053583:Interleukin-1beta / Q000494:pharmacology*; D016209:Interleukin-8 / Q000235:genetics* / Q000378:metabolism; D007422:Intestines / Q000166:cytology; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D009570:Nitriles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011759:Pyrrolidines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology; D013859:Thiocarbamates / Q000494:pharmacology*
C434003:3-(4-methylphenylsulfonyl)-2-propenenitrile; C114753:CXCL10 protein, human; C508606:CXCL8 protein, human; D054357:Chemokine CXCL10; D005419:Flavonoids; D005938:Glucocorticoids; D007093:Imidazoles; D053583:Interleukin-1beta; D016209:Interleukin-8; D016328:NF-kappa B; D009570:Nitriles; D011725:Pyridines; D011759:Pyrrolidines; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; D013450:Sulfones; D013859:Thiocarbamates; C020972:pyrrolidine dithiocarbamic acid; D003907:Dexamethasone; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one