Nitric oxide-induced down-regulation of beta-catenin in colon cancer cells by a proteasome-independent specific pathway.
OBJECTIVE
We have previously reported that nitric oxide could induce the death of colon cancer cells. Because an inappropriate activation of beta-catenin has been associated with intestinal cell malignant transformation, we explored whether nitric oxide could affect beta-catenin expression and function.


METHODS
Human colon cancer cell lines were treated with the nitric oxide donor glyceryl trinitrate (GTN) before analyzing beta-catenin expression by immunofluorescence, immunoblotting, and immunoprecipitation methods and its transcriptional activity using a luciferase reporter gene driven by a T-cell factor-responsive promotor.


RESULTS
GTN induces beta-catenin degradation and down-regulates its transcriptional activity in colon cancer cells. This effect is preceded by GTN-induced tyrosine nitration of beta-catenin, together with its dephosphorylation on serine 33, 37, and 45 and threonine 41. GTN-induced beta-catenin degradation involves proteases that are sensitive to a broad-spectrum caspase inhibitor, z-VAD-fmk, and to serine protease inhibitors N-tosyl-L-phenylalaline chloromethyl ketone (TPCK) and [4-(2-aminoethyl)-benzenesulfonylfluoride] (AEBSF), whereas the ubiquitin/proteasome pathway is not involved. Interestingly, only TPCK and AEBSF restore beta-catenin transcriptional activity and preserve beta-catenin nuclear localization in GTN-treated colon cancer cells.


CONCLUSIONS
Exposure of colon cancer cells to nitric oxide unraveled a so-far-unidentified mechanism of beta-catenin regulation. The protein is nitrated and dephosphorylated, and its transcriptional activity is reduced through degradation by a TPCK and AEBSF-sensitive protease.
Prévotat
Laurent
L
Laboratoire d'Immunologie et Immunothérapie des Cancers, EPHE/INSERM U517, IFR100, Faculty of Medicine, 7 bd. Jeanne d'Arc, 21079 Dijon, France.
Filomenko
Rodolphe
R
Solary
Eric
E
Jeannin
Jean-François
JF
Bettaieb
Ali
A
D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D020169:Caspases / Q000378:metabolism; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000503:physiopathology*; D015536:Down-Regulation / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D019073:HT29 Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D009368:Neoplasm Transplantation; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D005996:Nitroglycerin / Q000494:pharmacology; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013450:Sulfones / Q000494:pharmacology; D051820:TCF Transcription Factors / Q000378:metabolism; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D000073940:Transcription Factor 4; D014158:Transcription, Genetic / Q000502:physiology; D014162:Transfection; D051176:beta Catenin / Q000235:genetics* / Q000378:metabolism*
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D009419:Nerve Tissue Proteins; D020030:Nitric Oxide Donors; D015842:Serine Proteinase Inhibitors; D013450:Sulfones; D051820:TCF Transcription Factors; C090743:Tcf4 protein, mouse; D000073940:Transcription Factor 4; D051176:beta Catenin; D009569:Nitric Oxide; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D014108:Tosylphenylalanyl Chloromethyl Ketone; D020169:Caspases; D046988:Proteasome Endopeptidase Complex; D005996:Nitroglycerin