Citrobacter rodentium-induced NF-kappaB activation in hyperproliferating colonic epithelia: role of p65 (Ser536) phosphorylation.

1. The transcription factors of the NF-kappaB/Rel family form dimeric complexes that control expression of various genes involved in inflammation and proliferation. 2. During transmissible murine colonic hyperplasia (TMCH) induced by Citrobacter rodentium, nuclear translocation of NF-kappaB in isolated colonic crypts increased 3 day's post-infection and continued over 12 days paralleling peak hyperplasia. Antibody supershifts for both p65/p50 hetero- and p50/p50 homodimers occurred. Expression levels of both p50 and p65 subunits increased in cytosolic/nuclear extracts and correlated with NF-kappaB activation kinetics. IkappaB alpha levels decreased during this time. 3. Phosphorylation of IKK alpha (at Ser(176/180)) and -beta (at Ser(177/181)) increased significantly during TMCH suggesting activation in vivo. 4. p65-Ser536 (p65(536)) exhibited increased phosphorylation on immunoblotting and immunohistochemistry (IHC) both at day 6 and 12 TMCH. p65(536) translocated to nucleus and interacted with transcriptional coactivator CREB binding protein (CBP). 5. Proteasomal inhibitor bortezomib (Velcade) caused accumulation of Ser(32/36)-phosphorylated IkappaB alpha and significant inhibition of NF-kappaB activity in vivo. Velcade also blocked nuclear translocation of activated p65: both immunoblotting and IHC failed to detect p65(536) nuclear immunoreactivity. Velcade, however, did not abrogate TMCH. 6. p65 interacted strongly with ribosomal S6 kinase 1 (RSK-1) during coimmunoprecipitation but not with IKK alpha or -beta. 7. Thus, NF-kappaB activation during TMCH involves both IkappaB alpha degradation and p65-Ser536 phosphorylation. p65/RSK-1 interaction and concomitant increase in p65(536) complexed with CBP may be important in modulating NF-kappaB activity in vivo. Activated NF-kappaB, besides modulating proliferation, may aid in providing protective immunity against C. rodentium infection in vivo.