Citrobacter rodentium ‐induced NF‐ κ B activation in hyperproliferating colonic epithelia: role of p65 (Ser 536 ) phosphorylation

1 The transcription factors of the NF‐ κ B/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‐ κ B 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‐ κ B activation kinetics. I κ B α levels decreased during this time. 3 Phosphorylation of IKK α (at Ser 176/180 ) and ‐ β (at Ser 177/181 ) increased significantly during TMCH suggesting activation in vivo . 4 p65‐Ser 536 (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 I κ B α and significant inhibition of NF‐ κ B 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 α or ‐ β . 7 Thus, NF‐ κ B activation during TMCH involves both I κ B α degradation and p65‐Ser 536 phosphorylation. p65/RSK‐1 interaction and concomitant increase in p65 536 complexed with CBP may be important in modulating NF‐ κ B activity in vivo . Activated NF‐ κ B, besides modulating proliferation, may aid in providing protective immunity against C. rodentium infection in vivo .British Journal of Pharmacology (2006) 148 , 814–824. doi: 10.1038/sj.bjp.0706784