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Background: Epithelial cells(EC)-derived interleukin-7 (IL-7) plays a crucial role in control of development and homeostasis of neighboring intraepithelial lymphocytes (IEL), and keratinocyte growth factor (KGF) exerts protective effects on intestinal epithelial cells and up-regulates EC-derived IL-7 expression through KGFR pathway. This study was to further investigate the molecular mechanism involved in the regulation of IL-7 expression by KGF in the intestine. Methods: Intestinal epithelial cells (LoVo cells) and adult C57BL/6J mice were treated with KGF. Epithelial cell proliferation was studied by flow cytometry for BrdU-incorporation and by immunohistochemistry for PCNA staining. Western blot was used to detect the changes of expression of P-Tyr-STAT1, STAT1, and IL-7 by inhibiting STAT1. Alterations of nuclear extracts and total proteins of IRF-1, IRF-2 and IL-7 following IRF-1 and IRF-2 RNA interference with KGF treatment were also measured with western blot. Moreover, IL-7 mRNA expressions were also detected by Real-time PCR and IL-7 protein level in culture supernatants was measured by enzyme linked immunosorbent assay(ELISA). Results: KGF administration significantly increased LoVo cell proliferation and also increased intestinal wet weight, villus height, crypt depth and crypt cell proliferation in mice. KGF treatment led to increased levels of P-Tyr-STAT1, RAPA and AG490 both blocked P-Tyr-STAT1 and IL-7 expression in LoVo cells. IRF-1 and IRF-2 expression in vivo and in vitro were also up-regulated by KGF, and IL-7 expression was decreased after IRF-1 and IRF-2 expression was silenced by interfering RNA, respectively. Conclusion: KGF could up-regulate IL-7 expression through the STAT1/IRF-1, IRF-2 signaling pathway, which is a new insight in potential effects of KGF on the intestinal mucosal immune system. [ABSTRACT FROM AUTHOR]

Abstract: IκBα is a member of the NF-κB inhibitor family that inhibits NF-κB activity by sequestering NF-κB in an inactive form in the cytosol. Unlike mammalian IκBα, which has been extensively studied, very little is known about the function of fish IκBα. In this study, we identified and analyzed an IκBα homologue, CsIκBα from half-smooth tongue sole (Cynoglossus semilaevis), a marine flatfish with important economic value. The deduced amino acid sequence of CsIκBα contains 308 residues and shares 58–82% overall sequence identities with the IκBα of a number of teleosts. In silico analysis identified in CsIκBα conserved domains that in mammals are known to be involved in phosphorylation, ubiquitination, and degradation of IκBα. Quantitative real time RT-PCR detected constitutive expression of CsIκBα in gut, spleen, liver, gill, heart, brain, muscle, and kidney. Experimental challenge with a bacterial pathogen-induced significant inductions of CsIκBα expression in head and trunk kidney, which, however, were transient and much lower in magnitude than that of interleukin-1β. To examine the effect of unregulated overexpression of CsIκBα in a live fish model, tongue sole were administered via intramuscular injection with plasmid pCNCsIkBa, which constitutively expresses CsIκBα. PCR, RT-PCR, and immunohistochemistry analysis showed that pCNCsIkBa was able to translocate to internal tissues, where transcription and translation of the recombinant CsIκBα took place. Compared to control fish, fish administered with pCNCsIkBa were impaired in the ability to block bacterial dissemination and survival in kidney and exhibited significantly reduced expression of multiple immune genes. These results suggest the possible existence in tongue sole of a NF-κB–IκBα signaling pathway that is negatively regulated by CsIκBα and required for effective defense against bacterial infection. [Copyright &y& Elsevier]