title: Cholangiocyte N-Ras protein mediates lipopolysaccharide-induced interleukin 6 secretion and proliferation.
abstract: Cholangiocytes, the epithelial cells lining the bile ducts in the liver, are periodically exposed to potentially injurious microbes and/or microbial products. As a result, cholangiocytes actively participate in microbe-associated, hepatic proinflammatory responses. We previously showed that infection of cultured human cholangiocytes with the protozoan parasite, Cryptosporidium parvum, or treatment with gram-negative bacteria-derived LPS, activates NFκB in a myeloid differentiation 88 (MyD88)-dependent manner. Here, we describe a novel signaling pathway initiated by Toll-like receptors (TLRs) involving the small GTPase, Ras, that mediates cholangiocyte proinflammatory cytokine production and induction of cholangiocyte proliferation. Using cultured human cholangiocytes and a Ras activation assay, we found that agonists of plasma membrane TLRs (TLR 1, 2, 4, 5, and 6) rapidly (<10 min) activated N-Ras, but not other p21 Ras isoforms, resulting in the rapid (<15 min) phosphorylation of the downstream Ras effector, ERK1/2. RNA interference-induced depletion of TRAF6, a downstream effector of MyD88 and known activator of MAPK signaling, had no effect on N-Ras activation. Following N-Ras activation the proinflammatory cytokine, IL6, is rapidly secreted. Using a luciferase reporter, we demonstrated that LPS treatment induced IL6 promoter-driven luciferase which was suppressed using MEK/ERK pharmacologic inhibitors (PD98059 or U0126) and RNAi-induced depletion of N-Ras. Finally, we showed that LPS increased cholangiocyte proliferation (1.5-fold), which was inhibited by depletion of N-Ras; TLR agonist-induced proliferation was also inhibited following pretreatment with an IL6 receptor-blocking antibody. Together, our results support a novel signaling axis involving microbial activation of N-Ras likely involved in the cholangiocyte pathogen-induced proinflammatory response.
authors.affiliation: Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota 55905. Electronic address: ohara.steven@mayo.edu.
authors.forename: Steven P
authors.initials: SP
authors.lastname: O'Hara
authors.affiliation: Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota 55905.
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Splinter
authors.affiliation: Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota 55905.
authors.forename: Christy E
authors.initials: CE
authors.lastname: Trussoni
authors.affiliation: Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota 55905.
authors.forename: Gabriella B
authors.initials: GB
authors.lastname: Gajdos
authors.affiliation: Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota 55455.
authors.forename: Pooja N
authors.initials: PN
authors.lastname: Lineswala
authors.affiliation: Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota 55905.
authors.forename: Nicholas F
authors.initials: NF
authors.lastname: LaRusso
mesh_terms: D001652:Bile Ducts / Q000166:cytology* / Q000469:parasitology; D049109:Cell Proliferation; D016785:Cryptosporidium parvum / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000378:metabolism; D006801:Humans; D007113:Immunity, Innate; D007249:Inflammation; D015850:Interleukin-6 / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D020033:Protein Isoforms; D015398:Signal Transduction; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D051193:Toll-Like Receptors / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism*
chemical_list: D015850:Interleukin-6; D008070:Lipopolysaccharides; D016328:NF-kappa B; D020033:Protein Isoforms; C495345:TLR4 protein, human; D048029:TNF Receptor-Associated Factor 6; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D018631:ras Proteins