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Title: FLICE-LIKE INHIBITORY PROTEIN (FLIP) PROTECTS AGAINST APOPTOSIS AND SUPPRESSES NF-KB ACTIVATION INDUCED BY BACTERIAL LIPOPOLYSACCHARIDE

Author
item Bannerman, Douglas
item EITING, KRISTINE - UNIV OF WASH-SEATTLE
item WINN, ROBERT - UNIV OF WASH-SEATTLE
item HARLAN, JOHN - UNIV OF WASH-SEATTLE

Submitted to: American Journal of Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/10/2004
Publication Date: 10/1/2004
Citation: Bannerman, D.D., Eiting, K.T., Winn, R.K., Harlan, J.M. 2004. Flice-like inhibitory protein (flip) protects against apoptosis and suppresses nf-kb activation induced by bacterial lipopolysaccharide. American Journal of Pathology. 165(4):1423-1431.

Interpretive Summary: Bovine mastitis is an inflammatory disease of the mammary gland mainly caused by bacterial infection of the gland. Economic losses due to mastitis are among the most costly compared to all other animal diseases in the U.S. A key component of mammary gland inflammation is the activation of NF-kB, a protein that promotes the induction of pro-inflammatory cytokines that aid in the defense of the mammary gland against invading bacterial pathogens. In addition to the ensuing inflammation, mastitis is also characterized by apoptosis (also known as programmed cell death). The current manuscript characterizes a dual role for the protein FLIP in mediating both apoptosis and NF-kB activation. In addition, this research increases our understanding of the intracellular signaling pathways that are activated in response to the Gram-negative bacterial envelope constituent, LPS, the latter of which promotes the pro-inflammatory state that often develops during mastitis.

Technical Abstract: Bacterial lipopolysaccharide (LPS) via its activation of Toll-like receptor-4 contributes to much of the vascular injury/dysfunction associated with Gram-negative sepsis. Inhibition of de novo gene expression has been shown to sensitize endothelial cells (EC) to LPS-induced apoptosis, the onset of which correlates with decreased expression of FLICE-like inhibitory protein (FLIP). We now have data that conclusively establish a role for FLIP in protecting EC against LPS-induced apoptosis. Overexpression of FLIP protected against LPS-induced apoptosis in the presence of cycloheximide, whereas, downregulation of FLIP using anti-sense oligonucleotides sensitized EC to direct LPS killing. Interestingly, FLIP overexpression suppressed NF-kB activation induced by LPS, but not by phorbol ester, suggesting a specific role for FLIP in mediating LPS activation. Conversely, mouse embryo fibroblasts (MEF) obtained from FLIP -/- mice showed enhanced LPS-induced NF-kB activation relative to those obtained from wild-type mice. Reconstitution of FLIP-/- MEF with full-length FLIP reversed the enhanced NF-kB activity elicited by LPS in the FLIP -/- cells. Changes in the expression of FLIP had no demonstrable effect on other known LPS/Tlr-4-activated signaling pathways including the p38, Akt, and Jnk pathways. Together, these data support a dual role for FLIP in mediating LPS-induced apoptosis and NF-kB activation.