|Williams, John - ROSLIN INSTITUTE|
Submitted to: Cytogenetics and Genome Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 8, 2005
Publication Date: November 8, 2005
Citation: Szperka, M.E., Connor, E.E., Paape, M.J., Williams, J.L., Bannerman, D.D. 2006. Sequencing, chromosomal mapping, and functional characterization of bovine flice-like inhibitory protein (flip). Cytogenetics and Genome Research. 112(1-2):90-97. Interpretive Summary: Approximately 50% of all clinical cases of mastitis are caused by Gram-negative bacteria. All of these bacteria contain a highly pro-inflammatory molecule called endotoxin or lipopolysaccharide (LPS), which is shed from the bacterial surface during bacterial replication or death. LPS has been implicated in much of the inflammation and injury associated with mastitis caused by Gram-negative bacteria. Cellular activation by LPS enables the host organism to mount an innate immune response. This immune response can be beneficial to the host by promoting clearance of the bacteria, however, an excessive inflammatory response can develop leading to the development of shock. In addition, LPS induces tissue injury and cell death (apoptosis) that can result in tissue scarring. In the setting of mastitis, this injury may contribute to decreased milk output. The present study sequenced, mapped, cloned, and functionally characterized bovine FLIP, and demonstrated a role for bovine FLIP in protecting against LPS-induced injury to bovine cells. Further, this study demonstrated that bovine FLIP downregulates cellular pro-inflammatory responses elicited by LPS.
Technical Abstract: FLICE-like inhibitory protein (FLIP) has been shown in both humans and mice to inhibit apoptosis and NF-kappaB activation induced by pro-inflammatory mediators. The activation of NF-kappaB and the induction of apoptosis are critical events in the pathogenesis of a variety of disease states in cattle, including mastitis. Since FLIP is known to moderate these events in other species, we mapped the bovine FLIP gene, sequenced bovine FLIP cDNA, and characterized its expression in cultured primary bovine endothelial cells. Sequencing of bovine FLIP revealed approximately 83, 74, and 68% amino acid sequence identity to its porcine, human, and murine orthologs, respectively. Bovine FLIP was mapped to chromosome 2 by radiation hybrid mapping. In addition, the functionality of bovine FLIP was studied. Over-expression of bovine FLIP protected against bacterial lipopolysaccharide (LPS)- and TNF-alpha-induced apoptosis in bovine endothelial cells consistent with previous studies of human FLIP. In addition, elevated expression of bovine FLIP blocked LPS- and TNF-alpha-induced upregulation of NF-kappaB-dependent gene products as assayed by E-selectin expression. Only the full-length bovine FLIP protein could inhibit NF-kappaB activation induced by LPS, whereas, the death effector domain region alone was able to inhibit TNF-alpha-induced NF-kappaB activation. Together, these data demonstrate the conservation of FLIP's ability to inhibit apoptosis and to downregulate NF-kappaB activation across species.