Abstract
Backgroundp8 is a DNA-binding protein induced in many tissues in response to LPS treatment. Hence, p8 could be a mediator of LPS-associated effects or, on the contrary, p8 expression may be part of the protective mechanism of the tissues in response to LPS. Finally, p8 expression in response to LPS could also be a simple epiphenomenon.MethodsTo investigate the role of p8 in vivo, we generated p8-deficient mice by gene targeting. Because p8 is a stress protein, we analyzed the response of p8-/- mice to a systemic stress induced by LPS injection. Liver was chosen as model organ to monitor alterations in gene expression.ResultsLPS resulted in higher serum TNF-α concentration and higher mortality rate in p8-deficient mice than in wild-type. Also, liver and pancreas, but not lung, from p8-/- mice showed increased amounts of MPO and HPO. To gain insight into the molecular bases of such susceptibility, we used high density DNA microarrays consisting of ~6000 genes and ESTs to compare gene regulation in response to LPS in p8+/+ and p8-/- livers. In wild-type, 105 genes and 73 ESTs were up-regulated and 232 genes and 138 ESTs down-regulated. By contrast, 212 genes and 125 ESTs were found up-regulated and 90 genes and 85 ESTs down regulated in p8-/- mice. Among them, only 93 (51 induced and 42 repressed) corresponded to the wild-type pattern, demonstrating that p8 deficiency hinders the normal response to LPS, which may account for the increased sensitivity of p8-/-mice to the endotoxin.ConclusionsThe large number of genes showing abnormal regulation after LPS suggests that p8 is an important regulatory factor involved in many cellular defence pathways.
Highlights
We identified a new gene, called p8, whose expression is strongly induced during the acute phase of pancreatitis [1]
Overexpression of p8 in certain cell lines promotes growth [1,2] but it can inhibit growth when recruited by inhibitory signals [9]. These results suggest that p8 expression may regulate many cellular functions, in response to cellular injury or during tumour development, and that p8 may play different roles depending on the cell type and/or growth conditions
Targeted disruption of the p8 gene creates a null mutation To investigate the function of p8, a null allele was generated in 129/Sv embryonic stem (ES) cells by replacing exon 2 of the p8 gene, which encodes 60% of the coding sequence, with a neomycin-resistance cassette
Summary
We identified a new gene, called p8, whose expression is strongly induced during the acute phase of pancreatitis [1]. P8 contains a canonical bipartite signal for nuclear targeting suggesting that p8 should be located in the nucleus. Supporting this hypothesis we detected p8 within the nucleus of Cos-7 cells transfected with a p8 expression plasmid, it was partly located to the cytoplasm [2]. Analysis of p8 primary structure suggested that it should be a DNA-binding protein [1]. We performed biochemical and biophysical studies showing that human recombinant p8 was in many structural aspects very similar to the "high mobility group" (HMG) proteins, sharing with them little aminoacid sequence homology [3]. It is interesting to note that HMG1, a member of the HMG family of proteins, is a potent pro-inflammatory mediator released passively by necrotic cells but not by apoptotic cells [reviewed in [5]]
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