Interferon-induced RIP1/RIP3-mediated necrosis requires PKR and is licensed by FADD and caspases.

Interferons (IFNs) are cytokines with powerful immunomodulatory and antiviral properties, but less is known about how they induce cell death. Here, we show that both type I (α/ß) and type II (γ) IFNs induce precipitous receptor-interacting protein (RIP)1/RIP3 kinase-mediated necrosis when the adaptor protein Fas-associated death domain (FADD) is lost or disabled by phosphorylation, or when caspases (e.g., caspase 8) are inactivated. IFN-induced necrosis proceeds via progressive assembly of a RIP1-RIP3 "necrosome" complex that requires Jak1/STAT1-dependent transcription, but does not need the kinase activity of RIP1. Instead, IFNs transcriptionally activate the RNA-responsive protein kinase PKR, which then interacts with RIP1 to initiate necrosome formation and trigger necrosis. Although IFNs are powerful activators of necrosis when FADD is absent, these cytokines are likely not the dominant inducers of RIP kinase-driven embryonic lethality in FADD-deficient mice. We also identify phosphorylation on serine 191 as a mechanism that disables FADD and collaborates with caspase inactivation to allow IFN-activated necrosis. Collectively, these findings outline a mechanism of IFN-induced RIP kinase-dependent necrotic cell death and identify FADD and caspases as negative regulators of this process.

Analysis of the in vivo functions of Mrp3.

Multidrug resistance protein 3 (MRP3) is an ATP-binding cassette transporter that is able to confer resistance to anticancer agents such as etoposide and to transport lipophilic anions such as bile acids and glucuronides. These capabilities, along with the induction of the MRP3 protein on hepatocyte sinusoidal membranes in cholestasis and the expression of MRP3 in enterocytes, have led to the hypotheses that MRP3 may function in the body to protect normal tissues from etoposide, to protect cholestatic hepatocytes from endobiotics, and to facilitate bile-acid reclamation from the gut. To elucidate the role of Mrp3 in these processes, the Mrp3 gene (Abcc3) was disrupted by homologous recombination. Homozygous null animals were healthy and physically indistinguishable from wild-type mice. Mrp3(-/-) mice did not exhibit enhanced lethality to etoposide phosphate, although an analysis of transfected human embryonic kidney 293 cells indicated that the potency of murine Mrp3 toward etoposide ( approximately 2.0- to 2.5-fold) is comparable with that of human MRP3. After induction of cholestasis by bile duct ligation, Mrp3(-/-) mice had 1.5-fold higher levels of liver bile acids and 3.1-fold lower levels of serum bilirubin glucuronide compared with ligated wild-type mice, whereas significant differences were not observed between the respective sham-operated mice. Bile acid excretion, pool size, and fractional turnover rates were similar in Mrp3(-/-) and wild-type mice. We conclude that Mrp3 functions as an alternative route for the export of bile acids and glucuronides from cholestatic hepatocytes, that the pump does not play a major role in the enterohepatic circulation of bile acids and that the lack of chemosensitivity is probably attributable to functional redundancy with other pumps.

miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1.

These studies show that miR-122, a 22-nucleotide microRNA, is derived from a liver-specific noncoding polyadenylated RNA transcribed from the gene hcr. The exact sequence of miR-122 as well as the adjacent secondary structure within the hcr mRNA are conserved from mammalian species back to fish. Levels of miR-122 in the mouse liver increase to half maximal values around day 17 of embryogenesis, and reach near maximal levels of 50,000 copies per average cell before birth. Lewis et al. (2003) predicted the cationic amino acid transporter (CAT-1 or SLC7A1) as a miR-122 target. CAT-1 protein and its mRNA are expressed in all mammalian tissues but with lower levels in adult liver. Furthermore, during mouse liver development CAT-1 mRNA decreases in an almost inverse correlation with miR-122. Eight potential miR-122 target sites were predicted within the human CAT-1 mRNA, with six in the 3'-untranslated region. Using a reporter construct it was found that just three of the predicted sites, linked in a 400-nucleotide sequence from human CAT-1, acted with synergy and were sufficient to strongly inhibit protein synthesis and reduce mRNA levels. In summary, these studies followed the accumulation during development of miR-122 from its mRNA precursor, hcr, through to identification of what may be a specific mRNA target, CAT-1.