The Set1-containing complex COMPASS, which is the yeast homolog of the human MLL complex, is required for mono-, di-, and trimethylation of lysine 4 of histone H3. We have performed a comparative global proteomic screen to better define the role of COMPASS in histone trimethylation. We report that both Cps60 and Cps40 components of COMPASS are required for proper histone H3 trimethylation, but not for proper regulation of telomere-associated gene silencing. Purified COMPASS lacking Cps60 can mono- and dimethylate but is not capable of trimethylating H3(K4). Chromatin immunoprecipitation (ChIP) studies indicate that the loss subunits of COMPASS required for histone trimethylation do not affect the localization of Set1 to chromatin for the genes tested. Collectively, our results suggest a molecular requirement for several components of COMPASS for proper histone H3 trimethylation and regulation of telomere-associated gene expression, indicating multiple roles for different forms of histone methylation by COMPASS.

Background & Aims Exosomes are small membrane vesicles involved in intercellular communication. Hepatocytes are known to release exosomes, but little is known about their biological function. We sought to determine if exosomes derived from hepatocytes contribute to liver repair and regeneration after injury. Methods Exosomes derived from primary murine hepatocytes were isolated and characterized biochemically and biophysically. Using cultures of primary hepatocytes, we tested whether hepatocyte exosomes induced proliferation of hepatocytes in vitro. Using models of ischemia/reperfusion injury and partial hepatectomy, we evaluated whether hepatocyte exosomes promote hepatocyte proliferation and liver regeneration in vivo. Results Hepatocyte exosomes, but not exosomes from other liver cell types, induce dose-dependent hepatocyte proliferation in vitro and in vivo. Mechanistically, hepatocyte exosomes directly fuse with target hepatocytes and transfer neutral ceramidase and sphingosine kinase 2 (SK2) causing increased synthesis of sphingosine-1-phosphate (S1P) within target hepatocytes. Ablation of exosomal SK prevents the proliferative effect of exosomes. After ischemia/reperfusion injury, the number of circulating exosomes with proliferative effects increases. Conclusions Our data shows that hepatocyte-derived exosomes deliver the synthetic machinery to form S1P in target hepatocytes resulting in cell proliferation and liver regeneration after ischemia/reperfusion injury or partial hepatectomy. These findings represent a potentially novel new contributing mechanism of liver regeneration and have important implications for new therapeutic approaches to acute and chronic liver disease.

Recent studies have documented that remote organs are affected by ischemic injury to the kidney. Here we studied whether the liver also suffers damage during induction of renal ischemia-reperfusion in rats and compared this to bilateral nephrectomy. Hepatic levels of tumor necrosis factor-alpha increased significantly after 6 and 24 h of renal ischemia or nephrectomy. Malondialdehyde, an index of lipid peroxidation, increased while total glutathione was decreased in the liver in both the renal ischemia and nephrectomy groups, suggesting activation of oxidative stress. Expression of liver spermine-spermidine acetyl transferase, an enzyme upregulated in early phases of hepatic injury was significantly increased 6 h after either kidney ischemia or nephrectomy. Apoptosis was increased in hepatocytes 24 h after nephrectomy. We also found histological evidence of hepatocyte injury following both ischemia and bilateral nephrectomy. Infusion of reduced glutathione, before the induction of renal ischemia, significantly improved liver architecture and was associated with a reduction in hepatic malondialdehyde and serum alanine transaminase levels. Our study shows that acute kidney ischemia or renal failure activates oxidative stress and promotes inflammation, apoptosis, and tissue damage in hepatocytes.

Hepatic ischemia-reperfusion results in an acute inflammatory response culminating in the recruitment of activated neutrophils that directly injure hepatocytes. Recent evidence suggests that CD4+ lymphocytes may regulate this neutrophil-dependent injury, but the mechanisms by which this occurs remain to be elucidated. In the present study, we sought to determine the type of CD4+ lymphocytes recruited to the liver after ischemia-reperfusion and the manner in which these cells regulated neutrophil recruitment and tissue injury. Wild-type and CD4 knockout (CD4-/-) mice were subjected to hepatic ischemia-reperfusion. CD4+ lymphocytes were recruited in the liver within 1 h of reperfusion and remained for at least 4 h. These cells were comprised of conventional (alphabetaTCR-expressing), unconventional (gammadeltaTCR-expressing), and natural killer T cells. CD4-/- mice were then used to determine the functional role of CD4+ lymphocytes in hepatic ischemia-reperfusion injury. Compared with wild-type mice, CD4-/- mice had significantly greater liver injury, yet far less neutrophil accumulation. Adoptive transfer of CD4+ lymphocytes to CD4-/- mice recapitulated the wild-type response. In wild-type mice, neutralization of interleukin (IL)-17, a cytokine released by activated CD4+ lymphocytes, significantly reduced neutrophil recruitment in association with suppression of MIP-2 expression. Finally, oxidative burst activity of liver-recruited neutrophils was higher in CD4-/- mice compared with those from wild-type mice. These data suggest that CD4+ lymphocytes are rapidly recruited to the liver after ischemia-reperfusion and facilitate subsequent neutrophil recruitment via an IL-17-dependent mechanism. However, these cells also appear to attenuate neutrophil activation. Thus the data suggest that CD4+ lymphocytes have dual, opposing roles in the hepatic inflammatory response to ischemia-reperfusion.

CXC chemokines and their receptor, CXC chemokine receptor-2 (CXCR2), are important components of the hepatic inflammatory response to ischemia/reperfusion (I/R). However, direct effects of CXC chemokines on hepatocytes during this response have not been studied. Wild-type and CXCR2 ؊/؊ mice were subjected to 90 minutes of partial hepatic ischemia followed by up to 96 hours of reperfusion. CXCR2 ؊/؊ mice had significantly less liver injury at all reperfusion times compared with wild-type mice. Early neutrophil recruitment (12 hours) was diminished in CXCR2 ؊/؊ mice, but within 24 hours it was the same as that of wild-type mice. Hepatocyte proliferation and regeneration was accelerated in CXCR2 ؊/؊ mice compared with wild-type mice. These effects were associated with increased activation of nuclear factor B and signal transducers and activators of transcription-3, despite there being no difference in the expression of proliferative factors such as tumor necrosis factor ␣, interleukin-6, and hepatocyte growth factor. To establish whether the accelerated proliferation and regeneration observed in CXCR2 ؊/؊ mice was due to effects on hepatocytes rather than just a generalized decrease in acute inflammatory injury, mice were treated with the CXCR2 antagonist, SB225002, after neutrophil recruitment and injury were maximal (24 hours after reperfusion). SB225002 treatment increased hepatocyte proliferation and regeneration in a manner identical to that observed in CXCR2 ؊/؊ mice. Treatment of primary wild-type hepatocytes with macrophage inflammatory protein-2 revealed that low concentrations protected against cell death, whereas high concentrations induced cell death. These effects were absent in hepatocytes from CXCR2 ؊/؊ mice. Conclusion: Our data suggest that hepatocyte CXCR2 regulates proliferation and regeneration after I/R injury and reveal important differences in the role of this receptor in liver regeneration and repair induced under different conditions that may be related to ligand concentration. (HEPATOLOGY 2008;48:1213-1223