Ischemic tissues require mechanisms to alert the immune system of impending cell damage. The nuclear protein high-mobility group box 1 (HMGB1) can activate inflammatory pathways when released from ischemic cells. We elucidate the mechanism by which HMGB1, one of the key alarm molecules released during liver ischemia/reperfusion (I/R), is mobilized in response to hypoxia. HMGB1 release from cultured hepatocytes was found to be an active process regulated by reactive oxygen species (ROS). Optimal production of ROS and subsequent HMGB1 release by hypoxic hepatocytes required intact Toll-like receptor (TLR) 4 signaling. To elucidate the downstream signaling pathways involved in hypoxia-induced HMGB1 release from hepatocytes, we examined the role of calcium signaling in this process. HMGB1 release induced by oxidative stress was markedly reduced by inhibition of calcium/calmodulin-dependent kinases (CaMKs), a family of proteins involved in a wide range of calcium-linked signaling events. In addition, CaMK inhibition substantially decreased liver damage after I/R and resulted in accumulation of HMGB1 in the cytoplasm of hepatocytes. Collectively, these results demonstrate that hypoxia-induced HMGB1 release by hepatocytes is an active, regulated process that occurs through a mechanism promoted by TLR4-dependent ROS production and downstream CaMK-mediated signaling.

Pancreatic cancer has the lowest survival rate among human cancers, and there are no effective markers for its screening and early diagnosis. To identify genetic susceptibility markers for this cancer, we carried out a genome-wide association study on 981 individuals with pancreatic cancer (cases) and 1,991 cancer-free controls of Chinese descent using 666,141 autosomal SNPs. Promising associations were replicated in an additional 2,603 pancreatic cancer cases and 2,877 controls recruited from 25 hospitals in 16 provinces or cities in China. We identified five new susceptibility loci at chromosomes 21q21.3, 5p13.1, 21q22.3, 22q13.32 and 10q26.11 (P = 2.24 × 10(-13) to P = 4.18 × 10(-10)) in addition to 13q22.1 previously reported in populations of European ancestry. These results advance our understanding of the development of pancreatic cancer and highlight potential targets for the prevention or treatment of this cancer.

The chromatin-binding factor high-mobility group box 1 (HMGB1) functions as a proinflammatory cytokine and late mediator of mortality in murine endotoxemia. Although serine phosphorylation of HMGB1 is necessary for nucleocytoplasmic shuttling before its cellular release, the protein kinases involved have not been identified. To investigate if calcium/calmodulin-dependent protein kinase (CaMK) IV serine phosphorylates and mediates the release of HMGB1 from macrophages (Mφ) stimulated with LPS, RAW 264.7 cells or murine primary peritoneal Mφ were incubated with either STO609 (a CaMKIV kinase inhibitor), KN93 (a CaMKIV inhibitor), or we utilized cells from which CaMKIV was depleted by RNA interference (RNAi) before stimulation with LPS. We also compared the LPS response of primary Mφ isolated from CaMKIV+/+ and CaMKIV−/− mice. In both cell types LPS induced activation and nuclear translocation of CaMKIV, which preceded HMGB1 nucleocytoplasmic shuttling. However, Mφ treated with KN93, STO609, or CaMKIV RNAi before LPS showed reduced nucleocytoplasmic shuttling of HMGB1 and release of HMGB1 into the supernatant. Additionally, LPS induced serine phosphorylation of HMGB1, which correlated with an interaction between CaMKIV and HMGB1 and with CaMKIV phosphorylation of HMGB1 in vitro. In cells, both HMGB1 phosphorylation and interaction with CaMKIV were inhibited by STO609 or CaMKIV RNAi. Similarly, whereas CaMKIV+/+ Mφ showed serine phosphorylation of HMGB1 in response to LPS, this phosphorylation was attenuated in CaMKIV−/− Mφ. Collectively, our results demonstrate that CaMKIV promotes the nucleocytoplasmic shuttling of HMGB1 and suggest that the process may be mediated through CaMKIV-dependent serine phosphorylation of HMGB1.

ObjectiveTo determine that 1) an age-dependent loss of inducible autophagy underlies the failure to recover from AKI in older, adult animals during endotoxemia, and 2) pharmacologic induction of autophagy, even after established endotoxemia, is of therapeutic utility in facilitating renal recovery in aged mice.DesignMurine model of endotoxemia and cecal ligation and puncture (CLP) induced acute kidney injury (AKI).SettingAcademic research laboratory.SubjectsC57Bl/6 mice of 8 (young) and 45 (adult) weeks of age.InterventionLipopolysaccharide (1.5 mg/kg), Temsirolimus (5 mg/kg), AICAR (100 mg/kg). Measurements and Main Results: Herein we report that diminished autophagy underlies the failure to recover renal function in older adult mice utilizing a murine model of LPS-induced AKI. The administration of the mTOR inhibitor temsirolimus, even after established endotoxemia, induced autophagy and protected against the development of AKI.ConclusionsThese novel results demonstrate a role for autophagy in the context of LPS-induced AKI and support further investigation into like interventions that have potential to alter the natural history of disease.

Abstract. TGF-␤1 is a profibrotic cytokine that plays a central role in the onset and progression of chronic renal diseases. The activity of TGF-␤1 is tightly controlled by multiple mechanisms, in which antagonizing Smad-mediated gene transcription by co-repressors is an important regulatory component. This study examined the expression of Smad transcriptional co-repressors in the fibrotic kidney and investigated their potential functions in controlling TGF-␤1 response. Western blot analysis demonstrated that the protein levels of Smad transcriptional co-repressors SnoN and Ski were progressively reduced in a time-dependent manner in the fibrotic kidney induced by unilateral ureteral obstruction in mice, whereas renal Smad abundance was relatively unaltered. Consistently, SnoN and Ski staining was diminished in the nuclei of renal tubular epithelium and interstitium after obstructive injury. In vitro, knockdown of SnoN expression by RNA interference in tubular epithelial cells dramatically sensitized their responsiveness to TGF-␤1 stimulation. Conversely, ectopic expression of exogenous SnoN or Ski after transfection conferred tubular epithelial cell resistance to TGF-␤1-induced epithelial to myofibroblast transition. Both SnoN and Ski could block Smadmediated activation of TGF-␤1-responsive promoter and exhibited additive effect in abrogating the profibrotic actions of TGF-␤1. These results indicate that as a result of loss of Smad transcriptional co-repressors, the profibrotic TGF-␤1 signaling in diseased kidney is markedly amplified in a magnitude much greater than previously thought. Therefore, new strategy aimed to increase Smad transcriptional co-repressors expression may be effective in antagonizing TGF-␤1 signaling and thereby blocking the progression of chronic renal fibrosis.