FoxA2 involvement in suppression of protein C, an outcome predictor in experimental sepsis

Abstract

Low levels of protein C (PC) predict outcome as early as 10 h after insult in a rat polymicrobial sepsis model and were associated with suppression of PC mRNA, upstream transcription factor FoxA2, and cofactor hepatocyte nuclear factor 6 (HNF6). Small interfering RNA suppression of FoxA2 in isolated hepatocytes demonstrated regulation of both its cofactor HNF6 and PC. Our data suggest that reduced FoxA2 may be important in the suppression of PC and resulting poor outcome in sepsis.

FIG. 1.

Expression of protein C in rat CLP. (A) Analysis of protein C plasma levels at 10 and 22 h as a function of subsequent survival in a rat CLP model of sepsis. Blood draws were from the retro-orbital sinus, and the plasma was analyzed for PC concentrations by ELISA. Early deaths were examined for animals that died within either the 10- to 22-h or the 22- to 30-h time frames. (B) Effect of induction of sepsis by CLP on liver protein C mRNA expression. cDNA was synthesized by using total RNA isolated from the livers of control-sham and CLP animals sacrificed after final blood samplings. For determination of relative mRNA levels, the cDNA was analyzed by qPCR (TaqMan). An internal standard curve was generated by serial dilution of an appropriate cDNA reaction and used for relative quantification. The assay for 18S rRNA was the Eukaryotic Endogenous Control kit (ABI, Foster City, CA) and was used to normalize to the gene of interest (GOI). Results are from four independent experiments with 42 animals (20 sham and 22 CLP). (C) Relationship of liver PC mRNA to plasma PC concentration by ELISA at 22 h post-CLP.

FIG. 2.

Comparison of the PC promoter region of the human and rat. (A) Analysis of the upstream region of the human and rat PC genes with MatInspector, which identified the noted FoxA2 (perfect match to the core; matrix score of 0.993) and HNF1 binding sites. The numbering is relative to the transcription start site of the human PC gene. (B) Change in expression of transcription factors FoxA1, -2, and -3 and HNF1 at 22 h after induction of sepsis by CLP determined by qPCR (TaqMan). Eight animals per group were used for determining Fox factors, 13 animals were used for sham conditions, and 14 animals were used for CLP for HNF1 analysis. *, P < 0.05. (C) Inhibition of FoxA2 by immunoassay following CLP. *, P < 0.05. (D) Relationship of levels of FoxA2 and PC mRNA at 22 h after induction of sepsis by CLP. Values are the ratio of the GOI over the 18S control as described in the legend to Fig. 1.

FIG. 3.

Relationship of FoxA2 and PC expression. (A) Analysis of FoxA2 and PC expression levels and binding to the FoxA2 site in the PC promoter by electrophoretic mobility shift assay, following introduction of an siRNA for FoxA2 into rat hepatocytes (data are the results of four experiments). *, P < 0.01. Primary hepatocytes were obtained from Clonetics, Cambrex Bio Science Walkersville, Inc., Walkersville, MD. siGENOME SMARTpools for rat HNF3B (FoxA2), rat ONECUT1 (HNF6), and the nonspecific control pool were purchased from Dharmacon, Inc., Lafayette, CO. (B) Relationship between FoxA2, HNF6, and PC at 22 h after induction of sepsis by CLP. Values are the ratio of the GOI over the 18S control, as described in the legend to Fig. 1. (C) Analysis of the effect of an siRNA to FoxA2 on the expression of HNF6 in rat hepatocytes (data are the result of four experiments). *, P < 0.01.

FIG. 4.

FoxA2, PC, and mortality predictors. (A) Relationship of HNF6 and FoxA2 to predictive plasma PC cutoff values for outcomes. Data are means ± SE for 14 animals. The cutoff value of 60% of the pre-CLP baseline was determined as described above by receiver operator characteristic curve analysis generated from the logistic regression model in the JMP5.1 software package. (B) Levels of FoxA2 and HNF6 expression in animals predicted to be survivors or nonsurvivors by MIP2 levels. Plasma samples were analyzed at baseline and 22 h post-CLP for MIP2 by ELISA. Data are means ± SE for 14 experiments. (C) Analysis of MIP2 plasma levels as a function of PC plasma levels. The measurement of MIP2 was by immunoassay, as previously described (12).