Mitoneet mediates TNFα-induced necroptosis promoted by exposure to fructose and ethanol

Abstract

Fructose and ethanol are metabolized principally in the liver and are both known to contribute to the development of hepatic steatosis that can progress to hepatic steatohepatitis. The present study indentifies a synergistic interaction between fructose and ethanol in promoting hepatocyte sensitivity to TNFα-induced necroptosis. Concurrent exposure to fructose and ethanol induces the overexpression of the CDGSH iron-sulfur domain-containing protein 1 (CISD1 or mitoneet), which is localized to the outer mitochondrial membrane. The increased expression of mitoneet primes the hepatocyte for TNFα-induced cytotoxicity. Treatment with TNFα induces the translocation of a Stat3-Grim-19 complex to the mitochondria, which binds to mitoneet and promotes the rapid release of its 2Fe-2S cluster, causing an accumulation of mitochondrial iron. The dramatic increase of mitochondrial iron provokes a surge in formation of reactive oxygen species, resulting in mitochondrial injury and cell death. Additionally, mitoneet is constitutively expressed at high levels in L929 fibrosarcoma cells and is required for L929 cells to undergo TNFα-induced necroptosis in the presence of caspase inhibition, indicating the importance of mitoneet to the necroptotic form of cell death.

Keywords: Ethanol; Fructose; Mitoneet; Necroptosis; TNFα.

Fig. 1.

TNFα-mediated necroptosis in hepatocytes exposed to fructose and ethanol. Following 24 hours in culture, hepatocytes were exposed to 10 mM ethanol, 1 mM fructose or their combination. After an additional 24 hours, the hepatocytes were either left untreated or pre-treated for 30 minutes with 1 µM ZVAD-FMK or 10 µM necrostatin. Following the pre-treatment, the hepatocytes were treated with 10 ng/ml TNFα. Alternatively, hepatocytes were pre-treated with cycloheximide (1 µg/ml) for 30 minutes followed by the addition of 10 ng/ml TNFα. At the time points indicated, floating and attached cells were harvested. (A) The onset of necroptosis was determined by propidium iodide uptake. Values are the means ± s.d. of three independent experiments. (B) For assessment of necrosome formation, the hepatocytes were harvested at 4 hours after addition of TNFα and whole cell extracts prepared. RIPK-1 was immunoprecipitated from the whole cell lysates. The immunoprecipitates were then run on SDS-PAGE gels and blotted onto PVDF membranes. The western blots were developed with antibodies against RIPK-1 and RIPK-3. (C) At the time points indicated, floating and attached cells were harvested. The onset of apoptosis was determined by annexin V binding. Values are the means ± s.d. of three independent experiments.

Fig. 2.

Exposure to fructose and ethanol stimulates mitoneet expression, whose activation mediates the increased sensitivity to TNFα-induced necroptosis. (A) Hepatocytes were transfected with 50 nM of siRNA targeting mitoneet or a non-targeting control siRNA. After 24 hours, the hepatocytes were either left untreated or exposed to 10 mM ethanol, 1 mM fructose or their combination in the absence or presence of 10 µM pioglitazone for 24 hours. The hepatocytes were then harvested, mitochondria isolated, and the level of mitoneet determined by western blotting. The results are typical of three independent experiments. (B) Hepatocytes were pre-treated or not for 30 minutes with 10 µM of pioglitazone before treatment with 10 ng/ml of TNFα. The cells were harvested at the time points indicated and cell viability determined using propidium iodide. Values are the means ± s.d. of three independent experiments. (C) L929 cells were treated as in A. The results are typical of three independent experiments. (D) L929 cells treated as in A were harvested at the time points indicated and cell viability determined with propidium iodide. Values are the means ± s.d. of three independent experiments.

Fig. 3.

Mitoneet mediates the TNFα-induced translocation of Stat3–Grim-19 to the mitochondria. (A) Hepatocytes treated with siRNA targeting microneet or a non-targeting control siRNA (as in Fig. 2A) were pre-treated or not for 30 minutes with 10 µM of necrostatin and then treated with 10 ng/ml of TNFα. After 4 hours of TNFα treatment, the hepatocytes were harvested and mitochondria isolated. Mitochondrial extracts were prepared, separated by SDS-PAGE and then transferred to PVDF membranes. The blots were probed with antibodies specific for Grim-19, STAT3 or mitoneet. The blots were then stripped and re-probed with antibodies against VDAC-1. The results are representative of three independent experiments. (B) Mitoneet was immunoprecipitated from mitochondrial lysates from hepatocytes treated as in A. The immunoprecipitates were then separated by SDS-PAGE and blotted onto PVDF membranes. The blots were probed with antibodies against Grim-19 or STAT3. For the reverse immunoprecipitation, Grim-19 was immunoprecipitated from mitochondrial lysates and the immunoprecipitates separated by SDS-PAGE and blotted onto PVDF membranes. The blots were probed with antibodies against mitoneet or STAT3. The results are representative of three independent experiments. (C) Hepatocytes were transfected with siRNA targeting Grim-19 or Stat3 and then treated as in A. At the time points indicated, the cells were harvested and viability determined by uptake of propidium iodide. Values are the means ± s.d. of three independent experiments.

Fig. 4.

Stat3–Grim-19 interaction with mitoneet mediates mitochondrial iron uptake and ROS production. (A) Hepatocytes were transfected with siRNA targeting Grim-19 or Stat3 as in Fig. 2A. Some hepatocytes were pre-treated with 10 µM pioglitazone for 30 minutes prior to the addition of TNFα. At the times indicated following the addition of TNFα, the cells were harvested and mitochondria isolated. Mitochondrial lysates were prepared and mitoneet immunoprecipitated. The absorbance of the immunoprecipitates was measured at 458 nm. (B) Hepatocytes were transfected with siRNA targeting Grim-19, Stat3, mitoneet or a non-targeting control. At the times indicated following TNFα addition, the cells were harvested and the mitochondria isolated. The content of mitochondrial iron was determined. (C) ROS formation determined by measuring MitoSox fluorescence. All values are the means ± s.d. of three independent experiments.

Fig. 5.

The mitochondrial calcium uniporter is required for mitochondrial uptake of iron released by mitoneet. Hepatocytes were transfected with siRNA targeting MCU or a non-targeting control. After 24 hours, the hepatocytes were exposed to 10 mM ethanol in combination with 1 mM of fructose. Following an additional 24 hours, the cells were treated with 10 ng/ml of TNFα. Alternatively, where indicated, hepatocytes were pre-treated with 10 µM of Ru360 for 30 minutes prior to the addition of TNFα. (A) At the time points indicated, the cells were harvested and mitochondria isolated. The content of mitochondrial iron was determined. Values are the means of three independent experiments with the error bars indicating standard deviation. (B) Mitochondrial lysates were prepared and mitoneet immunoprecipitated. The absorbance of the immunoprecipitates was measured at 458 nm. (C) At the time points indicated, the cells were harvested and ROS formation was determined by measuring MitoSox fluorescence. (D) The cells were harvested at the time points indicated and cell viability determined by propidium iodide. All values are the means ± s.d. of three independent experiments.

Fig. 6.

Stat3–Grim-19 directly induces mitochondrial damage that is dependent on mitoneet activation. (A) Hepatocytes were transfected with siRNA targeting mitoneet or a non-targeting control siRNA. After 48 hours, hepatocytes were loaded with 200 nM TMRM for 30 minutes. The cells were then washed twice with PBS and incubated further for 5 minutes in respiratory buffer containing 20 nM of TMRM. Digitonin at 2.5 µg/ml was then added to permeabilize the plasma membrane. Where indicated, hepatocytes were pre-treated with 10 µM of Ru360 or 10 µM of pioglitazone for 5 minutes before the addition of the Stat3–Grim-19 complex. The recombinant complex of Stat3-Grim-19 was added at a final concentration of 100 µM. TMRM fluorescence was monitored over a 20 minute time course. 5 µM of CCCP was added at the 18 minute time point. The result is the average of three independent experiments. (B) Hepatocytes as in A were loaded with MitoSOX for 30 minutes. Digitonin (2.5 mg/ml final concentration) was then added to permeabilize the plasma membrane. Time-lapse microscopy was conducted over a 20 minute time course. Recombinant Stat3–Grim-19 complex was added at a concentration of 100 µM at the 2 minute time point with MitoSOX fluorescence intensity assessed. Values are the means ± s.d. of three independent experiments. (C) Hepatocytes treated as in A were labeled for 15 minutes with 1 mM RPA in Williams Medium E containing 10 mM HEPES buffer instead of Phenol Red. Where indicated, the cells were pre-treated for 10 minutes with 10 µM of pioglitazone or 10 µM of Ru360. Digitonin (2.5 mg/ml final concentration) was added to permeabilize the plasma membrane. Recombinant Stat3–Grim-19 complex was added at a concentration of 100 µM and fluorescence was monitored. At the end of the time course, 5 mM of FeCl₃-8-hydroxyquinoline (FHQ) was added so as to attain maximal quenching. The result is the average of three independent experiments.

Fig. 7.

Mitoneet mediates a necroptosis pathway.