Abstract
Effects of the environmental toxin and carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) include a wasting syndrome associated with decreased gluconeogenesis. TCDD is a potent activator of the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. The relationship between gene activation by the AHR and TCDD toxicities is not well understood. We recently identified a pathway by which the AHR target gene TiPARP (TCDD-inducible poly(ADP-ribose) polymerase) contributes to TCDD suppression of transcription of phosphoenolpyruvate carboxykinase (PEPCK), a key regulator of gluconeogenesis, by consuming NAD(+) and decreasing Sirtuin 1 activation of the peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), a transcriptional activator of PEPCK. We report here that TCDD-induced TiPARP also targets PEPCK for ADP-ribosylation. Both cytosolic and mitochondrial forms of PEPCK were found to undergo ADP-ribosylation. Unexpectedly, AHR suppression also enhanced ADP-ribosylation and did so by a poly(ADP-ribose) polymerase-independent mechanism. This report 1) identifies ADP-ribosylation as a new posttranslational modification for PEPCK, 2) describes a pathway by which transcriptional induction of TiPARP by the AHR can lead to a downstream posttranslational change in a TCDD target protein (PEPCK), and 3) reveals that the AHR exerts complex, previously unidentified modulatory effects on ADP-ribosylation.
Highlights
Effects of the environmental toxin and carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) include a wasting syndrome associated with decreased gluconeogenesis
We recently identified a pathway by which the aryl hydrocarbon receptor (AHR) target gene TCDD-inducible poly(ADPribose)-polymerase (TiPARP) (TCDD-inducible poly(ADP-ribose) polymerase) contributes to TCDD suppression of transcription of phosphoenolpyruvate carboxykinase (PEPCK), a key regulator of gluconeogenesis, by consuming NAD؉ and decreasing Sirtuin 1 activation of the peroxisome proliferator-activated receptor ␥ coactivator 1␣ (PGC1␣), a transcriptional activator of PEPCK
We recently reported that the AHR target gene, TiPARP (TCDD-inducible poly(ADP-ribose) polymerase, PARP7
Summary
The agarose beads were removed by centrifugation, and lysates were incubated with 5 g of an anti-PAR (polyADP-ribose) antibody (Trevigen, Inc., Gaithersburg, MD) or normal rabbit IgG (Cell Signaling) overnight at 4 °C with gentle rocking. Protein A-agarose beads previously washed 3 times with Cell Lysis Buffer and incubated overnight with 1 mg/ml BSA were added to the samples for 2 h at 4 °C. For primary CEH, rat hepatocytes, and cell lines, cells were scraped in 2ϫ sample buffer, or lysates were prepared in Cell Lysis Buffer with PARP and poly(ADP-ribose)glycohydrolase inhibitors as indicated above. The following primary antibodies were used: Oct-A probe (Santa Cruz) for detection of FLAGtagged TiPARP; anti-PAR, #4336-BPC (Trevigen) or #96-10-04 (Enzo Life Sciences); anti-PCK1 (Sigma) (an antibody that was sold for PEPCK-C but recognizes both isoforms in chick); acetyl (K9) histone 3 and histone 3 (Millipore, Billerica, MA); AHR (Santa Cruz Biotechnology); ␣-tubulin (Cell Signaling); porin (Abcam, Cambridge, MA); -actin (Sigma). Statistical Analysis—Differences between group means were evaluated using GraphPad software by unpaired, two tailed t tests; p values Յ 0.05 were considered statistically significant
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