2,3,7,8-Tetrachlorodibenzo-p-dioxin poly(ADP-ribose) polymerase (TiPARP, ARTD14) is a mono-ADP-ribosyltransferase and repressor of aryl hydrocarbon receptor transactivation

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP-ribose) polymerase (TiPARP/ARTD14) is a member of the PARP family and is regulated by the aryl hydrocarbon receptor (AHR); however, little is known about TiPARP function. In this study, we examined the catalytic function of TiPARP and determined its role in AHR transactivation. We observed that TiPARP exhibited auto-mono-ADP-ribosyltransferase activity and ribosylated core histones. RNAi-mediated knockdown of TiPARP in T-47D breast cancer and HuH-7 hepatoma cells increased TCDD-dependent cytochrome P450 1A1 (CYP1A1) and CYP1B1 messenger RNA (mRNA) expression levels and recruitment of AHR to both genes. Overexpression of TiPARP reduced AHR-dependent increases in CYP1A1-reporter gene activity, which was restored by overexpression of AHR, but not aryl hydrocarbon receptor nuclear translocator. Deletion and mutagenesis studies showed that TiPARP-mediated inhibition of AHR required the zinc-finger and catalytic domains. TiPARP and AHR co-localized in the nucleus, directly interacted and both were recruited to CYP1A1 in response to TCDD. Overexpression of Tiparp enhanced, whereas RNAi-mediated knockdown of TiPARP reduced TCDD-dependent AHR proteolytic degradation. TCDD-dependent induction of AHR target genes was enhanced in Tiparp−/− mouse embryonic fibroblasts compared with wildtype controls. Our findings show that TiPARP is a mono-ADP-ribosyltransferase and a transcriptional repressor of AHR, revealing a novel negative feedback loop in AHR signalling.

Aryl Hydrocarbon Receptor Activation by Dioxin Targets Phosphoenolpyruvate Carboxykinase (PEPCK) for ADP-ribosylation via 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-inducible Poly(ADP-ribose) Polymerase (TiPARP)

“…The lack of a mobility shift on SDS-polyacrylamide gel electrophoresis for PEPCK after TCDD or TiPARP treatment is consistent with other evidence that TiPARP is a mono-rather than a poly-ADP-ribosylase (56). Interestingly, several members of the PARP family previously considered to be poly-ADP-ribosylases are proving to catalyze mono-ADP-ribosylation (10).…”

Loss of the Mono-ADP-ribosyltransferase, Tiparp, Increases Sensitivity to Dioxin-induced Steatohepatitis and Lethality

“…Frozen livers were homogenized in TRIzol reagent (Life Technologies, Inc.), and total RNA was isolated using the Qiagen RNeasy Plus kit and reverse-transcribed as described previously (25). Primers used to amplify lipoprotein lipase (Lpl) were forward 5Ј-GGATGGACGGTAACGG-GAAT-3Ј and reverse 5Ј-GGCCCGATACAACCAGTCTA-3Ј; cluster of differentiation 36 (Cd36) were forward 5Ј-ACCCCT-CCAGAATCCAGACA-3Ј and reverse 5Ј-ACACAGGCT-TTCCTTCTTTGC-3Ј; phosphoenolpyruvate carboxykinase (Pck1) were forward 5Ј-CCTAGTGCCTGTGGGAAGAC-3Ј and reverse 5Ј-AAGTTGCCTTGGGCATCAAAC-3Ј; sterol regulatory element-binding transcription factor 1 (Srebp1) 5Ј-GCACACAAAAGCAAATCACTG-3Ј and reverse 5Ј-TCTC-CACCACTTCGGGTTTC-3Ј; fatty acid synthase (Fas) 5Ј-GC-TGCGGAAACTTCAGGAAAT-3Јand reverse 5Ј-AGAGAC-GTGTCACTCCTGGACTT-3Ј; Cyp1a1 5Ј-CGTTATGACC-ATGATGACCAAGA-3Ј and reverse 5Ј-TCCCCAAACTCA-TTGCTCAGAT-3Ј; Cyp1b1 forward 5Ј-GTGCGGCAAAAG-CATGTCTC and reverse 5Ј-GGGGAAAAGCAACGTTCT GAC-3Ј; Tiparp forward 5Ј-TCCCCGTGTCTGTGGAAAGC ATG-3Ј and reverse 5Ј-TTGACCGGAGGGGGCCTTCT-3Ј; tumor necrosis factor ␣ (Tnf␣) forward 5Ј-AGGGTCTGGGC-CATAGAACT-3Ј and reverse 5Ј-CCACCACGCTCTTCTGT-CTAC-3Ј; interleukin 1 ␤ (Il1␤) forward 5Ј-GGACCCATAT-GAGCTGAAAGCT-3Ј and reverse 5Ј-TGTCGTTGCTTGG-TTCTCCTT-3Ј; and chemokine CXC motif ligand 2 (Cxcl2) forward 5Ј-AAGTTTGCCTTGACCCTGAA-3Ј and reverse 5Ј-AGGCACATCAGGTACGATCC-3Ј.…”

“…Reporter Gene Assays-For luciferase reporter gene assays, HuH7 cells were transfected with pcDNA-TIPARP (25) and Cyp1a1 luciferase plasmid (pGudLuc; generously provided by Dr. Michael Denison, University of California at Davis) or pCMV-FLAG-MACROD1, pCMV-FLAG-MACROD1_G182E, pCMV-FLAG-MACROD1_D184A, or pCMV-FLAG-MACROD2 and treated with 10 nM dioxin for 24 h as described previously (25). Human MacroD1 and MacroD2 cDNAs were amplified by PCR using pCMV6 Myc-DDK MACROD1 or pCMV6 Myc-DDK MACROD2 variant 1 as templates, respectively (Origene, Rockville, MD).…”

“…ADP-ribosylation assays were carried out as described previously (25). Human PARP10 (ARTD10) cDNA was PCR-amplified using pEV-HA-RFO-ARTD10 (PARP10) as template (a gift from Professor Bernard Lüscher, IBMB Aachen, Germany).…”

“…We recently reported that TIPARP mono-ADP-ribosylates itself and core histones, and as part of a negative feedback loop it represses AHR activity by increasing its proteolytic degradation (25). However, the impact of TIPARP on AHR signaling and dioxin-induced toxicity in vivo has not been determined.…”