Abstract
BackgroundInitiation, amplitude, duration and termination of transforming growth factor β (TGFβ) signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We previously reported that ADP-ribosylation of Smads by poly(ADP-ribose) polymerase 1 (PARP-1) negatively influences Smad-mediated transcription. PARP-1 is known to functionally interact with PARP-2 in the nucleus and the enzyme poly(ADP-ribose) glycohydrolase (PARG) can remove poly(ADP-ribose) chains from target proteins. Here we aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGFβ signaling.MethodsA robust cell model of TGFβ signaling, i.e. human HaCaT keratinocytes, was used. Endogenous Smad3 ADP-ribosylation and protein complexes between Smads and PARPs were studied using proximity ligation assays and co-immunoprecipitation assays, which were complemented by in vitro ADP-ribosylation assays using recombinant proteins. Real-time RT-PCR analysis of mRNA levels and promoter-reporter assays provided quantitative analysis of gene expression in response to TGFβ stimulation and after genetic perturbations of PARP-1/-2 and PARG based on RNA interference.ResultsTGFβ signaling rapidly induces nuclear ADP-ribosylation of Smad3 that coincides with a relative enhancement of nuclear complexes of Smads with PARP-1 and PARP-2. Inversely, PARG interacts with Smads and can de-ADP-ribosylate Smad3 in vitro. PARP-1 and PARP-2 also form complexes with each other, and Smads interact and activate auto-ADP-ribosylation of both PARP-1 and PARP-2. PARP-2, similar to PARP-1, negatively regulates specific TGFβ target genes (fibronectin, Smad7) and Smad transcriptional responses, and PARG positively regulates these genes. Accordingly, inhibition of TGFβ-mediated transcription caused by silencing endogenous PARG expression could be relieved after simultaneous depletion of PARP-1.ConclusionNuclear Smad function is negatively regulated by PARP-1 that is assisted by PARP-2 and positively regulated by PARG during the course of TGFβ signaling.
Highlights
Signal transduction pathways, including transforming growth factor b (TGFb), are controlled by negative regulatory mechanisms [1,2,3]
Nuclear Smad function is negatively regulated by poly(ADP-ribose) polymerase 1 (PARP-1) that is assisted by PARP-2 and positively regulated by poly(ADPribose) glycohydrolase (PARG) during the course of TGFb signaling
Using proximity ligation assays (PLA) and immunoprecipitations, we demonstrate that TGFb induces endogenous PARP-1/Smad3 and PARP-2/ Smad2/3 complexes, while only having small effects on the poly(ADP-ribose) polymerase-1 (PARP1)/PARP-2 interaction
Summary
Signal transduction pathways, including transforming growth factor b (TGFb), are controlled by negative regulatory mechanisms [1,2,3]. We recently established a mechanism of negative regulation of Smad activity taking place in the nucleus, based on the finding that Smad and Smad can associate with the nuclear ADP-ribosyltransferase (ADP-ribosyltransferase diphtheria toxin-like 1, ARTD1), known as poly(ADP-ribose) polymerase-1 (PARP1) [9]. PARP-1 binds to Smad proteins and ADP-ribosylates them proximal to their DNA-binding domain, reducing their affinity to DNA and negatively regulating their transcriptional activity. A straightforward consequence of this biochemical modification is that PARP-1 negatively regulates gene responses to TGFb signaling [9]. Initiation, amplitude, duration and termination of transforming growth factor b (TGFb) signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGFb signaling
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