Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia

NF-κB is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-κB pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes IκBε, a negative regulator of NF-κB in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced IκBε protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that IκBε loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-κB deregulation during lymphomagenesis

Fine-Tuning of Smad Protein Function by Poly(ADP-Ribose) Polymerases and Poly(ADP-Ribose) Glycohydrolase during Transforming Growth Factor β Signaling

BACKGROUND: Initiation, 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. METHODS: A 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. RESULTS: TGFβ 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. CONCLUSION: Nuclear 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

PARG forms complexes with Smad proteins and de-ADP-ribosylates Smad3.

<p>(<b>a</b>) Immunoprecipitation of Flag-Smad2, Flag-Smad3 or Flag-Smad4 followed by immunoblotting for myc-PARG in cell lysates of transiently transfected 293T cells with the indicated plasmids and after stimulation with vehicle (-TGFβ, left panel) or 5 ng/ml TGFβ1 for 30 min (right panel). Expression levels of all transfected proteins are shown in the TCL immunoblot of the 293T cells. (<b>b</b>) Immunoprecipitation of Flag-Smad2/3/4 followed by immunoblotting for myc-PARG in cell lysates of transiently transfected 293T cells with the indicated plasmids and in the absence of stimulation with TGFβ. Expression levels of all transfected proteins are shown in the TCL immunoblot of the 293T cells. α-Tubulin immunoblot serves as protein loading control. Stars mark non-specific protein bands. (<b>c</b>) Immunoprecipitation of endogenous Smad2/3 followed by immunoblotting for transfected myc-PARG in 293T cells stimulated with vehicle (-TGFβ) or with 5 ng/ml TGFβ1 for 30 min. Negative control immunoprecipitation using non-specific IgG is shown. TCL shows the levels of endogenous Smad2/3 proteins and transfected myc-PARG before immunoprecipitation. Smad2/3 immunoblot also serves as protein loading control. (<b>d</b>) In vitro de-ADP-ribosylation assay of Smad3 using PARG. GST-Smad3 was first ADP-ribosylated using recombinant PARP-1. The proteins were pulled-down and washed, prior to reconstitution with PARG reaction buffer and increasing amounts of recombinant PARG (shown as milli-units (mU) of enzymatic activity). The ADP-ribosylated proteins are shown in the autoradiogram along with the CBB-stained input GST-Smad3 levels. Panels a–c show results from representative experiments that were repeated at least twice and panel d shows results from representative experiments that were repeated at least three times.</p

PLA of endogenous Smad3 and PARP-1 complexes in HaCaT cells.

<p>(<b>a</b>) HaCaT cells were analyzed with PLA using antibodies against Smad3 and PARP-1 after transfection with control or the indicated specific siRNAs and stimulation with vehicle (-TGFβ) or with 2 ng/ml TGFβ1 for the indicated time periods. Specific RCA signals were detected in the nuclei. Cells stimulated with 10 mM hydrogen peroxide for 10 min served as positive control. PLA with single antibodies against Smad3 or PARP-1 are shown as controls. PLA images are shown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g001" target="_blank">Fig. 1a</a>. (<b>b</b>) Quantification of the experiment shown in panel (a) following the histogram method of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g001" target="_blank">Fig. 1b</a>. The figure shows a representative experiment from three or more repeats.</p

PLA of endogenous PARP-1 and PARP-2 ADP-ribosylation after TGFβ stimulation in HaCaT cells.

<p>(<b>a, c</b>) HaCaT cells were analyzed with PLA using antibodies against PARP-1 and PAR chains (<b>a</b>) or antibodies against PARP-2 and PAR (<b>c</b>) after stimulation with vehicle (0 min) or with 2 ng/ml TGFβ1 for the indicated time periods. Specific RCA signals were detected in the nuclei. PLA with single antibodies against PARP-1 or PAR are shown as controls. PLA images are shown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g001" target="_blank">Fig. 1a</a>. (<b>b, d</b>) Quantification of the experiments shown in panels (a, c) following the histogram method of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g001" target="_blank">Fig. 1b</a>. The figure shows a representative experiment from three or more repeats.</p

PARG regulates transcriptional responses to TGFβ.

<p>(<b>a–c</b>) Real-time RT-PCR analysis of endogenous <i>fibronectin (FN1)</i> (a), <i>PAI-1</i> (b) and control <i>PARP-1</i> (c) mRNAs in HaCaT cells transiently transfected with the indicated siRNAs (bottom of panel c) prior to stimulation (or not) with 5 ng/ml TGFβ1 for 9 h. The data are graphed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g007" target="_blank">Fig. 7g, h</a>. (<b>d–f</b>) Real-time RT-PCR analysis of endogenous <i>fibronectin (FN1)</i> (d), <i>PAI-1</i> (e) and control <i>PARG</i> (f) mRNAs in HaCaT cells transiently transfected with the indicated siRNAs (bottom of panel f) prior to stimulation (or not) with 5 ng/ml TGFβ1 for 9 h. The data are graphed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g007" target="_blank">Fig. 7g, h</a>. Stars (panels b–g) indicate statistical significance, <i>p</i><0.05. The figure shows representative experiments from four or more repeats.</p