The functional role of poly(ADP-ribose)polymerase 1 as novel coactivator of NF-kappaB in inflammatory disorders.

Mammalian poly(ADP-ribose)polymerase 1 (PARP-1) is an abundant nuclear chromatin-associated protein and belongs to a large family of enzymes that catalyzes the transfer of ADP-ribose units from its substrate beta-nicotinamide adenine dinucleotide (NAD+) covalently to itself and other nuclear chromatin-associated proteins. PARP-1 knockout mice are protected against myocardial infarction, streptozotocin-induced diabetes, lipopolysaccharide-induced septic shock, and zymosan-induced multiple organ failure, indicating that PARP-1 is involved in the regulation of the pathogenesis of these disorders. PARP-1 and nuclear factor kappa B (NF-kappaB) have both been suggested to play a crucial role in inflammatory disorders. NF-kappaB encompasses a family of inducible transcription factors which play a crucial role in the regulation of genes involved in immune and inflammatory responses. Recent reports have shown that PARP-1 can act as a coactivator of NF-kappaB. These findings might provide new insights into the pathophysiology of different diseases such as type I diabetes and septic shock. The purpose of this review is to give a short overview of the current knowledge about PARP-1 and its functional and biochemical interactions with NF-kappaB. A more precise role for PARP-1 in NF-kappaB-dependent gene regulation and cellular metabolism during development of pathophysiological processes is discussed. Special considerations is given to the pathophysiological significance of these findings in terms of inflammatory disorders.

The enzymatic and DNA binding activity of PARP-1 are not required for NF-kappa B coactivator function.

Poly(ADP-ribose) polymerase 1 (PARP-1)-deficient mice are protected against septic shock, diabetes type I, stroke, and inflammation. We report that primary cells from PARP-1(-/-) animals are impaired in kappa B-dependent transcriptional activation induced by different stimuli involved in inflammatory and genotoxic stress signaling. PARP-1 was also required for p65-mediated transcriptional activation. PARP-1 enzymatic inhibitors did not inhibit the transcriptional activation of a kappa B-dependent reporter gene in wild type cells. Remarkably, neither the enzymatic activity nor the DNA binding activity of PARP-1 was required for kappa B-dependent transcriptional activation in PARP-1(-/-) cells complemented with different PARP-1 mutants. However, PARP-1 interacted in vitro directly with both subunits of NF-kappa B (p50 and p65), and mapping of the interaction domains revealed that both subunits bind to different PARP-1 domains. Furthermore, a PARP-1 mutant lacking the enzymatic and DNA binding activity interacted comparably to the wild type PARP-1 with p65 or p50. Finally, we showed that PARP-1 is activating the natural inducible nitric-oxide synthase and P-selectin promoter in a kappa B-dependent manner upon stimulation of the cells with inflammatory stimuli or cotransfection of p65. Our results provide evidence that neither the DNA binding nor the enzymatic activity of PARP-1 but its direct protein-protein interaction with both subunits of NF-kappa B is required for its coactivator function, thus expanding the role of PARP-1 as an essential and novel classical transcriptional coactivator for kappa B-dependent gene expression in vivo.

Regulation of human flap endonuclease-1 activity by acetylation through the transcriptional coactivator p300.

We describe a role for the transcriptional coactivator p300 in DNA metabolism. p300 formed a complex with flap endonuclease-1 (Fen1) and acetylated Fen1 in vitro. Furthermore, Fen1 acetylation was observed in vivo and was enhanced upon UV treatment of human cells. Remarkably, acetylation of the Fen1 C terminus by p300 significantly reduced Fen1's DNA binding and nuclease activity. Proliferating cell nuclear antigen (PCNA) was able to stimulate both acetylated and unacetylated Fen1 activity to the same extent. Our results identify acetylation as a novel regulatory modification of Fen1 and implicate that p300 is not only a component of the chromatin remodeling machinery but might also play a critical role in regulating DNA metabolic events.

Transcription coactivator p300 binds PCNA and may have a role in DNA repair synthesis.

The transcriptional coactivator p300 interacts with many transcription factors that participate in a broad spectrum of biological activities, such as cellular differentiation, homeostasis and growth control. Mouse embryos lacking both p300 alleles die around mid-gestation, with pleiotropic defects in morphogenesis, in cell differentiation and, unexpectedly, in cell proliferation because of reduced DNA synthesis. Here we show that p300 may have a role in DNA repair synthesis through its interaction with proliferating cell nuclear antigen (PCNA). We show that in vitro and in vivo p300 forms a complex with PCNA that does not depend on the S phase of cell cycle. A large fraction of both p300 and PCNA colocalize to speckled structures in the nucleus. Furthermore, the endogenous p300-PCNA complex stimulates DNA synthesis in vitro. Chromatin immunoprecipitation experiments indicate that p300 is associated with freshly synthesized DNA after ultraviolet irradiation. Our results suggest that p300 may participate in chromatin remodelling at DNA lesion sites to facilitate PCNA function in DNA repair synthesis.

A role of poly (ADP-ribose) polymerase in NF-kappaB transcriptional activation.

The transcription factor NF-kappaB plays a critical role in immune and inflammatory responses. Here we show that poly (ADP ribose) polymerase (PARP) is required for specific NF-kappaB transcriptional activation in vivo. The activation of the HIV-LTR promoter and an NF-kappaB-dependent artificial promoter was drastically reduced in PARP (-/-) cells, independently of the signaling pathway through which NF-kappaB was induced. Furthermore NF-kappaB-dependent gene activation was restored in vivo by the expression of PARP in PARP (-/-) cells. Finally, we show that both NF-kappaB and PARP formed a stable immunoprecipitable nuclear complex. This interaction did not need DNA binding. Our results suggest that PARP is an important cofactor in the activation cascade of NF-kappaB-dependent target genes.