Functional association between PARP-1 and NF-κB
The data presented in this study suggest that there is a defective NF-κB activation in the absence of PARP-1 that is not related to an impairment of the signal transduction pathway. The results in Figure 2A show that the rate of re-accumulation of the regulatory protein IκBα (whose gene is under the transcriptional control of NF-κB), is normal in PARP-1-deficient cells. This result suggests that the association of PARP-1 with NF-κB modulates the expression of certain genes under the control of NF-κB, but not all of them. Partial inhibition of NF-κB DNA binding alone seems not to be sufficient for down-regulation of the IκBα gene.
An earlier report suggested a decreased ability to trans-activate through NF-κB following inhibition of PARP-1 with 3-AB (Le Page et al., 1998), with consequences for iNOS gene transcription. In this respect, an increasing number of transcription factors or transcription coactivators have been reported to be associated with PARP-1, including AP-2 (Kannan, 1999), oct-1 (Nie et al., 1998), YY1 (Oei et al., 1997) and TEF-1 (Butler and Ordahl, 1999).
The molecular dissection of the interaction between PARP-1 and the transcriptional machinery of NF-κB still remains to be elucidated. Efforts to show a physical association between PARP-1 and p65 or p50 subunit by immunoprecipitation experiments were not conclusive (data not shown). One possibility might be that PARP-1 and NF-κB contact through a third protein involved in the architectural regulation of transcription, the high mobility group-I protein [HMG-I(Y)], which is a well known coactivator of NF-κB-dependent transcription (Thanos and Maniatis, 1995; Perrella et al., 1999) and a substrate for PARP-1 (Tanuma and Johnson, 1983). This is also an attractive possibility since PARP-1 has a high binding affinity for DNA bends (Gradwohl et al., 1987; Sastry and Kun, 1990) such as those induced in the NF-κB enhanceosome by HMG-I(Y) (Thanos and Maniatis, 1995).
While this publication was under review, a study by Hassa and Hottiger (1999) has also found a defective NF-κB activation in PARP-1−/− cells.
Resistance of PARP-1−/− mice to endotoxic shock
The response to endotoxin-induced septic shock has been tested in a number of genetically modified mice, including those for iNOS and TNF-α. The iNOS−/− mice were not resistant to LPS-induced death (Laubach et al., 1995; MacMicking et al., 1995), while TNF-α-deficient mice were resistant to the lethality of low doses of LPS (administered with D-gal), but not to high doses of endotoxin (Marino et al., 1997). In both cases, the persistence of LPS sensitivity could be explained by the existence of NO- and TNF-independent pathways to LPS-induced death. The resistance of PARP-1-deficient mice to septic shock is, however, more dramatic than that reported for iNOS and TNF-α knockout mice, and is similar to that found for interleukin-1β converting enzyme (ICE)-deficient mice (Li et al., 1995). Current hypotheses for the pathogenesis of septic shock are that microbial products such as LPS induce a massive production and release of TNF-α by macrophages, which in turn induce a cascade of cytokine production. TNF-α has profound effects on vascular endothelial cells, leading to cell adhesion, vascular leakage and shock (Gutierrez-Ramos and Bluethmann, 1997). As a consequence, neutralization of TNF-α prevents lethality in animal models of sepsis. The reduced synthesis of TNF-α (the gene for which is under the transcriptional control of NF-κB) in PARP-1-mutant mice might be relevant to explain the resistance to LPS-induced lethality in these mice.
Role of PARP-1 in the inflammatory response
Studies from several laboratories have noted the participation of PARP-1 in the inflammatory response, and different mechanisms have been proposed to explain that the inactivation of PARP-1 (either pharmacologically or using genetically engineered mice lacking PARP-1), improve in the animal the outcome of a variety of pathophysiological conditions associated with an exacerbated tissue or systemic inflammation (Szabo and Dawson, 1998).
The most extended model implicates PARP-1 in the following pathway: after an inflammatory stress (as LPS treatment induces in the model of endotoxic shock) or during reperfusion after cerebral ischemia, different cells, including macrophages and endothelial cells, activate a massive synthesis of NO, which is in turn converted into a cytotoxic derivative, peroxynitrite. Rapid DNA single-stranded breaks are induced by peroxynitrite, leading to over-activation of PARP-1 and depletion of cellular energy resulting in mitochondrial free radical generation and cell necrosis (Figure 6, ii) (Szabo et al., 1996, 1997).
Figure 6. PARP-1 is involved in the inflammatory response at two different levels: (i) activation of NF-κB and synthesis of proinflammatory factors and (ii) increasing the sensitivity of cells (particularly endothelial cells) to oxygen radicals produced during inflammation with consequences on DNA damage, energy depletion and necrosis (see Discussion). IKK, IκB kinase.
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In the present study, we have found a functional association between PARP-1 and NF-κB, a key regulatory molecule involved in multiple pathologies (Barnes and Karin, 1997; Kitamura et al., 1997), and particularly in inflammation (Wong et al., 1998). Through this association, PARP-1 may regulate NF-κB-dependent transcription, and the synthesis of inflammatory mediators (Figure 6, i). Thus, PARP-1 appears to promote inflammation at two levels, and through its effects on NF-κB and by mediating the cytotoxicity of NO-derivatives, one or both of these mechanisms might also explain the resistance of PARP-1−/− mice to brain ischemia, where synthesis of NO by different isoforms of NOS, TNF-α upregulation and NF-κB activation play a crucial role (Iadecola et al., 1995; Barone et al., 1997; Schneider et al., 1999).
Three recent studies carried out in PARP-1-deficient mice generated in two different laboratories have also shown an increased resistance of PARP-1-mutant mice to streptozotocin-induced diabetes (Burkart et al., 1999; Masutani et al., 1999; Pieper et al., 1999). Interestingly, as an autoimmune disease, the pathogenesis of diabetes is also related to inflammatory damage of pancreatic islet β-cells. Consequently, we propose that a common mechanism involving NF-κB activation is a critical molecular event involved in the pathogenesis of different diseases such as septic shock, brain ischemia, type-1 diabetes or arthritis, from which PARP-1-deficient mice are largely protected.
In conclusion, the data presented here, as a whole, demonstrate that PARP-1 is involved in the regulation of the NF-κB signalling pathway leading to synthesis of inflammatory mediators, and in the development of LPS-induced endotoxic shock. This finding might help to design new therapeutic strategies for treatment of septic shock, based on the combined pharmacological inhibition of NF-κB and PARP-1 (Szabo and Dawson, 1998), allowing the effective down-regulation of pro-inflammatory cytokines by turning off NF-κB-dependent transcription (Liu et al., 1997), and reducing cell injury through inhibition of PARP-1. In any case, all approaches that aim to eliminate PARP-1 or PARP-1 activity from the cell or the organism should take into account that since this enzyme is involved in genomic surveillance, its long-term inhibition might lead to the accumulation of DNA damage, mutations and oncogenic transformation.