Interaction between PARP-1 and HIF-2α in the hypoxic response.

Hypoxia-inducible factors (HIFs) mediate the transcriptional adaptation of hypoxic cells. The extensive transcriptional programm regulated by HIFs involves the induction of genes controlling angiogenesis, cellular metabolism, cell growth, metastasis, apoptosis, extracellular matrix remodeling and others. HIF is a heterodimer of HIF-α and HIF-ß subunits. In addition to HIF-1α, HIF-2α has evolved as an isoform that contributes differently to the hypoxic adaptation by performing non-redundant functions. Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear protein involved in the control of DNA repair and gene transcription by modulating chromatin structure and acting as part of gene-specific enhancer/promoter-binding complexes. Previous results have shown that PARP-1 regulates HIF-1 activity. In this study, we focused on the cross-talk between HIF-2α and PARP-1. By using different approaches to suppress PARP-1, we show that HIF-2α mRNA expression, protein levels and HIF-2-dependent gene expression, such as ANGPTL4 and erythropoietin (EPO), are regulated by PARP-1. This regulation occurs at both the transcriptional and post-trancriptional level. We also show a complex formation between HIF-2α with PARP-1. This complex is sensitive to PARP inhibition and seems to protect against the von Hippel-Lindau-dependent HIF-2α degradation. Finally, we show that parp-1(-/-) mice display a significant reduction in the circulating hypoxia-induced EPO levels, number of red cells and hemoglobin concentration. Altogether, these results reveal a complex functional interaction between PARP-1 and the HIF system and suggest that PARP-1 is involved in the fine tuning of the HIF-mediated hypoxic response in vivo.

Modulation of transcription by PARP-1: consequences in carcinogenesis and inflammation.

Post-translational modification of proteins by poly(ADP-ribosyl)ation is involved in the regulation of a number of biological functions. While an 18 member superfamily of poly(ADP-ribose) polymerases (PARP)s has been described PARP-1 accounts for more than 90% of the poly(ADP-ribosyl)ating capacity of the cells. PARP-1 act as a DNA nick sensor and is activated by DNA breaks to cleave NAD(+) into nicotinamide and ADP-ribose to synthesize long branching poly(ADP-ribose) polymers (PAR) covalently attached to nuclear acceptor proteins. Whereas activation of PARP-1 by mild genotoxic stimuli facilitate DNA repair and cell survival, severe DNA damage triggers different pathways of cell death including PARP-mediated cell death through the translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus. PAR and PARP-1 have also been described as having a function in transcriptional regulation through their ability to modify chromatin-associated proteins and as a cofactor of different transcription factors, most notably NF-kappaB and AP-1. Pharmacological inhibition or genetic ablation of PARP-1 not only provided remarkable protection from tissue injury in various oxidative stress-related disease models but it result in a clear benefit in the treatment of cancer by different mechanisms including selective killing of homologous recombination-deficient tumor cells, down regulation of tumor-related gene expression and decrease in the apoptotic threshold in the co-treatment with chemo and radiotherapy. We will summarize in this review the current findings and concepts for the role of PARP-1 and poly(ADP-ribosyl)ation in the regulation of transcription, oxidative stress and carcinogenesis.

PARP-1-dependent 3-nitrotyrosine protein modification after DNA damage.

3-nitrotyrosine (NO2-Tyr) is thought to be a specific marker of cell injury during oxidative damage. We have evaluated the role of poly(ADP-ribose)polymerase-1 (PARP-1) in protein nitration after treatment of immortalized fibroblasts parp-1+/+ and parp-1-/- with the alkylating agent 2'-methyl-2'-nitroso-urea (MNU). Both cell lines showed increased iNOS expression following MNU treatment in parallel with a selective induction of tyrosine nitration of different proteins. PARP-1 deficient cells displayed a delayed iNOS accumulation, reduced number of nitrated proteins, and a lower global nitrotyrosine "footprint." We have identified the mitochondrial compartment as the major site of oxidative stress during DNA damage, being MnSOD one of the NO2-Tyr-modified proteins, but not in parp-1-/- cells. These results suggest that NO-derived injury can be modulated by proteins involved in the response to genotoxic damage, such as PARP-1, and may account for the limited oxidative injury in parp-1 knockout mice during carcinogenesis and inflammation.

Role of poly-(ADP-ribose) polymerase in transplant acute tubular necrosis and its relationship with delayed renal function.

The enzyme poly(ADP-ribose) polymerase (PARP-1) participates in the repair of DNA damaged by genotoxic agents such as oxygen-derived free radicals. If the allograft suffers pretransplant cold ischemia and subsequent ischemia-reperfusion injury (IR), overactivation of PARP-1 can be induced, which may lead to an increase in acute tubular necrosis (ATN) and a delay in total recovery of renal function (RRF) of the transplanted organ. We studied the nuclear expression of PARP-1 in tubular cells by immunohistochemistry with the monoclonal antibody PAR01 in 104 kidney transplant biopsies from allografts with ATN. In 50% of biopsies with ATN, >50% of tubular nuclei were PARP-1+; only 9.6% of biopsies were negative. The increase in the immunohistochemical expression of PARP-1 showed a statistically significant relationship with the duration of cold ischemia, with serum creatinine levels, and with the time required to achieve effective diuresis (P < .0001, Spearman test). Cold ischemia of >24 hours and serum creatinine levels >1.7 mg/dL showed a statistically significant relationship with the highest PARP-1 expression levels (2.83 +/- 0.4 vs 1.36 +/- 0.8, P < .0001, Mann-Whitney U test). We conclude that PARP-1 plays an important role in ATN and RRF and is related to the extent and severity of ATN and to the renal allograft function.

Role of poly (ADP-ribose) polymerase in kidney transplant and its relationship with delayed renal function: multivariate analysis.

UNLABELLED: Kidney allografts undergo pretransplant cold ischemia and consequent ischemia-reperfusion injury (IR). Poly (ADP-Ribose) polymerase (PARP-1) overactivation leads to massive NAD+ consumption and ATP depletion with induction of cellular necrosis under ischemic conditions, which may lead to an increase in acute tubular necrosis (ATN) and a delay in total recovery of renal function (RFR) of the transplanted organ. MATERIALS AND METHODS: Nuclear PARP-1 immunohistochemical expression (clone: PARP01) was studied in 155 paraffin-embedded renal biopsies from suboptimal donors and 95 kidney allograft biopsies with histopathological diagnosis of ATN. RESULTS: In 50% of ATN biopsies, more than 50% of tubular nuclei were immunostained for PARP-1. PARP-1 expression was higher in ATN biopsies than in those from suboptimal donors (2.40 +/- 0.74 vs 0.92 +/- 1.13, P = 0.0001 Mann-Whitney). PARP-1 showed a statistically significant relationship with the time required to achieve effective diuresis (Rho:0.779), with serum creatinine, and with duration of cold ischemia (Rho:0.803). These relationships were stronger in the biopsies with ATN. In conclusion, multivariate analysis demonstrated that PARP-1 expression and cold ischemia duration in kidney biopsies with ATN predicted the short-term delay in total recovery of renal function and serum creatinine in the first month.

Correlation of morphological findings with functional reserve in the aging donor: role of the poly (ADP-ribose) polymerase.

INTRODUCTION: The enzyme poly (ADP-ribose) polymerase (PARP-1) participates in the first events of DNA repair in higher organisms. Under conditions of tissue ischemia, this action can lead to significant decreases in NAD(+), massive adenosine triphosphate (ATP) depletion, and cell death. In renal grafts with pretransplantation cold ischemia and subsequent ischemia-reperfusion injury, overactivation of PARP-1 may lead to a higher index of acute tubular necrosis, a delay in total recovery of the function of the transplanted organ, and an early progression to chronic graft nephropathy. The present study examined whether increased tubular expression of PARP-1 in kidneys from aged donors contributed to recipient renal function. MATERIAL AND METHOD: We studied the nuclear expression of PARP-1 using immunohistochemistry with monoclonal antibody PAR01 in 75 kidney biopsy specimens from 40 aged donors. RESULTS: Immunohistochemical expression of PARP-1 showed a statistically significant relationship with donor age (r =.408, P =.006, Spearman test), with time required to achieve effective diuresis (r =.386, P =.01, Spearman test) and with creatinine levels in the first 3 months. We also highlighted a greater intensity of PARP-1 expression in suboptimal donor kidneys that failed to reduce the serum creatinine levels to <1.7 mg/dL (creatinine <1.7 PARP: 1.29 +/- 1.49 vs creatinine >1.7 PARP: 2.29 +/- 1.33, P =.047, Mann-Whitney U test). CONCLUSION: We conclude that the determination of PARP-1 in biopsy specimens from aged donors may be a useful predictive factor for renal graft function.